METHODS OF DIAGNOSING INCREASED RISK OF DEVELOPING MRSA

Abstract

A method for diagnosing increased risk of developing Methicillin-resistant Staphylococcus aureus (MRSA) hospital-acquired (HA-MRSA) or community-acquired MRSA (CA-MRSA) which includes obtaining a biological sample from a subject, detecting in the sample a single nucleotide polymorphism (SNP) in the FAM129B gene at position 17 of SEQ ID NO 1, and comparing the nucleotide at position 17 of SEQ ID NO. 1 in the sample with the nucleotide at position 17 in SEQ ID NO. 1, wherein an adenine at position 17 of SEQ ID NO. 1 in the sample indicates an increased risk of developing MRSA or CA-MRSA in the subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application No. 61/779,307, filed Mar. 13, 2013, the entire contents of which are incorporated by reference.

SEQUENCE LISTING

[0002] This application incorporates in its entirety the Sequence Listing entitled "2014-03-10_5475-353958_056-US_SEQ_LISTING_ST25.txt" (96,447 bytes), which was created on Mar. 10, 2014, and filed electronically herewith.

BACKGROUND OF THE INVENTION

[0003] Staphylococcus aureus (staph) bacteria are a common component of the skin surface and lining of the nasal passageways in humans and other animals, and are usually spread by skin-to-skin contact. Methicillin-resistant S. aureus (MRSA) is a strain of S. aureus that has become resistant to methicillin, an antibiotic commonly used to treat ordinary S. aureus infections. As such, MRSA is particularly hard to treat. When limited to the skin surface and lining of the nasal passageways, S. aureus bacteria are normally harmless. However, S. aureus infections can occur in situations where the bacteria enter into the skin subsurface or body cavity, normally through wounds (including, e.g., surgical incisions) or other sites such as hair follicles.

[0004] MRSA infections often occur in patients having weakened immune systems who have been exposed to MRSA, such as patients in long term care, patients undergoing kidney dialysis, or patients recovering from recent surgery or medical treatments such as chemotherapy that weaken the immune system. Skin wounding events or other forms of compromise to skin integrity (e.g., intravenous drug use) are another major risk for MRSA infection, which risk may or may not coincide with the exposure risk. MRSA is often acquired or develops in medical care facilities, such as hospitals. This type of MRSA is known as health care-associated MRSA (HA-MRSA). But MRSA can also develop in otherwise healthy people not exposed to hospital situations. In these situations, it is termed community-acquired MRSA (CA-MRSA). CA-MRSA has often been found to be acquired by athletes (who may share towels and razors), children in day care, members of the military and people obtaining tattoos, for example.

[0005] Symptoms of staph infections may include red, swollen and painful boil-like symptoms, which may be treated locally. In severe cases and in hospital situations, staph infections may be systemic and must be treated with systemically administered antibiotics.

[0006] Some individuals seem to be uniquely susceptible to staph infections and developing recurrent CA-MRSA skin infections. Such populations may also have an elevated risk for developing MRSA if admitted to medical care facilities. Treatment for these individuals involves attempts to decolonize the skin and nasal passages of the patient with topical antibiotics, which can be temporarily effective, especially when coupled with improved sanitation such as frequent hand washing and isolation from other patients. Thus, improved methods of recognizing persons susceptible to recurrent MRSA and/or CA-MRSA are needed and can improve patient care and reduce the incidence of MRSA in these individuals.

SUMMARY OF THE INVENTION

[0007] We describe methods of predicting or assessing the level of risk of a subject developing a Methicillin-resistant S. aureus (MRSA) infection comprising: obtaining a biological sample from the subject, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; and predicting and assessing the level of risk of the subject developing a MRSA, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a high risk of developing a MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1, indicates that the subject has a low risk of developing a MRSA infection.

[0008] We describe embodiments wherein the detecting step further comprises hybridizing at least one oligonucleotide occupying a locus corresponding to position 17 of either SEQ ID. NO: 1, to an oligonucleotide probe comprising a sequence that is complementary or identical to SEQ ID NO. 1, under stringency conditions than can detect the presence of different alleles at position 17 of said oligonucleotide or hybridizing at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO: 2, to an oligonucleotide probe comprising a sequence that is complementary or identical to SEQ ID NO: 2, under stringency conditions that can detect the presence of different alleles at position 17 of said oligonucleotide.

[0009] We describe embodiments wherein the detecting step further comprises evaluating the hybridization of at least one oligonucleotide from the biological sample which corresponds to position 17 of SEQ ID. NO 1. and wherein the detecting step further comprises sequencing the oligonucleotide from the biological sample.

[0010] In other embodiments the detecting step further comprises amplifying the oligonucleotide from the biological sample. In some embodiments the amplifying step uses at least one oligonucleotide primer and at least one oligonucleotide from the biological sample occupying a locus corresponding to position 17 of SEQ. ID. NO. 1. The oligonucleotide primer may be comprised of DNA.

[0011] We describe methods to detect, identify and treat MRSA in all it various stages and forms including treatments make after it is determined whether the patient has a high or low risk of developing a MRSA, CA-MRSA infection or HA-MRSA and it is determined that the patient has a high risk of developing a MRSA infection, the patient is given anti MRSA antibiotics, and may also be given decolonization treatments. Once it is determined that the patient has a high risk of developing a MRSA infection, the patient may be given more than one course of anti MRSA antibiotics, decolonization treatments and may be put on high infection alert for any future surgeries.

[0012] In other embodiments after it is determined that the patient has a low risk of developing a MRSA infection, the patient is not given any antibiotics and is only treated with incision and drainage, sometimes such a low risk patient is treated with incision and drainage and given a routine antibiotic treatment that does not include anti MRSA antibiotics.

[0013] This application also describes and claims one or more primers or probes to be used to amplify or detect at least one or more nucleotides from a biological sample, wherein one or more nucleotides occupy a locus corresponding to position 17 of SEQ. ID. NO. 1. There may be one or more primers that spans the nucleotide positions about position 17 of SEQ. ID. NO. 1. The primers may be from about 8 to about 44 nucleotides in length, or from about 14 to about 50 nucleotides in length. Described are probes wherein said one or more probes span the nucleotide positions about position 17 of SEQ. ID. NO. 1. Described are probes having a different disruption energy for one allele as compared to another allele; two probes, wherein the first probe is a sensor probe and the second probe is an anchor probe; and a SNP-specific probe, in addition to methods to make and use these and other primers and probes.

[0014] In some embodiments one or more primers or probes are in a kit designed for use by a caregiver who seeks to predict or assess the level of risk of a subject developing Methicillin-resistant S. aureus (MRSA) infection, the kit and its instructions comprise obtaining a biological sample from the subject, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; and predicting and assessing the level of risk of the subject developing a MRSA infection, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a high risk of developing a MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a low risk of developing a MRSA infection.

[0015] Disclosed are methods to monitor and prepare a patient for surgery, wherein said monitoring and preparing comprising obtaining a biological sample from the patient, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of the at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; predicting and assessing the level of risk of the patient developing a MRSA infection, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the patient has a high risk of developing a MRSA or CA-MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the patient has a low risk of developing a MRSA or CA-MRSA infection; and wherein the predication and assessment indicates that the patient is at high risk for a MRSA infection, taking appropriate steps and care as one normally skilled in the art would take when operating on a person at high risk of developing a MRSA infection.

[0016] One aspect of the inventive method of determining whether a subject is at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA, may include: obtaining a biological sample from the subject, wherein said biological sample contains at least one oligonucleotide comprising a loci corresponding to position 17 of SEQ ID. NO 1; detecting the identity of each nucleotide that occurs at a loci corresponding to position 17 of comparison SEQ ID. NO 1 in said at least one oligonucleotide; and comparing the identity of each nucleotide that occurs at a loci corresponding to position 17 of comparison SEQ ID. NO 1 in said at least one oligonucleotide to the identity of the nucleotide at position 17 of SEQ ID. NO 1, wherein the subject is at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA if one or more nucleotides at the loci corresponding to position 17 of SEQ ID. NO 1 is the same as the identity of the nucleotide at position 17 of SEQ ID NO 1.

[0017] Another aspect of the inventive method of determining whether a subject is at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA, may include: obtaining a biological sample from the subject, wherein said biological sample contains at least one oligonucleotide comprising a loci corresponding to position 17 of SEQ ID. NO 2; detecting the identity of each nucleotide that occurs at a loci corresponding to position 17 of comparison SEQ ID. NO 2 in said at least one oligonucleotide; and comparing the identity of each nucleotide that occurs at a loci corresponding to position 17 of comparison SEQ ID. NO 2 in said at least one oligonucleotide to the identity of the nucleotide at position 17 of SEQ ID. NO 2, wherein the subject is not at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA if the one or more nucleotides at the loci corresponding to position 17 of SEQ ID. NO 2 in said at least one oligonucleotide is the same as the identity of the nucleotide at position 17 of SEQ ID NO 2.

[0018] In other aspects, the detecting step may further comprise hybridization of said at least one oligonucleotide comprising a loci corresponding to position 17 of SEQ ID NO. 1 to a probe comprising an oligonucleotide comprising a sequence complementary or identical to SEQ ID NO: 1, under stringency conditions than can detect the presence of different alleles at position 17 of said oligonucleotide. In other aspects, the detecting step may further comprise hybridization of said at least one oligonucleotide comprising a loci corresponding to position 17 of SEQ ID NO. 2 to a probe comprising an oligonucleotide comprising a sequence complementary or identical to SEQ ID NO. 2, under stringency conditions than can detect the presence of different alleles at position 17 of said oligonucleotide. In other aspects, the detecting step further comprises evaluating the hybridization of an oligonucleotide containing a locus corresponding to position 17 of comparison SEQ ID NO: 1 or 2 derived from said subject to a probe comprising a sequence complementary or identical to SEQ ID NO: 1 or 2, under stringency conditions that can determine the presence of different alleles at position 17 of said oligonucleotide.

[0019] In other aspects, the detecting step may further comprise amplifying at least one oligonucleotide from said biological sample containing a locus corresponding to position 17 of comparison SEQ ID. NO 1, wherein said amplifying step uses at least one oligonucleotide primer. In other aspects, the detecting step further comprises amplifying at least one oligonucleotide from said biological sample containing a locus corresponding to position 17 of comparison SEQ ID. NO 2, wherein said amplifying step uses at least one oligonucleotide primer. In some aspects, the oligonucleotide primer comprises DNA.

[0020] In other aspects, the determining step may further comprise using a probe to detect the presence of a locus corresponding to position 17 of comparison SEQ ID. NO 1 in the biological sample. In other aspects, the determining step may further comprise using a probe to detect the presence of a locus corresponding to position 17 of comparison SEQ ID. NO 2 in the biological sample.

[0021] In other aspects, the probe may be labeled with a detection signal. In other aspects, the probe may comprise an oligonucleotide having a sequence that is complementary or identical to a region flanking the locus corresponding to position 17 of comparison SEQ ID. NO 1 and/or 2, or the probe may be complementary or identical to SEQ ID NO 1 and/or 2.

[0022] In other aspects, the method may further comprise treating the subject with an antibiotic effective against MRSA, when the subject is found to be at increased risk of developing MRSA, CA-MRSA or having a recurrence of MRSA or CA-MRSA. In other aspects, the method may further comprise treating the subject to remove or prevent colonization by skin-surface or intranasal populations of MRSA, when the subject is found to be at increased risk of developing MRSA, CA-MRSA or having a recurrence of MRSA or CA-MRSA.

[0023] Embodiments of the present invention may comprise a kit for determining whether a subject is at increased risk of developing MRSA, CA-MRSA, or a recurrence of MRSA and/or CA-MRSA, comprising at least one primer for amplification of one or more nucleotides that occur at a loci corresponding to position 17 of comparison SEQ ID. NOS. 1, 2 or a combination thereof from a biological sample from the subject.

[0024] Other embodiments of the present invention may comprise a kit for determining whether a subject is at increased risk of developing MRSA, CA-MRSA, or a recurrence of MRSA and/or CA-MRSA, comprising at least one probe for detection of one or more nucleotides that occur at a loci corresponding to position 17 of comparison SEQ ID. NOS. 1, 2 or a combination thereof from a biological sample from the subject.

DETAILED DESCRIPTION

[0025] Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0027] All references, patents, patent publications, articles, and databases, referred to in this application are incorporated herein by reference in their entirety, as if each were specifically and individually incorporated herein by reference. Such patents, patent publications, articles, and databases are incorporated for the purpose of describing and disclosing the subject components of the invention that are described in those patents, patent publications, articles, and databases, which components might be used in connection with the presently described invention. The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.

[0028] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, embodiments, and advantages of the invention will be apparent from the description and drawings, and from the claims. The preferred embodiments of the present invention may be understood more readily by reference to the following detailed description of the specific embodiments and the Examples included hereafter.

[0029] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

DEFINITIONS

[0030] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[0031] In this specification and the appended claims, the singular forms "a," "an" and "the" include plural reference unless the context clearly dictates otherwise.

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press; DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).

[0033] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular DNA, polypeptide sequences or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0034] In describing the present invention, the following terms will be employed and are intended to be defined as indicated below.

[0035] The expression "anti-MRSA antibiotics" refers to antibiotics that one ordinarily skilled in the art would understand to be antibiotics usually used to treat antibiotic resistant infections like MRSA. Examples of such drugs include Vancomycin, Daptomycin, Linezolid, Ceftaroline, and Telavancin, among other antibiotics.

[0036] The term, "biological sample" means any material or fluid (blood, lymph, etc.) derived from the body of a subject, that contains or may contain genomic DNA (chromosomal and mitochondrial DNA) or other oligonucleotides such as, for example, mRNA that derive from genomic DNA. Also included within the meaning of the term "biological sample" is an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated. Methods of obtaining biological samples and methods of obtaining oligonucleotide molecules such as DNA and RNA from a biological sample are well known in the art.

[0037] As used herein, the terms "complementary" or "complementarity" are used in reference to oligonucleotides related by the base-pairing rules for DNA-DNA, RNA-DNA and RNA-RNA pairing. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A." Complementarity may be "partial," in which only some of the nucleic acid base pairs are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Under "low stringency" conditions, strands with a lower degree of complementarity will hybridize with each other. Under "high stringency conditions," only strands with a higher degree of complementarity will remain hybridized with each other.

[0038] "Complementary" may be modified, as in the term "completely complementary," refers to an oligonucleotide where all of the nucleotides are complementary to a target sequence (e.g., a miRNA). A completely complementary oligonucleotide may be shorter than the target sequence, thus, only hybridizing to a portion of the target.

[0039] "Complementary" may be modified, as in the term "partially complementary" refers to an oligonucleotide where at least one nucleotide is not complementary to (i.e., one or more "mismatches" with) the target sequence. Preferred partially complementary oligonucleotides are those that can still hybridize to a target sequence under physiological conditions. A particular partially complementary oligonucleotide may have a `random` pattern of one or more mismatches with the target sequence throughout the oligonucleotide (although the pattern of mismatches is preferentially constrained by retention of the ability to still hybridize to the target sequence under physiological conditions). A particular partially complementary oligonucleotide may have regions where the oligonucleotide sequence is highly, or even completely complementary to a target sequence, and regions where the oligonucleotide is not complementary, or is less complementary to the target sequence.

[0040] "Complementary" is illustrated, for example, partially complimentary oligonucleotides may have one or more regions that hybridize to a target sequence, and one or more regions that do not hybridize to the target sequence. Thus, a partially complementary sequence (such as a PCR or reverse transcriptase (RT) primer) may hybridize to a portion (i.e., the middle, the 5', or 3' end) of a particular target sequence, and not hybridize with the rest of the target sequence. Oligonucleotides with mismatches at the ends may still hybridize to the target sequence. Partially complementary sequences may be capable of binding to a sequence having less than 60%, 70%, 80%, 90%, 95%, to less than 100% identity to the target sequence. For purposes of defining or categorizing partially complementary sequences, a partially complementary sequence or region of a sequence becomes more complementary or becomes "highly complementary" as it approaches 100% complementarity to a target sequence. Thus, a highly complementary sequence may have 60%, 70%, 80%, 90%, 95%, to 99% identity to all or a portion of a target sequence. The exact percentage identity of the highly complementary sequence may depend on the length of the highly complementary sequence and the desired stringency and specificity of hybridization. Partially complementary sequences may hybridize to one or more target sequences. As we note, partially complementary sequences may be completely complementary or highly complementary to a portion of the target sequence, such that they are completely or highly complementary to, e.g., 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, 99% of the target sequence. Similarly, 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 95%, 99% of the partially complementary sequence may be completely complementary or highly complementary to all or a portion of the target sequence.

[0041] A "cyclic polymerase-mediated reaction" refers to a biochemical reaction in which a template molecule or a population of template molecules is periodically and repeatedly copied to create a complementary template molecule or complementary template molecules, thereby increasing the number of the template molecules over time.

[0042] By the term "detectable moiety" is meant, for the purposes of the specification or claims, a label molecule (isotopic or non-isotopic) which is incorporated indirectly or directly into an oligonucleotide, wherein the label molecule facilitates the detection of the oligonucleotide in which it is incorporated, for example when the oligonucleotide is hybridized to amplified gene polymorphic sequences. Thus, "detectable moiety" is used synonymously with "label molecule". Synthesis of oligonucleotides can be accomplished by any one of several methods known to those skilled in the art. Label molecules, known to those skilled in the art as being useful for detection, include chemiluminescent, fluorescent or luminescent molecules. Various fluorescent molecules are known in the art which are suitable for use to label a nucleic acid for the method of the present invention. The protocol for such incorporation may vary depending upon the fluorescent molecule used. Such protocols are known in the art for the respective fluorescent molecule.

[0043] A "diagnosis" of MRSA or CA-MRSA may include the early detection of the disease or a confirmation of a diagnosis of the disease that has been made from other signs and/or symptoms. A "diagnosis" can include a diagnosis of increased risk of development or recurrence of MRSA or CA-MRSA. A diagnosis may include a "prognosis," that is, a future prediction of the progression of MRSA or CA-MRSA, based on the presence or absence of one or more SNPs associated with MRSA or CA-MRSA. A diagnosis or prognosis may be based on one or more samplings of DNA or RNA from a biological sample obtained from a subject. An "increased risk" of developing MRSA or CA-MRSA may be diagnosed by the presence of one or more SNPs characteristic of a phenotype of susceptibility to recurrent CA-MRSA in otherwise asymptomatic or undiagnosed subjects.

[0044] "DNA amplification" as used herein refers to any process that increases the number of copies of a specific DNA sequence by enzymatically amplifying the nucleic acid sequence. A variety of processes are known. One of the most commonly used is polymerase chain reaction (PCR). PCR involves the use of a thermostable DNA polymerase, known sequences as primers, and heating cycles, which separate the replicating deoxyribonucleic acid (DNA), strands and exponentially amplify a gene of interest. Any type of PCR, such as quantitative PCR, RT-PCR, hot start PCR, LAPCR, multiplex PCR, touchdown PCR, real-time PCR, etc., may be used. In general, the PCR amplification process involves a cyclic enzymatic chain reaction for preparing exponential quantities of a specific nucleic acid sequence. It requires a small amount of a sequence to initiate the chain reaction and oligonucleotide primers that will hybridize to the sequence. In PCR, the primers are annealed to denatured nucleic acid followed by extension with an inducing agent (enzyme) and nucleotides. This results in newly synthesized extension products. Since these newly synthesized sequences become templates for the primers, repeated cycles of denaturing, primer annealing, and extension results in exponential accumulation of the specific sequence being amplified. The extension product of the chain reaction will be a discrete nucleic acid duplex with a termini corresponding to the ends of the specific primers employed.

[0045] A DNA "coding sequence" or a "nucleotide sequence encoding" a particular protein is a DNA sequence that is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. "Non-coding" genomic sequences may include regulatory, RNA transcription sequences (rRNA, tRNA, miRNA, etc.), introns and other non-gene sequences, such as structural sequences, putatively non-functional sequences ("junk DNA") and the like.

[0046] The terms "enzymatically amplify", "enzymatically amplifying", "amplify" and "amplifying" is meant, for the purposes of the specification or claims, DNA amplification, i.e., a process by which nucleic acid sequences are amplified in number. There are several means for enzymatically amplifying nucleic acid sequences. Currently the most commonly used method is the polymerase chain reaction (PCR). Other amplification methods include LCR (ligase chain reaction) which utilizes DNA ligase, and a probe consisting of two halves of a DNA segment that is complementary to the sequence of the DNA to be amplified, enzyme QB replicase and a ribonucleic acid (RNA) sequence template attached to a probe complementary to the DNA to be copied which is used to make a DNA template for exponential production of complementary RNA; strand displacement amplification (SDA); Q.beta.-replicase amplification (Q.mu.RA); self-sustained replication (3 SR); and NASBA (nucleic acid sequence-based amplification), which can be performed on RNA or DNA as the nucleic acid sequence to be amplified.

[0047] A "fragment" of a molecule such as a protein or nucleic acid is meant to refer to a portion of a longer or larger amino acid or nucleotide genetic sequence.

[0048] The term "genome" refers to all the genetic material in the chromosomes of a particular organism. Its size is generally given as its total number of base pairs. Within the genome, the term "gene" refers to a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions. "Non-gene regions" of the genome have or appear to have no functional role, but may have a structural (e.g., regions near the centromere) or unknown regulatory function. The physical development and phenotype of organisms can be thought of as a product of genes interacting with each other and with the environment. A concise definition of "gene" taking into account complex patterns of regulation and transcription, sequence conservation and non-coding RNA genes has been proposed by Gerstein et al. (Genome Research 17 (6), 669-681, 2007) "A gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products". In general, an individual's genetic characteristics, as defined by the nucleotide sequence of its genome, are known as its "genotype," while an individual's physical traits are described as its "phenotype."

[0049] By "heterozygous" or "heterozygous polymorphism" is meant that the two alleles of a diploid cell or organism at a given locus are different, that is, that they have a different nucleotide exchanged for the same nucleotide at the same place in their sequences.

[0050] By "homozygous" or "homozygous polymorphism" is meant that the two alleles of a diploid cell or organism at a given locus are identical, that is, that they have the same nucleotide for nucleotide exchange at the same place in their sequences.

[0051] By "hybridization" or "hybridizing," as used herein, is meant the formation of A-T and C-G base pairs between the nucleotide sequence of a fragment of a segment of a oligonucleotide and a complementary nucleotide sequence of an oligonucleotide. By complementary is meant that at the locus of each A, C, G or T (or U in a ribonucleotide) in the fragment sequence, the oligonucleotide sequenced has a T, G, C or A, respectively. The hybridized fragment/oligonucleotide is called a "duplex."

[0052] A "hybridization complex", such as in a sandwich assay, means a complex of nucleic acid molecules including at least the target nucleic acid and a sensor probe. It may also include an anchor probe.

[0053] A hybridization complex may be related to where two nucleic acid fragments are considered to be "selectively hybridizable" to a oligonucleotide if they are capable of specifically hybridizing to a nucleic acid or a variant thereof or specifically priming a polymerase chain reaction: (i) under typical hybridization and wash conditions, as described, for example, in Sambrook et al. supra and Nucleic Acid Hybridization, supra, (ii) using reduced stringency wash conditions that allow at most about 25-30% base pair mismatches, for example: 2.times.SSC, 0.1% SDS, room temperature twice, 30 minutes each; then 2.times.SSC, 0.1% SDS, 37.degree. C. once, 30 minutes; then 2.times.SSC room temperature twice, 10 minutes each, or (iii) selecting primers for use in typical polymerase chain reactions (PCR) under standard conditions (described for example, in Saiki, et al. (1988) Science 239:487-491).

[0054] A hybridization complex may be ratted to the term "capable of hybridizing under stringent conditions" as used herein refers to annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. Stringent hybridization conditions typically permit the hybridization of nucleic acid molecules having at least 70% nucleic acid sequence identity with the nucleic acid molecule being used as a probe in the hybridization reaction. For example, the first nucleic acid may be a test sample or probe, and the second nucleic acid may be the sense or antisense strand of a nucleic acid or a fragment thereof. Hybridization of the first and second nucleic acids may be conducted under stringent conditions, e.g., high temperature and/or low salt content that tend to disfavor hybridization of dissimilar nucleotide sequences. Alternatively, hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g. low temperature and/or high salt content that tend to favor hybridization of dissimilar nucleotide sequences. Low stringency hybridization conditions may be followed by high stringency conditions or intermediate medium stringency conditions to increase the selectivity of the binding of the first and second nucleic acids. The hybridization conditions may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO) or formamide to disfavor still further the hybridization of dissimilar nucleotide sequences. A suitable hybridization protocol may, for example, involve hybridization in 6.times.SSC (wherein 1.times.SSC comprises 0.015 M sodium citrate and 0.15 M sodium chloride), at 65.degree. Celsius in an aqueous solution, followed by washing with 1.times.SSC at 65.degree. C. Formulae to calculate appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch between two nucleic acid molecules are disclosed, for example, in Meinkoth et al. (1984) Anal. Biochem. 138: 267-284; the content of which is herein incorporated by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and standard molecular biology manuals may be consulted to select a suitable hybridization protocol without undue experimentation. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, the contents of which are herein incorporated by reference in their entirety.

