3.4 KB MITOCHONDRIAL DNA DELETION FOR USE IN THE DETECTION OF CANCER

Abstract

A method for detecting cancer in an individual comprising detecting a deletion in the nucleic acid sequence between residues 10743 and 12125 in mitochondrial DNA, obtaining a biological sample from the individual, extracting the mitochondrial DNA (mtDNA) from the sample, quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence between residues 10743 and 14125 of the mtDNA genome, and comparing the amount of mtDNA in the sample having the deletion to at least one known reference sample.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 14/507,027, filed Oct. 6, 2014, which is a continuation of U.S. patent application Ser. No. 12/748,120, filed Mar. 26, 2010, which is a continuation-in-part of U.S. patent application Ser. No. 11/975,390, filed Oct. 18, 2007, which is a continuation of PCT/CA2006/000652, filed Apr. 18, 2006, which PCT application claims the benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional Patent Application No. 60/672,016, filed Apr. 18, 2005; 60/721,522, filed Sep. 29, 2005; and 60/789,872, filed Apr. 7, 2006, which applications are hereby incorporated by reference in their entireties. Additionally, this application is a continuation of U.S. patent application Ser. No. 14/507,027, filed Oct. 6, 2014, which is a continuation of U.S. patent application Ser. No. 12/748,120, filed Mar. 26, 2010, which is a continuation of PCT/CA2007/001711, filed Sep. 26, 2007, which applications are hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE IDENTIFICATION LISTING

[0002] The present application includes a sequence identification listing in .txt format as follows:

[0003] Filename: Sequence_Listing re PCT International Patent Appl. No. PCT_CA2007_001711.txt

[0004] Size: 26.8 KB

[0005] Date Created: Mar. 22, 2010 This sequence identification listing is hereby expressly incorporated by reference in its entirety in the present application.

FIELD OF THE INVENTION

[0006] This invention is related to the field of mitochondrial genomics. In particular it is related to a 3.4 kb deletion in the mitochondrial genome and its utility as an indicator of cancer.

BACKGROUND OF THE INVENTION

[0007] Mitochondrial DNA (MtDNA) as a Diagnostic Tool

[0008] MtDNA sequence dynamics are important diagnostic tools. Mutations in mtDNA are often preliminary indicators of developing disease, often associated with nuclear mutations, and act as biomarkers specifically related to: disease, such as but not limited to, tissue damage and cancer from smoking and exposure to second hand tobacco smoke (Lee et al., 1998; Wei, 1998); longevity, based on accumulation of mitochondrial genome mutations beginning around 20 years of age and increasing thereafter (von Wurmb, 1998); metastatic disease caused by mutation or exposure to carcinogens, mutagens, ultraviolet radiation (Birch-Machin, 2000); osteoarthritis; cardiovascular, Alzheimer, Parkinson disease (Shoffner et al., 1993; Sherratt et al., 1997; Zhang et al, 1998); age associated hearing loss (Seidman et al., 1997); optic nerve degeneration and cardiac dysrhythmia (Brown et al., 1997; Wallace et al., 1988); chronic progressive external exophthalmoplegia (Taniike et al., 1992); atherosclerosis (Bogliolo et al., 1999); papillary thyroid carcinomas and thyroid tumours (Yeh et al., 2000); as well as others (e.g. Naviaux, 1997; Chinnery and Turnbull, 1999).

[0009] Mutations at specific sites of the mitochondrial genome can be associated with certain diseases. For example, mutations at positions 4216, 4217 and 4917 are associated with Leber's Hereditary Optic Neuropathy (LHON) (Mitochondrial Research Society; Huoponen (2001); MitoMap). A mutation at 15452 was found in 5/5 patients to be associated with ubiquinol cytochrome c reductase (complex III) deficiency (Valnot et al. 1999).

[0010] Specifically, these mutations or alterations include point mutations (transitions, transversions), deletions (one base to thousands of bases), inversions, duplications, (one base to thousands of bases), recombinations and insertions (one base to thousands of bases). In addition, specific base pair alterations, deletions, or combinations thereof have been found to be associated with early onset of prostate, skin, and lung cancer, as well as aging (e.g. Polyak et al., 1998), premature aging, exposure to carcinogens (Lee et al., 1998), etc.

[0011] Prostate Cancer

[0012] Prostate cancer is a frequently diagnosed solid tumour that most likely originates in the prostate epithelium (Huang et al. 1999). In 1997, nearly 10 million American men were screened for prostate specific antigen (PSA), the presence of which suggests prostate cancer (Woodwell, 1999). Indeed, this indicates an even higher number of men screened by an initial digital rectal exam (DRE). In the same year, 31 million men had a DRE (Woodwell, 1999). Moreover, the annual number of newly diagnosed cases of prostate cancer in the United States is estimated at 179,000 (Landis et al., 1999). It is the second most commonly diagnosed cancer and second leading cause of cancer mortality in Canadian men. In 1997 prostate cancer accounted for 19,800 of newly diagnosed cancers in Canadian men (28%) (National Cancer Institute of Canada). It is estimated that 30% to 40% of all men over the age of forty-nine (49) have some cancerous prostate cells, yet only 20% to 25% of these men have a clinically significant form of prostate cancer (SpringNet--CE Connection, internet, www.springnet.com/ce/j803a.htm). Prostate cancer exhibits a wide variety of histological behaviour involving both endogenous and exogenous factors, i.e. socio-economic situations, diet, geography, hormonal imbalance, family history and genetic constitution (Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNA alterations have been previously associated with prostate cancer, the need exists for further markers for the detection of prostate cancer.

[0013] 3.4 kb MtDNA Deletion and the Detection of Prostate Cancer.

[0014] In the applicant's pending PCT application bearing publication no. WO/06/111029 (the entire contents of which are incorporated herein by reference) a deletion of a 3379 bp segment of mtDNA was identified through full mitochondrial genome amplification of prostate tissue. The 3379 bp deletion (referred to as the 3.4 kb deletion) was determined to be located between nucleotides 10744-14124 of the mitochondrial genome. It was determined that the detection of this deletion could be used in the diagnosis of prostrate cancer when tissue samples are tested.

[0015] The 3.4 kb deletion removes all or part of the following genes from the mtDNA genome: (i) NADH dehydrogenase subunit 4L, (ii) NADH dehydrogenase subunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNA histidine, (v) tRNA serine2, and (vi) tRNA leucine2.

[0016] Breast Cancer

[0017] Breast cancer is a cancer of the glandular breast tissue and is the fifth most common cause of cancer death. In 2005, breast cancer caused 502,000 deaths (7% of cancer deaths; almost 1% of all deaths) worldwide (World Health Organization Cancer Fact Sheet No. 297). Among women worldwide, breast cancer is the most common cancer and the most common cause of cancer death (World Health Organization Cancer Fact Sheet No. 297). Although certain mtDNA alterations have been previously associated with breast cancer, for example, in Parrella et al. (Cancer Research: 61, 2001), the need exists for further markers for the detection of breast cancer.

BRIEF SUMMARY OF THE INVENTION

[0018] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;

[0019] a) obtaining a biological sample from the individual;

[0020] b) extracting mitochondrial DNA, mtDNA, from the sample;

[0021] c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;

[0022] d) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value.

[0023] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;

[0024] a) obtaining a biological sample from the individual;

[0025] b) extracting mitochondrial DNA, mtDNA, from the sample;

[0026] c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;

[0027] d) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid;

[0028] wherein an elevated amount of the deletion in the biological sample compared to the reference sample is indicative of cancer.

[0029] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;

[0030] a) obtaining a biological sample from the individual;

[0031] b) extracting mitochondrial DNA, mtDNA, from the sample;

[0032] c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;

[0033] d) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid;

[0034] wherein a similar level of the deletion in the biological sample compared to the reference sample is indicative of cancer.

[0035] In one embodiment, the present invention provides a method of monitoring an individual for the development of a cancer comprising;

[0036] a) obtaining a biological sample;

[0037] b) extracting mtDNA from the sample;

[0038] c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;

[0039] d) repeating steps a) to c) over a duration of time;

[0040] e) wherein an increasing level of the deletion over the duration of time is indicative of cancer.