[0055] A hybridization complex may refer to "stringent conditions" which typically will be those in which the salt concentration is less than about 1.5 M sodium ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) from about pH 7.0 to about pH 8.3 and the temperature is at least about 30.degree. Celsius for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree. Celsius, and a wash in 1-2.times.SSC at 50 to 55.degree. Celsius. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. Celsius, and a wash in 0.5-1.times.SSC at 55 to 60.degree. Celsius. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. Celsius, and a wash in 0.1.times.SSC at 60 to 65.degree. Celsius.

[0056] An "isolated" oligonucleotide or polypeptide is one that is substantially pure of the materials with which it is associated in its native environment. By substantially free, is meant at least 50%, at least 55%, at least 60%, at least 65%, at advantageously at least 70%, at least 75%, more advantageously at least 80%, at least 85%, even more advantageously at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, most advantageously at least 98%, at least 99%, at least 99.5%, at least 99.9% free of these materials.

[0057] An "isolated" nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences (as defined below) in association therewith.

[0058] As used herein, the term "locus" or "loci" refers to the location of a coding, regulatory or non-coding region on a chromosome. Absolute location of a region may be known to more or less precision (i.e., a locus is known to be located within a chromosome, chromosome arm, chromosome band, or to the nearest kilobase or base pair of a chromosome) due to the fact that genome length may differ slightly between individuals or the precise location of a locus is not known. A relative location may also be specified where a locus is located within a sequenced fragment of a chromosome. Pairs of genes, known as "alleles" may be present for a particular locus in organisms, such as humans, that are diploid (usually contain two copies of most chromosomes) in most cells and tissues. An individual's particular combination of alleles is referred to as its "genotype". Where both alleles are identical the individual is said to be homozygous for the trait controlled by that gene pair; where the alleles are different, the individual is said to be heterozygous for the trait. While inclusive of loci within coding regions, an "allele" may also be present at locations in non-coding regions. Certain organisms, cells or tissues may be haploid or polyploid (triploid, etc.) and have more or less than two alleles at a particular locus.

[0059] A "melting temperature" is meant the temperature at which hybridized oligonucleotide duplexes dehybridize and return to their single-stranded state. Likewise, hybridization will not occur in the first place between two oligonucleotides, or, herein, an oligonucleotide and a fragment, at temperatures above the melting temperature of the resulting duplex. It is presently advantageous that the difference in melting point temperatures of oligonucleotide-fragment duplexes of this invention be from about 1 degree C. to about 10 degrees C. so as to be readily detectable.

[0060] The term, "MRSA" means Methicillin-resistant S. aureus, it is a strain of S. aureus that has become resistant to methicillin, an antibiotic commonly used to treat ordinary S. aureus infections. When MRSA is acquired or develops in medical care facilities, such as hospitals, it is known as health care-associated MRSA (HA-MRSA). When MRSA develops in otherwise healthy people not exposed to hospital situations it is termed community-acquired MRSA (CA-MRSA). Here it should be understood that Methicillin-resistant S. aureus or MRSA includes HA-MRSA and CA-MRSA.

[0061] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but advantageously is double-stranded DNA. "DNA" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.

[0062] A "nucleoside" refers to a base linked to a sugar. The base may be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugar may be ribose (the sugar of a natural nucleotide in RNA) or 2-deoxyribose (the sugar of a natural nucleotide in DNA). A "nucleotide" refers to a nucleoside linked to a single phosphate group.

[0063] The term "oligonucleotide" refers to a series of linked nucleotide residues. The series of nucleotide residues are connected by a phosphodiester linkage between the 3'-hydroxyl group of one nucleoside and the 5'-hydroxyl group of a second nucleoside which in turn is linked through its 3'-hydroxyl group to the 5'-hydroxyl group of a third nucleoside and so on to form a polymer comprised of nucleosides linked by a phosphodiester backbone. Oligonucleotides may be used, for example, as primers in a PCR reaction, or as probes to detect the presence of a certain sequence in or within a nucleic acid molecule. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides may be chemically synthesized and may be used as primers or probes. A "modified oligonucleotide" refers to an oligonucleotide in which one or more natural nucleotides have been partially, substantially, or completely replaced with modified nucleotides.

[0064] The term "oligonucleotide encoding a protein" as used herein refers to a DNA fragment or isolated DNA molecule encoding a protein, or the complementary strand thereto; but, RNA is not excluded, as it is understood in the art that thymidine (T) in a DNA sequence is considered equal to uracil (U) in an RNA sequence. Thus, RNA sequences for use in the invention, e.g., for use in RNA vectors, can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

[0065] The following are non-limiting examples of oligonucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant oligonucleotides, branched oligonucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A oligonucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracil, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the oligonucleotide to proteins, metal ions, labeling components, other oligonucleotides or solid support.

[0066] "Percent identity" can be determined by hybridization of oligonucleotides under conditions that form stable duplexes between similar regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al. supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0067] A "polymerase" is an enzyme that catalyzes the sequential addition of monomeric units to a polymeric chain, or links two or more monomeric units to initiate a polymeric chain. The "polymerase" will work by adding monomeric units whose identity is determined by and which is complementary to a template molecule of a specific sequence. For example, DNA polymerases such as DNA pol 1 and Taq polymerase add deoxyribonucleotides to the 3' end of a oligonucleotide chain in a template-dependent manner, thereby synthesizing a nucleic acid that is complementary to the template molecule. Polymerases may be used either to extend a primer once or repetitively or to amplify an oligonucleotide by repetitive priming of two complementary strands using two primers. A "thermostable polymerase" refers to a DNA or RNA polymerase enzyme that can withstand extremely high temperatures, such as those approaching 100.degree. C. Often, thermostable polymerases are derived from organisms that live in extreme temperatures, such as Thermus aquaticus. Examples of thermostable polymerases include Taq, Tth, Pfu, Vent, deep vent, UlTma, and variations and derivatives thereof.

[0068] A "primer" is an oligonucleotide, the sequence of at least of portion of which is complementary to a segment of a template DNA which is to be amplified or replicated. Typically primers are used in performing the polymerase chain reaction (PCR). A primer hybridizes with (or "anneals" to) the template DNA and is used by the polymerase enzyme as the starting point for the replication/amplification process. The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.

[0069] "Probes" refer to oligonucleotides nucleic acid sequences of variable length, used in the detection of identical, similar, or complementary nucleic acid sequences by hybridization. An oligonucleotide sequence used as a detection probe may be labeled with a detectable moiety.

[0070] "Sequence identity" refers to the percent identity between two oligonucleotide or two polypeptide moieties. Genes that share a high sequence identity or similarity support the hypothesis that they share a common ancestor and are therefore homologous. Sequence homology may also indicate common function. Two DNA, or two polypeptide sequences are similar to each other and may be homologous when the sequences exhibit at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, preferably at least about 90%, 91%, 92%, 93%, 94% and most preferably at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity over a defined length of the molecules. As used herein, sequence identity also refers to sequences showing complete identity (100% sequence identity) to the specified DNA or polypeptide sequence.

[0071] A "restriction fragment" refers to a fragment of a oligonucleotide generated by a restriction endonuclease (an enzyme that cleaves phosphodiester bonds within a oligonucleotide chain) that cleaves DNA in response to a recognition site on the DNA. The recognition site (restriction site) consists of a specific sequence of nucleotides typically about 4-8 nucleotides long.

[0072] A "template" refers to a target oligonucleotide strand, for example, without limitation, an unmodified naturally-occurring DNA strand, which a polymerase uses as a means of recognizing which nucleotide it should next incorporate into a growing strand to polymerize the complement of the naturally-occurring strand. Such a DNA strand may be single-stranded or it may be part of a double-stranded DNA template. In applications of the present invention requiring repeated cycles of polymerization, e.g., the polymerase chain reaction (PCR), the template strand itself may become modified by incorporation of modified nucleotides, yet still serve as a template for a polymerase to synthesize additional oligonucleotides.

[0073] A "thermocyclic reaction" is a multi-step reaction wherein at least two steps are accomplished by changing the temperature of the reaction.

[0074] A "variance" is a difference in the nucleotide sequence among related oligonucleotides. The difference may be the deletion of one or more nucleotides from the sequence of one oligonucleotide compared to the sequence of a related oligonucleotide, the addition of one or more nucleotides or the substitution of one nucleotide for another. The terms "mutation," "polymorphism" and "variance" are used interchangeably herein. As used herein, the term "variance" in the singular is to be construed to include multiple variances; i.e., two or more nucleotide additions, deletions and/or substitutions in the same oligonucleotide.

[0075] A "single nucleotide polymorphism" or "SNP" refers to a variation in the nucleotide sequence of an oligonucleotide that differs from another related oligonucleotide by a single nucleotide difference. For example, without limitation, exchanging one A for one C, G or T in the entire sequence of oligonucleotide constitutes a SNP. It is possible to have more than one SNP in a particular oligonucleotide. For example, at one position in an oligonucleotide, a C may be exchanged for a T, at another position a G may be exchanged for an A and so on. When referring to SNPs, the oligonucleotide is most often DNA. SNPs can be found in coding regions of the genome (i.e., within an exon) or non-coding intragenic (i.e., in an intron) or intergenic regions.

[0076] RNA sequences within the scope of the invention are derived from the DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

[0077] "Subject" or "Patient" as used herein refers to a mammal, preferably a human, in need of diagnosis and/or treatment for a condition, disorder or disease.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Single Nucleotide Polymorphisms

[0079] Analysis of single nucleotide polymorphisms (SNPs) have proved to be effective in discovering genomic differences (i.e., genotypes) in individuals or populations exhibiting different phenotypes, such as susceptibility or increased risk of contracting certain diseases or syndromes. With the advent of rapid sequencing, amplification and high throughput screening of oligonucleotides, analysis of SNPs can be used to probe individual and population genomes for one or more SNPs that correlate with (are "markers" for) the presence of a certain phenotype. Discovery of one or more reliably correlating markers (among other uses) allows for early diagnosis of potential susceptibility or risk for certain diseases, even where a phenotype is not yet being exhibited in a particular individual, e.g., a late-onset cancer or susceptibility to a disease organism prior to exposure. Here, we disclose that a SNP at a loci corresponding to position 17 of SEQ ID. NOs 1 or 2 (the FAM129B gene) can be manipulated, evaluated and then used to differentiate, identify and in some cases treat subjects who are at increased risk of MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA.

[0080] Often, SNP detection methods can distinguish between homozygous and heterozygous individuals. The presence of zero, zero or one, one, one or two, or two copies of a particular base substitution allele may correlate with a particular phenotype. "Dominant," "recessive" and "intermediate dominance"/"incomplete dominance" of a particular allele at a locus can be defined as the relative contribution of each allele to the phenotype of a heterozygous individual. For example, where a heterozygous individual carrying one copy of allele A and one copy of allele A' has the same phenotype as a homozygous AA individual, and a different phenotype from a homozygous A'A' individual, allele A is dominant over allele A'. Where a heterozygous individual AA' exhibits a different or intermediate phenotype between the homozygote phenotypes, allele A' and A are said to exhibit an intermediate or incomplete dominance.

[0081] While SNPs occur at particular locations in the genome, presentation, identification and comparison of the location of particular SNPs is aided by inclusion of the sequence of nucleotides immediately upstream and downstream in the genome. SNP detection methods using oligonucleotide hybridization methods may use the same sequence as presented herein as all or part of a primer or probe sequence, and thus such sequences may serve as examples of an appropriate primer or probes for these methods. Persons of skill in the art realize that design of appropriate primers and probes, examples of which are provided below, are not necessarily limited to the sequences listed herein for purposes of presentation, and may be longer or shorter, and include more or less of the upstream and downstream flanking sequence(s), as long as they encompass the location of a SNP.

[0082] Methods and materials of the invention may be used more generally to evaluate a DNA sample from a subject, genetically type the subject, and detect genetic differences between subjects. In one embodiment of the invention, a biological sample which includes DNA from a subject is evaluated to detect the genotype of the subject for a nucleotide that occurs at a loci corresponding to position 17 of SEQ ID. NOs 1 or 2. A sample of genomic DNA from a subject may be evaluated by reference to one or more controls to determine if a SNP or group of SNPs is present. With this present invention, any method for determining genotype can be used for determining the genotype of the subject. Such methods include, but are not limited to, amplimer sequencing, DNA sequencing, fluorescence spectroscopy, fluorescence resonance energy transfer (or "FRET")-based hybridization analysis, high throughput screening, mass spectroscopy, microsatellite analysis, nucleic acid hybridization, polymerase chain reaction (PCR), RFLP analysis and size chromatography (e.g., capillary or gel chromatography), all of which are well known to one of skill in the art. In particular, methods for determining nucleotide polymorphisms, particularly single nucleotide polymorphisms, are described in U.S. Pat. Nos. 6,514,700; 6,503,710; 6,468,742; 6,448,407; 6,410,231; 6,383,756; 6,358,679; 6,322,980; 6,316,230; and 6,287,766 and reviewed by Chen and Sullivan, Pharmacogenomics J 2003; 3(2):77-96, the disclosures of which are incorporated by reference in their entireties. Genotypic data useful in the methods of the invention and methods for the identification and selection of genes associated with CA-MRSA are based on the presence of SNPs.

A Single Nucleotide Polymorphism Associated with CA-MRSA

[0083] Genomic DNA was obtained from a population of patients with recurrent CA-MRSA as well as from healthy spouse controls, who were likely to have been exposed to the same MRSA bacteria as the recurrent CA-MRSA patients. A microarray hybridization assay for single nucleotide polymorphism (SNP) alleles that segregated between the CA-MRSA and control populations was performed that was capable of detecting the presence of 906,000 known polymorphisms as well as their copy number in each subject (i.e., that could detect, whether the subject had multiple gene copies, and whether the subject was homozygous or heterozygous for particular allele(s) at a particular locus.)

[0084] A highly segregated SNP was found in the FAM129B gene, where all CA-MRSA subjects tested had two copies (homozygous) of one allele and control subjects had two copies (homozygous) of another allele. This SNP is present at a loci corresponding to position 17 of SEQ ID NOs. 1 and 2, wherein each of SEQ ID NOs. 1 and 2 identify an alternate oligonucleotide at that position. More specifically, SEQ ID NO. 1 identifies an "A" (adenine) at position 17 and SEQ ID NO. 2 identifies a "C" (cytosine) at position 17. This SNP is located in an intron of the FAM129B gene. SEQ ID NOs. 1 and 2 generally correspond with positions 17444-17464 of SEQ ID NO. 3, the full-length DNA sequence of the FAM129B gene.

[0085] Subjects with at least one or more copies of the allele corresponding to position 17 of SEQ ID. NO 1 are at increased risk of developing at developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA. And subjects with at least one or more copies of the allele corresponding to position 17 of SEQ ID. NO 2 are not at increased risk of developing at developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA.

[0086] The FAM129B gene encodes a protein that has a predicted molecular mass of 83 kDa, and contains a pleckstrin homology domain and a proline-rich region that contains six serine phosphorylation sites (Chen et al (2011) J. Biol. Chem. 286(12):10201-10209; Old et al. (2009) Mol. Cell 34: 115-131). Phosphorylation has been associated with MAP kinase signaling cascade; in melanoma cells the MAP kinase pathway was active and the FAM129B protein was localized throughout the cytoplasm. When the MAP kinase pathway was inhibited, the FAM129B protein migrated to the cell membrane and melanoma cell migration through a collagen matrix was inhibited. (Old et al., p. 125). Subsequent work found that FAM129B was cytoplasmically localized in actively growing HeLa cells, but appeared to be localized at cell-cell junctions on the plasma membrane when the HeLa cells achieved confluence, and throughout the cell membrane during telophase. (Chen et al. pp. 10203-10204.) FAM129B also inhibited apoptosis in HeLa cells treated with TNF.alpha. or CHX, compared with knockdown FAM129B HeLa cells silenced with siRNA sequences specific to FAM129B. A recent investigation of the corresponding Fam129B protein in mice showed that Fam129B is expressed in the epidermal keratinocytes in embryonic and adult mice. Fam129B-knockout mice exhibited delayed wound healing and had altered expression of several wound-repair and cell-motility related genes (Oishi et al. (published online Sep. 11, 2012), J. Biochem. doi:10.1093/jb/mvs 100).

Methods of Diagnosing Increased Risk of Developing MRSA

[0087] Aspects of the present invention comprise methods of determining whether a subject is at increased risk of developing MRSA or CA-MRSA, or a recurrence of MRSA or CA-MRSA, comprising: obtaining a biological sample from a subject; obtaining at least one oligonucleotide from said biological sample that contains a loci corresponding to position 17 of SEQ ID. NOs 1 and 2; detecting in the oligonucleotide the identity of a nucleotide that occurs at a loci corresponding to position 17 of SEQ ID. NOs 1 and 2; and comparing the identity of the nucleotide that occurs at a loci corresponding to position 17 of SEQ ID. NOs 1 and 2 in the oligonucleotide to the identity of a nucleotide at position 17 of SEQ ID. NO 1 and/or SEQ ID. NO 2, wherein the subject is at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA if the nucleotide that occurs at the loci corresponding to position 17 of SEQ ID. NOs 1 and 2 in the oligonucleotide is the same as the identity of the nucleotide at position 17 of SEQ ID NO 1, and wherein the subject is not at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA if the nucleotide that occurs at the loci corresponding to position 17 of SEQ ID. NOs 1 and 2 in the oligonucleotide is the same as the identity of the nucleotide at position 17 of SEQ ID NO 2.

[0088] Obtaining Oligonucleotides from Subjects.

[0089] Biological samples may be any material or fluid (blood, lymph, etc.) derived from the body of a subject, that contains or may contain genomic DNA (chromosomal and mitochondrial DNA) or other oligonucleotides such as, for example, mRNA that derive from genomic DNA, or an organ or tissue extract and culture fluid in which any cells or tissue preparation from a subject has been incubated. Methods of obtaining biological samples and methods of obtaining oligonucleotide molecules such as DNA and RNA from a biological sample are well known in the art, such as blood draws, cheek cell swabs, biopsies and the like.

[0090] For purposes of obtaining at least one oligonucleotide from said biological sample that contains a loci corresponding to position 17 of SEQ ID. NO 1 and 2, DNA or other oligonucleotides, such as pre-mRNA, can be extracted or partially purified from the biological sample for further processing by techniques known to those skilled in the art (see, e.g., U.S. Pat. Nos. 6,548,256 and 5,989,431; Hirota et al. (1989) Jinrui Idengaku Zasshi. 34: 217-23 and John et al. (1991) Nucleic Acids Res. 19:408, the disclosures of which are incorporated by reference in their entireties). For example, high molecular weight DNA may be purified from cells or tissue using proteinase K extraction and ethanol precipitation. DNA, however, may be extracted from an animal specimen using any other suitable methods known in the art.

[0091] Alternatively, a purification step may be not be needed where probes such as those described below may operate to detect the presence of a SNP by directly hybridizing to genomic DNA in situ in the biological sample, such that obtaining at least one oligonucleotide from said biological sample that contains a loci corresponding to position 17 of SEQ ID. NO 1 and 2 may occur without an oligonucleotide extraction step from the biological sample. The biological sample may be partially processed (i.e., homogenization, partial purification) prior to hybridization to facilitate the hybridization step.

Detecting SNP Polymorphisms

[0092] Any method of detecting the identity of individual nucleotides at SNP loci may be used to practice this invention.

[0093] In one aspect, detecting the identity of the SNP corresponding to position 17 of SEQ ID NOs:1 and 2 of the present invention may be performed by sequencing the region of the genomic DNA sample that spans the FAM 129B polymorphic locus. Many methods of sequencing genomic DNA are known in the art, and any such method can be used, see for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. For instance, as described below, a DNA restriction fragment spanning the location of the SNP of interest can be amplified using the polymerase chain reaction, then subjected to further genomic sequencing methods.

[0094] In other aspects, detecting the identity of the SNP corresponding to position 17 of SEQ ID NOs:1 and 2 of the present invention may be performed by the use of allele-specific probes that hybridize to a region of DNA containing the allele of interest. The probes may be further tagged with a detection signal to aid in detecting the presence of the allele in the biological sample. Probes and detection signals are described below.

A. Amplification

[0095] A genomic oligonucleotide spanning the location of the SNP of interest in the FAM129B gene may also be amplified as part of the detection step. More specifically, detecting the identity of SNP of the present invention may comprise DNA amplification to amplify specific, genomic sequences containing the SNP correlated to healthy and/or recurrent CA-MRSA subject phenotypes, by one of several known methods of DNA amplification, such as PCR. As noted above, the PCR amplification process involves a cyclic enzymatic chain reaction for preparing exponential quantities of a specific nucleic acid sequence. It requires a small amount of a sequence to initiate the chain reaction and oligonucleotide primers that will hybridize to the sequence. In PCR the primers are annealed to denatured nucleic acid followed by extension with an inducing agent (enzyme) and nucleotides. This results in newly synthesized extension products. Since these newly synthesized sequences become templates for the primers, repeated cycles of denaturing, primer annealing, and extension results in exponential accumulation of the specific sequence being amplified. The extension product of the chain reaction will be a discrete nucleic acid duplex with a termini corresponding to the ends of the specific primers employed.

[0096] The methods of the present invention may use oligonucleotide primers to amplify specific, genomic sequences containing the SNP correlated to healthy and/or recurrent CA-MRSA subject phenotypes. Such primers should be of sufficient length to enable specific annealing or hybridization to the nucleic acid sample. The sequences typically will be about 8 to about 44 nucleotides in length. Longer sequences, e.g., from about 14 to about 50, may be advantageous for certain embodiments. The design of primers is well known to one of ordinary skill in the art. Primers may comprise sequences upstream or downstream of the location of the SNP, but not contain the SNP itself (begin or end at, e.g., 1-1000 base pairs upstream or downstream of the location of the SNP), or comprise a sequence comprising the SNP. Such primers may be used to specifically amplify one allele or another at that SNP location. In any case, primers should be designed such that the SNP is contained within the amplified sequence. For instance, suitable primers may be designed using sequences within SEQ ID NO:3 (the FAM129B gene) upstream or downstream from the location of the SNP at position 17 of SEQ ID NOs 1 and 2.

[0097] Where it is desired to amplify a fragment of DNA that comprises a SNP according to the present invention, the forward and reverse primers may have contiguous stretches of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any other length up to and including about 50 nucleotides in length. The sequences to which the forward and reverse primers anneal are advantageously located on either side of the particular nucleotide position that is substituted in the SNP to be amplified (e.g., position 17 of SEQ ID NOs 1 and 2).

[0098] Oligonucleotide primers can be produced by a conventional production process for general oligonucleotides. They can be produced, for example, by a chemical synthesis process or by a microbial process that makes use of a plasmid vector, a phage vector or the like. Further, it is suitable to use a nucleic acid synthesizer.

B. Oligonucleotide Sequencing

[0099] As noted above, detecting the identity of the SNP corresponding to position 17 of SEQ ID NOs. 1 and 2 of the present invention may be performed by sequencing the region of the genomic DNA sample that spans the FAM 129B polymorphic locus. Reagents allowing the sequencing of reaction products can be utilized herein. For example, chain-terminating nucleotides will often be incorporated into a reaction product during one or more cycles of a reaction. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. PCR exonuclease digestion methods for DNA sequencing can also be used. Many methods of sequencing genomic DNA are known in the art, and any such method can be used, see for example Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. For example, as described below, a DNA fragment spanning the location of the SNP of interest can be amplified using the polymerase chain reaction or some other cyclic polymerase mediated amplification reaction. The amplified region of DNA can then be sequenced using any method known in the art. Advantageously, the nucleic acid sequencing is by automated methods (reviewed by Meldrum, (2000) Genome Res. 10: 1288-303, the disclosure of which is incorporated by reference in its entirety), for example using a Beckman CEQ 8000 Genetic Analysis System (Beckman Coulter Instruments, Inc.). Methods for sequencing nucleic acids include, but are not limited to, automated fluorescent DNA sequencing (see, e.g., Watts & MacBeath, (2001) Methods Mol. Biol. 167: 153-70 and MacBeath et al. (2001) Methods Mol. Biol. 167:119-52), capillary electrophoresis (see, e.g., Bosserhoff et al. (2000) Comb Chem High Throughput Screen. 3: 455-66), DNA sequencing chips (see, e.g., Jain, (2000) Pharmacogenomics. 1: 289-307), mass spectrometry (see, e.g., Yates, (2000) Trends Genet. 16: 5-8), pyrosequencing (see, e.g., Ronaghi, (2001) Genome Res. 11: 3-11), and ultrathin-layer gel electrophoresis (see, e.g., Guttman & Ronai, (2000) Electrophoresis. 21: 3952-64), the disclosures of which are hereby incorporated by reference in their entireties. The sequencing can also be done by a commercial company. Examples of such companies include, but are not limited to, the University of Georgia Molecular Genetics Instrumentation Facility (Athens, Ga.) or SeqWright DNA Technologies Services (Houston, Tex.).

C. Oligonucleotide Hybridization

[0100] Detecting the identity of a SNP corresponding to position 17 of SEQ ID NOs. 1 and 2 of the present invention may be performed by the use of allele-specific probes that hybridize to a region of DNA containing the allele of interest.

[0101] One example method for determining the genotype at the polymorphic locus encompasses obtaining a biological sample that includes a nucleic acid sample, hybridizing the nucleic acid sample with a probe, and disrupting the hybridization to determine the level of disruption energy required wherein the probe has a different disruption energy for one allele as compared to another allele. In one example, there can be a lower disruption energy, e.g., melting temperature, for an allele that harbors a cytosine residue at a polymorphic locus, and a higher required energy for an allele with a different residue at that polymorphic locus. This can be achieved where the probe has 100% sequence identity with one allele (a perfectly matched probe), but has a single mismatch with the alternative allele. Since the perfectly matched probe is bound more tightly to the target DNA than the mismatched probe, it requires more energy to cause the hybridized probe to dissociate.

[0102] In a further step of the above method, a second ("anchor") probe may be used. Generally, the anchor probe is not specific to either allele, but hybridizes regardless of what nucleotide is present at the polymorphic locus. The anchor probe does not affect the disruption energy required to disassociate the hybridization complex but, instead, contains a complementary label for using with the first ("sensor") probe.

[0103] Hybridization stability may be influenced by numerous factors, including thermoregulation, chemical regulation, as well as electronic stringency control, either alone or in combination with the other listed factors. Through the use of stringency conditions, in either or both of the target hybridization step or the sensor oligonucleotide stringency step, rapid completion of the process may be achieved. This is desirable to achieve properly indexed hybridization of the target DNA to attain the maximum number of molecules at a test site with an accurate hybridization complex. By way of example, with the use of stringency, the initial hybridization step may be completed in ten minutes or less, more advantageously five minutes or less, and most advantageously two minutes or less. Overall, the analytical process may be completed in less than half an hour.

[0104] In one mode, the hybridization complex is labeled and the step of determining the amount of hybridization includes detecting the amounts of labeled hybridization complex at the test sites. The detection device and method may include, but is not limited to, optical imaging, electronic imaging, imaging with a CCD camera, integrated optical imaging, and mass spectrometry. Further, the amount of labeled or unlabeled probe bound to the target may be quantified. Such quantification may include statistical analysis. The labeled portion of the complex may be the target, the stabilizer, the probe or the hybridization complex in toto. Labeling may be by fluorescent labeling selected from the group of, but not limited to, Cy3, Cy5, Bodipy Texas Red, Bodipy Far Red, Lucifer Yellow, Bodipy 630/650-X, Bodipy R6G-X and 5-CR 6G. Colorimetric labeling, bioluminescent labeling and/or chemiluminescent labeling may further accomplish labeling. Labeling further may include energy transfer between molecules in the hybridization complex by perturbation analysis, quenching, electron transport between donor and acceptor molecules, the latter of which may be facilitated by double stranded match hybridization complexes. Optionally, if the hybridization complex is unlabeled, detection may be accomplished by measurement of conductance differential between double stranded and non-double stranded DNA. Further, direct detection may be achieved by porous silicon-based optical interferometry or by mass spectrometry. In using mass spectrometry no fluorescent or other label is necessary. Rather detection is obtained by extremely high levels of mass resolution achieved by direct measurement, for example, by time of flight (TOF) or by electron spray ionization (ESI). Where mass spectrometry is contemplated, probes having a nucleic acid sequence of 50 bases or less are advantageous.

[0105] The label may be amplified, and may include, for example, branched or dendritic DNA. If the target DNA is purified, it may be un-amplified or amplified. Further, if the purified target is amplified and the amplification is an exponential method, it may be, for example, PCR amplified DNA or strand displacement amplification (SDA) amplified DNA. Linear methods of DNA amplification such as rolling circle or transcriptional runoff may also be used.

[0106] A detectable label can be incorporated into a nucleic acid during at least one cycle of an amplification reaction. Spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means can detect such labels. Useful labels in the present invention include fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase etc.), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. The label is coupled directly or indirectly to a component of the assay according to methods well known in the art. As indicated above, a wide variety of labels are used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. Non-radioactive labels are often attached by indirect means. Polymerases can also incorporate fluorescent nucleotides during synthesis of nucleic acids.

[0107] To label an oligonucleotide with the fluorescent dye, one of several conventionally known labeling methods can be used (Tyagi & Kramer (1996) Nature Biotechnology 14: 303-308; Schofield et al. (1997) Appl. and Environ. Microbiol. 63: 1143-1147; Proudnikov & Mirzabekov (1996) Nucl. Acids Res. 24: 4532-4535). Alternatively, the oligonucleotide may be labeled with a radiolabel e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, etc. Well-known labeling methods are described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press. The label is coupled directly or indirectly to a component of the oligonucleotide according to methods well known in the art. Reversed phase chromatography or the like used to provide a nucleic acid probe for use in the present invention can purify the synthesized oligonucleotide labeled with a marker. An advantageous probe form is one labeled with a fluorescent dye at the 3'- or 5'-end and containing G or C as the base at the labeled end. If the 5'-end is labeled and the 3'-end is not labeled, the OH group on the C atom at the 3'-position of the 3'-end ribose or deoxyribose may be modified with a phosphate group or the like although no limitation is imposed in this respect.

[0108] During the hybridization of the nucleic acid target with the probes, stringent conditions may be utilized, advantageously along with other stringency affecting conditions, to aid in the hybridization. Detection by differential disruption is particularly advantageous to reduce or eliminate slippage hybridization among probes and target, and to promote more effective hybridization. In yet another aspect, stringency conditions may be varied during the hybridization complex stability determination so as to more accurately or quickly determine whether a SNP is present in the target sequence.

[0109] A SNP-specific probe can also be used in the detection of the SNP in amplified specific nucleic acid sequences of the target gene FAM129B, such as the amplified PCR products generated using the primers described above. In certain embodiments, these SNP-specific probes consist of oligonucleotide fragments. Advantageously, the fragments are of sufficient length to provide specific hybridization to the nucleic acid sample. The use of a hybridization probe of between 10 and 50 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 12 bases in length are generally advantageous, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of particular hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having stretches of 16 to 24 nucleotides, or even longer where desired. A tag nucleotide region may be included, as at the 5' end of the primer that may provide a site to which an oligonucleotide sequencing primer may hybridize to facilitate the sequencing of multiple PCR samples.

[0110] The probe sequence must span the particular nucleotide position that may be substituted in the particular SNP to be detected, here, position 17 of SEQ ID. NOs 1 and 2. Advantageously, two or more different "allele-specific probes" may be used for analysis of a SNP, a first allele-specific probe for detection of one allele, and a second allele-specific probe for the detection of the alternative allele. For example, one probe could be used for detection of the adenosine at position 17 of SEQ ID NO. 1 and another probe could be used for detection of cytosine at position 17 of SEQ ID NO. 2.

[0111] It will be understood that this invention is not limited to the particular primers and probes disclosed herein and is intended to encompass at least nucleic acid sequences that are hybridizable to the nucleotide sequence disclosed herein, the complement or a fragment thereof, or are functional sequence analogs of these sequences. Homologs (i.e., nucleic acids derived from other species) or other related sequences (e.g., paralogs) can be obtained under conditions of standard or stringent hybridization conditions with all or a portion of the particular sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

[0112] Advantageously, probes may be affixed to substrates and used in "microarray" and other high-throughput detection applications such as those used in the Example below and which are well known in the art. Microarrays can show the presence of one or both SNP alleles, copy number (such as whether an individual is homozygotic, or heterozygotic for a particular polymorphism), and thus provide a genotype for an individual subject.

D. Subjects at Increased Risk of CA-MRSA

[0113] In one embodiment of the present invention, subjects with at least one or more copies of the allele corresponding to position 17 of SEQ ID. NO 1 are at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA. In another embodiment of the present invention, subjects with at least one or more copies of the allele corresponding to position 17 of SEQ ID. NO 2 are not at increased risk of developing at developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA.

[0114] Other SNPs, or other biomarkers, such as gene or protein biomarker, miRNA and the like, the levels or presence/absence of which are correlated with increased risk of developing MRSA or CA-MRSA, or the occurrence of recurrent MRSA or CA-MRSA may also be used alone or in conjunction with the SNP of the present invention to diagnose subjects who are at increased risk of developing MRSA and/or CA-MRSA, or a recurrence of MRSA or CA-MRSA. Other SNPs or biomarkers may identify a structurally or functionally abnormal FAM129B gene caused by a point mutation(s), a deletion, a truncation, or a translocation of at least a portion of the FAM129B gene. An exemplary biomarker may identify and/or detect the presence of (a) a decrease or an increase in expression of the FAM129B gene (as compared to a control group which is not at an increased risk of developing MRSA or CA-MRSA, or recurrent MRSA or CA-MRSA) or (b) the abnormal methylation of at least a part of the FAM129B gene. In one embodiment, methods to detect a structurally or functionally abnormal FAM129B gene may include using an oligonucleotide primer that is complementary to or identical to a portion of SEQ ID NO: 3 to amplify an oligonucleotide sample from a subject (and the amplified oligonucleotides may then be sequenced); or hybridizing oligonucleotides in a sample from a subject to an oligonucleotide probe having a sequence that is complementary to or identical to a portion of SEQ ID NO: 3.

Treatment of Patients at Increased Risk of Recurrent CA-MRSA

[0115] In some aspects of the method, subjects found to be at increased risk of developing MRSA, HA-MRSA or CA-MRSA or having a recurrence of MRSA, HA-MRSA or CA-MRSA may be treated with an antibiotic effective against MRSA. In other aspects, subjects found to be at increased risk of developing MRSA or having a recurrence of MRSA can be treated with appropriate topical and/or nasal treatments to remove surface colonies of or prevent colonization by skin-surface or intranasal populations of MRSA. Appropriate treatments may be increased sanitation (more frequent hand washing with regular or antibiotic soaps such as Hibiclens (4% clorhexidine), topical antibiotic treatments, and oral antibiotics, mouth rinses and nasal ointments containing antibiotics. See, e.g., Buehlmann, M. et al. "Highly effective regimen for decolonization of methicillin-resistant Staphylococcus aureus carriers" Infect. Control. Hosp. Epidemiol. (2008) 29(8); 510-6.

[0116] A more aggressive treatment of a MRSA, HA-MRSA or CA-MRSA patient may involve the administration of an antibiotic regime including the repeated and/or prophylactic use of one or more anti-MRSA antibiotics such as Vancomycin, Daptomycin, Linezolid, Ceftaroline, Telavancin, Bactrim and the like. Treatment of MRSA patients often includes decolonization efforts, frequent monitoring, long term follow-up and special treatment for any further surgeries (e.g. surgical prescreening for MRSA and antibiotic treatment for prophylaxis) including long duration evaluation and monitoring for infection. Treatment for patents at low risk of MRSA infection could involve as little as incision and drainage followed by administration of a common antibiotic or in some cases with no antibiotic at all.

[0117] Kits comprising the methods and devices of the MRSA risk assessment described here in are also described and would be known to one skilled in the art given the descriptions provided.

[0118] Some embodiments of the present invention may comprise a kit for determining whether a subject is at increased risk of developing MRSA, or a recurrence of MRSA comprising at least one primer for amplification of one or more nucleotides that occur at a loci corresponding to position 17 of comparison SEQ ID. NOS. 1, 2 or a combination thereof from a biological sample from the subject.

[0119] Other embodiments of the present invention may comprise a kit for determining whether a subject is at increased risk of developing MRSA or CA-MRSA, or a recurrence of MRSA and/or CA-MRSA, comprising at least one probe for detection of one or more nucleotides that occur at a loci corresponding to position 17 of comparison SEQ ID. NOS. 1, 2 or a combination thereof from a biological sample from the subject.

[0120] Some embodiments of the invention may comprise one or more probes for use in determining whether a subject is at increased risk of developing MRSA or CA-MRSA, or a recurrence of MRSA or CA-MRSA, wherein the one or more probes comprise the oligonucleotide(s) described by SEQ ID NOs. 1 and/or 2. Other embodiments of the invention may comprise an amplification product for use in determining whether a subject is at increased risk of developing MRSA or CA-MRSA, or a recurrence of MRSA or CA-MRSA, wherein the amplification product comprises an oligonucleotide sequence comprising SEQ ID NO. 1 and/or 2. Other embodiments of the invention may comprise amplification primers for use in determining whether a subject is at increased risk of developing MRSA or CA-MRSA, or a recurrence of MRSA or CA-MRSA, wherein the amplification primers comprise oligonucleotide sequences in SEQ ID NO:3 immediately flanking the location of SEQ ID NO:1 and 2; comprise SEQ ID NO:1 or 2 and oligonucleotide sequences in SEQ ID NO:3 immediately flanking the location of SEQ ID NO:1 and 2; or comprise SEQ ID NO:1 or 2.

[0121] The invention will now be further described by way of the following non-limiting examples.

EXAMPLES

Example 1

Study Design

[0122] Fourteen participants were contacted and consented to collection of blood samples for analysis. Collection and analysis were approved through the Beaumont Institutional Review Board. Eleven participants were patients who were seen for recurrent community acquired MRSA skin infections (CA-MRSA) but had no known specific risk factors for developing recurrent infection. Three participants were controls and were cohabiting spouses of three of the patients. This gave controls who were directly and closely exposed to the patient (i.e., shared a bed) and were thus within the same environment but did not become infected with CA-MRSA.

Example 2

Methods

[0123] Collection and analysis was done via the Beaumont BioBank. Analysis was performed in an automated and blinded manner. Genomic DNA from all participants was prepared for analysis using Affymetrix Genome-wide Human SNP 6.0 microarrays. Each array contains more than 946,000 probes for detection of copy number variation and more than 906,000 single nucleotide polymorphism (SNP) probes for genotyping. One array per patient sample was prepared according to the manufacturer's protocol and scanned with an Affymetrix GeneChip.RTM. Scanner 3000. Affymetrix Genotyping Consol software and the Partek Genomics Suite were used for analysis and visualization of the data.

[0124] Data was subjected to per SNP and per sample quality control to minimize false positives. None of the remaining samples from individuals were excluded based on the expression data. SNPs from X and Y chromosomes were excluded from further analysis. SNPs with no call rates <5% and minor alleles frequencies >5% were included for further analysis. The final number of SNPs included in the analysis was 633,268.

[0125] A chi-square test was used to set the phenotype to be tested for association with the SNPs. Three models were tested:

[0126] 1. Allele: frequencies of alleles (A vs. A') were compared between CA-MRSA subjects and the control subjects

[0127] 2. Genotype: frequencies of three possible genotypes (AA, AA', and A'A') were compared between CA-MRSA subjects and the control subjects

[0128] 3. Dominant/Recessive: two combinations of genotypes are compared between CA-MRSA subjects and the control subjects--Dominant (AA+AA' vs. A'A', with A as the causal variant) and Recessive (AA vs. AA'+A'A', with A as the causal variant)

Example 3

Results

[0129] The analysis using each model revealed several potential SNPs of interest, but the most significant (p value 1.21.times.10-7) was located within the open reading frame of a gene identified as FAM129B. This SNP (SNP_A 8307872, rs2249861) was present with two copies of a single form in all 11 MRSA patients and with two copies of another form in all three controls. There were no participants who were heterozygous (one copy of the gene in each form).

[0130] The particular SNP in gene FAM129B which segregated between the control and CA-MRSA populations was located in an intron sequence. The SNP has the following sequence in CA-MRSA subjects: GGGGGCAAGTTAGTCAACCTGTCTGAGTCTTAG [SEQ ID NO:1] with the SNP location at position 17 underlined. Control populations had the alternate allele: GGGGGCAAGTTAGTCACCCTGTCTGAGTCTTAG [SEQ ID NO. 2] at position 17.