[0041] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;

[0042] a) obtaining a biological sample from the individual;

[0043] b) extracting mitochondrial DNA, mtDNA, from the sample;

[0044] c) quantifying the amount of mtDNA in the sample having a sequence corresponding to the sequence identified in SEQ ID NO: 1;

[0045] d) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 to at least one known reference value.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0046] An embodiment of the invention will now be described by way of example only with reference to the appended drawings wherein:

[0047] FIG. 1 is a schematic diagram showing the design and sequence of a primer useful for the detection of the 3.4 kb deletion. The primer (SEQ ID NO: 2) binds to bases 10729-10743/14125-14139 of the mtDNA genome, wherein the portion of the primer that binds to bases 10729-10743 is depicted in gray in the upper left corner of the figure (nucleotides 1-15 of SEQ ID NO: 2) and the portion of the primer that binds to bases 14125-14139 is depicted in white in the lower right corner of the figure (nucleotides 16-30 of SEQ ID NO: 2);

[0048] FIG. 2 is a graph showing a comparison of cycle threshold between malignant and symptomatic benign participants in the 3.4 kb study;

[0049] FIG. 3 is a graph showing cycle threshold as related to Example 1;

[0050] FIG. 4 shows a ROC curve illustrating the specificity and sensitivity of one embodiment of the present invention;

[0051] FIG. 5 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention;

[0052] FIG. 6 shows real-time PCR data relating to 3.4 kb mtDNA deletion levels associated with breast cancer; and,

[0053] FIG. 7 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0054] As used herein, "cycle threshold" (C.sub.T) is the point at which target amplification using real-time PCR rises above background, as indicated by a signal such as a fluorescence signal. The C.sub.T is inversely related to the quantity of the sequence being investigated.

[0055] As defined herein, "sensitivity" refers to the fraction of true positives (true positive rate) results obtained using the method of the present invention.

[0056] As defined herein, "specificity" refers to the fraction of false positives (false positive rate) results obtained using the method of the present invention.

[0057] In one embodiment of the present invention, methods are provided for monitoring and diagnosing cancer through the detection and quantification of the aforementioned 3.4 kb mtDNA deletion. For example, the present invention may be used for detecting the presence of pre-neoplasia, neoplasia and progression towards potential malignancy of prostate cancer and breast cancer. In one aspect, the present invention involves the detection and quantification of the 3.4 kb mtDNA deletion (SEQ ID NO:1) for the detection, diagnosis, and/or monitoring of cancer. In this method, mtDNA is extracted from a biological sample (for example body tissue, or body fluids such as urine, prostate massage fluid). The extracted mtDNA is then tested in order to determine the levels (i.e. quantity) of the 3.4 kb deletion in the sample. In tests conducted by the present inventors, the levels of the deletion were found to be elevated in samples obtained from subjects with cancer when compared to samples obtained from subjects without cancer. Based on the information and data supplied below, the inventors have concluded that elevated levels of the 3.4 kb deletion in the mtDNA is indicative of cancer.

[0058] As disclosed in PCT WO/06/111029, the 3.4 kb deletion spans approximately nucleotides 10744 to 14124 of the mtDNA genome. The mtDNA genome is listed as SEQ ID NO:8 (Genbank accession no. AC_000021). The inventors have determined, as provided by example below, that this deletion is also associated with cancer and in particular prostate and breast cancer. Therefore, such deletion provides an accurate biomarker and, therefore, a valuable tool for the detection, diagnosis, or monitoring of cancer in at least these tissues.

[0059] The deletion results in the creation of two deletion monomers, one of 3.4 kb in size (small sublimon) and one of approximately 12.6 kb in size (large sublimon). The occurrence of the deletion may be detected by either identifying the presence of the small sublimon, or by determining that the 3.4 kb sequence has been deleted from the large sublimon.

[0060] As discussed above, the deletion is approximately 3379 bp, and comprises genes encoding NADH dehydrogenase subunit 4L, NADH dehydrogenase subunit 4, NADH dehydrogenase subunit 5, tRNA histidine, tRNAserine2, and tRNA leucine2.

[0061] In one embodiment, samples of, for example prostate tissue, prostate massage fluid, urine or breast tissue, are obtained from an individual and tested over a period of time (e.g. years) in order to monitor the genesis or progression of cancer. Increasing levels of the 3.4 kb deletion over time could be indicative of the beginning or progression of cancer.

[0062] Age related accumulation of the 3.4 kb mtDNA deletion may predispose an individual to, for example, prostate cancer or breast cancer, which is prevalent in middle aged and older men, and middle aged and older women, respectively. According to one aspect of the invention, a method is provided wherein regular cancer screening may take place by monitoring over time the amount of the 3.4 kb deletion in body tissues such as breast tissue or body fluids such as prostate massage fluid, or urine.

[0063] The system and method of the present invention may be used to detect cancer at an early stage, and before any histological abnormalities. For example, the system and method of the present invention may be used to detect pre-neoplasia in breast tissue.

[0064] The following primer sequences are preferred for the detection of the 3.4 kb deletion:

TABLE-US-00001 3.4 forward (binds to bases 10729-10743/ 14125-14139 of the mtDNA genome) (SEQ ID NO: 2) 5'-TAGACTACGTACATACTAACCCTACTCCTA-3'; 3.4 reverse (binds to bases 14361-14379 of the mtDNA genome) (SEQ ID NO: 3) 5'-GAGGTAGGATTGGTGCTGT-3'.

[0065] In one embodiment of the present invention, a pair of amplification primers are used to amplify a target region indicative of the presence of the 3.4 kb deletion. In this embodiment, one of the pair of amplification primers overlaps a spliced region of mtDNA after deletion of the 3.4 kb sequence has occurred (i.e. a splice at a position spanning approximately residues 10744 and 14124 of the mtDNA genome). Therefore, extension of the overlapping primer can only occur if the 3.4 kb section is deleted.

[0066] In another embodiment of the present invention, a pair of amplification primers are used to amplify a target region associated with the deleted 3.4 kb sequence. The deleted 3.4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. In this embodiment, one of the pair of amplification primers overlaps the rejoining site of the ends of the 3.4 kb sequence. Thus, an increase in the amount of the 3.4 kb molecule detected in a sample is indicative of cancer. The below primer pair is preferred for the detection of the deleted 3.4 kb nucleic acid.

TABLE-US-00002 Forward 14115/10755 (SEQ ID NO: 9) 5'-CCCACTCATCACCTAAACCTAC-3' Reverse 10980R (SEQ ID NO: 10) 5'-GGTAGGAGTCAGGTAGTTAG-3'.

[0067] In one aspect of the invention, a kit for diagnosing cancer, for example prostate or breast cancer, comprising means for extraction of mtDNA, primers having the nucleic acid sequences recited in SEQ ID NOS: 2 and 3, or SEQ ID NOS: 9 and 10, reagents and instructions, is provided.

[0068] Another aspect of the invention provides methods for confirming or refuting the presence of a cancer biopsy test from a biopsy sample (e.g. prostate or breast cancer), comprising: obtaining non-cancerous tissue from a biopsy sample; and detecting and quantifying the amount of the 3.4 kb mtDNA deletion in the non-diseased tissue.

[0069] In one embodiment the present invention provides a method for screening individuals for prostate or breast cancer from a body fluid sample comprising; obtaining a body fluid sample, and detecting and quantifying the level of the 3.4 kb mtDNA deletion in the body fluid.

[0070] Although real-time quantitative PCR methods, as described in the examples below, represent the preferred means for detecting and quantifying the presence or absence of the 3.4 kb deletion, other methods that would be well known to an individual of skill in the art could also be utilized. For example quantification of the deletion could be made using Bio-Rad's Bioplex.TM. System and Suspension Array technology. Generally, the method requires amplification and quantification of sequences using any known methods.

[0071] The examples provided below illustrate that not only can this deletion be used for the detection of prostate cancer in prostate tissue, but can also be used to detect the presence of cancer in other biological samples, for example prostate massage fluid, urine, and breast tissue. Based on the findings in these examples, the 3.4 kb mtDNA deletion may be used as a biomarker for cancer.

[0072] The various examples provided illustrate a difference in the amount of mtDNA having the 3.4 kb deletion between samples obtained from subjects having cancer, and subjects without cancer. The amount of the 3.4 kb deletion was found to be higher in the samples obtained from subjects having cancer. This determination was made by comparing the amount of the 3.4 kb deletion in the test samples with amounts from known cancer cells and/or known non-cancer cells.