Sequence CWU 1

1

3133DNAHomo sapiens 1gggggcaagt tagtcaacct gtctgagtct tag 33233DNAHomo sapiens 2gggggcaagt tagtcaccct gtctgagtct tag 33373651DNAHomo sapiens 3caggaactgg gccagttccg gtcccttcct tttggggctc tcactctgga ggatggggtg 60gatgggaggt aagacttgtg cacaagtccc caggacacaa ggcacacggg gttctttttt 120gtttgtttgt ttagagagga ggttttgctc tgtcactcag gctggagtgc agcggaatga 180tcatagctca atgcagcctc aaactcctgg gctcaaggga tcctcacttc ttgacctccc 240aaagcgtcat aggcttaagg cactacagca cccactgaca aggggttctt tttctttttc 300ttttcttttc tttttttttt ttaggcggag tttcactctt gtcaccccgg ctggactgca 360atggcgagat ctcgcctcac tctcactgca acctccgcct cccagattca agcgattctc 420ctgcctcagc ctcctgagta gctgggatga cagggcccca ccgccatgcc cagctaattt 480ttttgtattt ttagtagaga cggtttcacg atgttggcca ggctggtctt gaactcctga 540gctcagatga tccacctgcc tcggtctcct aaagtgctgg gattacaggt gtaagccatg 600gcgcctggcc actttttttt tttttttttt tttttgagac caagtctcac tctgttgccc 660aggctggagt gcagtggctg gatcttggct cactgcatcg tctgcctcct gggttcaagg 720gattcctcat gcctcagcct cccggatggg atttcaggca caggccacta cgcccagcta 780aatttttttt tttttttttg agatggagtc ttgctctgtc tctcaggctg gagtgcagtg 840gcgcgatctc agctcactgc aacctccgcc tcccagatac aagtgattct cctgcttcag 900cctcccaagt agatgggatt tcaggcaccc accaccacac ccagctaatt tttttgtgtt 960tttagtagag acagggtttc actgtgttgg ccaggctggt ctcaaattcc tgacattgtg 1020agccacctgc ctcagcctcc caaagtgctg agattacaag tgtgagccac tgtgactggc 1080ccaacatttt tttttaatta aaaaattaaa gccaggtgta gtggtgcatg cctgtagtcc 1140cagctactca ggaggctggg tcgggaggat tgtttgagcc caggagtttg aggctgcagc 1200aagccatgat caactcactg cactccagcc tgggttacag agcaagaggt cttatccctg 1260aaacaaacaa acaaacaaaa agatatagca gagaagccca ttttgtgttt gagaaagagc 1320tgaactctag gattggatct ggaggatggt gtgggaagcg tggggtattg gatctggagg 1380atggtgtggg aagcgtgggg tattggatct ggaggatggt gtgggaagcg tggggtattg 1440gatctggagg atggtgtggg aagcgtgggg tattggatct ggaggatggt gtgggaagtg 1500tggggtattg gatctggagg atggtgtggg aagtgtgggg tattggatct ggaggatggt 1560gtgggaagtg tggggtattg gatctggagg atggtgtggg aagcgtgggg tattgcagct 1620ctaaagtagg cagagctatg tatgggtctc atcctcatct cttaattttt ttggggggag 1680ggtggacaca gtctcgctct gtcacccagg ctgcagtaca gtggcgcaat ctcggctcac 1740tgcaacccct gcctctctgg ttcaagtgat tcttgtgcct cagcctcccg agtagctggg 1800attacagacg tgcaccacca cgcctggcta atttttgtat ttttagtaga gacgggcttt 1860caccatgttg gccaggctgg tctcaaactc ccgacctcag gtgatccgcc cacctcggcc 1920tctgaaagtg cgtgagccac tgcacctggc ctcatctgtt actttaaaat aaaataacaa 1980taaattattt aaaaatagag gctgggcatg gtgccttaca cctgtaatcc cagcactttg 2040ggaggccgag gcaggtggat cacaaggtta ggagtttgag actagtctgg tcaacatggt 2100gaaaccccgt ctctactaaa aatacaaaaa ttagccggac gttgtggcac ttggggaggc 2160tgaggttgca gtaagctgag atggcgccac tacactccag cctggtgaca gagcaagact 2220ctgtcttggg aaaaaaaaaa atagagacga ggtcttgcta tgttgcccag gctggtctca 2280aactcctggg ctcaagcgat cctcctgcct cagcctccca aagtgttggg attacaggcg 2340tgagccactg tgcctgatct catcctcatc tcttacttat acctcatgaa gcctcagttt 2400tctctcctgt aaagtgggtg ttccccaaaa gttgtgacca ttcaatgagg cagaagctat 2460aaagcactga actgagcaca ggagcctgca atgaacgggc gcttccatta tctttatcat 2520cctgaagtta tttctcactc cctgcctgtc tgctggcact tcagcggccc cctgcctgac 2580ttcagtgctg ttggatccaa ccacggaatt tgtgcctggg aaggcggtaa ggcaccccct 2640tcctggaagt ttacaagcag aagctctgca cagtagcaga ggcccacatg cactatagaa 2700gggactttgt accccatgat gtgtcatggc cactgcaggc atgagtgttt cagtataggt 2760ctatgaacaa acctgtgagg tcccaaggac caggtgggtc cctcactgtc acgttcccct 2820ccagtgccca ggatagtgtt ctgcacatca tacgtgctcc ctcaagctcc cattgcgact 2880gacgctagtg atgtcatgtc gaaggaggac agtcctggtc ctcagagaga aagatttatt 2940taaacagata attgcacaca cacaaaatca tgttgaatgt gatgcttgcc atgaaggagg 3000agtttaggac actggggagg gggtgataat aatggcaggc ctgacctagt aggggggcag 3060ggtgcagaaa tgtcctctct gatgaagggt aattagctga ggttgaaggc tgaatgctgc 3120ttggtggggc aagatgcggg aaaaaaatcc atgtttcaca cagccaggca cggtggctca 3180cgcctgtaat cccagcactg tgggagccaa ggcgggcgga tcacctgagc tcaggagttt 3240gagaccagcc tgatcaatat ggtgaaaccc tgtctctact aaaaatacca gaattaacca 3300ggcatagtgg cgggtgcctg taatcccagc tactcaggag gctgaggcag gagaatcgct 3360tgagcctggg aggtggaggt tgcagtgagc agagatcccg ccactgcact ccagcctgga 3420cgacagagca agactccatc tcaaaagaaa aaaaaaaaaa aaagggccca gcacagtggc 3480tcatgcctgt agtcccagca ctttgggaag ctggggtggg tggatcacga ggtcaggagt 3540tcaagaccat cctggccaag atggtaaaac cccgtctcta ctaaaaatac caaaattagc 3600caggcgtggt ggcaggaacc tgtaatccca gctacttgag agtctgaggc agagaattgc 3660ttgaacccag gaggctgagt ttgcagtgag ccgagatcgc accactgcac tccagcctgg 3720gtgacagagc gagactccat ctcaaaaaaa aaaaaaaaag aatccatatt tcataagctg 3780tgaggtggga tggagcaggg ctagtttgag gatgtgaaat ctcagtgtgg ctagagaatg 3840gggaggggaa gtggggggag ggactggcag gggcccccag tgccatgtct ggagtctttc 3900tctccaggac agtggaagcc acagaaggtt agaagtgggg gaatggcaag gtcgtatctg 3960tgtttacaac aattcctctg gctcccagtg aggaatgcat ggggggctgg aggcagggag 4020gccagttccc tggacttggg gtggcagtgg agctggggag aggtgacagc ttggccactg 4080aaagagtaag agggagttta gcagcctgag gccaggtgac agacggaaca ggctgtaagc 4140tacggacctt tccaccaccc aacagttaat aaagagagga ggtccgggct gggtgccagg 4200gtcccttctg cacaatgggc catgccaact gggtaaacag aggtagatac atgatattcc 4260ttaaaaacaa aatcctgatt cattcacact ccttgctgtt tactggaggt gtttgttttt 4320tttctcctgg tctatttcag gtttgacagg tttgtgggga gagcagagct gggactccgt 4380gccaactggg agggagaggg actcatgggg agagaaaggg cctctccttc ttcctacagg 4440actagcaagg ggtcttgggc tgggctcctg cggaatgctc ttgggcaaat tgctttcctt 4500ctctgagtct cggtccaagc tctggctctg accgtctgaa ccctcccttt taaagccaaa 4560tattattatt cttcgccctt ggatccctgc ttccaaatat gcttgaggat gtggtgggca 4620ggggaggggc ggggcaggtt tgcagggagg cagtgggcag aagccacttc cagagaaagg 4680cctgatgagt caggacctgt aggtggcatt ctctgttcaa gaggggcagg cccagcaagt 4740gggcagggag aacctttcct ctctgctcca gatgtagacc attgccaccc tttgtccaca 4800gccctggcat tttcaaccag gttctggggc ctgagccaag aaaagggagt gggagcccct 4860ggtgaataga gacagcgtcc agagtcttag aaaaagaaca gatatccctc tgccttgcag 4920atttcagaaa tccttgctat agaaagaact cttacaaatc aataagaaaa agatagacat 4980tcccacagaa aagcaggcag aggatataaa ttttaaaaac cacaaatggc caggctgggt 5040acggtggctc atacctgtaa tcccagcact ttgagaggcc aagggaggca gatcacttgt 5100ggtcaggagt tccagaccag cctggccaac atagcaaaac cccatttcca ctaaaaatac 5160aaaaattagc caggcgtggt ggcaggtgcc tgtaatccca gctacttggg aagctgaggc 5220aggagaattg cttgaaccca gaaggtaggt agagattgca gtgagcagag atcgtgccac 5280tgcactctag cctgggcaac agaacgagac cctgtctcga aataaacaaa ataaaaataa 5340aacccacaaa tggccactaa atgtgtgaaa agatgtttcc atttactcaa aagtaacaag 5400gggacatcaa aatggcctcc agcgtgaggg caggggtttc cagcatttta gagttgagaa 5460attctctgat gtcaaacttc ctggaatcct tcatagtgct gatgactatg tttatttttc 5520tttccttgaa aaaaaaaaaa gggcaaataa cttcattcct tccccaccca aagactgtaa 5580ctttttttta ttacaataat tgatttctta acatatagta gagagagttg agaaagttta 5640aagagcttta caatctttcc agagtgaaaa aaaaaaacaa caaattctgt tctggatgct 5700caggctgcct ccccatctgg ggtcccacca gtccatgcca gggcccaccc ctccttccct 5760gcaggaagtc ctagatcagc catctggctt aggccccaca aggactcacc ttgcttcctt 5820ttgctcacac tcacgccttt acctgtcacg ccttctctgt gcctacctgt tccagtgttt 5880gaacaaggat tgctgtatac atacaatgga atatatatac atacgtacat acatatatct 5940ataaagaggg aaggtctcac tctgtcaccc aagctggagt gtagcggcga gatcacagca 6000cactgcagcc tcaaactcct gggctcaagg tcttcctgct ttggcctcct agtagctggg 6060actacaagca tgcaccacca ggcccagcta atttttttaa aaaattattt aacgacgggg 6120tctcattctg ttgcccatgc tcgtctcaaa ctcctggcct caagcaatcc tcttgcctca 6180gcttcccaga gtgctggcat tatgagtgtg agccaccgtg ctcagcctga atatcattaa 6240aaaaaaaatc ctcgcaacaa tttattcaac tctaaaatgg gctttatcat tactacccca 6300gaagctgtga ggacagtagg aggcagaaaa gtccttggtg tgacacctgg tctgtggtcc 6360atctccataa atggcagctc ttcctgaacc ttcttcaagc aggctccttc ttgccaatca 6420ggtcccaagg catcatctcc tcctccttcc tagaccactt tcctagatgg catcccaccc 6480caaccccact tgtcacccta tcccatctta atgtttatta ctctctgaag tcattttatt 6540tatttgttgt attttttcat aagactataa cctctatgaa gacgaggacc gtgactttct 6600tgcatactga tttatcccca atacctagaa tagcatccaa cacatagtag gtgcttgcat 6660gtttattttt tcactttaaa ataaatggtg gctcacacct gtaatcccag cactctggga 6720ggcttaggcg ggaggatccc ttgagctcag gagttggaga ccagcctggg cagcatggtg 6780agatcccatc tctatgaaaa acatttttta attagctggg tgtggtaacc caagggtgag 6840attggaggat gccttgagcc caggaggcca aggctgcact gagtccatgt ttgcaccact 6900gcactccagc ctgggcgaca cagagagacc ttgtctcaaa aataaaaata aaataaactt 6960tgttgaagta taattttgat ataataaaat gcatccgttt gatgcatctg tacagtttga 7020taaatttaga taaatatgtt ctcttaccaa cacctcagtc aagacatagg accattttta 7080tccccctaaa gttccctcca gatttttttt tgccatcaat tctccttcat cccatcccta 7140gacaaggcag tttttttttg gtgtgaaatg aatgaatctt gaattgtcag ggactggccc 7200cagatttgcg gagagctatt cacatgtgtg catagcaccc tactgttcac aatgcctttc 7260cactttctgt ttttttcttt ggttttgttt tgtttttaat ggagtctcac tctgtcacca 7320ggctggagtg cagtggcacg atcttggctc actgcaacct ctgcctcccg ggttcaagcg 7380attcttctgc ctcagccttc tgagcagctg ggactacagg cgcgtgccac catgcccagc 7440taatttttgc atttttaata gagatggggt ttcaccatgt tggccagact ggtctcagac 7500tcctgatctc gtgatctgcc tgcctcggcc tcccaaaatg ctgagattac aggcgtgagc 7560cattgcgccc ggccgccttt ccactttcat ctcatgtgcc tctcacatat gaggccatgg 7620atgctcagat ggattaggtg acatgtgaca gaaggtctcc aaagcaagag gagacctttt 7680ggatgctcaa gttgcctccc catctggggt cccaccctgg gccagggagg aacccgtccc 7740tgggccaggg aggaacccgt ccctgggcca ggggcctcac atggtaggca ggctgtgggt 7800gttccaggct tgggtgtgcc acgaagcctg gggaaaggcc actgtggccc catctctaac 7860ccccctggcc taggcctcca gcccagccaa gaagatgcct ctggtgtcca gatgccagct 7920agctccagct agctcctccc tgggcagaac agaccctgca gtttgggcta tgagaaaaat 7980gaaaagaggt tatcagcatc acttgcttct ggttgctcag agagaatcag acactgggtg 8040ccaggagact tggcctttgg ccaggttctc cctctgcctc actgtgtgcc cttagcaagt 8100cactttccct ctctgggcct cagtttccac ctctataaaa ggaaactagt gatccctaac 8160cagcatgact tcaactccac ctgatgatga ctctttgacg catgaatgca tcccggccct 8220caaaggctgg gcccctgccc atgctgtccc cactccctgg aatgctgttt ctcagtatct 8280gctgtcaaaa gtcttgtttt tcaacttcca gttctggtcc taccccctct gtgattttcc 8340ttaaaacaga atgttctgct ccttcctcaa gcttctctaa gctcttgagg gcagggacta 8400tgaataatag aatgacacgt tctgttcatt gagagatgat ggaccctggt gccaggcact 8460atgctgggca ctttgtgata tcatttaacc ctcccgcaaa aagcatcatt ctcttttgac 8520agatgaggta gctgaagttc aaaggggtaa tgccacttgt ccagggtcac agaggggagg 8580gcagcttggg cttccttctc ttttcacccc cagcatctag cagagggcct ggcacacagg 8640tgttgaccac ccattgctga atgactggta agtgactgaa tgcatgaatg aatgcctgca 8700tcactttaat ccatttcaaa cctacctcct gtggtgtgca caggcctgaa gtacaggtgc 8760tcagcaaatt taactgttat catcatcact tcctgcctta tgccacagtc acactaagag 8820ctgcccaata acaacgtgaa atgcatgaga gaagtggtga gtgcttagtt accaggggtc 8880aacaagcaga aagtggatgg ccttgtccca gtgcagatca ggagatctga gcaccagcag 8940ccaaaggatt gagatctggg gatcccactg gttaacctga ctgtctcccc tgccagcttg 9000ggaggtcctg aaactgaaac acattcccct ggcactgcga cccacccatg cccagcccca 9060taggctggtg cctgtcggtc tccgggtttt gattctggta gtcttttgca aactgtaaag 9120tgctactgct ttttcctttt ctgatttgct ctgtccccca agccgcctcc cgagtgaaat 9180gcccatatgc tgagcctgag gcaagcccgc ctggcactgg ggcgggggtg ccttcgggcc 9240cgagccttcc ccaccccctt cctatagctg ttgtgcaaga ccagcgggca caccttctgg 9300ggcaggtcga atcaaggcca gggaacttcc tgagcgcggg cagtggcggg cgggacttgg 9360gggggcaggg ggcactgttc agggtagagg agggggcgaa cgccgaattc cggcccgtag 9420gtccaggcgt cccctgccag tgccccacat cctcctcggc gcgattcttt tcccgcgcgg 9480gcaaggttgg ggaggggagg ggggtgacac tcaggctgga gtcccccaga gcacccccag 9540tttttcagat gtccccacca gccccgtctc tttctctgct taggtacagg gcgggtgcgc 9600tcttcgcagt ctctccgcgg tctccctttc tgggaccttc tctttgtctc cttctccagg 9660gcggtcccgg cgtccaccgc ccctgtgccc cgcccgccgg aggcagcgga gggggcgggg 9720catacggggc gggccctgga ggggctgggt ggggcgcagg ggggtgcagg accgagggcg 9780gggcggggcg gggcggggtg ggacgcggcg ggcgcagggc agcgggcggg gagggggcgg 9840ggttggcctg cgggggcgtg gtctggtggg gcggagcggg gcgccggggt gcagggccga 9900gggcagggag ggggccaagg cggccggctg gagggacggg gcccgaccgg gagcggagcc 9960ggagcggaag ccgcagccgg gcggcgggag cggcgggagc ggcgggagcg ggggaagcag 10020ggcgggccgg gctccatggc gccagcggcg tccgcctgag cagcgcgggc aacagcggcg 10080gcgtcggccg gatcgggccg cgacacctcc tggccatggg ggacgtgctg tccacgcacc 10140tggacgacgc ccggcgccag cacatcgcag gtgagggtcg cgccgcgcca cgcgcgccca 10200ggggcctgcc ggaacccccg gaccacgccc ccgccccggc cagcagttgg cgccggggcc 10260gggggctcgg gggagtcagg ccgggccggt gggagctgcc agggagcgtg ccccgcgtcc 10320gagctcgttc cctctgagcc tgggcgcccc ttcccgcgct cttctgagcg ccacctttcc 10380cggctcaagg ggactcggaa ctccttctct cccggaaccc cttcggagcc cgggcttggc 10440ggacccccag cccacgaccc ctggccgagc tggggcaggg gtcgcagcct gctgcccacc 10500cttggctgtc cccgcccaga agcgttgcaa gcccatccgc taccagcttt gggatctccc 10560agacccagcc agctcgcgct caccttcctc cccagccgtc ccgtccccag ctccgccggg 10620ccccagccct ggtacacgtg gcggaggctt cctgaaggac gcgcccatga aggcccggga 10680ctgattctgg ggagtggggc cagggatcga tggggcgccc cggaggcggt ggggttgcat 10740gtcctttggc gcagacagca ggctgcccct gctagcagca ggggcccagg gaccggtcac 10800cagagtgggg ggccctcagg gccagtcgcc agccccttca accctgttct tcagccaacc 10860ctactctcag cctattactg cacagtggcc acttctccca gggacagctg tccccaaggc 10920tcctggggaa gccggagctg cagagggggc ctgggttcct tttccccaca gactctggag 10980aacagtgaga taggatgagg atctcggctg cctgagaaac tcctctctgg ccgatctctc 11040ctggctggtg gagctgccct gggtggaatc ccagcatttg cttcccagac atctgtcctg 11100tacaccttgg ttttctcctt tttgaaatgg ggtcagtgat ttcttcctta ttaatgagtt 11160gttgggaggc ttacgtgaag taactcatgt ggcttgctta gcacagagcc tggcatagga 11220gaggcacttg tatgtggggt gacggttcag gtgggtcgcc cctcgctgtc tagtgggccc 11280tgagcaccgc gtccgcctgc acttattttt gtgtcctgta gaatttcacc accgtgttct 11340ccacgctccc ttgagagcag gaagccctga gagcagtggc caggtgtcct ttaactccat 11400gtctccaggg ccgcacccat gctgggcacc agggaggtgc tggggagatc tgccaagtga 11460ctgtggtccc gggccaagtc ccaaggccag agtccctccc ttgtgccagg cctggggctg 11520gctgggcaca ggggtgactg gggtgatgtg aaaatgaatc ggaccctctc tagcctctga 11580ccagcgtggg gttaagtcct ggaaagcctc cttatccgac caggcctgga ctgtgggagg 11640aggaagggcc agggcggagt cttcctgtcc cctccctgtc gcagcaagct cctggggctc 11700tgtaacagtc cctccattca ggacctcctg ttgaggcccc tcgaggggcc tgggtggggt 11760gtgccttgca ttcggtgggg tttttcctct ccccatactc agtgctgggc ctccctgtct 11820tcccgtctcc tgggctgaga attgccagcc caccctctct cccctggcat gttacagggg 11880aggggactga gaccacagaa ggtgaagtgt cctttactgg agagggttgg atttattttc 11940tggataccag gctgggtgga agaaagttgt gcccactcag agcctgctgg tctctctctt 12000ctccacactt cagtgctatt tataatcctc tttaaatgcc tcgaattcct ggtccagact 12060cacctgaggg gagttcctga tgtggggggg tggggtgttg gaagggggca gcggggaagc 12120ctcccagtct ccccagagga agtgagctgc ctttgaaatc tctgctggaa gggaatgagg 12180gcgtgtattt gcctcctcca gtgatccagg caccctcctc accccaccca aggagcagta 12240ggtactctca aagctggcaa ttaaaattcc agaaccctcc ctacctgagg ccttgttctc 12300tgccccagta gaaatgtcag agttagagac atcccttccc acagtgctct gagttcccag 12360gcctgttagg gttccaaggt ttgacctgca gctttgtgcc cgccccaagc aggtgagcag 12420gtgaatgcca gcctgcctgc aagtcctggc aaaggtcaag cgtgctcccg gccacggtgc 12480tgttttgctg ccacggcacc tttcatcctg ggatttcaaa gcccctggca agcagaggct 12540tgtttccaca ccaggagccc cacggggctg gtggcaggat gactcctgcc tccttacacg 12600aggcagtgtg ccgtgaggaa gaatccctgg cctgggatct gggaggcctg aggatccttg 12660ggtaggccac ttcctctccc tgggtctcag tttccctagt tgtgaaccga agtgggtggg 12720ccactcgatg gctgaagtcc ctttcagctc tgagggccta acaatcccaa attttcccca 12780caaatctccc ccagactgaa gccagctcaa ggtcccagta taatgcctgc aaccttggag 12840taatatcttt ttttttttga gacaaggtct tgctctgtca cccaggctgg agtgcagtgg 12900catcatcata gctaattgca gcctcgacct cctgggctca agtgatcctc ccacctcagc 12960ctcccaagta gctgggacta caggcatgcg ccaccatgcc gtgctaattt gtgtgtgtgt 13020gtgtgtgtgt gtgtgtgtgt gtgagagaga gagagagaga cagggtctcc ctatgttgcc 13080caggttggtc ttgaactcct gggctcaacc aatcctcctt tgggagcctt ggcctcccaa 13140agtgctagga ttacaggtgt gaacttcccg cccatgaact aacatctttt tttttttttt 13200tttttttgag acagagtctc actctctgtc ccaggctgga gtccagtggc acaatctcag 13260cccactgcaa cctccacctc ctgggttcaa gtgattctcc tgcttcagcc ctctgttaca 13320ggtacacacc accatgcttg gctgattttg gtatttctag tagagacggg ggtttcacca 13380tattggtcag gctggtctcg aactcctgac ctcaggtgat ccaccctcct cagcctccca 13440aagtgccaag attataggcg tgagccacca cgcccagcca aaaataatag ctttattgag 13500gcgtaattca cctaccatac aattcagcca cctaaagtgt acacttcagc ggtttttagt 13560atattcacag aattgcgcaa ccattgccac catcaatgtt ggagcatttt catcagcccg 13620gaaagaaatc ctctactcct tagtagtcgc tcctgtttcc tcctagtccc tcctaaatct 13680aggcaaccac tgaacatttt gtctaattgc aaaagtaatg tgtgcacagg ccaggcgtgg 13740tggctcacgc ctgtaatctc agcactttgg gaggctgagg agggcggatt gcttgagctg 13800gggagttcaa gaccagcgtg ggcaacatgg caaagccccg tctctagtaa aaatacaaaa 13860aattagccgg gcattgtcgt atgcgcctgt ggttccagct aattgggagg ctgagttgga 13920aggattgcct gagcaaaagt aacacatgta caaacaattt aggagggtaa aataaaacat 13980gaaaacaccc tccatctttc tccgttaaat cccacttccc agaagaagtc acttaagtgt 14040ggtgtgtcac catttccaaa cctacctctc tctgtacatg ctcataatag cttggtttat 14100aaaaacagat tttttttttt tttttgagat ggcgtctcac tcaggctgga gtgcagtggc 14160aagatctcag ctcactacaa cctccgcctc ccaggttcaa gcaattctcc tgcctcagcc 14220tcctgagtag ctgggattac aggcgtgcac caccatgccc ggctaatttt ggtattttta 14280gtagagacag ggtttcacca tgttggccag gctggtcttg aactcctgac ctcaggtgat 14340ccacccacct cgacctccca aagttctggg attacaggca tgagccacca cgcccaacct 14400ataaaaagag atattactct atgcttctgt ggtttgattt ttttcacttg gtaaattggt 14460ggacatcttt ctgtgtcaga acacacaaga ctacctcatt cttttcttgg tgagttcgtt 14520tctgtgcatg gatggtttgt ctacccagcc ctctgtgaat gggcatttgg gttgtttcca 14580ctattttgct agtattcaca gtgctgcttg aacctgctgg tacaaaccct ctgtaatatt 14640ccagtatcag agaggccctg aaatggaact cctgggtcac cttttttttt tttttttgat 14700acagagtctc attttgttgc ccaggctaga gttcagtggc atgatctcgg ctcattgcaa 14760cctccgcctc ctgggttcaa gcaattctcc tgtctcagcc tcctgagtag ctgggactac 14820aggtgcccac caccacacct ggctaatttt tgtatcttta gtagagatgg ggttttacca 14880tattggtcaa