EXAMPLE 1

3.4 kb Deletion in the MtDNA of Prostate Tissue

[0073] A deletion of approximately 3.4 kilobases (kb) was identified through full mitochondrial genome amplification of fresh frozen prostate tissue. Using linear regression, the size of the deletion was estimated to be between 3000 base pairs (bp) and 3500 bp. Two possible candidate deletions were identified using Mitomap.TM. (Brandon, M. C., Lott, M. T., Nguyen, K. C., Spolim, S., Navathe, S. B., Baldi, P. & Wallace, D. C., MITOMAP: a human mitochondrial genome database--2004 update. Nucleic Acids Research 33 (Database Issue): D611-613, 2005; www.mitomap.org), the 3397 bp deletion at 9574-12972, and the 3379 bp deletion at 10744-14124. In order to determine which of the two deletions was associated with prostate cancer, if either, a forward primer which bridged the deletion junction was developed for each of the two candidates, ensuring that the primer extended further than the repeat regions that flank the deletions. FIG. 1 is a schematic diagram showing the design and sequence of the primer (i.e. SEQ ID NO: 2). Positive amplification results for the amplicon corresponding to the 3379 bp deletion (referred to as the 3.4 kb deletion) at 10744-14124 were obtained.

[0074] As indicated above, the 3.4 kb deletion removes all or part of the following genes: (i) NADH dehydrogenase subunit 4L, (ii) NADH dehydrogenase subunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNA histidine, (v) tRNA serine2, and (vi) tRNA leucine2.

[0075] The 3.4 kb deletion was determined to be present in 91% of 33 fresh frozen prostate samples. With the specific deletion primers, formalin fixed tissues were tested in order increase the n value.

[0076] The present investigators sequenced entire mitochondrial genomes from 32 tissue samples microdissected by laser capture microdisection and 12 needle biopsies from histologically normal prostates. Archived tissue sections from each of these samples were used for the following study. 1-2 serial sections were removed from each sample. DNA was extracted from each sample in its entirety rather than as a microdissection. Thus, each sample consisted of a mixture of glandular prostate tissue as well as stromal prostate tissue. This extraction was performed using Qiagen's QIAamp.TM. DNA Mini Kit (Cat #51304). Following extraction the samples were quantified using a Nano-Drop.TM. spectrophotometer and the concentrations were subsequently normalized to 2 ng/ul. Each sample was amplified using 20 ng input DNA and an iQ.TM. SYBR Green Supermix.TM. kit (Bio-Rad Laboratories Inc.) Reactions were run on an Opticon.RTM. 2 two colour real-time PCR system (MJ Research).

[0077] As shown in FIG. 2, a distinct difference was observed in cycle threshold and, by extension, quantity of the deletion between the malignant prostate samples and the symptomatic benign prostate samples. Malignant samples exhibited a consistently earlier cycle threshold than the benign samples.

EXAMPLE 2

3.4 kb Deletion Blinded Study--Comparison of Cycle Threshold

[0078] An additional 21 prostate tissue samples were selected, 10 of which were benign and 11 of which were malignant. The pathological status was determined by needle biopsies conducted by a qualified pathologist. The samples were blinded such that the present investigators were unaware of their pathological status when they conducted this test. The present investigators were able to predict pathological status correctly in 81% of the cases by examining the cycle threshold. Of the 4 incorrect calls, two were malignant samples that were determined to be benign and 2 were benign samples that were determined to be malignant. Follow-up clinical information for the 2 individuals in the latter scenario was requested from the physician to determine if they had been diagnosed with prostate cancer subsequent to the needle biopsy results used for this study. One of the individuals who originally produced a benign sample but was predicted by this study to have a malignancy subsequently produced a malignant sample. As a result, one of the false positives became a true positive. Therefore, pathological status was predicted correctly in 86% of the cases examined in this study. The ultimate positive predictive value (PPV, where PPV=true positives/(true positives+false positives)) for this study was 91% and the negative predictive value (NPV, where NPV=true negatives/(true negatives+false negatives)) was 80%.

EXAMPLE 3

3.4 kb Deletion Study--Methods (n=76)

[0079] Seventy-six prostate tissue samples were examined for the 3.4 kb deletion in this study. All tissue samples were formalin-fixed, 25 being malignant, 12 being normal, and 39 having benign prostatic disease as shown histologically. Of the latter group more then half had hyperplasia. All specimens were needle biopsies taken from the investigators' tissue archives.

[0080] Prostate Specimens

[0081] A tapelift was performed on each slide using Prep-Strips (Catalogue Number LCM0207) from Arcturus Bioscience Inc. This allowed the removal of any particulate matter or non-adhering tissue from the slide prior to DNA extraction. With the tissue still on the slides, the slides were rinsed with PBS (Phosphate Buffered Saline Solution) to remove as much fixative as possible. The 1-2 needle biopsy sections on the slides were scraped into sterile microcentrifuge tubes using individually wrapped, sterilized surgical razor blades. DNA was then isolated and purified using a QIAamp.RTM. DNA Mini Kit (Qiagen, Cat. #51304) according to manufacturer's specifications. A negative extract control was processed in parallel with the slide extractions as a quality control checkpoint. The total concentration of DNA and purity ratio for each sample was determined by spectrophotometry (Nano-Drop.TM. ND-1000) and dilutions of 2 ng/.mu.l were prepared for the purpose of Quantitative Polymerase Chain Reaction (qPCR).

[0082] Primers (Oligonucleotides)

[0083] Purified oligonucleotide primers were chemically synthesized by Invitrogen (California, USA). The sequences of the primers and the expected sizes of the PCR products amplified are listed in Table 1. In addition, PCR analysis for mtDNA deletions included positive controls (DNA from a source known to carry the mutant mtDNA). Each primer set with the exception of TNF (tumor necrosis factor) were checked against a mitochondria-free rho 0 cell line to confirm the absence of pseudogene coamplification.

TABLE-US-00003 TABLE 1 Amplification Primers. Length of Position amplified Amplified product Primer Pair 5'-3' (base pairs) 3.4 Deletion 10729-14379 273 Real-Time (less 3379 bp at 10744-14124) 12 s mtDNA 708-945 238 TNF 3756-3886 131 3.4 forward (10729-10743-14125-14139) SEQ ID NO: 2 5'TAGACTACGTACATACTAACCCTACTCCTA-3' 3.4 reverse (14361-14379) SEQ ID NO: 3 5'-GAGGTAGGATTGGTGCTGT-3' 12 s forward (708-728) SEQ ID NO: 4 5'-CGTTCCAGTGAGTTCACCCTC-3'' 12 s reverse (923-945) SEQ ID NO: 5 5'-CACTCTTTACGCCGGCTTCTATT-3' TNF forward (3756-3775) SEQ ID NO: 6 5'-CCTGCCCCAATCCCTTTATT-3' TNF reverse (3866-3886) SEQ ID NO: 7 5'-GGTTTCGAAGTGGTGGTCTTG-3'

[0084] Real-Time Polymerase Chain Reaction

[0085] Three separate PCRs were performed on each sample. Each reaction was 25 .mu.l total volume and included template DNA, one pair of primers (12 s or 3.4 Deletion or TNF), an iQ.TM. SYBR Green Supermix.TM. kit (Catalogue Number 170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water (ddH.sub.2O). The TNF (tumor necrosis factor) comprised single copy nuclear gene primers, and 12 s comprised total mitochondrial genome primers. The volume and concentrations for template DNA, primers, and reaction buffer are listed below.

TABLE-US-00004 TABLE 2 qPCR Components. Concentration per Volume per Reagent Reaction Reaction Reaction Buffer 1X 12.5 .mu.l Primer (forward 250 nM 0.0625 .mu.l of each and reverse) 100 umole stock ddH.sub.2O N/A 2.375. .mu.l Template DNA 20 ng 10.0 .mu.l Total 25 .mu.l

[0086] The cycling parameters for each amplicon are listed in Table 3.

TABLE-US-00005 TABLE 3 Cycling Parameters. Step Temperature (.degree. C.) Duration 1 95 3 min 2 95 30 sec 3 66 (3.4 deletion primers) or 30 sec 61.5 (12 s primers) or 61.5 (TNF primers) 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve 50.degree. C.-110.degree. C. reading every 1.degree. C. 3 sec Repeat steps 2-5, 44 times for a total of 45 cycles.

[0087] Thermal cycling, real-time detection and analysis of the reactions was carried out using a DNA Engine Opticon.RTM. 2 Continuous Fluorescence Detection System equipped with Intuitive Opticon Monitor.TM. software (MJ Research Inc.). The standard curve method was utilized for DNA quantification. A set of serial dilutions (10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10.sup.1) of three purified PCR generated templates, one product for the 3.4 deletion, one for the 12 s primers, and one for TNF. From this, three different standard curves were generated showing the number of copies of total mtDNA (12 s amplicon-total mitochondrial genome primers), the amount of mtDNA having the 3.4 kb deletion, or total nuclear DNA (TNF-single copy nuclear gene primers). The C.sub.T values of the samples were then converted to the number of DNA copies by comparing the sample C.sub.T to that of the standards. The 3.4 deletion was considered to be absent or at low levels if the deletion was not detected within 37 cycles.

[0088] The determination of malignancy is based upon the quantity of the 3.4 kb deletion present in the normalized sample as indicated by the location of the cycle threshold. This location may be either absolute, as in greater than 25 cycles but less than 35 cycles, or more likely a ratio between the total mitochondrial DNA present as indicated by the 12 s amplicon, and the 3.4 kb deletion. This may be expressed as a per cent of the total mitochondrial DNA. The number of cells, as represented by the TNF amplicon, may be incorporated to refine the distinction between benign and malignant tissues.

[0089] In order to automate the analyses of these samples, bioinformatics tools were employed. The three variables that were considered for these analyses were the cycle threshold C.sub.T of Tumour Necrosis Factor (TNF), total pieces of mitochondria that contain those specific primer sites, and those mitochondria that harbour the deletion of interest.

[0090] Cluster Analysis

[0091] The clustering was not normalized nor were logarithmic functions used due to the similar and small range of data.

[0092] FIG. 3 shows the actual movement and trends of the data. The x-axis is the patient number and the y-axis is the cycle threshold obtained from real time PCR.

[0093] It is important to note that the higher the cycle threshold is, the lower amount of the deletion is present.

[0094] The general trend shown in FIG. 3 is based upon the differences/ratios between the variables of Deletion, Total, and TNF. The deletion is low to absent for the benign/normal samples (right side) and increases (toward the left) with abnormal benign and malignant samples. The abnormal benign and malignant samples begin to differentiate themselves from each other based on the cycle threshold ratio of Deletion to TNF.

[0095] Supervised Learning

[0096] Supervised learning is based on the system trying to predict outcomes for known samples. Half of the data was used to train and the other half to test the algorithm. Supervised learning compares its predictions to the target answer and "learns" from its mistakes. But, if the predicted output is higher or lower than the actual outcome in the data, the error is propagated back through the system and the weights are adjusted accordingly.

[0097] Data SET: 5% to 35%--Benign

[0098] 35% to 65%--Hyperplasia

[0099] 65% to 95%--Malignant

[0100] Artificial Neural Network (ANN) Algorithm (shown schematically below):

##STR00001##

[0101] Supervised Learning of Deletion Data using Artificial Neural Network (ANN)

[0102] Three Classifications:

[0103] Benign

[0104] Hyperplasia

[0105] Malignant

[0106] Three variables for each classification were used based on Real Time PCR Cycle Threshold C.sub.T:

[0107] Tumour Necrosis Factor (TNF)--Nuclear copy control.

[0108] Total Mitochondria--Mitochondria copy control

[0109] Deletion--Mitochondria in the deleted state.

[0110] Results:

[0111] Half of data set is used to train the ANN, and the remaining half is used to compare the accuracy.

[0112] Three Classification Accuracy=86.6%

[0113] Positive Predictive Value (PPV);

[0114] Benign to Malignant=88.2%

[0115] Negative Predictive Value (NPV)

[0116] Benign to Malignant=76.5%

EXAMPLE 4

3.4 kb Deletion in MtDNA Associated with Breast Cancer

[0117] 18 samples were tested from malignant and benign breast tissue, 9 being malignant and 9 being benign, for the presence of the aforementioned 3.4 kb deletion. Samples were classified as either malignant or benign using conventional histopathological analysis.

[0118] DNA was isolated and purified from the samples using a QIAamp.RTM. DNA Mini Kit (Qiagen, Cat. #51304) according to manufacturer's specifications.

[0119] Purified oligonucleotide primers were chemically synthesized by Invitrogen (California, USA). The sequences of the primers and the expected sizes of the PCR products amplified are listed in Table 1 above.

[0120] Real-Time Polymerase Chain Reaction

[0121] Three separate PCRs were performed on each sample. Each reaction was 25 .mu.l total volume and included template DNA, one pair of primers (12 s or 3.4 Deletion or TNF), an iQ.TM. SYBR Green Supermix kit (Catalogue Number 170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water (ddH.sub.2O). The TNF (tumor necrosis factor) comprised single copy nuclear gene primers, and 12 s comprised total mitochondrial genome primers. The volume and concentrations for template DNA, primers, and reaction buffer are listed below:

TABLE-US-00006 TABLE 4 qPCR Components. Concentration per Volume per Reagent Reaction Reaction Reaction Buffer 1X 12.5 .mu.l Primer (forward 250 nM 0.0625 .mu.l of each and reverse) 100 .mu.mole stock ddH.sub.2O N/A 2.375. .mu.l Template DNA 20 ng 10.0 .mu.l Total 25 .mu.l

[0122] The cycling parameters for each amplicon are listed in Table 5.

TABLE-US-00007 TABLE 5 Cycling Parameters. Step Temperature (.degree. C.) Duration 1 95 3 min 2 95 30 sec 3 66 (3.4 deletion primers) or 30 sec 61.5 (12 s primers) or 61.5 (TNF primers) 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve 50.degree. C.-110.degree. C. reading every 1.degree. C. 3 sec Repeat steps 2-5, 44 times for a total of 45 cycles.

[0123] Thermal cycling, real-time detection and analysis of the reactions was carried out using a DNA Engine Opticon.RTM. 2 Continuous Fluorescence Detection System equipped with Intuitive Opticon Monitor.TM. software (MJ Research Inc.). The standard curve method was utilized for DNA quantification. A set of serial dilutions (10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3, 10.sup.2, 10.sup.1) of three purified PCR generated templates were performed, one product for the 3.4 deletion, one for the 12 s primers, and one for TNF. From this, three different standard curves were generated showing the number of copies of total mtDNA (12 s amplicon-total mitochondrial genome primers), 3.4 deletion or total nuclear DNA (TNF-single copy nuclear gene primers). The C.sub.T values of the samples were then converted to the number of DNA copies by comparing the sample C.sub.T to that of the standards.

[0124] The determination of malignancy was based upon the quantity of the 3.4 kb deletion present in the normalized sample as indicated by the location of the cycle threshold. This location may be either absolute, as in greater than 25 cycles but less than 30 cycles, or more likely a ratio between the total mitochondrial DNA present as indicated by the 12 s amplicon, and the 3.4 kb deletion. This may be expressed as a percent of the total mitochondrial DNA.

[0125] In order to automate the analyses of these samples, bioinformatics tools were employed. The three variables that were considered for these analyses were the cycle threshold C.sub.T of Tumour Necrosis Factor (TNF), total species of mitochondria that contain those specific primer sites, and those mitochondria that harbour the deletion of interest.

[0126] Table 6 and FIG. 7 show the difference in the mean C.sub.T scores for samples from malignant tissue and benign tissue. The mean C.sub.T value for normal tissue was 30.5889, while the mean C.sub.T for malignant tissue was 27.8533 thereby illustrating a difference in the quantity of mtDNA having the 3.4 kb deletion in malignant breast tissue compared to normal breast tissue.

TABLE-US-00008 TABLE 6 Mean values for C.sub.T scores Group Statistics Std. Error GRP N Mean Std. Deviation Mean del3.4 normal 9 30.5889 2.53897 .84632 malignant 9 27.8533 2.52253 .84084

[0127] FIG. 8 is an ROC curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for breast cancer when testing breast tissue. These results were obtained using a cutoff C.sub.T of 29.1900. The sensitivity of the marker at this C.sub.T was 77.8%, while the specificity was 77.8%.

[0128] Table 7 shows the calculation of the area under the curve for the present example. As a measure of the accuracy of the test.

TABLE-US-00009 TABLE 7 Results Showing Area Under the Curve Area Under the Curve Test Result Variable(s): del3.4 Asymptotic 95% Confidence Asymptotic Interval Area Std. Error.sup.a Sig..sup.b Lower Bound Upper Bound .790 .112 .038 .570 1.010 .sup.aUnder the nonparametric assumption .sup.bNull hypothesis: true area = 0.5

[0129] The determination of the cutoff C.sub.T of 29.1900 is shown in table 8 below. The results listed in table 8 show that a cutoff C.sub.T of 29.1900 provided the highest sensitivity and specificity at 78% and 78% respectively.