gttggtctcg aactcttgac ctcaggtgat acatctgcct cagcctgcca 14940aattgctgag attataggcg tgagccacca tgccctgcct gggtcacctt ttgatagatg 15000gacacatagc ctttaagaag gcagcaggcg tttgacactc tcctgctaag atggacctta 15060gcattgagca ttttcatcct tttgacaacc agatatgaga agaagggtgt cttaattatt 15120tattgagcaa aacaaataat gcttctttcc aaagtgattt taaaaactta cctctctgct 15180gggcgcggtg actcacgctt gtaattccag cgctttggga gacaggcagg cagatcacct 15240gaggtcaaga gtttgagacc agcctggcaa acatgatgaa accacgtctc tactaaaaaa 15300ttagccgggc ctggtggtgc gtgcctgtag tcccagctac ttgggaggct gcggcacaag 15360aatcgcttga acccgggagg tggaggttgc agtgagacga gatcgcacca ttgcactcca 15420gcctgggtga cagagtgaga ctctgtctca aaaaaaaaaa aaatctacct ctcaacattt 15480gggaagtggc tgtagatgcc aagagtgaag ggatggaggg gttccacctt ctgccacccc 15540tcaccctgct gtgtggcttc ctctttgctc ccctggactt tgcagagaga ccaggctggc 15600cggaagcagt gcctgggcct gggccgctgc ccttagccaa ggtccgggtc agcccctggg 15660ggcgaagtgt ctggggatga tgctgtctct ctgttgggtc ttatataaca tgcttttgtt 15720ggctttgacc ctgccttccc ctccatccag cctcctctag tccccctgac tctgttcctt 15780ttccactgct ggggcctgag tgccggcctc gcctactcag agctgctcca gcctcctcgg 15840agcctctagt ccttcccaca cacgctgcca aagggacctt tctaaaccca cgcggggcca 15900tggctgccct ccctgctgtc caaggagtga agcggaaaaa caggactagg aaccactcac 15960ccgccgtcct gcgataacca cttcatcctc tgggagtact tcccatctgt gtcgatcatt 16020acactttacc gggaccatga ctcaagtttg tgtcttgctt ctttcactac atgatctata 16080gtaaacatct ctcccaggtc attaaaacct ccctggaagc atccccaaag gctgcatttt 16140attccatttg aggacagagg gtcactttcc cagctttttc cctaatgctg gacatgaggt 16200tgtttctcac atgttggttt gtaggggcaa acagagccac tgtgtgtgtt tcttttctgg 16260ctccagtcaa cttccctgtc gaagaccgat tcatctccca gcccaactct tcccgtgctc 16320tgcttccgcc aacctgggca tttagaggga ggctccttcc tgccatctct gagcatcgtt 16380cggggctagt gggcttgaag gggctgcctg tgtactgggg acggggactg atgaccatgg 16440cctctgggtg tgtggccata gagtctagcc tgcggtgtgt aggaatggat atatattttc 16500tgtctgaaga catggttgat agagcaggga ggggcagtaa tcaggctcgg agtgataact 16560gtaaacatta gcaacggttt aactgagcac ttgctatgtg ccaagccttc ctttgtctgt 16620atcatcttat tgaattccca cctcagtggc aggcctggat cggatagtcc aaggccttag 16680ctatttaaac cacgttctgc tattagtatg tcatccgtgg tgcccagact gggcaggggg 16740acactgaaaa aggcctccaa gagaggtaag ctttcggagg cggggagagg aagtgggagg 16800aggagccgag tgggaataga gcagcttgtg tcagtctccc tcccataggt tgcacaaatc 16860tatcccaccc gtcattactc agtctgagac tcacagcctc tgaccagccc cattctccgt 16920ccctgcgccc ccctcctcct tccccgcggg cagagctcat gactcactgc accccgggct 16980cctgcaagca tcatcctgcg gttgcagaaa ttcctgcact gctttttcct tcccctgccc 17040caccccatcc acttctgtgc tggaggaaga agggtattct gtagacgctg ctgggcagag 17100gtttaaggaa ggagtccggg cagatgccta ggtttaaaac ccgtgagggt ggagcaaagc 17160cattctcttc tcctggtaga atcttagggc ctgagtcatg gaagggtaca atcttttgat 17220ctcaaaaatt ttaaaactgg aagaacagga agtgtccacc ccctgggact ggtcccattt 17280ttttggagct gggctgcttc tggcccacga aggtgaaggg gcctgcctgg tgacgtggtg 17340gatggtaagc ctgaagaagc agggagccca gaggttttgg ggtaaggctt tgcagtcaga 17400ctgggtttga gtcctggctc tgccactgga tggactggtg ttcgggggca agttagtcaa 17460cctgtctgag tcttagagcc ctccttacat gaggatgaga gttctggcat ctcaaggtat 17520gaaggtcaag tcccctggtc agcgcagaac agtgttggtg ggaggtgaca atatgtgagt 17580ggtagctgtt atgattgtga ctttattgag ctaacttgac acccagggag atgtgtctgg 17640tctgtctgtg gcctcgtgca tcccctgctc cctgccatgt gctccaggaa gttgacatga 17700cccactcctt tcctaagcag ccctaaatat gagaatcttc ttcccttcca aggtcagagg 17760agcaccagcc tatggccctg gacccctggg gtattcagcg agttcctgga ggacggtggg 17820atggggctgt ggttccagca aggtcagtca ctccgcagca gcatccactc tgtgcctggc 17880atttggcagt gcacagagga aaagggttag aagatggatg ggaacatgct ccctccttgc 17940ccacccatgt gtcaagaact agacttgtaa gccttaagcc tggtcccatt gaccacttgc 18000tgtgtgactc tggacccaat ggccatgcct ctctgtgcct tttctccctg tctgtgacct 18060caaggccagt atctactctt gggaattctc tgcctctgaa aacccttttg taggggtctc 18120aagatgcctt ccaaaggcag ggctggtggc ttctatgtag aaatagaggt gcatgcctgt 18180gagaccatca aggggtgtga tgagctctgg cccccaacac acgtagtcct tatttcttgt 18240atgaattcaa tcaatagtta ttgaacacct tctgtgtgcc cggcgctgtg ctgggcacca 18300gggacacgca gctacacaat cacatggtgt ctgcctgtgt gtgcttcctg gctggctgtc 18360cctcctcagg gcaagcaatg ttcccaaagg gaaaggcctt ccttgggaag gtgtaaggga 18420gactggcctg gggtgggcca gcctgctggg actcaggatg ggggaggtag agggaaacac 18480aaaggctggt cttggcgagg aagggccttt gtccttaggg tcccagggag gccttggagg 18540cattttcagg cagaagtggc accggtgggt cagctgtgag tgagtgtgga tgggaggagg 18600tgaaccccat ggtgagaggg actggacctg cgtgtgtggg ggtcgggtgc aatgagggat 18660atgtgtgtac agggtgggtg gcgtggccag gcctcagtga tggtgctgtg cagggctggg 18720gcgagctgat gtacgtgact gttggctaca gggaaacctg tctggattct gtaggttgtt 18780ttatcaaggc agaccttcaa ccactggtct tgcagcctct gctcccactc ctctagtgac 18840aggaaactca gtcctttccc aggcaaccct gaaaatgaga accttcttcc cctccaaggg 18900cagagtagaa agaggccggg agtcagagga cctggtttga gatgctgtca ggactgccct 18960gtcatttggc agctgccctc ccttctctgg acctcaaatt cctcatctgt aaagtggaca 19020cagcagtatc ccaggcccct gaggctgttg tgcagggtcg gtgagatgat gtggccttag 19080ctctttgtgc ttagggagca tcggctcctg gttcatccac tgttaccgtt ctatgtggtt 19140cacataggac attggccaag ggaggccctc ctcttgcaga tcagaagtgc cgggtgtggg 19200tcagggtgct gggtagaggg tccacgtgca ggggtggggg cccagggagt aactgggcat 19260ccaggagggg aggaagaatg aactgtggtt tgcaaagtgc agaccccact ttcccacctg 19320cccatctgtg gccaagccaa ctgcagctgc tgtccatctt tcccatgccc catgctgagc 19380agaactattt gtgtccagcc gaatttgtga agcttagcac cttctagaag aggagtccct 19440tgggaagaag ctgcccaccc actcgtgctc cagtcacctc tgatttattc cacctgccag 19500gaaccactgg tgggtgcggg ctaggaggca ggctccccag gatgggtgcc tggaggcctc 19560tgacaagccc ttccttttct ctgtgcctcc tcctcacttt gcctatctgt gcaacgggca 19620tgcacttcca gctgtgaccc cttaggaacc tgtgggcctt aggctgcact gaaggcacag 19680agcaggtgca gcacctccac attgcaggag gccatcttgc cgttgaagga agagcttgct 19740ctgaaagctg tcctgttcct ggcctgagct gggaactggg gacaaagcag tgacatgagc 19800cctgatctca tggaactcat gccagccaag aaggttccat caagctggat gtgctgtggc 19860tcatgttaca aagcccgtgg atctttgttt cccagagtgt atgttggttt cagcactcag 19920tcggcccatc tcaagaggca gcgtgtctgg ctttgacctg gtggcagtat gcttctgtca 19980ctgaaagctg tgtggccttc ggcacacagc tctcctcctt ggaggtccag ttgtcgctaa 20040gatgggattg gtgctccctg cctttgggct gggtgaaggt tctgggaggt gcactgcgca 20100gttcctggcc tgtgcagggt gctcagtaaa tggcaattgc gttttagctg aattcttagg 20160gaagctgtgt gtcctttgcc cggcctgtca gagatttaag gggtgattca ggcatctaga 20220ggaggaggga gctgacaatt gcacttctgt tttgcttttt gtttttgctt tttttgagag 20280cagtattctc tgggaggcga agggtagaaa agagagctga gtttgagctt tggggtgggc 20340aggcctgatg ggctggggaa ggtgtgtgcc tctcacccta ccacacctcc cctagccctg 20400ccttgatttt tgaggtgctg atgagaagtg tctgcttttc tttttcaggc ccagaccctt 20460cctcgtccta gttctggagg accccatgag ctcctctaac caatcccctc ctttttctca 20520tgccccagta aactgaggcc caggaagatg atgggacatg cctatgctct caggggcacc 20580agagcttggc tggaacccag ggccttgacc ccagcatgaa gcttttccct ctccgacttc 20640gtactcccac cccccgcacc cgtgcagggc cccagacggc aagggatggt gagggaagga 20700atgattgggc cgggtggggc tgcagaactt ataattatga aaactgggta aacaaacaag 20760ctctctggga gagaagcaag ccctgtgcgg gcctcacaga tgggcctgtg tccgaggaga 20820cagcccaggc ccgtctatgc gcagacagtg aggggctgtg ttggacttaa aaagccaaag 20880ccacactccc ctcccacccc cactcctata tttcaggctg ccaggaaaag aggaggggga 20940aggggcagcc ctttcttcca aggaggtagg ggctgacccc ctaggactct cctggggtca 21000gccctcctgg cccagcttgg gacagtcaca gctcactgtg tgaccttggt caaggggcgt 21060ctctcctctg ggcctcagtt ccccatctct agagtggagg tcaccccatc tctcaaggag 21120ggtggcagga tggggaaaga tttgagccaa ctggttgaat acactgagcc ctttcactca 21180ccctgacccg aggccaactg ggatctcaga aactcttcat ttctctctgg ccagaggcag 21240gggcccctct tggagcagag gagtgacttt ctcagtgacc tgagagccgg cctggtcctg 21300ggaggccaaa cctccaatcc tggcctgact ttagcaggct gcccagctca gctttcctgt 21360cttcctgctg agattgtggc agaatccgag aggagctgtt tctggtgcct gccctgcagg 21420ctgggggctg ggcagcttgc cctgcctgga ggcccccggg tggagcctgg cagacttcct 21480gtgcaattgg ggctgagccc ccgccctccc tcccctcttc ctgcagtccc ctgtgtggct 21540tctgggagtc cagctctgta gatgggaggc cggtcctgct tcagacctct gtggccttgg 21600gtgaactcct gcctctcctc caagcctcag tttcccccat ggttcaatgg aggggttgga 21660taagagcctc tggtagtaga gtgaccttgc tactaaccat gtgtcaccct ccttcagact 21720gacccttcca ggctgtcctg tgccctcgag gtcaagccca gaccctgaaa tgccctcaca 21780gaccaagccc tgcagccagt agccccactc ctggtaggct ggatcaggct cttcctctga 21840gctcccacag ccccttggcc acccgtgtgt acctgtagcc cttctcacat ggttgtgatt 21900catttttgtt tttgagacag ggtcttgcac tgttgcccag gctggagtgc agtattgcaa 21960ttatagctta ctgcagcctc aacctcccgg gctccggcaa tccttctgcc tcagtctccc 22020cagtagctgg gaccccaggt gtgcaccacc acatggggct aatttatttt tatattttgt 22080ataggcagag tctccctgtg ttacccaggc tggtctcaaa ctcctgagct aagccatcct 22140cctgcctggc ctcccaaagt gctgggatta caggtgtttg agccaccacg cccagcccgt 22200ggttgtgatt ctaattccca tctgtgatat gagtacctgc gggctaactc accatgggct 22260cccctgaggt ggacaggtgg cctcacagct cagacttgct ccaccagcag ctgaacaagt 22320accctaggtc aggctttatg ccttagtttc attggctgta aaatgggcat gatgatgctt 22380ctactccact ggattgtaaa ggttattaaa ttatgtgtgt aaagcacaag aaatgctgag 22440gagtcaataa ctgtaatgaa catttattag tgcaaattgt tttagttccc tagcaggaat 22500gaatgaatga atgaatgaat gaatgaatga atgccattgc aaggtcctac atcctgggag 22560catggggatc ccaaagctct ctggatgaag gtcctggtag gtgcagaatg tgtgtgtgtg 22620tggttggtgg gggttggtta cagaggggca gggcagatgt gggaggggct tggggtggtg 22680acgtaccccc tgcccttgtg gaaggagtgg tttgggcccc tccgtgtctc agacccgcag 22740gcacgaagcc atgtggccat ccacaacttc cttcctcctg cctgtattcc tcgaagcgtg 22800actcactggc tttgagccct tcattcttgt ccttgagccc cgagggtgag gcgggtagag 22860tctttcttgg aagggcggct ctctctctgg gttcctgtct attcctggcc cttctgcagc 22920ttctgagcat gacaggcagg gatggagggg tgcgggtagg gtggggtgga cacctgggcc 22980ctggtcctgg tggccccact gaccccatgt gtgtcaggct tggggtgggc acccactgag 23040ggcccaccca gggcttggat ctgagctggt gtggctgaaa cagcccagtc aagctcacct 23100gggccctgcc cctgtagagc tcctgtctgg tcaggagaca cacagaacaa ctaccactca 23160gtcattaatt aaaatcatta ccggagggcg aattgctata aagaaatggc cactcttccc 23220tgggagctca cgtgtcctgg gatctcccca gcagtgcctg tggtttgctc tcctgtcaca 23280gaggaggaaa cacagcccac tgagtgggaa agggagcagg ctcagctctg gggcaagtcc 23340tgtgcccacc tggggcctca ggatctcgtc acccatctta gaggtgtgct gttcacgtga 23400ccctcctgcc ttggcctccc aaagtgctgg gattacagac gtgagctgcc gctccccact 23460agggaatgct gttcaggagg cccagggtcc gccgctggcc ccccatggtt ctgggtgggc 23520gggctggctt cggggtttta gcctggggac tgtggaactg cagcgagagc tgggtttgtt 23580gtctttaaat ccctcctttg tgtgtcccta gacagaaacc actgtgacta aagtattggg 23640atcttctaaa ggctaggcag gaaatggggg ctggtaaggg tggtcgtccc ccttcccact 23700ccctccacca atacacagga cagcagaaag gtggcccaaa gccctgggag aaacccaccc 23760caacatttca catctagaat gtaccgcgtt aggtaacaaa atggttacat tacagcatgg 23820gggatggaat ggaaacaatg tattaacagg cattgttcaa tggcataata tgaagccatt 23880aaaagtgatg attcagtttt gtatgtctga aagtgtgtat gtttgtatag gcatagaaca 23940aaataaggaa agaaattcac caaaatataa atagaggtta ttttggagtg gtgggataat 24000ggattatttt aattttttta ttctctcctg atttttgttt tttgtttttt gtttttttac 24060agtgagtgga ttaaatcaaa aagaataacc tgggtgggtg cagtggttca ggcctgtaat 24120cccagcattt tggtggctca cgcctgtaat cccagcactt tgggaggctg aggtgggcag 24180atcacctgag gtcaggagtt tgagaccagc ctggccaaca tggtgaaacc cccacccccc 24240gtctctacta aaaatacaaa aattagccgg gcatggggtt gcacacctgt ggtcccagct 24300actcgagagg ctgaggcagg aggattgctt taacttggga ggctgagatt gcagtgagcc 24360aagatcgtgc catagcactc cagcctgggt gacagagcaa gaccctgtct caaaaagcaa 24420aaaaaaaaaa aaaaaaaaaa agaaaaagaa aaagaaaaga ataaccctat tgactgtatt 24480tagtggaaca atttgattcc tgtctgactt cagaatctaa gccattctgg cccgactgga 24540gccccaaggg ggttgcaagc acagggcagg ctccggagcg gggaagaggg aagagaaaat 24600tctggaagag gggtgtttga gctcaatctt ggaggacagg agggattttt aaccagcaga 24660cttggggtaa gggtgagtgg agggcatggc ctggcataga cccttgctta tccaaggcag 24720ggagctgagc acagccttga gtgccaggca aaggggttgg ggaatggaga gtccttccca 24780tgaacacttg ttagctggac tttagaatga ggactcagtt tctgtgatga gggcaaggag 24840agcaacaaat ttgtgttagg taggctctgt ctgtgtgccc agcctctgag cccccagtgg 24900ctgtgagcag ataggactgt ggtagtttag cccagagatg gcagatggat tccacaaata 24960aagagtctct ctccgtgatg agtgctgaag gccgggcgtg ggtgggggca ttggggtggt 25020gaggagtggt tgggtaccat ccaactggga aagcctatga cccatcagac ctccagtccg 25080gaggtgtccc tgctaagtaa aaaaaaatta gccaggcatg gtggcacaag cctatagtcc 25140cagctactct ggaggctgag gtgggaggat tgcttgagcc caggaggttg aggctgcaat 25200tagctggatc atgctacggc tctccagcct gggcaactga gcaagatagg gctccaggcc 25260agacagagct ccaggtcccc agggtaatat gcacaggatg ctggtacctg gcatagaact 25320tgggtggcca gaagcatgcg ctgcacgagg ctgagcacga agaccccatt ctgctgtgtt 25380tagggcttcc cttccgcctg tctccatgat gacctcagct ctctgctttc ccttacccat 25440ctgtagcaca gggatggttg cagctacctt caagagggtt gttgtgagct tcatgccagg 25500gccacccctg gtacctggca cacagcaaat actcaggaag agtttccgtc ctcggggttc 25560agtccccagc cagggaactc cttccctcct ggaggagcct cagcagcctg aacttgatgg 25620ccaggatggt attatatcag gttgcttggc ggtgagctct ggcctccccc tcagacccac 25680tgtggctccc acctggcagc atctgccaaa ggtcaggttc tcacagctct ggaggtttgg 25740cctttcaact ttgagcttcc ccagtcttcc ccctttattg tttttcacct tttattatta 25800ttgttacata agcaatagct agtggtcaaa gaaaattaga aaataacaga taagtaatag 25860gaagaaaatg aaaatcaccg ataatcctac cacccagaaa agactttcag gcaccttcct 25920gatacataat ttgttcttct accttaatag tgagtagtgg gcctcctcct tgtgaatgga 25980tacagaatat tccattatgg ctgagcaagg aggctaatgc ctgtaatccc agcattttgg 26040gaggctgaga caggcggatc acctgaggtc cggagttgga gaccagcctg gccaacatag 26100tgaaaccctg tctctactta aaatacaaaa attagccagg cgtggtggtg gtggacacct 26160gtaatctcag ctacttggga ggctgatgca ggagaattgc ttgaacccag gaggcagagg 26220ttgcagtgag ctgagatcac accactatac tccagcctgg gtgacagagc aagactctgt 26280ctcaaaaaaa aaaaaaaaaa aaaaacgaat attccattac gcctcaatag tctatatttt 26340aacttactca gtcaaagccc tattgccagg cagttaggct ttttctattt ttcttttttg 26400tttgtttgtt tttgagatgg catcttgctc tgttgcccag gcttgagggc agtgtcacca 26460tcatggccaa ttgcagcctc aatctcctgg gctcaagcaa tcctcccacc tcaacctccc 26520aagtagctgg gactacaggc ttgtgccacc atgcctggct aattttttaa aaaaattttc 26580gtagagatgg attctcactt tgtcatccag actggtctga aactcctggc cttaagtgat 26640cctcctgcct cagcctccca aagtgctgcg attacaggca tgagccatgg tgtccggctc 26700tattttttgt tgttgtaaac agtgctgtgt tggaaagctt aaatcaatta atgggattga 26760gccattgatt ccgcatggtt tgagagccat ctctgctcag tgtcaggctg tgtgctggga 26820ctgggggtgc tgggtgggga ggcggccatg gtcctcaacc tcttggggac ccctgtctgg 26880ggaagacagg cccagcatgg cacgccagca gaggtggggg tttagggcat ctttgggagc 26940cagggagcag ccataaccca gacctggaga gtcatgggag gcccagctga ggacacctgc 27000agctggtgac ctgagggtgg ggcaagccct gcacctgctg ctggctgagc tcatgggagg 27060ggacctctca ggtgagccac aaaggataca ggaggagaga gtttcaggca gagggcaggt 27120ctgcagaggc ccagagtaga cagagcatgg gcattgaggg aaccaaaact tcccagagat 27180cggtgctgtg gttcttgcca gcctggcagc agctcccatg tgcatgacac tgtggtagtt 27240cttttctttt tctttctttc tttctttttt ttttaagatg gagttttgct cttgttgccc 27300aggctggagt gcagtgacgt gatctcagct cactgcaacc tccgcctcct gggttctagc 27360aattctcctg cctcagcctc ccaagtagct gagactacag gcatgtgcac cacgcctggc 27420taattttgta tttatttatt tatttatatt tttatttatt tatttttgag acagagtctc 27480gctctgttgc ccaggctgga gtgcagtggc gcaatctcta ctcactgcaa cctctgcctc 27540ttgggttcaa gcgattctcg tgcctcagcc tcccaagtag ctgggattac aggtgcccac 27600cactgtaccc agctaatttt tgtgttttta gtagagacgg ggtttcaccg tgttgcccag 27660gccggtcttg aactcctgat ctcaggtgat ctgcccatct cagcctccca aagtgctggg 27720attacaggtg tgagccacag caccctgcca attttgtatt tttagtagag acggggtttc 27780actatgttgg ccaggctggt ctcgaactcc tgacctcagg tgatccaccc accttggcct 27840cctaaagtgt tgggattaca gacatgagcc accgcaccta gccggtagtt ttttttcaaa 27900gtgccgttcc atctgtccct ttgtgcggtg gggatcactg gcttcccttt tcttaatggg 27960aacacagaag cagagtgacg tgcgtgcccc agggaagtgg ctggcggacc caggcctttc 28020accggcccag cacacgggac agggggttga agtgcaggat ccgccagctc tggtttcccg 28080gctctttgca ggtgctggat cacttgacag gatccaagca ggaagaggac gcggaggatc 28140tgagcttagg tgaccgcctt agtttccatg aggaggtagt gaccttttct gctgctgata 28200tgtgaccttg gcctggccca gccccaaacc cagccctata tttgcaggaa gggtcctgcc 28260tcttgggcca tactgggatt ggagaggccc tggtttatat ggaccttggg ggctgggaag 28320tgtctgtccc caagctggag ttaacctttc tgggaatatg ccatcctcct ctggtgtctt 28380aacatgactg ggagactgaa ctgcagagct ctggtctaat gtcacatagt caggggataa 28440atatttctgt gcaaagtgct gtgctagatg ctgggcatat gttatgagtg agacagacag 28500ggtccctgcc ctcctagggc aaatggagat ggaacaatga attattaaag caaagttgtg 28560ctaatgacta acaggggtct gacctgtgtg agcgagctgg caacagtcag ggacagtgct 28620tttcagcacc ctaggctggc agttaggact caggtcagtt ctatcaaggc ttaatggtgg 28680gctcctctgg gcttcccaat gtgtcaggaa ggagcgttgt ctcatgtccc aggtggaggg 28740catcagggcg ttgagggtcc agcaccatgg tgtccagtcc cttcacggta cggggaggag 28800acagggagcc aggaagggtg gggggcttgt ccagggtcac tcagcagcag tcaggtacat 28860ggatttgggg cccagtgctc ttagccctgt atcaccaccc catttcactg gccagtgatg 28920gggagacact gaggcggtcc ccactcaccc tcaagggact cccccgggga agcagaccaa 28980gaccacagag gctgttggct gcgcagggtc tctcagggcg cacaaatggg gtcacgcagg 29040cacctgggac agagagtggg aggctggcgg ggctgggagg ggagccgttc tcccacctcc 29100ccttctggcc ccctctggag cctgaggaca cctgcagctg gtgacctgag ggtggggcaa 29160gccctgcacc tgctgctcgc tgagcttaga gtacagacca agggagtctt gttatttgaa 29220taaactttcc ttctatccat gtaaagggtc cctggcattc caatctggga ttgggtttcc 29280tctggcccag cagcaagcac acgagtggat tttcatattg gctcatcttt cttgttctct 29340tcttcccctt ctttccctcc ctggctttga gaaggcagaa tcgcccctcc ccagtcccac 29400tcactgagag gaccctgcaa gccagaagct gcaggcagca gcatgcatgt gtgtgcacat 29460gtgtaagtgt gcgtatgtgt gtgagtggat gcacggtgtg tgagtgtaca tgtgtgtggg 29520catgcacatg tgtgaatgtg cgtgtgtgtg cctgcacatg tgagtgcatg cgtgtgggcg 29580tgcatgtgag tgtgtgtgcg tgcatgtgca tgcacatgtg agtgcatgtg tgtggcatgc 29640atgtgagtgt gagtgtgcat gcgtgtgggc atacacgtgt gtgtgaatgg tgggtgagca 29700ggggtcacga ggcaacttgg gaagaacctg acttgggctt agggaaagga tattagcagt 29760agagggtgct tggctgatga gatctgtggg gtttgtgtat ccgagtttag tccttttcca 29820ttggggttgg gccatggtga ctggcactta ttagctgtta gttcttattg gaacagaagc 29880tggcatttat tgggctcttc ctgggagtca ggcagggtga caggtgcatc tcacacatct 29940tatttagtcc