TABLE-US-00010 TABLE 8 Determination of C.sub.Tcutoff. Coordinates of the Curve Test Result Variable(s): del3.4 Positive if Less Than or Equal To.sup.a Sensitivity 1 - Specificity 24.6000 .000 .000 25.6800 .111 .000 25.7700 .222 .000 25.9250 .333 .000 26.2050 .444 .000 26.8400 .556 .000 27.4800 .556 .111 28.1600 .556 .222 28.8800 .667 .222 29.1900 .778 .222 29.4600 .778 .333 29.8750 .778 .444 30.5850 .778 .556 31.2200 .778 .667 31.5000 .889 .667 31.7650 .889 .778 32.9900 1.000 .778 34.3350 1.000 .889 35.6400 1.000 1.000 .sup.aThe smallest cutoff value is the minimum observed test value minus 1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.

EXAMPLE 5

The 3.4 kb Deletion in the Prostate Massage Fluid of Individuals with Prostate Cancer as Compared to the Fluid from those without Histological Evidence of Prostate Cancer

[0130] Forty prostate massage fluid samples were collected by urologists from patients who were either subsequently diagnosed with prostate cancer or showed no histological evidence of prostate cancer following a prostate needle biopsy procedure. The sample was deposited on a IsoCode Card.TM. (Schleicher & Shuell), dried, and then extracted according to the manufacturer's protocol. All DNA extracts were quantified using a NanoDrop.TM. ND-1000 Spectrophotometer and the DNA concentration normalized to 2 ng/ul. Each sample was then amplified according to the following parameters:

TABLE-US-00011 1X iQ SYBR Green Supermix .TM. (Bio-Rad P/N 170-8880) 150 nmol forward primer (SEQ ID NO: 2) (5'-TAGACTACGTACATACTAACCCTACTCCTA-3'). 150 nmol reverse primer (SEQ ID NO: 3) (5'-GAGGTAGGATTGGTGCTGT-3') 20 ng template DNA in a 25 ul reaction.

[0131] Reactions were cycled on an Opticon.TM. 2 DNA Engine (Bio-Rad Canada) according to the following protocol:

[0132] 1. 95.degree. C. for 3 minutes

[0133] 2. 95.degree. C. for 30 seconds

[0134] 3. 66.degree. C. for 30 seconds

[0135] 4. 72.degree. C. for 30 seconds

[0136] 5. Plate Read

[0137] 6. Repeat steps 2-5 44 times

[0138] 7. 72.degree. C. for 10 minutes

[0139] 8. Melting Curve from 50.degree. C. to 105.degree. C., read every 1.degree. C., hold for 3 seconds

[0140] 9. 10.degree. C. Hold

TABLE-US-00012

[0140] TABLE 9 Results showing the mean C.sub.T Values for Prostate Massage Fluid Test Group Statistics Std. Error Group N Mean Std. Deviation Mean DEL3.4 benign 25 37.1869 3.18495 .63699 malignant 15 33.7712 3.98056 1.02778

[0141] Tables 9 and 10 show a significant difference between the mean C.sub.T values obtained for the benign sample and the malignant sample groups (p=0.005).

TABLE-US-00013 TABLE 10 Results Showing Difference (p = 0.005) for C.sub.T values of samples. Independent Samples Test Levene's t-test for Equality of Means Test for 95% Confidence Equality Interval of the of Variances Mean Std. Error Difference F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper DEL3.4 Equal variances 1.251 .270 2.989 38 .005 3.41570 1.14283 1.10217 5.72923 assumed Equal variances 2.825 24.696 .009 3.41570 1.20917 .92382 5.90758 not assumed

[0142] FIG. 5 is a Receiver Operating Characteristic (ROC) curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when testing prostate massage fluid. These results were obtained using a cutoff C.sub.T of 37.3683. The sensitivity of the marker at this C.sub.T is 87%, while the specificity is 64%.

[0143] The accuracy of the test depends on how well the test separates the group being tested into those with and without the prostate cancer. Accuracy is measured by the area under the ROC curve. Table 11 shows the calculation of the area under the curve for the present example.

TABLE-US-00014 TABLE 11 Results Showing Area Under the ROC Curve Area Under the Curve Test Result Variable(s): DEL3.4 Asymptotic 95% Confidence Asymptotic Interval Area Std. Error.sup.a Sig..sup.b Lower Bound Upper Bound .768 .074 .005 .622 .914 .sup.aUnder the nonparametric assumption .sup.bNull hypothesis: true area = 0.5

TABLE-US-00015 TABLE 12 Determination of Specificity and Sensitivity Coordinates of the Curve Test Result Variable(s): DEL3.4 Positive if Less Than or Equal To.sup.a Sensitivity 1 - Specificity 26.2992 .000 .000 27.3786 .067 .000 28.2484 .133 .000 29.5193 .200 .000 30.1757 .200 .040 30.4580 .200 .080 30.5980 .267 .080 31.5709 .333 .080 32.5712 .333 .120 32.9500 .333 .160 33.3314 .400 .160 33.6547 .467 .160 33.9247 .533 .160 34.3554 .533 .200 34.9056 .533 .240 35.4650 .533 .280 35.9172 .533 .320 36.0648 .600 .320 36.3616 .667 .320 36.6421 .733 .320 36.8531 .733 .360 37.1188 .800 .360 37.3683 .867 .360 37.5200 .867 .400 37.8341 .867 .440 38.2533 .867 .480 38.5198 .933 .480 38.6519 .933 .520 38.8552 .933 .560 39.1258 .933 .600 39.2734 .933 .640 39.4952 .933 .680 39.7323 1.000 .680 39.6956 1.000 .720 41.0000 1.000 1.000

The smallest cutoff value is the minimum observed test value-1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the average of two consecutive ordered, observed test values.

[0144] The determination of the cutoff C.sub.T of 37.3683 is shown in table 12 above. The results listed in table 12 illustrate that a cutoff C.sub.T of 37.3683 provided the highest sensitivity and specificity.

EXAMPLE 6

The 3.4 kb Deletion in the Urine of Individuals with Prostate Cancer as Compared to the fluid from those without Histological Evidence of Prostate Cancer

[0145] Urine samples were collected from 5 patients who were diagnosed with prostate cancer and 5 who have had a needle biopsy procedure which was unable to detect prostate malignancy. These samples were collected following a digital rectal exam (DRE) to facilitate the collection of prostate cells.

[0146] Upon receipt of the samples a 5 ml aliquot was removed and then 2 mls were centrifuged at 14,000.times.g to form a pellet. The supernatant was removed and discarded. Pellets were resuspended in 200 ul phosphate buffered saline solution. Both the resuspended pellet and the whole urine sample were subjected to a DNA extraction procedure using the QiaAMP.TM. DNA Mini Kit (Qiagen P/N 51304) according to the manufacturer's directions. The resulting DNA extracts were then quantified using a NanoDrop.TM. ND-1000 Spectrophotometer and normalized to a concentration of 0.1 ng/ul.

[0147] Samples were analyzed by quantitative real-time PCR with the 3.4 kb deletion specific primers according to the following:

TABLE-US-00016 1X iQ SYBR Green Supermix .TM. (Bio-Rad P/N 170-8880) 100 nmol forward primer (SEQ ID NO: 2) (5'-TAGACTACGTACATACTAACCCTACTCCTA-3') 100 nmol reverse primer (SEQ ID NO: 3) (5'-GAGGTAGGATTGGTGCTGT-3') 1 ng template DNA in a 25 ul reaction.

[0148] Reactions were cycled on an Opticon.TM. 2 DNA Engine (Bio-Rad Canada) according to the following protocol:

[0149] 1. 95.degree. C. for 3 minutes

[0150] 2. 95.degree. C. for 30 seconds

[0151] 3. 69.degree. C. for 30 seconds

[0152] 4. 72.degree. C. for 30 seconds

[0153] 5. Plate Read

[0154] 6. Repeat steps 2-5 44 times

[0155] 7. 72.degree. C. for 10 minutes

[0156] 8. Melting Curve from 50.degree. C. to 105.degree. C., read every 1.degree. C., hold for 3 seconds

[0157] 9. 10.degree. C. Hold

TABLE-US-00017 TABLE 13 Mean values for C.sub.T scores Group Statistics Std. Error GRP fluid 38 N Mean Std. Deviation Mean CTf Benign 5 33.2780 1.10900 .49596 Malignant 5 30.6980 2.55767 1.14382

[0158] Tables 13 and 14 show a significant difference between the mean C.sub.T values obtained for benign sample and the malignant sample groups (p=0.005).

TABLE-US-00018 TABLE 14 Results Showing Difference (p = 0.005) for C.sub.T values of samples. Independent Samples Test Levene's t-test for Equality of Means Test for 95% Confidence Equality of Interval of the Variances Mean Std. Error Difference F Sig. t df Sig. (2-tailed) Difference Difference Lower Upper CTf Equal variances 1.272 .292 2.069 8 .072 2.58000 1.24672 -.29494 5.45494 assumed Equal variances 2.069 5.453 .089 2.58000 1.24672 -.54639 5.70639 not assumed

[0159] FIG. 6 is a Receiver Operating Characteristic (ROC) curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when testing urine. These results were obtained using a cutoff C.sub.T of 31.575. The sensitivity of the marker at this C.sub.T is 80%, while the specificity is 100%.

[0160] The determination of the cutoff C.sub.T of 31.575 is shown in table 15. The results listed in table 15 show that a cutoff C.sub.T of 31.575 provided the highest sensitivity and specificity.

TABLE-US-00019 TABLE 15 Determination of C.sub.Tcutoff. Coordinates of the Curve Test Result Variable(s): CTf Positive if Less Than or Equal To.sup.a Sensitivity 1 - Specificity 26.2900 .000 .000 28.4950 .200 .000 30.3850 .400 .000 31.0800 .600 .000 31.5750 .800 .000 32.1400 .800 .200 32.8150 .800 .400 33.8700 .800 .600 34.3350 .800 .800 34.3550 1.000 .800 35.3700 1.000 1.000 .sup.aThe smallest cutoff value is the minimum observed test value minus 1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.

EXAMPLE 7

Detection of Re-Circularized 3.4 kb Deleted Sequence in Prostate Malignant and Benign Tissue

[0161] In this example, the amount of re-circularized 3.4 kb deleted mtDNA molecules in samples was tested as an indicator for prostate cancer. As mentioned above, the 3.4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. Amplification of a target region from the deleted 3.4 kb mtDNA sublimon was conducted using a primer pair (SEQ ID NOS: 9 and 10). The forward primer (SEQ ID NO: 9), overlaps the rejoining site of the ends of the 3.4 kb sequence.

[0162] Prostate tissue was formalin-fixed paraffin embedded prostate tissue needle biopsies.

[0163] The reagent setup used for this example was as follows:

[0164] 250 nmol each primer

[0165] 12.5 ul of 2.times. reaction mix,

[0166] 20 ng (10 ul of 2 ng/ul) template in 25 ul reaction volume.

[0167] The cycling parameters were as follows:

[0168] 1. 95 degrees Celsius for 3 minutes

[0169] 2. 95 degrees Celsius for 30 seconds

[0170] 3. 62 degrees Celsius for 30 seconds

[0171] 4. 72 degrees Celsius for 30 seconds

[0172] 5. Plate Read

[0173] 6. Repeat steps 2-5 44 times

[0174] 7. 72 degrees for 10 minutes

[0175] 8. Melting Curve from 50-100 degrees, reading every 1 degree for 3 seconds

[0176] 9. 4 degrees HOLD.

[0177] Amplification of a target region from the deleted 3.4 kb mtDNA sublimon was conducted using a primer pair (SEQ ID NOS: 9 and 10).

[0178] Table 16 below provides a summary of testing conducted for the detection of the actual 3.4 kb deleted in mtDNA obtained from malignant and benign prostate tissue. Using a C.sub.T score of 30.0, a clear identification of malignant and benign tissue was possible. As such, an increase in the amount of the 3.4 kb molecule present in a sample was indicative of cancer.

TABLE-US-00020 TABLE 16 C.sub.T scores for Detection of Cancer in Prostate Tissue Description C.sub.T Benign sample 1 33.75 Malignant sample 1 28.79 Benign sample 2 30.96 Malignant sample 2 28.4 Benign sample 3 32.19 Malignant sample 3 27.38

[0179] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto.

REFERENCES

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[0181] Birch-Machin M A, Taylor R W, Cochran B, Ackrell B A C, Tumbull D M. Ann Neurol 48: 330-335, 2000(b)

[0182] Birch-Machin, M. A. (2000). Mitochondria and skin disease. Clin Exp Dermatol, 25, 141-6.

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[0186] Huoponen, Kirsi, Leber hereditary optic neuropathy: clinical and molecular genetic findings, Neurogenetics (2001) 3: 119-125.

[0187] Hayward S W, Grossfeld G D, Tlsty T D, Cunha G R., Int J Oncol 13:35-47, 1998

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[0192] Mitochondrial Research Society http://www.mitoresearch.org/diseases.html.

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[0209] Zhang, C., et al., BioChem. BioPhys. Res. Comun., 195: 1104-1110, 1993