tcacaccaac tcttggaggt gggtgctctt tgaccctcca gcctctgctt 30000acacacctcc agtgatgagg ccttccctac ccgccagggt cccttatcct gggcatctga 30060ggccgttaga aagcccttgc tgggatcttg tgtgtcttct gtggatgact tgagaatggg 30120ctctgcttgg caatagggcg tggctacggc tggacttccc cagcgtctgt tttccaggga 30180actgtccaac tctaaggttt ggggagagga acaagtcctc cctcctttcc ctgagcagcc 30240cctatgtggt acttagcccg tcacagcttc attcatcaca taccaagcaa gtctgaagct 30300taatgttgaa accctagagg tattttcatt agcatcgatc tggatgtctt agtcaatgca 30360ataagagaag agagaaaaaa agagaagtac aaatgcaaga cagcgggaaa gccctcatgg 30420agttcaggga ctggtgacaa caaagcactg ttggtctttg gacttctcca ccccctaagg 30480ctgcctacag gaagcagagt cttggagaga ccagaacttg cccagggcca cacgtgagtt 30540ggcggcagag ctggacgagg actcagggcc cttgattttc ccaaccagca tctctggtga 30600tggatgcagc agctaaggga gaggcggcaa ggccagagtg ttctccatgc cagtgtcaca 30660gagcaggggg gctgtggtcg gtcgaggttc tcaggggtgc ggtacgtttg ggtgttggct 30720ttcagaagtc ttcattgatg tttcttgctc cacttctcct tcctgatgaa ttaacctgag 30780agatgagggt ccagaaagat tggaccccaa gcatgtcacc ttttaatctc atgacccacc 30840agctgaggga ccagggaggt gctgttccag tcaccctttg ctggttccca ttgcgtggcg 30900tgaaacatgg gacagagaac tgccatgagc actgatgaga gatgccactg ggcaccctga 30960ccatttatgt cctggccacc aatactggga cagatggagc tccttcgaga ggttccatca 31020ctgccattga taacaccagc ccacatttgc ccaggctctg tgggtttatt catttcattg 31080aatccttatg acaccttgca agatggggga tcgtaaagtt gctgggtttt ttttgtttgt 31140ttttttgttt ttgaggtgga gtttcgctct tgttgcccag gctggagtgc aatggtgcga 31200tctctgctca ctgcaacctc cacctctagg gttcaagcga ttctcctgcc tcagcctcct 31260gagtagctag gattacaggc atgcgccacc acacctggct acttttgtat atttagtaga 31320ggcagggttt ctccatgttg gtcaggttgg tctcgaactc ccgacctcag atgatctgcc 31380caccttggcc tcccaaagtg ctgggattac aggcgtgagc caccgtgccc ggtggtgctg 31440ttttttgttg ttgttgttgt tgttgtgttt tttgagatgg agtctcgctc tgtagcccag 31500gctggagtgc agtggcgcga tctcagctca ctgcaacctc cacttcctag gttcaagcga 31560ttctcctgcc tcaaccttcc gagcagttgg gattataggc atgcgccacc atgcctagct 31620aatttttgta tttttagtag agacggtgtt tcaccatgtt ggccaggctg gtctcaaact 31680cctgacctta ggtgacctcc caaagtgctg ggattatagg cgtgagccac cgcacctggc 31740caggttgctg ttactcccat gttacagctg aggaaacaga ggttcagaga gaattgagcc 31800atgtgttgct gaaagtccta tagccagcaa agggtggagt taggatttga actgagatct 31860ggttccagga ccttgacact gcactgttga agggtccttc ttgacctagg acgtgctgat 31920aaatctttgt ggaatggatt aactctttca tgcctgtccg aatcttggcc tttctcgggt 31980cttctgcttg gcacacactc ccttcccggt gtcctccctc ccctagcagg gactctttgt 32040gtctcagctg ctacctccct ctgggaggct tccgtgaccc tgcaggcttg gtcagtcacc 32100ttcctgggct cctgtgacct cctgtgcttc tcccagttgg ggctcctagt aattgtagtt 32160tttgttgaca atctgcctcc ctcattaggg tactctctcc tgagtaccca gtggccagga 32220ttgctgaatg aatgagtgag agcaacataa accctttctt ggccatcgtg atgaagcctc 32280agagcctcgc ctgaacttgg tgactcgcct gacatctgcc attttggctt ggcccgcgct 32340ggctgatgga ggacatggga tctgggctgg gtcctgtcct cagccctgcc ccagtggttg 32400tgacctgtct gatggggcgg ggctgagtgg ttgacactgc ggacatcttg ggggaagggc 32460ttgaccacag gctcttcctg catatctcaa ggtccaacac caggctggac gctgagggga 32520acattggtgg tgggttctca ggcccagggc aggtgtccag gcccgtgggg ggcacctgcc 32580aatgtgcacg agccacagca gagcaccctg ggttcccagt ccagcctcgg ccctggacgt 32640gactgagaga aggggtatga ggagcacctg tggacaggcg tggggatgct cctctggggc 32700ctgggcagtt ttgacaggtg gctggctaag gctcaggctc ctgtgggggt ggtgaggatg 32760acaagaaagg aatgaaggcc agagacattg tggagaaaaa gtcaacagga aagggtgact 32820ccatgtgaga ggaaaggagg gtcgtgtcta cacaatcact gtcatagatc agcctcctcc 32880ttgggcgtgg ccttggccag ggggccgcag gatggcagcc ttgggtgccc accccctgga 32940gtgtgggtcc tgctctgggt ggtgacgctg ctgggggcct cggcaccccc tgggctgccc 33000tcccagcgag gatctgtgtt cccagacggc aggcatgcct catcctgggc tcccagaaca 33060agctgccact gtgggctgtg ggctggggcc gctgccaggc aggcggggtg gggggcagga 33120ggggatgaga gggcttttgt ttccttctgt ttgtgtcaag atgggaatgg acttcctccc 33180accaggcggc ctcgaggtgt aaaggaaagg gcagccccag tgggcgggtt ttgggtcacc 33240cgtttttttg ggtgcatttc tctgggctcc cgcctcagcc ccgccctctc ctcccagccc 33300tcctgcctca aagctcaccc ttccccacca gacctgttgt ggcccagccc tggttaaata 33360tttgccaacc gagaagagtt ttcaaggcag ccttgtgaaa aaccccagct gactcctggc 33420tctgttgggc ctgacctgcc tctgtgatga gacccagacg gcctgtgccc tcagactgag 33480tccctgccca ccatggcctc cctggtgacc tgtttggcgc ctgaggctgg gtgacctaac 33540acttgttcgg cacttgttta tcaagcaccc ccctgggcct ggcgggagct gggtgctggg 33600gagacagcgg agagcagtct cagtccgggg agcgtctcag atgaggcatt aactgtggtt 33660gaggggagtg ccggggagca ggccaggggc tgtgggagtg gaggccaggg aggctgtgga 33720gacctgggag gccgggactt gtctggaggg agggttccga ggaggtggca cttgagcctg 33780gggaggagaa agaggagttg gccaggcaca aggggtggac gctaggaggg cactgtagac 33840gtgggacccc aaggccagag gccagatttc ctccgctgag gaggaagctg aggggctgag 33900gttgcggttc gcttgggttc gctgggtgtg aaagccccat tcacctgttt ctccccaaga 33960tggtctcaga ggagctggga cactaacagg gcaatgggac cctgggcagc ttttccccat 34020ttccccagcc tcagtttccc catctgtcca aaagatagtt tggacttgat cttggaggtc 34080ccttcccagg tccagtggcc tcttaggagt atgtggctgg cccatgaatg agggaatgtg 34140ccatggcagg gattctacca gcaccaaagg ccaagataac agccatggcc gggtgcggtg 34200gctcatgcct gtaatcctag cacttcggga ggccgaggcg ggtggatcac ctgaggtcag 34260gaattcgaga ccagcctgac caacgtggtg aaaccccgtc tctactaaaa atacaaaaat 34320cagctgggtg tggtggcaca tgcctgtaat cccagctgct tgggaggctg aggcaggaga 34380attgtttgaa cctgggaggc ggagattgca gcgagctgag atcatgccac tgcactccag 34440cctgggcaac agagtgggga ctccatctca aaaaaaaaat taattaatta attaaaaaaa 34500taaaagaaaa aaagaataac agccatgtgc ccttataggg ttcatgcagc cctatcctgg 34560tgttagcaac aaaagccaac atctcctgag tgctccctgc cctagcaacc ttgtgaggtg 34620gtggtggagg actatgacat tatacccatt ttacagataa ggaaactgag gcccagagag 34680gctgttctga gctcagggtt tgcttcttgt tttaagcatg gctccagaaa tacccgtctc 34740tgtgagaaac ccaccctcct ctcccaaggc cagcgccatg ttaggtacca gggatacgga 34800agtgaacaag ataccattcc aacctgggaa gctgccagtc gagcaaggaa tgtcccagca 34860gagagtcagc aaatggttct agctcttctc tcttccagca cagtttgagc agcgaacaga 34920agcaggcttg ccttcacatc ccagttttgc tcttccctgt gtagtatcgg gaaagtcact 34980taacttctca ggagcctcag atcccttatc cataaagtgg gtttggcagt acgtcccagt 35040gagggttaaa tgaaacaatg ttgactataa tttttagcat atatgggatc tgcctctgtg 35100cctggcacag aactaactgt tacatgcatt agctcatgtt gcaattatga ttataatttt 35160ctgcaagtgg ggaactgaag aacagtgtga tggaggagag gatgggtgag cagggccttg 35220aaagtctggc taagattgtg aactgtggaa ttgaagggaa gggcggtcca gccagaggac 35280acaacatgga tgaagatgtg gaggtaggaa atgtgtaatg gaggcgagag acgcccaggg 35340tgtggggatg ggaaggagag cggggctgag gttgcggatc ttggatgtga ttctgtgtaa 35400catgcttgtg ggcttcagtg tgccaggcct gtggtgacaa gttagtccat agtctcttgg 35460ggccatcaga gcccagagag atcctttaca aagtgcttca tgcaaccatc ttccagatgt 35520ttccacagcc cgtggagtct aggttcagcc agcctgggct cacaccccag cttggatttt 35580ggctccctgc ttgacagccg gctgacctgg gacaaggccc tggaccttcc agcctctgtc 35640ttgatcgggg gtgattcctg taccacttcc tgcagctgtt gtccgggtct atgtcagatg 35700gcgtgaggtg tccccagcca gcgtgacacc cattagcttc ggggtaggag ggctccctaa 35760ctgttctgtg cctttttgtt cctggtgtgg agacagagcc tttctccctg ggccattttg 35820agagtcgaga atgatctcgc actggaagtc cctagctgag ggctggctca cgcagggtca 35880cacggctgtc attgtcgggc cttggcgtgc tgagggatgc tgtctttaag ggaagaggtt 35940tttccttgtt aataactggc cttggggcca ggcacagtgg ctcacgcctg taaccccagc 36000actttgggag gctgaggcgg gtggatcacc tgaggtcagg agttcgaggc cagcctggcc 36060aacatggtga aacccccatc tctactaaaa atacaaaaaa ttagccaggt gtgatggtgg 36120gtgcctgtaa tcccagctac tggggaggct gaggcagaag aattgcttga atctgggagg 36180cagaggttgc agtgagccaa gatcgtgcca ctgcactcca gcctgggcaa cagagccaga 36240ctccgtctca aaacaaaaca aaacaaaact ggcctcaggg aagagtgacg cagccaaggg 36300ccccttcagg gcacctgagg ccagcagggg gggaaatgtc ggtggcagcc accttcccaa 36360ggaaggacag cacagtgtac atttcctcat ccccaggacc cagccaaggg cctgctccga 36420tgtcagcctc cgtttccgtg gtgaggaatc tccggggtgt gtgggggctt attgctcagt 36480ggcttcgtgg ctctcttttg ggggtgggtc tggcctgaga ttggcacttc tggcctgttg 36540ctgtgggctt cccttccttt tccactgcgt gccaggccct gccccaggag caggggtgct 36600gcctgtggga gccacatgag gaacagacct ttacccacca ggaggccagc ggtgggctgc 36660gggaataggt ggagggctcc cacccagcct ggggctttcc agttgaaaga cgcatggtgg 36720tgggctgagg agtgagcttg gcggaagact gtattccgga catggggagc agcctctgcg 36780agaccccaga tgacttgggc ctgcgggcat ggtaaggagc tcagttccat cccagaggca 36840gtaggagctg taccactgga aggtttctga gccaggaaac gttgggcaac tcccctgtgg 36900cccctgtgtg tgggaaggat cagaggcagc caggatggag taggcaggac ttcagtggct 36960gcggcgggca caggacggga gtggcctggc tggggggcag tgactagagg cacaggctgg 37020gtggccactt gactggcccc tgggtgctgg ggagcctgcc tcaccctgtt gggcttgtgc 37080tagggggcag gtagtggtgg ggtgggaatc ccataggccc tgtttctctc aggcccagaa 37140ggggggtccc aggggtctgc tgttgtaccc cttctgccaa ggatggctca tggagggacc 37200ctgaatccgt cccttgcctt ctctggtatg gtccctcttc tggacaggaa ttgattgggt 37260gggggacact cagctgtcct cctccttcct ggcctgccag attctggctg ctggctcagg 37320ctcttctcgt ggatgggctg ctggtccccc tcctgctact tgatggagcc tgtcattacc 37380cctcccaccc ccaacttccc acctgattac aggtcctgag caggagccct gtgtctgccc 37440ttcccgtgga ggccacccac aggctggtgc tgcggaggcc agggtgagat ggttttgccc 37500aaacgttggt tccctgttca gagttcatgt catttacaag ctgtgtgtcc ctggagaagt 37560cccttaacct ctctgagcct cagtgttctc tgggaagtga ggacagtgcg tgaaataatg 37620agtgtgcatg tgcagtgccg tgctgggcac agaggtggtg ctgacaagtg gctgctgcta 37680ttgtgtgtcg attgtgacag taggtggggg gcaagtagaa agtagaaagg ccctgagcga 37740ggcactggtc tatgccaacc cagctctgtt ggatgtgctg tgtggcccta ggagagtgtc 37800accacctttc tggaccccaa gctctccctg ctgtctcagg cacatgatgg agatggtggt 37860gcttgggaca actctgagac ctgcgagaat ggagtccctg ccccgactct gcctctgctg 37920gtccctggac tgtagggtgg gtggggtcgg cacccccctc tttcccccac ctccccatgc 37980tggacctgct ccctatgccc tgggaaccag gcgggcaggt tctgagcctg ccccacccta 38040cagggtgctg ctacctgcct ggtttcagtc ggcaaacctc tcagccctga aaccctgaaa 38100ccgggctggg cctcgtgggt gaggtgtgtc cctggagccc actggagaga ccggatgagc 38160caggaacggg tctggcacag cgccctccac tggggccaaa ccagggctgg cgccggcgtg 38220cagcccccat ggtggacatg agctggatgt ggtcgcagct ggagatgact ctcttcctct 38280tgggtccccc gtccgtgtgg aacagggttt caggcttagg catcaggtcg ggaccagggt 38340gcagatccta cctctgcctc ttctcagcag agtgatttgg cagaggtacc cagttctcgg 38400tcacctggtc tgaaatgttc tgccagtacc aataccacct ggcccgaggt tgagttgaag 38460aatcgggggc acgtgtccaa gcaaaatgct tagcacagga gcggcacatg gtggctttgg 38520ggagcgtcat gacaaggcaa agaaggaact ccttagcttg gcattcgagg ccctccggga 38580tacgcctttt taggtccatt ttctgagaac ccctggactc cttcctccat tctcagctca 38640gcaagcctcc tctcctctct gagcctcagt tcccctatct gcacagtggg cctggtaccc 38700catcccccag ctccccaagc ctccaggcca ccctggggcc tccccagttc ttgcccagcc 38760tcactatctg cacaggaagt agcgtggccc ttaacgttgt cagtttggaa aatcagagat 38820taattaacgt tccggccgcc ttggctgctg ttggagctgg tcccagggtg ggccaggctg 38880catgctcagc ctctgtttca agggcctcac tgtgggttag aggaaccttg acaagaggca 38940cttcttagga gcccccattg gtgaagtgcc cccttcttgc cagggtaaag ggcttcatgc 39000tcagtagccc gtttgtctca taacccattc aggcagatac tgttgtgcca ttttgcagag 39060gggaaaataa aggctcagag aggagaagtg ccttagccaa ggccacacag ctcataggtg 39120gcagagctgg gacttggacc cagagcctgt gtgtccatgt agaaatggtg ccctgtttgc 39180aggtgatggg gggtgcctag ttctcccccc gggtcctttt gtctggcccg agggggctgt 39240gatcagctgc tggtcagagc tgactcacca gcagctcaca ggaggggctg aggccagcat 39300cccggtcagt tcctggggcc agggcagcac tgattagatt gtcgttgaga ttttcggtca 39360gctgagctga cagggaccca ccctcccatt ttaaaggtgg gaaaattagg gcccagtgag 39420gatgaagaag taaggctcca gtgggtcgga tgattctcca ccagctccga cccgacaggg 39480gccagtggag ctggaaaccc tagacctcag tcccggctct gacacctgcc agctgttgcc 39540tcgggaaagt cactttgccc tctgagcctc ggtttcctcg tctgtaagat gaggatagta 39600aagccttgtg ttgcctcatt cttgggattg ggcaagaccc cagcaagaga gtggaaaacc 39660aggggtttga aagcggcatt gcgggtcctc gtgaggggtc actgggatga gaggctttgc 39720ggggtagggc tctgtgcagg ctttggtggg agacaaggtc caaaacccac ctctttcccc 39780tccgttgtgt gtctttgagt gagtcagtga gtctctctga gcctcacttt tcttcctttc 39840tttttttttg actgacccaa attagatctt tattgtattt ttccacatat ctatcattcc 39900caaaagatca gtcagctcga tccttaattt ttcttactat gagatgtccc taatacacag 39960tctttgtata aattatccat tttaagtata tagtgaattc cagatgtgat cacaactgca 40020tttctttctt tttttttttt tttttgagat ggagtctcac tgtgtctccc aggctagagt 40080acgatggcgc aatctcagct cactgcagcc tccatctccc aggttcaagc aattatcctg 40140cctcagtctc ccaagtagct gggattacag gcgcccacca ccatgcctgg ctaatttttg 40200tatttttggt agagatgggg tttcaccatg ttggccaggc tggtctcaaa ctcctgacct 40260caagtgatct gcctgcctcg gcctcccaaa gtgctgggat tacaggcgtg agccactgtg 40320cccggccgca caactgcatt tctttttttt tttttttttc tttgagatgg agtctcactg 40380tgtctcccag gttggaatgc agtggcgcga tcttggctca cagcaacctc tgcctctcag 40440attgaagtga ttctcctgcc tcagcctcct gagtagctgg gattgcaggc acgcatcacc 40500atgcctggcc acacaactgc gtttctaaat agccatgtga cactcactga gtgcttcctg 40560ggtggcagcc gctgtgttta gcctgagtct cacttttctt atctgtcaga tgggatacta 40620atacctgtta ggattaaaca ggctgtggag catgcagtaa gttgtcggtg gataggcacc 40680tatccctggc aaactctggg gatccagggg cagagttagg aggactggct tgtgaggggg 40740tccttctcag tcaccctcaa taacccgata ggatgcagcc cttggagaca gggttccagt 40800catatgggga gcagagcccc cctgagatcc acagagcacc cccttattca accaacttgc 40860tgaaagggtt ggctctcagc tccacaacaa acccacaagt gtgtgttgtg agttttgccc 40920tggacctgca tccccaaaag tggcctcttc agaggtggat gcagcccagt acccatagag 40980acaccaggtg gagacagagg cctgggatgg gagtggcttc ctgaggtccc tgggcacaga 41040tcaggactca actccactcc cagaattcac acactgccct cttgggctaa gaagggaacc 41100gggaccccaa caggcaggcg ccttttgtga caacctctgt gaggggcctc ggcaggtttt 41160gtggactgac agagtggcct ttcctgagct gtttaattgt tctgagcctc ggttcataca 41220gctgtaaagt gagggttgta tttccctctg ggccatttga agaatggtgt gtgttggggc 41280aaagaacagg tgggccctgt gtctgatggg cgtgggctta cctcctgctg gcagaaagcg 41340aaaagggggc aaccaacggc tctgggcgcc agcagaggct aagggttatg ccaggggatc 41400ccagccacgg ttaggcagga ccccccatca aacgcaggga cctgctgtcc cccttggctt 41460ccccgcatct gtgatctttg tttgtggaag gcctcccatc attatcatta ttattttgtg 41520attcttgtcc cggttcttca gaaaacaaat ctttattgga ttatttcaaa ctataaaagt 41580tatactgcta ataaacattt caaacagtct cttatgactg ctgttctcag ctgaggacac 41640tgatgattaa cagtgacgtt tatggaggcc tggagctgag cacctcaaac gctatccggt 41700cctgcagagc tcagagctcg ggaacagctt cggagaggcc catttacaaa tgaagaatgt 41760gagtctctga gagctaggcg cttgcagggc tggcatatgg cagggcgggg actctggtcc 41820tgacttcaag ctcctcacca ggaggctggg cacctggggc cgcagggcag gctctgtgag 41880tcctgcccca ctccattccc tgccttctca cctagcagca cctggggcag agtcaatgca 41940gggaggcaga atggatgaga taatgtggaa agtgacgaag ctggcacaca ccttcacacc 42000accccacccc agcctctctg tagctcacag aagcgagtga tttctaccac cagctggagc 42060cccccagttc ctgggcccag gacctgcggg ccagccctgc ctctgtagat gggggcacag 42120ccagtttcct ctctgccctc taaggggagg tagggggcct gtggggccct gtagacaatg 42180gccagagcta ggaccaggag ggtggcatgc agccgactat ggctcgaacc taattgcctc 42240catctggctg tgtgacctca gctgagctgc tcaccctctc tggctcacat tcccctaaca 42300gggtggtaag cctgagtgtt ggaggccctg tccaacatct gccccaaatc cgtggcgtgt 42360ggtgtctcca gaggctgcgg caggaaatgg aaagagctgc tgtgggcaag tatggaggaa 42420agaggggtgc ccgggctcag gacccccaca gacacccagg ccgggacgcc atgtgggaat 42480ttgagttgta aaatcctgaa gcaacaatgc cgggggagag ccactgcagg gagcaagaag 42540gaaaagtggg gtactgggtg gggcctgcct ggactgcagc cccagagctc agcaggtaag 42600gcggtggcca ggctgggtcc tctggctgag cccccaccag ggctgcagtg ctcgcctggg 42660tcctcttcca gcagggtcca gccaggaagg ccctgcaagc cctttgtcca ggctggcccc 42720ctcccaatgc tgctggcctt ttgggtccct ctggcctcca cacctggggt ccccagcacc 42780cgagtcctca tcttctgcgt ctctgggtta cttcctctgt acaaagcacg tggcggttcc 42840ttccatgctg gctgaggaca tctctcaaga atgtgctcgg ggctacccct gccaaggagc 42900catccgtggc cctgtttccc atggtcctcc ctttctagct ctcccgtggt cgccaccctt 42960cttctgagcg gagctggctt cccggtgctg cctgggctcc gtccttaagg tccttaaccc 43020cttgctaatg ctgtccccct acctgcagca tggcgctccc tcagtacccc ctccgatgcc 43080tgccccacgg ccttcccttg tttggactta gtaactattt ggctgctgga ccaaaccccc 43140actattcctc cccttgccga catctgcttg tgccaggcct ggtgcccagg atccagtggt 43200gagcaaaagg agaccagacc cctccagcag agttcctggc cctccctacc tgacccattt 43260ggctcctgaa ttctgagagc aagcctggat agtgagcgac tccagcccca cctggggcca 43320acgtgacaaa cctagaggga actgcatcct ggacccctgg cccttgcctg ggtcccctca 43380gcccgaggcc ctaggcctgt gggcagcact gcgctggtcc agggacctga ccttcagcct 43440tgaccttcag ctcccaccct gacaggcagc ggggtccctg cactcccatg tctcccttcc 43500tcagagcttc ctgcagcttc ctctccagtc cacagcctgc ccaacttggg ccttaactgt 43560ccctcggtgg gacatgcctg ggccttctcc tggctcagtc ttgccctctt ttgtcctccc 43620cagcccagcc caggggcctt tctgaaccca gctcctccct gcctggacac catatctgga 43680ctgtgctgtc cgggcagttg agcacgggct gcggtcaggc agacctgtgt tcgagtcctc 43740acagcaatgg cagctccctc cagggcctgc tccgtgaggg ccagggtggc agactccagc 43800cctttgggat ctgtctgtgg gctgggtgca ctggggctgg ctgggggccc tggagccacg 43860gccaaaggaa ccctgagacc tgcaggccca agcctgccca tcctgccgac cacgttgata 43920gaatgcctgt ggggtgcgtg gcttgactcc tcatccctga aataggaaaa caacagcact 43980ggcttcctgg ggttctcatg gagattctct gggttcacat acgtaaaacc tgccgtagca 44040cgggtttaat gctctgcaag tggtggccat gacgatgaca accatggtac cgatgttatt 44100gtcagcttcc atcctctttt ctctattatc cccgatgtat gactctcttc catttgcccc 44160ctctctgcct catgattttt caaaacaatt cccatggtct cagagagagt tctggttgtt 44220tatggttttt aaaaaatagg cattcaaagc aaattcctca tttgttctcc ccttcctccc 44280cccaaactgg aatctggaca gacatgacct cactccagga aaggggagta cagggaggga 44340cagggtctgt ggagaggggt tggggggcat ccctccaccc ccatcactga ggcagggccc 44400ggggtgtggg cagacaccca ggaaggtcac atgcctggga ccctggttat gagctgtgag 44460gcatggtgtg tgacatgccc tttcagccct cctcccaggc caccacctcc tggtggggac 44520ccaggactgc atcccagctc taccacccac tggctgtgtg accttatgcc agtgacttaa 44580cctccctggg cctcagttac ctcctctgta aagtggggat aacacaggtt ctccctcaca 44640gaactataga gaggatggaa tgaaatgtca tcctagccag gtgccctcgg tacctggaga 44700ctggccatgg gaggggcagg ttcctgccag ctcctgtttt gagaaaggac caggtctttg 44760aaacctggaa tccaagactg tcagagatgg cagggcctgt ccgtcatcga atgcagcctc 44820tcagtgcatg ggggaaaccg aggctcagag aggggagccg cacagcgggc cagcacaaat 44880cccagttggg acgcgagctg cagcttctta ggaccaggaa gcctctcccc tcatgggtcc 44940gagaccctct ctgggcctcc tccttcatct gaacatgggg gcacccagga gcacacgtgc 45000tgagcctgat