Sequence CWU 1

1

1013379DNAHomo sapiens 1cctaaaccta ctccaatgct aaaactaatc gtcccaacaa ttatattact accactgaca 60tgactttcca aaaaacacat aatttgaatc aacacaacca cccacagcct aattattagc 120atcatccctc tactattttt taaccaaatc aacaacaacc tatttagctg ttccccaacc 180ttttcctccg accccctaac aacccccctc ctaatactaa ctacctgact cctacccctc 240acaatcatgg caagccaacg ccacttatcc agtgaaccac tatcacgaaa aaaactctac 300ctctctatac taatctccct acaaatctcc ttaattataa cattcacagc cacagaacta 360atcatatttt atatcttctt cgaaaccaca cttatcccca ccttggctat catcacccga 420tgaggcaacc agccagaacg cctgaacgca ggcacatact tcctattcta caccctagta 480ggctcccttc ccctactcat cgcactaatt tacactcaca acaccctagg ctcactaaac 540attctactac tcactctcac tgcccaagaa ctatcaaact cctgagccaa caacttaata 600tgactagctt acacaatagc ttttatagta aagatacctc tttacggact ccacttatga 660ctccctaaag cccatgtcga agcccccatc gctgggtcaa tagtacttgc cgcagtactc 720ttaaaactag gcggctatgg tataatacgc ctcacactca ttctcaaccc cctgacaaaa 780cacatagcct accccttcct tgtactatcc ctatgaggca taattataac aagctccatc 840tgcctacgac aaacagacct aaaatcgctc attgcatact cttcaatcag ccacatagcc 900ctcgtagtaa cagccattct catccaaacc ccctgaagct tcaccggcgc agtcattctc 960ataatcgccc acgggcttac atcctcatta ctattctgcc tagcaaactc aaactacgaa 1020cgcactcaca gtcgcatcat aatcctctct caaggacttc aaactctact cccactaata 1080gctttttgat gacttctagc aagcctcgct aacctcgcct taccccccac tattaaccta 1140ctgggagaac tctctgtgct agtaaccacg ttctcctgat caaatatcac tctcctactt 1200acaggactca acatactagt cacagcccta tactccctct acatatttac cacaacacaa 1260tggggctcac tcacccacca cattaacaac ataaaaccct cattcacacg agaaaacacc 1320ctcatgttca tacacctatc ccccattctc ctcctatccc tcaaccccga catcattacc 1380gggttttcct cttgtaaata tagtttaacc aaaacatcag attgtgaatc tgacaacaga 1440ggcttacgac cccttattta ccgagaaagc tcacaagaac tgctaactca tgcccccatg 1500tctaacaaca tggctttctc aacttttaaa ggataacagc tatccattgg tcttaggccc 1560caaaaatttt ggtgcaactc caaataaaag taataaccat gcacactact ataaccaccc 1620taaccctgac ttccctaatt ccccccatcc ttaccaccct cgttaaccct aacaaaaaaa 1680actcataccc ccattatgta aaatccattg tcgcatccac ctttattatc agtctcttcc 1740ccacaacaat attcatgtgc ctagaccaag aagttattat ctcgaactga cactgagcca 1800caacccaaac aacccagctc tccctaagct tcaaactaga ctacttctcc ataatattca 1860tccctgtagc attgttcgtt acatggtcca tcatagaatt ctcactgtga tatataaact 1920cagacccaaa cattaatcag ttcttcaaat atctactcat cttcctaatt accatactaa 1980tcttagttac cgctaacaac ctattccaac tgttcatcgg ctgagagggc gtaggaatta 2040tatccttctt gctcatcagt tgatgatacg cccgagcaga tgccaacaca gcagccattc 2100aagcaatcct atacaaccgt atcggcgata tcggtttcat cctcgcctta gcatgattta 2160tcctacactc caactcatga gacccacaac aaatagccct tctaaacgct aatccaagcc 2220tcaccccact actaggcctc ctcctagcag cagcaggcaa atcagcccaa ttaggtctcc 2280acccctgact cccctcagcc atagaaggcc ccaccccagt ctcagcccta ctccactcaa 2340gcactatagt tgtagcagga atcttcttac tcatccgctt ccacccccta gcagaaaata 2400gcccactaat ccaaactcta acactatgct taggcgctat caccactctg ttcgcagcag 2460tctgcgccct tacacaaaat gacatcaaaa aaatcgtagc cttctccact tcaagtcaac 2520taggactcat aatagttaca atcggcatca accaaccaca cctagcattc ctgcacatct 2580gtacccacgc cttcttcaaa gccatactat ttatgtgctc cgggtccatc atccacaacc 2640ttaacaatga acaagatatt cgaaaaatag gaggactact caaaaccata cctctcactt 2700caacctccct caccattggc agcctagcat tagcaggaat acctttcctc acaggtttct 2760actccaaaga ccacatcatc gaaaccgcaa acatatcata cacaaacgcc tgagccctat 2820ctattactct catcgctacc tccctgacaa gcgcctatag cactcgaata attcttctca 2880ccctaacagg tcaacctcgc ttccccaccc ttactaacat taacgaaaat aaccccaccc 2940tactaaaccc cattaaacgc ctggcagccg gaagcctatt cgcaggattt ctcattacta 3000acaacatttc ccccgcatcc cccttccaaa caacaatccc cctctaccta aaactcacag 3060ccctcgctgt cactttccta ggacttctaa cagccctaga cctcaactac ctaaccaaca 3120aacttaaaat aaaatcccca ctatgcacat tttatttctc caacatactc ggattctacc 3180ctagcatcac acaccgcaca atcccctatc taggccttct tacgagccaa aacctgcccc 3240tactcctcct agacctaacc tgactagaaa agctattacc taaaacaatt tcacagcacc 3300aaatctccac ctccatcatc acctcaaccc aaaaaggcat aattaaactt tacttcctct 3360ctttcttctt cccactcat 3379230DNAArtificial Sequencesynthetic primer 3.4 kb deletion forward 2tagactacgt acatactaac cctactccta 30319DNAArtificial Sequencesynthetic primer 3.4 kb deletion reverse 3gaggtaggat tggtgctgt 19421DNAArtificial Sequencesynthetic primer mtDNA genome forward 4cgttccagtg agttcaccct c 21523DNAArtificial Sequencesynthetic primer mtDNA genome reverse 5cactctttac gccggcttct att 23620DNAArtificial Sequencesynthetic primer TNF nuclear gene forward 6cctgccccaa tccctttatt 20721DNAArtificial Sequencesynthetic primer TNF nuclear gene reverse 7ggtttcgaag tggtggtctt g 21816569DNAHomo sapiensmisc_feature(3107)..(3107)n is a, c, g or t 8gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720tcaccctcta aatcaccacg atcaaaagga acaagcatca agcacgcagc aatgcagctc 780aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctacnttc aaattcctcc 3120ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc ccgtaaatga 3180tatcatctca acttagtatt atacccacac ccacccaaga acagggtttg ttaagatggc 3240agagcccggt aatcgcataa aacttaaaac tttacagtca gaggttcaat tcctcttctt 3300aacaacatac ccatggccaa cctcctactc ctcattgtac ccattctaat cgcaatggca 3360ttcctaatgc ttaccgaacg aaaaattcta ggctatatac aactacgcaa aggccccaac 3420gttgtaggcc cctacgggct actacaaccc ttcgctgacg ccataaaact cttcaccaaa 3480gagcccctaa aacccgccac atctaccatc accctctaca tcaccgcccc gaccttagct 3540ctcaccatcg ctcttctact atgaaccccc ctccccatac ccaaccccct ggtcaacctc 3600aacctaggcc tcctatttat tctagccacc tctagcctag ccgtttactc aatcctctga 3660tcagggtgag catcaaactc aaactacgcc ctgatcggcg cactgcgagc agtagcccaa 3720acaatctcat atgaagtcac cctagccatc attctactat caacattact aataagtggc 3780tcctttaacc tctccaccct tatcacaaca caagaacacc tctgattact cctgccatca 3840tgacccttgg ccataatatg atttatctcc acactagcag agaccaaccg aacccccttc 3900gaccttgccg aaggggagtc cgaactagtc tcaggcttca acatcgaata cgccgcaggc 3960cccttcgccc tattcttcat agccgaatac acaaacatta ttataataaa caccctcacc 4020actacaatct tcctaggaac aacatatgac gcactctccc ctgaactcta cacaacatat 4080tttgtcacca agaccctact tctaacctcc ctgttcttat gaattcgaac agcatacccc 4140cgattccgct acgaccaact catacacctc ctatgaaaaa acttcctacc actcacccta 4200gcattactta tatgatatgt ctccataccc attacaatct ccagcattcc ccctcaaacc 4260taagaaatat gtctgataaa agagttactt tgatagagta aataatagga gcttaaaccc 4320ccttatttct aggactatga gaatcgaacc catccctgag aatccaaaat tctccgtgcc 4380acctatcaca ccccatccta aagtaaggtc agctaaataa gctatcgggc ccataccccg 4440aaaatgttgg ttataccctt cccgtactaa ttaatcccct ggcccaaccc gtcatctact 4500ctaccatctt tgcaggcaca ctcatcacag cgctaagctc gcactgattt tttacctgag 4560taggcctaga aataaacatg ctagctttta ttccagttct aaccaaaaaa ataaaccctc 4620gttccacaga agctgccatc aagtatttcc tcacgcaagc aaccgcatcc ataatccttc 4680taatagctat cctcttcaac aatatactct ccggacaatg aaccataacc aatactacca 4740atcaatactc atcattaata atcataatag ctatagcaat aaaactagga atagccccct 4800ttcacttctg agtcccagag gttacccaag gcacccctct gacatccggc ctgcttcttc 4860tcacatgaca aaaactagcc cccatctcaa tcatatacca aatctctccc tcactaaacg 4920taagccttct cctcactctc tcaatcttat ccatcatagc aggcagttga ggtggattaa 4980accaaaccca gctacgcaaa atcttagcat actcctcaat tacccacata ggatgaataa 5040tagcagttct accgtacaac cctaacataa ccattcttaa tttaactatt tatattatcc 5100taactactac cgcattccta ctactcaact taaactccag