gtggcctggg cctgccttca aggccatagc actcacctct attcagaaac 45060cgcgcagaag acagaagcaa atggccagtg aaagcagaaa ggttgttcac ttggtagagg 45120cattaatttt agaatgttta ttttttatta tgaaaggaat catgattata aaataattca 45180aatgctataa aaggaaataa accaaaaatg aagttcactc tgtactcccc actcccacac 45240cagaggtcct gccttcctag cctggcatgc tgccttccag acctcctcca cgcacccaag 45300cctaagtaag ctatatgcac aaataggagg agttttggtt ttgttatgtg tgtgttgaga 45360cagggtctcg ctctgtcgcc caggctggag tgcagttgca cgatcatagc tcgctgcagc 45420ctcgacctcc ctgggcttaa gtgaccctcc cacctcagcc tcctgaatag ctgggaatac 45480aggcacacgc caccacacct ggctaatttt tgttttgttt tgttttgttt tttgagatgg 45540agtctcgctc tgtcgcccag gctggagtgc agtggcacga tctcggctca ctgcaagctc 45600cgcctcccgg gttcatgcca ttctcctgcc tcagcctccc gagtagctgg gactacaggc 45660gcccgccacc atgcctggct aattttttct attttttagt agatacaggg tttcaccgtg 45720ttagccaggg tggtctctat ctcctgacct tgtgatctgc ccacctcggc ctcccaaagt 45780gctgggatta caggcctaag ccaccgtgcc cagccttttg ttttgttttt ttaagataga 45840gtcttgctct gtcacccagg atggggtgta gtggtacgat cttggttcat tgcaacctct 45900gcctcctggg ttcaagtgat tctcctgtct cagcctcctg agtagccggg attacaggta 45960cccgccacca tgcctggcta atttttctat ttttagtaga gatggggttt tgccatgttg 46020gccaggctgg tctcgaactc ctgacctcag gtgatctgtc taccttgtcc tcccaaagtg 46080ctgtgaacca ccgcacctgg ccagactttt gtattttttt gtagggatga ggtcttgctg 46140tgttgctcag gctggtctca aactcctggg ctcaagcgat ccacctgcct tagtctccca 46200aagtgctggg attataggtg tgagccactg cacctggcca gaaagcgatt tttttttttt 46260taaatgagct gatagtcaac aggtgactct gtcacttgct ttctttcctt aatggagcat 46320gagccctttc cgtttgttgg cacctatccg gataggtccc tccatttttt taaatttttt 46380tattatatgg agtctcgctc tgtcgcccag gctggagtgc agtggtgcaa tctcgactca 46440ctgaaacctc cgccttccac caaacttcca ggttcaagtg attctcctgc ctcagtctcc 46500tgagtagctg gccttacagg cgcctgccac catgccctgc tgatttttgt atttttagta 46560gagacaggat ttcgccatgt tggccacatt gggcttgaac tcctgacctc aggtgatcca 46620cctaccttgg ccttgaaaag ttctgggagc caccatgcct ggcccccctc catcttttta 46680ctttttactg acctgggtga atgaaccaat gtataccgat catccctaga gatcacattc 46740agctgctccc cgctatggcc tcagtgcaca ttcgcctttg tgtcctgcga atcgttctat 46800gggggtagat tcctagaggt gggagcatcc aaggacagga cacccaccct ccgaaatggc 46860tgtccccatt cctggcgccc ccagctctga gtgacatggc ctgtttttgc atgtccccac 46920cagtgtgacg acaggaaatg gagtcttggt tctgtttgca tttgtttgat tctctgtcta 46980tggccattac ccgctttccc atctggaggt tcaccttttc caaggcagtt ggttttcaca 47040ggaaacgcgg ggctctggcc agaaatgcag cgcagatgag cgcgaggagc ctgtgcaaac 47100aattccacat gcaggaattt ggggctttgc agccactcct gcctagggga aggatcgtga 47160tgcatcagca tccctgagga cctcaagctc ctgcttgtcc ttctcttcca gaaaaaaccg 47220ggaagatcct gacggagttc ctccagttct atgaagacca gtatggcgtg gctctcttca 47280acagcatgcg ccatgagatt gagggcacgg ggctgccgca ggcccagctg ctctggcgca 47340aggtgagagg tgctgggagg gctcgggtat gttctgctac ttctcgccat gtggcctcgg 47400gggagtgacc ttacttttct gggcctcagt ttcccattgt tactgtgatg gcctctaagg 47460gtctccccag cttctttttt taaaattaat tatttttttc ttttttgaga cagagtctcg 47520ctctgtcact caggctggag tgcagtggca caatctcagc tcactgcaac ctccgcctcc 47580caggttcaag gcactctcct gactcagcct cccgagtagc tgggaccaca ggcgtgcacc 47640accatgcccg gctaagtttt tgtattttta gtagagatgg ggttttacca tgttggccag 47700gctggtctca aactcctgac ctcaagccat ctgactgcct tagcctccca aaatgctggg 47760attacaggca ggtgtgagcc actatgtgcg gcttattcaa tttttttttt ttcagactgg 47820gtcttgctgt tgcccaggct ggagtgcagt ggcgcaatca tgcctcactg cagcctcaac 47880cttcctggct caagtgatcc ttccacctca gtctccccag ttactgggac tacaggtgca 47940caccatcact ctaggctaat ttttgcattt tttgtagaga tggggttttg ctgtgttgcc 48000caggctggtc tcaaactcct gggctcaagt gatcctccag cctcagctcc ccaaaagtgc 48060tgggattaca ggcgtgagcc accatgccta gcctccttcc cacttctgaa taggtgggta 48120acccaggtcg tgttgttgag accccttcag tcaccgtggg tgactcctcc tagctcacct 48180cctccaccat cagagctggg gctctagagt ctggatagtc tccaattcaa ggcaagctcc 48240tccagtaaga gcctcagttt actcatctgt caaatgggat gagaacacct ccttcacagg 48300gtggttgtgc ctgaagcagg gaaggcactg tcactggctc catcatgatc tgctatgata 48360taaatagtag gatgctgttt gctaaggaca tatttgttga gtgagagatt agaagctgcc 48420tggctccagc ctgagggtca gcgtgggcca gagggggctg gcaaagcctt ctgggaagag 48480gaacctgtga ttgactcgga cacggggccg ggggcgggac tcaagagatc ctcccacctt 48540ggcctcccaa agtgctggga ttacaggcct cagctgccgc acctggccaa ggctgagcct 48600ttaagtggct cattcaggat ggcggaggct ggaccagccc agggcccttt ctgctgtgac 48660tgcaacccgc ccggtggagg gaggtcagga ccgtgtgtcg gagctgctca gagcatcacc 48720tttgcttttt gacccagaaa aggttgcctc ttccccagtg tcatggtaac ctcttgaccc 48780tgtggcagca gagcgtcaga ggggcctgct gtggcccggc ctgggtgtct ggcaggtgcg 48840tatctgtgtg gtaggaaagg aaagaaaagg aggccttgga cgcagcccag gtgagcgggg 48900ccagaatggc tcccctgtct ggaaagaaat gtcatgggca gcccaagtcc ctataatcca 48960agacaagtgg gcactggctg agggcgtgaa gcatttcctc tgcgtgaggc ctcatttaag 49020tctggggagg caggagtgca tatttatttg tagaaaaata gcaacatgta agcaaggaga 49080gtgaaatgag atcactcacc cacagtccct gaaaagcagt atttttggtc tcattttggc 49140cactcaggaa accgatcatc acaatgcaaa gttatgataa catcataata ctggaaataa 49200tattaatcac cagtggtgat caaaacaata gtggttttta ttagaaacaa taatagaata 49260caaataaaaa tagtgataat aatgataacg taataataca acagaaataa gaataataaa 49320acagcagttg tctttttgtt tgtttttgct ttttcagagt ctcactctgt tgcccaggct 49380ggagtgcagt ggcacagtct cggctcactg caacctccac ctccctggtt caagcgattc 49440tcatacctca gcttccccag tagctgggat cacaggcatc caccaccaca ccccgctaat 49500tttttatatt tttagtagag acagggtttc accatgttgg ccaggctggt ctcgaactcc 49560tgaccttagg tgatccacct gcctcggcct cccaaagtgc tgggattacg ggcgtgagcc 49620acagcgcccg gctaaaacag cagttatctt atgccggtac tgtttgctca gccccgtggc 49680cccactctga gggaggggct atgatgatgc ctgcatcaca gatgtagaca ttgaggctca 49740gagatgtgga gctacttgtc caggatctgg cagtcggtgg tgccgacaca ggagttccag 49800ttatctgacc ttggggcctg tgctgtctct gaccgaggca ttggagagct tcgcagagtc 49860taaaatattt tgcctgggct gtcgagttct gtttcatttc ctgcaggcct tccaattgaa 49920cacgactctg ccatttgaaa gtcttaaaaa taaaacccaa acccacagcc agccacccat 49980tcgagctccc caggatctgg gggcggactg ggtggtgggg gttgttgagc ctgcctgggc 50040cacaagcctg cttctgcaca gcctcctgct gggctgggac tggccctgtc ccggagtgcc 50100cgtccttgtg tcccggggct cctgccctaa gcagggatga agatgaaaac taggggcctt 50160ttgctagggt gttatgagag ctctgtctgc tctttctaga aaagacaggg atttgagctg 50220ggtgtgggca gtttatacat agacggctga gggcagctct gtgacttggg caaagcccac 50280tgtgtgtggg ggtggtaggg gaaccatttt cttttcctgt aaatattttt atacccggta 50340ggaagaagcc tgcaggtcct attgtgtgag cacaatgggc tctccttctc caggagtgtt 50400ttgtgtttcc ccttgaagag cagcagggag ggagctgggg agagggggac atggtgaagg 50460gggtggagag acgggactgc agggatgggc cagcgcagag ccaggaagcc aggaaggtgc 50520aggcatgatg gcaatcaccc tttctccatg agtgcatctg agaaatgttt ctttttgcaa 50580aaggcacctt atttattgta gaacaaatag aatttgcaga ggaacactgt actctcctgg 50640gttccctccc atccaagacg aagcctcttc cactgtggtc tatctccttc cagaagcttt 50700ctggtgcttg tctgctcagg acgccttgca cctggccata gcttcgcttg ggcccccaac 50760cctagtttaa agatctccct tctccttccc catccccaga taagtcagtc cctgaggatt 50820ggaggtcgtc acagacgctg agtcctaccg tagtggtcat ggcggtcctg tgcattatgc 50880acctgcctgt caggtgccaa ctcttactgt tcccatttta cagatgagga aactgatgct 50940caaagtgact agacacactg tgagtcaggc agagctaggg ctttgtagga aggtccgggc 51000gtttctgcag cccatgtagg tcactagcct gccaggacgc gggtcagaca tcaaacaccc 51060ccagacagag gccccctcca ttccccctac tcctttttag gaaggcgtct catcagccat 51120cagagctgac aggctctctg gagccacctt gttcaagtta tcattgccct gaggggaaac 51180agaagcctag agctggggag aggcttgccc aggcactggg gccctcgcag ttctggcctg 51240tgggtgtgtc ccccgtgccc ggagcgacct cggggcacca gtgctgtagc tgtaggtggg 51300tggggagcaa ccatctcccc agacccttcc aaggtagcgg tgacagccca aggctgggat 51360ggagagggag agcctggctg tggccaggcc ctacatcagt gcctgtcctg ggttcaagtc 51420ctggcatcac caccctgcct ggatgctggg tgagcccctc cacggctccg agtctcgctc 51480ctcctctgca gaatggatga gatgcagccc ggcctcagag gccttgggag gacttgggga 51540gacccaaaat ggaggacatg ctaggcaggg ggtgcgcaca caggctctgc tggctgggcc 51600atcagcttca tggggagcac caccccccca gacctgggcc tcactcccca cccgctcctg 51660cccacaggtg ccactggacg agcgcatcgt cttctcgggg aacctcttcc agcaccagga 51720ggacagcaag aagtggagaa accgcttcag cctcgtgccc cacaactacg ggctggtgct 51780ctacgaaaac aaagcggtga ggcctggccc cgggcgccac actgagcctc cccgctttct 51840ccatgtgtgc cccctccccc aactcggaaa cttaggcccc agacgccacc gtcacagcct 51900gcacgcggtc accagggcca gagcaccacc tcagcccctc ccctccccca aactgggctg 51960ccctgggtgc cctctctgga cggcagctca ttcttcatct ctggggaata tcgctgactc 52020cttctgtccc tcataaacca actgtcactc actcctcccc atccctccta atctctcttc 52080agtccctcca gctggctcca gggcgttcct cctgccacct ccttcctcac tgggcttcct 52140gcctctcctc cgacccctcc caccctcgac agccacagag agctgttgga ggcacaatct 52200ggccattcct tgcggtggct tgcccctgcc ctacaggtgg gtaggtgtcc gagaaggaca 52260tcctatggga gcattgtgcc ttatcttttc cctgagccct cagggaagcc ccacctggtt 52320gagccacaga cctgatttct tcaattgctc atttctccag agcagccaga aggacaggcc 52380tgggccctgt atttcacgga agatcaatgt tagagcaaca taaagtcaag agcaacctcg 52440tgccccagag tagtggttct caactggtag cacgtccaaa tcacctgaag ggcttgctga 52500aagacacggt gctgggcttt cccccagagt ttgattcagg ttgggagcca agatttgcat 52560ctcttttttt tttctttttt tttttttttt tgagatgaag tctagctctg tcgccaggct 52620ggagtgcagt ggcgccatct cagctcactg caacctccac ctccctggct caagtgattc 52680tcctgcctca gcctcaggag tagctgggac tacaggcgtg caccaccatt cccagctaat 52740ttttgtattt ttagtagaga ctggttttca ccatgttggc ccggatggtc ttgatctcct 52800gacctcatga tctgcccacc tcggcctccc aaagtgctgc gattacaggc gtgagccact 52860gcacccggac tgtaaagatt tgcatttcta gtaagttctc gggtggtgtt gaagatcaga 52920gaccatgttt tgcaaatcag tgccctacag gttctgagct actttcagga actgtgttgt 52980atagtgacac ctagtggcag ggagtagcat ggtagccctg ctgtatgagc tattccatca 53040gaactgcttt gccccctgct actggtgtaa gtttctagac ctaattctcc agaggacgct 53100tgtgtgatgc tcagaaccta ggaaattggt gggggcagtt cagggtaatg gtcaaatacc 53160ccaggctctg gagatagata gccctggttc atgtcctaca tctgctactt cccaactgtg 53220tgaagttggg ggtgatccac cttttgaatt gggaaaattc tgtgaaatta agcatgcaat 53280gcccttagca cagggcctgg tatgtaagaa atgcttaact agtggagctg ctgggaacag 53340aggcaatagc agcagtagaa acagaacaac aacctcggcc aagctcgctg agtccttgtg 53400gtgtgccagg cactgcagat tatctcactc agtcctcaca tggctctgtg aggcaggaat 53460ggttattacc ctggttttgt ttttatttta aaatgttttc cccatcctga atattcttga 53520ttccctggtt ttatagatga agaagctgaa gtttaggcat ttaaggaatt ttatcctcag 53580catcacacag acactaccca gtgaagccag gaattgagcc cagagctggg gtctaggttg 53640ctgtggatgc cacactgccc caatcaccat gatagtgaca ttctcccctc tgttcctctc 53700ttgcctcttc ttcctttccc ttctcctccc cactttgggc cacactgagt agggccttga 53760agccaggtgg gatttgggct tggccgcacc ttaaccctca gggaccctct ctcttgcctc 53820cctcaggcct atgagcggca ggtcccacca cgagccgtca tcaacagtgc aggctacaaa 53880atcctcacgt ccgtggacca atacctggag ctcattggca actccttacc aggtaaagga 53940gcacccatcc caggccacaa tgccctgcac aggcatctgg agctggccag gggaggggtg 54000gccctgagcc cattgctttc agccccatgt ttgcggagag accaactagg cttagagctg 54060tggatggaga cctcattgtt tgcctttcgc atctccctaa gtccttgaga gcctcctgga 54120agatgttcag tgaccttccc tcgtaaacct gtgggtcagc actgtgttcg tcaggagtta 54180ctcttcttgg catcctgctt ttgcttcacc ttggctgaaa ttccctagaa gccttacggg 54240aacaacattg gcctctctga tgatccctgg gaactagagg gcaggggaca tgcctgcctg 54300gggggcaggt catgccccag ggacttgaga gtagagtcat gccttcattc tgtggtgatg 54360gagggcctgg gcattcctgg attcgagcgg cattcctgga ttccagcagc attcctggat 54420tccagcgtga ctctggcttt gtttcactgt gtctgcttgg gcgagtcacc ccccctcccc 54480tgaatctcag cctcttcagt gtctacaaag cacaggcact aagaggactg ccctcggggt 54540gttgtgtgga ccctggaggg gcacaggcca acccagagcc agggcagaaa gaagaactgt 54600tattgtcata tcattaacaa cagtggcagg gtcacagccc aggaccagtt atctcgctgg 54660atcttacata tccttgagaa tgaaattgaa ggagggaaaa attccttttg ctgtattttg 54720tccctcccca catgcctccc tccccctgtg gacacttaag ccacactttg gcctccacag 54780ccgaaatctg aagggaaacc caagaaggta gtgcttgacc cttgaacctg ggggctctga 54840ccttgtgttg tttgtgcttt acagagttta aaacctaacc caagagtggg gtggatgtga 54900cttattaccc actctaagaa atgggtctgc acggatgctg gactcaggtt ttgggctgag 54960gagaacacag ccagaaagaa ggctgctccc acccccgccg gcttgctggt caccttggga 55020caggctggcc ttctctgggc ctgagcctga cctcagagct gatgccaagg gatcagttct 55080ctgacatcct ctggtggagg gcaaccacag agggcagctg ctcatcaggc actgtctcca 55140cagggaccac ggcaaagtcg ggcagtgccc ccatcctcaa gtgccccaca cagttcccgc 55200tcatcctctg gcatccttat gcgcgtcact actacttctg catgatgaca gaagccgagc 55260aggacaagtg gcaggctgtg ctgcaggact gcatccggca ctgcaacaat ggtgagtggc 55320ctgtgggtgc agggtcggtg ggagggaggg gcaggacctg ggttattgct gaatggtgac 55380acctagtggc tgactatggt gtggcaaccc tgctctacag gctgttctaa tttcccttct 55440ggggttataa gaaaccatgt aataccaccc ttagtcttct ttcttgatga aaacagtttt 55500gtgctgctca ggaaaaaagt aggaaattat tcacagaggg gtgtggttgt tgagcccttg 55560gaccctgagt cacaaagtca caatcctgat tctgccattt tccagctacg tgaccttagg 55620gaactgactt acctcttgga ggctctgtaa agtggggata atgccttgtg tgtcttaggg 55680gggttatgag gatttagagt ctacctcatg cttggtaaag attttttttt taattttata 55740tatatatata tatatatata tatatatata tatatatata tatatatata tatatatttt 55800tttttttttt tttttttttt aaaaccacca acctgcaggg tgatttggaa acttcactgc 55860ctccctcagg gcctcattgc tctcatctgt aaacttatgg ggtgactctt ccagctcact 55920catttattca acaaacattt ttagtgtcta ctctgaggag gcccagattt aggcactggg 55980tgtgcaacaa gggagcaaga aacattccct gccctcctac aattggcagt ctggtagggg 56040agacagatga tagacagatg aggatgccag cacgatggct cacagctgtg ctgggtgctg 56100ggtggagcca acctgggagc ctgagggtgt gtgtgtgttg ggggaggggt cgatcagggc 56160aggctccctt gaggaggtta tggttcagct tcagcagaga cctgaaggag aagtcccaga 56220cctgaaggag gagatgccag aagtcccagg gcccctcata tcctgcatag aggagagggg 56280aggagtgttc caggcagagg taggcagtca gcatgaaggg agacccacag gtggtagggg 56340taggcccgga ggaggaactg cacgaggccc tgaagcaggg agggggtgac ctgaaatggg 56400gctggagggg ggcctctggg gtcaggtctg gaaaagcctt gaaggcctct aaggaatttg 56460aacagaaagg ctgaggatgg tcttccaagg tgagggtggg tccctggggg tctttctgcc 56520cctagggaag gaacaaccca gtcctggaca ctgaactgct tccaggtgat ctggcctgag 56580ggaagctcct gcccccgccc cacccttggg cgcagtgcca catggtgcag aggtggaagg 56640ttgcagggga cggagtgggc gccaatggcc tctgggctgc tgccgtggga ctcagctcac 56700cccacccctc ctgctcgcct ctccagagcc acctctgccc tgtctggaat ctgccaagca 56760ggaggaacca ggtcttcaat caggccagcc cctgccacct gggcccagct gcccacaaag 56820ggcctcctgt tcccaggtcc cggggactgt gggctgggac tgggtggagg cactcaaggc 56880tctggctgcc tggcccaggg ccctcccgcc tccccagccc ctggcctggt gccatctctc 56940caggtttcca aggctaacgg ctgacctggc ctggcagggt gggtgacctg cgccagatag 57000ggaaggggtg ggcacagaac aaggtgggga agggccacct cagtccccca tgtgtgcagt 57060ggggatgatg ataaacttga gcttgagaga tgattgggaa gagctggtct gggtggggca 57120cttggccgtg ggcctggcac gcacagtgag cactcagtgg ggaggccatg ggggagttct 57180tggttctaag gtgcagtgga ccagagggag gcccaggaga gctgctgggc cagggaatga 57240aggggtgtcc tgtgccagga aggtgtcctg ccgggtggcc ccttctcccc agggcgggag 57300ctgacgaggc tggtttcagg ccttccttcc accctgcgga ctgtgagaaa gttctctgct 57360tcctccgtct tggaggctgg gcttggaatt tgtgaagcct ggaattgctc actccggggg 57420ctgccaggaa gtggggggtg gggggaagga ggccaactgg gcggacacca gcggtcccag 57480ggaccccttc acatcctgca ttcctggagc agctgggggt ggcaggaggc agctggggca 57540ctggctgtgc cgggtcctca gtgttggcct ggggtgcaga ggtcaaggct tcttcctgtc 57600tgaccgtggt ccccctctgt ctgctgcctg ggagtgagga atgcagtttc ctttcttgga 57660aggtctccag gtctggctag agtcagctgg tcctgggttc aagccccgct ctgccatcca 57720ctagctgagt agcctcgagc agtaactgcc tctctgagcc tcagttcttt ccctgggaaa 57780agtagggcac agtgagctga ggattcaacg agctgatgtg tgtgcagcct cagccagggc 57840tggggcctca gtttcccaag actgccataa caaataccac agaccgggca gctacacagc 57900agaaatatgt tctctcccag ctggaggtct gagtcgaggg caaggtgtca gcagggctgg 57960tttctccgag acctctctcc ttggctggtg gccggccgcc ttctccctga gtcctcacag 58020ggtcatgcct ctgtgtgcct gtgttttctc ttctttcaag aagaaatgta cttttctttt 58080tttctttttc ttttttcttc ttttttgaga cagaggcttg ctctgtcacc caggctggag 58140tgcagtggcg caatctcggc tcactgcaac ctccgcctcc caggttcaag tgattctcct 58200gcctcagcct cccgagtagc tgggactaca ggcgtgtgcc accacacctg gctaattttt 58260tgtagccaga atggtcttga tctcctgatc tcgtgattct gccgcctcgg cctcccaaag 58320tgctgggatt acaggcgtga gtcactgtgc ccggccaaaa tgtactattc ttttaagggc 58380actagttgta ttggatttgg tcccatcctc atcccctctg tttaacttaa ttctttaaga 58440ctctaggacc aagggcgggt gcggtggctc aggcctgtaa tcccagtgct ttgggaggct 58500gaggtgggtg gatctcttga ggtcaagagt ttgagaccaa cctggccaac atggtgaaaa 58560cccatctcta ctaaaaatac aaaaattagc tggacatggt ggcgggtgcc tatagtccca 58620gctacttggg agctgaagcg ggagaatggc ttgaacctgg gaggcggagg ctgcagtgag 58680ccaagattgc accactgaac tccagcctgg gcgacagagt aagactcttc tcagaaaaaa 58740aaaaaaaaaa aaaaaaaaaa gaccctgggt ccaaatataa tcacgttctg gggtactggg 58800ggttaggact ccagcatatg aatttggggg gtgtggagac acacttcagc ccctaacagc 58860taggctcacg ggaagagctc aggaggggtg accctgaaac ttgtcacact cagcggctgg 58920gtgtgcagaa agagagaatc ccaagggtca aggctggctg gcctccctcc cgctgtcccg 58980gccctctgcc tcaccttctc gccacctgga aagcaggcct gccctgcaca cctgacttgg 59040tgcatgcagg atgaaaattc cctctccccg tgggagtgcc tgcaggtaac gcactgtcca 59100gattttccag tcaaaacttg gtttctagga aaccatgccc ttctgagagg cgggtggggt 59160ggcgggggga cctccttccc tggcccaggt gggcagcccc cacccagggc tctgcatgct 59220gctccagctg gaacatccct gccctgctcc tgtcactcct gcctgacttc aggtctctga 59280aatgcaggag tgagactccc acaggccccc gtccccatcc aggcagctgc gctgtaggga 59340tgagggctgg gcctccccct gctcagctcc ctcccggctc cgacaggcag tgatttctgt 59400ggtcagctgg gtgtggggag aggagggacc cagagagggg ctttttctct gccggtggtg 59460actcagccca tagtcatggc agtgggagcc ttaggtttct gatgcacagg cttccgtttc 59520cccttcctgg gctttgggga tgcggtggtc agaggcacgc ggccagggga gtctgcctgc 59580tgcctggctc ggtggcgttt gaccaccttc ttaccccaag tcagattttg taacttcctg 59640ccaattgcct cccgggcact gaatctggag gtccacgcct cactcagggg tttaccttgc 59700tggcgtttgg gccactgaca gcaccaacag tgacaaaaat tcagaaatgg tgttcttcta 59760gctgttatta atagcaaact cttccattct tttttttttt tttttttttt tttttgagac 59820agagtcttac tctgttgccc aggctagagt gcagtggcgc aatctcggct cactgcaacc 59880tctgcctcct gggttcaagc gattctcctg cctcagtctc cctagtagct gggattacag 59940gtgcctgcca ccatgcccag ctaatttttg tatttttagt agagatgggg ttttgccatg 60000ttagccagac tggtctcgaa ctcctgacct caagtgattt cccccacctc agcctcccaa 60060agtactggga

ttgcaggcat gagccaccac acccagcttc attctttaac aaagttattc 60120aagcatctgt gccaggtact gacctggact tagcagtaac aggacagacc ccgggcccag 60180tggggcaggc agctggctcc tcagttattg ataaaaagtg tggcgcagag cagtgctgga 60240ccctgatctc ctgtccctac actgtggccc agggagcccc gcagtggacc tgtgaggcca 60300ttgttccatt gttcagaaga ggaaacaggc catttcgggt tcagtgatct agaaccaggc 60360tctgaaacct atggcctatg ggcccagatc agccaattgc ctgtttctgt caacaaagtt 60420tattggagca gaggcacgct catttgtttc tggatggtcc gtggctgttt ttgtgctgtg 60480actgtagagg cgagcattgc gggaggggtt ttatggcctg ccgggctgag aatatctgct 60540gcctagcctg ttccaggcaa agtttgaagg gccctgatct agcagtaagg gaggaactgg 60600ggctcgaaca agggtctctg ggtgcctcta gggccattct gggtggctgc ttatgggacg 60660ggagctttta gtcctggctc tgccactccc tgctctgtga ccctgggccc gtggcctgtc 60720ccctaacggt tgtctcttca tctgtaatca tctgtaaagc aataaggtga atgatagtgc 60780ccaggtcttg gggtggttgg gaggatttca ggagctaagg aggaggcaag ggggctgcct 60840tgtcactgtt ggtggtgggg tcattactgc ttttgcctat cagatacaac catgactgct 60900ctgatgacga ggaaagtgag ggtcccagct gcccctcctg aggtgcatgc catgtgtgtg 60960gggggggttt tggtcagttc tcagttggtc ttggtagagg ccactgggcc atctgctgac 61020ctcctctctc cctccgtctc ctgggcagga atccctgagg actccaaggt agagggccct 61080gcgttcacag atgccatccg catgtaccga cagtccaagg agctgtacgg cacctgggag 61140atgctgtgtg ggaacgaggt gcaggtgagg ccagacgggc tggtgaggaa acctcagaag 61200tcacccaagg acccagcagg ggagggcagg gggtggggta cagagatggg gctctggggc 61260gctgccggtt tggaaggaga tgacatgctt agatttggac aagtgcaggg ccagtgcacg 61320tggagatact cgctgtctta ttcatcactt actgggcatc tgagcaccta ccctggacga 61380ggcactgggg tgcagctggg aatgatgtgg acaaattcct ggccctcagg gagctctccg 61440tatagcagga gaaacagaca tttggcaaat aaatgtacac ttccatgtgc aggcaaagca 61500gggagggagt atggtattct aggtagggaa ggcagcctgt gcaaaggccc tggggctggc 61560ccttgagtct gaggatccta caggacaccc aggcagatga gttgggacct gtgggaatgg 61620agctcaggag agagggcagc agggagggtg aagcaatggg ccagtcgcgg aggaggtggg 61680ggctgcagcg gcctggcttg tgggaatgca gagagaaaca ggccagctga gcgctctcca 61740gcccctggcc tggcttgtgc ctcctcaacc tagatcctga gcaacctggt gatggaggag 61800ctgggccctg agctgaaggc agagctcggc ccgcggctga aggggaaacc gcaggagcgg 61860cagcggcagt ggatccaggt gggtgggcct ggggccctgg agcggggacg ggacctgcag 61920ctggtgcggt gccctcagcc ctggtgccaa ttcagctgct ctgcccacag atctcggacg 61980ccgtgtacca catggtgtac gagcaggcca aggcgcgctt cgaggaggtg ctgtccaagg 62040tgcagcaggt gcagccggcc atgcaggccg tcatccgaac tgacatggac caaattatca 62100cctccaagga gcaccttgcc agcaagatcc gaggtaggca gccacccccg tgctcgacgg 62160gccctccagg ggccaaggtg gactttcttt ttatttcatg attcatgtaa atttcacagc 62220aatgacttgc tcattgatca tttgtaagaa ttagagccaa atctgcactg tggtactcca 62280acccctgtga gttgttttgt ggctattatt tctataagcc cagaacttga agagaaaaat 62340ttttgttctg gaggttcaag ccttcttttc atctctgact cgtcacagat ttttaccatg 62400tgtcatttta aagtacattt acgttcagca agtatagctt tcccaggtca gtttattctg 62460tcacccaggc tggaatgtag tggtgcgatc tcagctcacc acaacctccg cctcccggat 62520tcaaacaatt ctcctgcctc agcctcctga gtagctggga ctacaggcac acgcgcgcca 62580ccatgcccgg ctaacttttg tgtatttagt agtgatgggg tttcaccatg ttggccaggc 62640tgatcttgaa ctcctgacct cgtgatccgc tcaccttggc ctcccaaagt cctgggatta 62700caggcatgag ccactgtgcc cagactgtac gcctttctct tcccagccag atgggatcct 62760ttcaccagct tggtccccat aacacatctg cagagctcag ttttagcaat tgtcagttgc 62820ccatgggacg ttctttttat tttattttat ttcattttat tttatttgag acagggtttc 62880actctgtcgc ccaggctgca gtgcaatggc acgaccttgg ctcactgcag tctctaccat 62940cctcccactt caacccccca agtagctggg actacaggca tgtgccacca cgcccagcac 63000atttttgtat tttttgtaga gacagggttt tgccatgttg cccaggctgg tctcaaatgc 63060ctgagctcaa gcaatccacc caccttggct tcccaaagtg ctaggattac agatgtgagc 63120gactgtgccc agccattatt attattatta ttttgagatg gagtcttgat ctgttgccca 63180ggctggagtg cagtggcaca atctgggctc actgcaacct tgcctctcgg gttcaagcaa 63240ttctcctgcc tcagcccctc aagtagctgg gattacaggc acctgctacc atgcccagct 63300aatttttgta tttttagtag agacagggtt ttgccatgtt ggccaggctg gtcttgaatt 63360cttaacctca agtgatccac ctgcctcggc ctcccaaagt gctgggatta caggtgtgag 63420ccactgtgcc tggcctgttt tttttgtttg tttttttttt tttttttttg agacggagtc 63480tcgctctgtc gcccaggctg gagtgcagtg gcgtgatctc ggctcactgc aagctccacc 63540tcccgggttc acaccattct cctgcctcag cctcctgagt agctgggact acaggcgccc 63600gccaccatgc ctacctaatt ttttgtattt tttagtacag acaaggtttc accgtgttag 63660ccaggatggt ctcgatctcc tgacctcatg atccgcccac ctcggcatcc caaaatgctg 63720ggattatagg cgtgagccac cgcgcctggc cgcccggcct gttttttaag agacagagtc 63780tcgctctgtc ttccaggtag agagcagtgg tacaatcatg gttcactgcg tgacctcctg 63840agctcaagta gtcctcccac ctcagcctcc tgcgtaactg ggactacagg tgcaggccac 63900cacgcctggc taatttttaa attgtttgta gagaaagggg tcttggctgg gcaaaactct 63960atctctttta aaaatttttg aaagagagag agagaagggg gtcttactgt gttgcccagg 64020ctggtctcga actcctggcc tcagacaatc ctcctgcctc agcctcccaa agcactggga 64080ttacaggcat gagtggccgt gcctggtctc ctgtaggtta ttctttacta gccagatcct 64140gagtattggt tgacagctgg ataggacact gagcattgac atttcttgtg tggaacaaag 64200aacgttagct gggctgggat gggggtggac atctaaatat tcttgagtga aaatgctttg 64260gtcagaatgt tctggagatg gtccccaccc aaaggaagaa ggcttttaca taattatgca 64320gagccagcca cactattcca gagaggatca gcccagtttt tgtaaaacca agaggctcat 64380tgaatctact aaggtatttt tcattgctgt tgttcttttt gttcaaacga agaagcaaga 64440cccatcctaa aggagttttt gtctacttct agctaagcag aacactgctg acacctctgg 64500gctggcagtt ttcttatttt attttatttt cattttcatt tttaattttt gtgggtacat 64560agtaggtgtg catatttatg gggtacatga gatgttttga tcaggcatgc aatgtgtaat 64620agtcacatca tgggaaatgg ggtatctatc ccctcaagca tttatccttt gtgttacaaa 64680caatccaagt atattctttt agtttttttt tttttttttg gacacagagt ctcactcttc 64740ttgcccaggc tggagtgcag tggtgccatc tcagctcacc gcaacccctg cctcccaggc 64800tcaagtgatt cttctgtctc agcctcccaa gtagctggga ttacaggcac ataccaccac 64860gcccggctaa tttttgtatt tttagtagaa atgggatttc accatgttgg ccaggctggt 64920ctcaaactcc tgacgtcaag tgatcttcct gcctcggcct cccaaagtgc tgggattaca 64980ggcctgagcc actgcacctg tgcagcctct tttagttact tttaagtgta caattaaatt 65040attattgatt atagtcaccc tgttgcgcta tcaaatacta gggcttattc attctgtaac 65100cagaaagggg tcccgatcca gaccccgaga gagggttctt ggatcttggg caagaaagaa 65160ttcaaggtga gtccatagag ttaaagcaag tttattaaga aagcgaagga ataaaagaat 65220ggctactgcg tagacagagc acgagggctg ctggttgccc atttttatgg ttatttctga 65280tgacatgcta aacaaggggt ggattattca tgcctccctt tttagataga gcatataggg 65340agacttcctg acgttgccat ggcatctgta acctgtcatg gtgctggcgg gagtgtagca 65400gtgaggacga ccgaggtcac tctcatcacc atcttggttt tggtgggttt tgggccacct 65460tctttactgc aacctgtttt atcagcatgg tctttatgac ctgtatcttg tgccgaccta 65520tctcatccca tgacttagaa tgccttcacc gcctggcagt gcagcccagt aggtctcagc 65580ctcattttac ccagctccta tttaagatac agttgctctg attcaaatgc ctctgacatt 65640tctacttttt gtgcccatta accattccca tctccccctc gccctcgact accctctcca 65700gcctctagta accatctttc tactctatct ccatgggctc aattgctttg ttttctgctt 65760gtttatttgt ctgtttttac cttttttatt gctcccacag gggaggcccc ctccaaaatg 65820tcaatttgct ttaattttta gatcccacaa ataagtgagg acatgtgata tttgtctttc 65880tgtgcctggc tgaatataat gacctccagc tccaaccatg ttgttgcaga tgactgaaac 65940tcgttctttt ttacggctga atagtacccc atcgtgtata tgtaccgcat tttctttatc 66000cattcgtctg ttatggacac ttaggttgct tccaaatctt ggttgttgtg aacagagccg 66060caacaaacac gggagtgcag atatcgctgc gatgggctga tttcctttat ttgggtatat 66120acccagcagt gggattgctg gattgtgtgg tagctctatt agttttttga ggaacctcca 66180aactgttctc catagtggtt gtactcattt acattcccac tgtgaaccct gaaaatttga 66240ggcaggtctc agttaaatta gaaagttgat tttgccaagt tggggacacg cactcgtgac 66300acagcctcag gaggaactga tgacatgtgc ccaggtggtc agagcacagc ttggttttat 66360acattttagg gaaacctgag ccatcaatca acatacgtaa aatgggccgg gcacagcagc 66420tcaagctgta atcccagcac tctgggaggc cgaggcgggt ggatcacttg aggtcaggag 66480ttcgagacca gcctggccaa catggtgaaa ccccgtctct attaaaaata caaagcttag 66540ctggatgtgg tggcgcatgc ctgtagtccc agctgctcta ggaggctgag gcatgagaat 66600tgcttgaacc tgggaggcag aggctgcagt gagccgagat cgagccacta tactccagcc 66660tggtcaacag agtgagaccc tgtctcaaaa aaaaaaaaaa aatgtaaaat gaacatggtt 66720cagtccggaa aggcgggaga acttgaagga aaagcaggac aactcaatgc aggcatgagg 66780cttccaggtc ataggaagaa aagagacaaa tagttgcatt cttttgagtt gatgattagc 66840ctctccaaag gagggaagca gatatgcatt aatctcagtg agcggagggg tgactttgaa 66900tagaacggga ggtgggtttg ctttaagcaa ttcccagctt gacttttccc tttagcttcg 66960taattctggg ggcctaagat attttccttt cacaccacca gcagtgtacg gaggttccct 67020tttctccaca tcctcgctag catttgttat tttctttttt ttggataaag ccattttaac 67080tggggtgacg tggcatctca ttgtagtttt gatttgcagt tctctgatca gtgatgtcaa 67140gtaccttttc atatgcctgt tttgggttga gtacctttca tatgcctgtt tgccatttat 67200atgtcttctt tggggaaata tgtattcaaa tcttttgccc atttttaatt ggattactag 67260gatttttttc ctatagactt gtttgagatc cttatatttt ctgattatta atctcttgtc 67320agatggcagt tttcttagtt ttttgccatc atggtttcag aaagatcaga gtccaatctc 67380agaacacctt gtcatttttc ctgcatggat ttctcatctt tatccactat ttctctgaaa 67440tcattcagct tgctaatgtt gcccccagtg gttttttttt ttaaataata ttggccaggt 67500acggtggctc atgcctgtaa tcccagcact tgggaggcca aggcaggagg atcacttgag 67560cccagggagt ttgagaccag cctggataac acagtgagac tctgcctctt aaaataataa 67620taataataaa agtattgatg ggggctgagc gcggtggctc acgcctataa tcccagcact 67680ttgggaggcc gaggtgggcg ggtcacctga gatcaggagt ttgagaccag cctggccaat 67740atggtgaaac cctgtctcta ctaaaaatac aaaaaaaaat caccgggcat ggtggtgcac 67800acctgtaatc ccagctactt gggaggctga ggcatgagaa tcacttgaac ctgggaggca 67860gtggttgcag tgagctgaga tcgtgccatt acattccaga ctgggcgaca gcacgagact 67920ctgtctcaaa aattaataac aataataata tgatgggaag agagagttgg gagagggctg 67980aaaaattact ttttgggcac tgtgctcatt acttgggtga tgagatcaat cctacccaaa 68040acctcagggt cacacaatgt acccatggaa caagcctgca gtgtaccccc tgcatctaaa 68100ataaaagttg aaataaaaat gataataata ttaggaggct gaagtgggag gattgcttga 68160gcccaggagt ttgaggctac agtgagcttt gattattcca ctgcactccg gcctggacaa 68220cagagtaaga ccctgtctca aataaaataa taataggccg ggcatggtgg ctcatgcctg 68280taatcccagc actttgggag gccgaggcgg gtggatcacg aggtgaggag tttgagacca 68340gcctgaccaa catggtgaaa ccccgtctct actaaaaata taaaaattag ctgggcacag 68400tggcacgcgc ctgtagtccc agctactcag gaggctgagg caggagaatt gcttgaacct 68460gggaggtgga ggttgcagtg agctgagatc gcgccactgc actccagcct gggcgacaga 68520gcgagactcc gtctcaaaaa aaatataata ataatgataa taatagtaat acagtagcct 68580ccagcacctg ggctctggga tggctgggag gtgctccttg gggagcccct gctgacccgg 68640ctccctcggg gcacagcctt catcctcccc aaggcagagg tgtgcgtgcg gaaccatgtc 68700cagccctaca tcccatccat cctggaggcc ctgatggtcc ccaccagcca gggcttcact 68760gaggtgcgag atgtcttctt caaggaggtc acggacatga acctgaacgt catcaacgag 68820ggcggcattg acaagctggg cgaggtgagg ccggcaccgc ccctaggagg gggtctcctg 68880agagtaggtc tggtctgtcc cagagggccc cggctgcgtg actccgggca gccgttcccc 68940tccaaggccg tccctgtaga cggggagggg ctgctgggga ctaggggcag aggctgtgag 69000gccagggctg ttggagtcct cgtggggcct gcagtttgag gaaagaggga acattagctc 69060ccggccacca gcctttagaa accaagagtg gtgctaaaat agaaaagaat ttaacagaag 69120agagggaagg agggaaggag aggaggggag agggaaggag ggaaggagag gagggaagga 69180gggaaggaga ggagggaagg agggaaggag aggaggggag agggaagggc gggcttcagg 69240tggagtaatg agattgataa ggaagaattc acaagtcacg cgccctccaa atggagacag 69300ccgttgccct tacttgacct cagttatctg gctctcgggg tgaacgtcct tccacagtct 69360ccatcttggg ctggctgccc ggagcccgca tagttgtcag attgaattta tcctgtatct 69420tagtttcctc ctttatactc tgaaatgggg agtccacaga ccataataat cccagaatct 69480gagacctgga gaggaccttg gagagcagga aagctctctc ccaattttga agctgaggaa 69540actgaggctc agagagggtg aatgacttgc ccaaggtcac ccagcacctc agaagggatg 69600agtgccttct gccggcaccg aggctgcctc tccacagcgc tgtgccactg gactgcgccc 69660tccatgcctt cagagcctca tgcttctgtc ctgggccctc ctgggtttac ctttgtggcc 69720atgagcacca ggtggccatc cctggtagca ggcgccagcc ccagggcctg gacttcgagg 69780acccaggcaa agtgtgtggg gggtgctgcc tggccacagg gcggccctgc tcagcccccg 69840cggccccggc ccctgcagta catggagaag ctgtcccggc tggcgtacca ccccctgaag 69900atgcagagct gctatgagaa gatggagtcg ctgcgactgg acgggctgca gcagcgattt 69960gatgtgtcca gcacgtccgt gttcaagcag cgagcccaga tccacatgcg ggaggtagac 70020ccgaggctac gacccccacc ccacaccctg tccactgcct tccacaggcc ccctcgccca 70080gggggtccct tctgctccgt cccgtcatgc catagccacg cttgcctggc acgctctctc 70140ctcctctgct tggaacatct gtgcctctgc ccacctggct aattctgact cacttcccag 70200gtctcagctt agagacccct tcctccagga agctctcctt gcgcccccag gccccttccc 70260atatttcctg ttgccttgag gcagggactt gctccagcgt gcagagctgt gagtgggcat 70320agaccgcttg ccattcctcc ctggcaggcc tcggggtggg gggctggttg aggatgagga 70380cccctcagcg gcacccagga aggggctgtg gtcaccctgg gggcttcaca tgtccgcggt 70440atgccctgca gcaaatggac aatgccgtgt atacgttcga gaccctcctg caccaggagc 70500tggggaaggg gcccaccaag gaggagctgt gcaagtccat ccagcgggtc ctggagcggg 70560tgctgaaggt gagcggcccc acgccctgcc ccggaccgtc ctaggcgagg ccgagccccg 70620agcctcatcc ccggccacca ccaggattgc tgtccctgca aggaggcgcc atggtcctct 70680tgtgcggatg aggactcgga ggcttgggtc agaccactgg cctcgtccta ccctcagggc 70740caatggagcc cactgacctg cagacccccg acccccttgt tccctgcaga aatacgacta 70800cgacagcagc tctgtgcgga agaggttctt ccgggaggcg ctgctgcaga tcagcatccc 70860gttcctgctc aagaagctgg cccctacctg caagtcggtg agcagccccc acctgctcca 70920gccgtttggg ccctaagttg ggggatggag cccacaggcc tccctctacc cttggccctg 70980acctctcatc tctgccgccc cacccgggca ggagctgccc cggttccagg agctgatctt 71040cgaggacttt gccaggttca tcctggtgga aaacacgtac gaggaggtgg tgctgcagac 71100cgtcatgaag gacatcctgc agggtgggtg ccgtcccggg gtggggactg ttgccaggag 71160gatgctgggg gcgcagctga gaagtggtgg cagctgaggg ggtcagctaa ggctcaaaga 71220aaagcccaga cttgcacctg gggtctgggt gaagcccact cttcgctgtg tggcctccgg 71280agcagcctta gccttgcgtc tctgtgcttt agtttcttct cgcgtccttg gctttgctgg 71340gagggtcaga ggagtgatta gatgacatgt ggtcagcagc ctgggcctgg agatagcatc 71400acggccttgc tgctttcagc aacgaggcct ggaatgagtc acatcccaac cacagacccc 71460caaggcggga tgagacgggt cttctttgag cagagtccag cacaggggcg gctgtgggat 71520cagtgctgtg tcctgaccct tcccctgtgg ccctgtagct gtgaaggagg ccgcggtgca 71580gaggaagcac aacctctacc gggacagcat ggtcatgcac aacagcgacc ccaacctgca 71640cctgctggcc gagggcgccc ccatcgactg gggcgaggag tacagcaaca gcggcggggg 71700cggcagcccc agccccagca ccccggagtc agccaccctc tcggaaaagc gacggcgcgc 71760caagcaggtg gtctctgtgg tccaggatga ggaggtgggg ctgccctttg aggctagccc 71820tgagtcacca ccacctgcgt ccccggacgg tgtcactgag atccgaggcc tgctggccca 71880aggtctgcgg cctgagagcc ccccaccagc cggccccctg ctcaacgggg cccccgctgg 71940ggagagtccc cagcctaagg ccgcccccga ggcctcctcg ccgcctgcct cacccctcca 72000gcatctcctg cctggaaagg ctgtggacct tgggcccccc aagcccagcg accaggagac 72060tggagagcag gtgtccagcc ccagcagcca ccccgccctc cacaccacca ccgaggacag 72120tgcaggggtg cagactgagt tctaggccag tgggtccctg actgctgcac atggcacagg 72180ccgttccctt ccggacccag gcaggctcag ctctggggag ggcaccctgg tctgtgcctt 72240gtgggtggag gcggggcagg gctgtgtggc accgccaggg agcgggccca cctgagtcac 72300tttattgggt tcagtcaaca ctttcttgct ccctgttttc tcttctgtgg gatgatctca 72360gatgcagggg ctggttttgg ggttttcctg cttgtgccaa gggctggaca ctgctggggg 72420gctggaaagc ccctcccttc ctgtccttct gtggcctcca tcccctcatg ggtgctgcca 72480tccttcctgg agagagggag gtgaaagctg gtgtgagccc agtgggttcc cgcccactca 72540cccaggagct ggctgggcca ggaccgggag agggagcact gctgccctcc tggccctgct 72600ccttccgcag ttaggggtgg accgagcctc gctttcccca ctgttctgga gggaagggga 72660aggagggggt cttcaggctg gagccaggct gggggtgctg ggtggagaga tgagatttag 72720ggggtgcctc atggggtggg caggcctggg gtgaaatgag aaaggcccag aacgtgcagg 72780tctgcggagg ggaagtgtcc tgagtgaagg aggggacccc catcctgggg gatgctggga 72840gtgagtgagt gagatggctg agtgagggtt atggggagcc tgaggtttta tgggcctgtg 72900tatccccttc tcccggcccc agcctgcctc cctcctgccc gcctggccca caggtctccc 72960tctggtccct gtccctctgg tggttgggga tggagcggca gcaaggggtg taatggggct 73020gggttctgtc ttctacaggc caccccgagg tcctcagtgg ttgcctgggg agccggacgg 73080ggctcctgag gggtacaggt tgggtgggcc ctccctgagg gtctggggtc aggctttggc 73140ctctgctgcc tctcagtcac caagtcacct ccctctgaaa atccagtccc ttctttggat 73200gtccttgtga gtcactctgg gcctggctgt cgtccctcct cagcttcttg ttcctgggac 73260aagggtcaag ccaggatggg cccaggcctg ggatccccca ccccaggacc cccaggcccc 73320ctcccctgct gctttgcggg gggcagggca gaaatggact ccttttgggt ccccgaggtg 73380gggtcccctc ccagccctgc atcctccgtg ccctagacct gctccccaga ggaggggcct 73440tgacccacag gacgtgtggt ggcgcctggc actcagggac ccccagctgc cgcagccctg 73500gtctctggcg catctcttcc ctcttgtccc gaagatctgc gcctctagtg ccttttgagg 73560ggttcccatc atccctccct gatattgtat tgaaaatatt atgcacactg ttcatgcttc 73620tactaatcaa taaacgcttt atttaaagcc a 73651

Claims

1. A method of predicting or assessing the level of risk of a subject developing a Methicillin-resistant S. aureus (MRSA) infection comprising: obtaining a biological sample from the subject, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; and predicting and assessing the level of risk of the subject developing a MRSA, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a high risk of developing a MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1, indicates that the subject has a low risk of developing a MRSA infection.

2. The method of claim 1, wherein the detecting step further comprises hybridizing at least one oligonucleotide occupying a locus corresponding to position 17 of either SEQ ID. NO: 1, to an oligonucleotide probe comprising a sequence that is complementary or identical to SEQ ID NO. 1, under stringency conditions than can detect the presence of different alleles at position 17 of said oligonucleotide or hybridizing at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO: 2, to an oligonucleotide probe comprising a sequence that is complementary or identical to SEQ ID NO: 2, under stringency conditions that can detect the presence of different alleles at position 17 of said oligonucleotide.

3. The method of claim 2, wherein the detecting step further comprises evaluating the hybridization of at least one oligonucleotide from the biological sample which corresponds to position 17 of SEQ ID. NO 1.

4. The method of claim 3, wherein the detecting step further comprises sequencing the oligonucleotide from the biological sample.

5. The method of claim 4, wherein the detecting step further comprises amplifying the oligonucleotide from the biological sample.

6. The method of claim 5, wherein the amplifying step uses at least one oligonucleotide primer and at least one oligonucleotide from the biological sample occupying a locus corresponding to position 17 of SEQ. ID. NO. 1.

7. The method of claim 6, wherein the oligonucleotide primer comprises DNA.

8. The method of claim 1, wherein a treatment step is taken after it is determined whether the patient has a high or low risk of developing a MRSA or CA-MRSA infection.

9. The method of claim 8, wherein after it is determined that the patient has a high risk of developing a MRSA infection, the patient is given anti MRSA antibiotics.

10. The method of claim 8, wherein after it is determined that the patient has a high risk of developing a MRSA infection, the patient is given anti MRSA antibiotics and is given decolonization treatments.

11. The method of claim 8, wherein after it is determined that the patient has a high risk of developing a MRSA infection, the patient is given more than one course of anti MRSA antibiotics, decolonization treatments and is put on high infection alert for any future surgeries.

12. The method of claim 8, wherein after it is determined that the patient has a low risk of developing a MRSA infection, the patient is not given any antibiotics and is only treated with incision and drainage.

13. The method of claim 12, wherein, after it is determined that the patient has a low risk of developing a MRSA infection, the patient is treated with incision and drainage and given a routine antibiotic treatment that does not include anti MRSA antibiotics.

14. One or more primers or probes to be used to amplify or detect at least one or more nucleotides from a biological sample, which one or more nucleotides occupy a locus corresponding to position 17 of SEQ. ID. NO. 1.

15. The one or more primers of claim 14 wherein said one or more primers spans the nucleotide positions about position 17 of SEQ. ID. NO. 1.

16. The one or more primers of claim 15 that is from about 8 to about 44 nucleotides in length.

17. The one or more primers of claim 16 that is from about 14 to about 50 nucleotides in length.

18. The probes of claim 14 wherein said one or more probes span the nucleotide positions about position 17 of SEQ. ID. NO. 1.

19. The probe of claim 18 selected from a probe having a different disruption energy for one allele as compared to another allele; two probes, wherein the first probe is a sensor probe and the second probe is an anchor probe; and a SNP-specific probe.

20. The one or more primers or probes of claim 14 in a kit designed for use by a caregiver who seeks to predict or assess the level of risk of a subject developing Methicillin-resistant S. aureus (MRSA) infection comprising: obtaining a biological sample from the subject, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; and predicting and assessing the level of risk of the subject developing a MRSA or CA-MRSA infection, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a high risk of developing a MRSA or CA-MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the subject has a low risk of developing a MRSA or CA-MRSA infection.

21. A method of monitoring and preparing a patient for surgery, wherein said monitoring and preparing comprises: obtaining a biological sample from the patient, wherein said biological sample includes at least one oligonucleotide occupying a locus corresponding to position 17 of SEQ ID. NO 1; detecting the identity of said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1; determining whether said at least one oligonucleotide occupying the locus corresponding to position 17 of SEQ ID. NO 1 is an adenine or a cytosine; predicting and assessing the level of risk of the patient developing a MRSA infection, wherein an adenine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the patient has a high risk of developing a MRSA infection and a cytosine occupying the locus corresponding to position 17 of SEQ ID. NO 1 indicates that the patient has a low risk of developing a MRSA infection; and wherein the predication and assessment indicates that the patient is at high risk for a MRSA infection, taking appropriate steps and care as one normally skilled in the art would take when operating on a person at high risk of developing a MRSA infection.

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