caccacgacc ctactactat 5160ctcgcacctg aaacaagcta acatgactaa cacccttaat tccatccacc ctcctctccc 5220taggaggcct gcccccgcta accggctttt tgcccaaatg ggccattatc gaagaattca 5280caaaaaacaa tagcctcatc atccccacca tcatagccac catcaccctc cttaacctct 5340acttctacct acgcctaatc tactccacct caatcacact actccccata tctaacaacg 5400taaaaataaa atgacagttt gaacatacaa aacccacccc attcctcccc acactcatcg 5460cccttaccac gctactccta cctatctccc cttttatact aataatctta tagaaattta 5520ggttaaatac agaccaagag ccttcaaagc cctcagtaag ttgcaatact taatttctgt 5580aacagctaag gactgcaaaa ccccactctg catcaactga acgcaaatca gccactttaa 5640ttaagctaag cccttactag accaatggga cttaaaccca caaacactta gttaacagct 5700aagcacccta atcaactggc ttcaatctac ttctcccgcc gccgggaaaa aaggcgggag 5760aagccccggc aggtttgaag ctgcttcttc gaatttgcaa ttcaatatga aaatcacctc 5820ggagctggta aaaagaggcc taacccctgt ctttagattt acagtccaat gcttcactca 5880gccattttac ctcaccccca ctgatgttcg ccgaccgttg actattctct acaaaccaca 5940aagacattgg aacactatac ctattattcg gcgcatgagc tggagtccta ggcacagctc 6000taagcctcct tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca 6060tctacaacgt tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca 6120tcataatcgg aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg 6180cgtttccccg cataaacaac ataagcttct gactcttacc tccctctctc ctactcctgc 6240tcgcatctgc tatagtggag gccggagcag gaacaggttg aacagtctac cctcccttag 6300cagggaacta ctcccaccct ggagcctccg tagacctaac catcttctcc ttacacctag 6360caggtgtctc ctctatctta ggggccatca atttcatcac aacaattatc aatataaaac 6420cccctgccat aacccaatac caaacgcccc tcttcgtctg atccgtccta atcacagcag 6480tcctacttct cctatctctc ccagtcctag ctgctggcat cactatacta ctaacagacc 6540gcaacctcaa caccaccttc ttcgaccccg ccggaggagg agaccccatt ctataccaac 6600acctattctg atttttcggt caccctgaag tttatattct tatcctacca ggcttcggaa 6660taatctccca tattgtaact tactactccg gaaaaaaaga accatttgga tacataggta 6720tggtctgagc tatgatatca attggcttcc tagggtttat cgtgtgagca caccatatat 6780ttacagtagg aatagacgta gacacacgag catatttcac ctccgctacc ataatcatcg 6840ctatccccac cggcgtcaaa gtatttagct gactcgccac actccacgga agcaatatga 6900aatgatctgc tgcagtgctc tgagccctag gattcatctt tcttttcacc gtaggtggcc 6960tgactggcat tgtattagca aactcatcac tagacatcgt actacacgac acgtactacg 7020ttgtagccca cttccactat gtcctatcaa taggagctgt atttgccatc ataggaggct 7080tcattcactg atttccccta ttctcaggct acaccctaga ccaaacctac gccaaaatcc 7140atttcactat catattcatc ggcgtaaatc taactttctt cccacaacac tttctcggcc 7200tatccggaat gccccgacgt tactcggact accccgatgc atacaccaca tgaaacatcc 7260tatcatctgt aggctcattc atttctctaa cagcagtaat attaataatt ttcatgattt 7320gagaagcctt cgcttcgaag cgaaaagtcc taatagtaga agaaccctcc ataaacctgg 7380agtgactata tggatgcccc ccaccctacc acacattcga agaacccgta tacataaaat 7440ctagacaaaa aaggaaggaa tcgaaccccc caaagctggt ttcaagccaa ccccatggcc 7500tccatgactt tttcaaaaag gtattagaaa aaccatttca taactttgtc aaagttaaat 7560tataggctaa atcctatata tcttaatggc acatgcagcg caagtaggtc tacaagacgc 7620tacttcccct atcatagaag agcttatcac ctttcatgat cacgccctca taatcatttt 7680ccttatctgc ttcctagtcc tgtatgccct tttcctaaca ctcacaacaa aactaactaa 7740tactaacatc tcagacgctc aggaaataga aaccgtctga actatcctgc ccgccatcat 7800cctagtcctc atcgccctcc catccctacg catcctttac ataacagacg aggtcaacga 7860tccctccctt accatcaaat caattggcca ccaatggtac tgaacctacg agtacaccga 7920ctacggcgga ctaatcttca actcctacat acttccccca ttattcctag aaccaggcga 7980cctgcgactc cttgacgttg acaatcgagt agtactcccg attgaagccc ccattcgtat 8040aataattaca tcacaagacg tcttgcactc atgagctgtc cccacattag gcttaaaaac 8100agatgcaatt cccggacgtc taaaccaaac cactttcacc gctacacgac cgggggtata 8160ctacggtcaa tgctctgaaa tctgtggagc aaaccacagt ttcatgccca tcgtcctaga 8220attaattccc ctaaaaatct ttgaaatagg gcccgtattt accctatagc accccctcta 8280ccccctctag agcccactgt aaagctaact tagcattaac cttttaagtt aaagattaag 8340agaaccaaca cctctttaca gtgaaatgcc ccaactaaat actaccgtat ggcccaccat 8400aattaccccc atactcctta cactattcct catcacccaa ctaaaaatat taaacacaaa 8460ctaccaccta cctccctcac caaagcccat aaaaataaaa aattataaca aaccctgaga 8520accaaaatga acgaaaatct gttcgcttca ttcattgccc ccacaatcct aggcctaccc 8580gccgcagtac tgatcattct atttccccct ctattgatcc ccacctccaa atatctcatc 8640aacaaccgac taatcaccac ccaacaatga ctaatcaaac taacctcaaa acaaatgata 8700accatacaca acactaaagg acgaacctga tctcttatac tagtatcctt aatcattttt 8760attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac cacccaacta 8820tctataaacc tagccatggc catcccctta tgagcgggca cagtgattat aggctttcgc 8880tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac accccttatc 8940cccatactag ttattatcga aaccatcagc ctactcattc aaccaatagc cctggccgta 9000cgcctaaccg ctaacattac tgcaggccac ctactcatgc acctaattgg aagcgccacc 9060ctagcaatat caaccattaa ccttccctct acacttatca tcttcacaat tctaattcta 9120ctgactatcc tagaaatcgc tgtcgcctta atccaagcct acgttttcac acttctagta 9180agcctctacc tgcacgacaa cacataatga cccaccaatc acatgcctat catatagtaa 9240aacccagccc atgaccccta acaggggccc tctcagccct cctaatgacc tccggcctag 9300ccatgtgatt tcacttccac tccataacgc tcctcatact aggcctacta accaacacac 9360taaccatata ccaatgatgg cgcgatgtaa cacgagaaag cacataccaa ggccaccaca 9420caccacctgt ccaaaaaggc cttcgatacg ggataatcct atttattacc tcagaagttt 9480ttttcttcgc aggatttttc tgagcctttt accactccag cctagcccct accccccaat 9540taggagggca ctggccccca acaggcatca ccccgctaaa tcccctagaa gtcccactcc 9600taaacacatc cgtattactc gcatcaggag tatcaatcac ctgagctcac catagtctaa 9660tagaaaacaa ccgaaaccaa ataattcaag cactgcttat tacaatttta ctgggtctct 9720attttaccct cctacaagcc tcagagtact tcgagtctcc cttcaccatt tccgacggca 9780tctacggctc aacatttttt gtagccacag gcttccacgg acttcacgtc attattggct 9840caactttcct cactatctgc ttcatccgcc aactaatatt tcactttaca tccaaacatc 9900actttggctt cgaagccgcc gcctgatact ggcattttgt agatgtggtt tgactatttc 9960tgtatgtctc catctattga tgagggtctt actcttttag tataaatagt accgttaact 10020tccaattaac tagttttgac aacattcaaa aaagagtaat aaacttcgcc ttaattttaa 10080taatcaacac cctcctagcc ttactactaa taattattac attttgacta ccacaactca 10140acggctacat agaaaaatcc accccttacg agtgcggctt cgaccctata tcccccgccc 10200gcgtcccttt ctccataaaa ttcttcttag tagctattac cttcttatta tttgatctag 10260aaattgccct ccttttaccc ctaccatgag ccctacaaac aactaacctg ccactaatag 10320ttatgtcatc cctcttatta atcatcatcc tagccctaag tctggcctat gagtgactac 10380aaaaaggatt agactgaacc gaattggtat atagtttaaa caaaacgaat gatttcgact 10440cattaaatta tgataatcat atttaccaaa tgcccctcat ttacataaat attatactag 10500catttaccat ctcacttcta ggaatactag tatatcgctc acacctcata tcctccctac 10560tatgcctaga aggaataata ctatcgctgt tcattatagc tactctcata accctcaaca 10620cccactccct cttagccaat attgtgccta ttgccatact agtctttgcc gcctgcgaag 10680cagcggtggg cctagcccta ctagtctcaa tctccaacac atatggccta gactacgtac 10740ataacctaaa cctactccaa tgctaaaact aatcgtccca acaattatat tactaccact 10800gacatgactt tccaaaaaac acataatttg aatcaacaca accacccaca gcctaattat

10860tagcatcatc cctctactat tttttaacca aatcaacaac aacctattta gctgttcccc 10920aaccttttcc tccgaccccc taacaacccc cctcctaata ctaactacct gactcctacc 10980cctcacaatc atggcaagcc aacgccactt atccagtgaa ccactatcac gaaaaaaact 11040ctacctctct atactaatct ccctacaaat ctccttaatt ataacattca cagccacaga 11100actaatcata ttttatatct tcttcgaaac cacacttatc cccaccttgg ctatcatcac 11160ccgatgaggc aaccagccag aacgcctgaa cgcaggcaca tacttcctat tctacaccct 11220agtaggctcc cttcccctac tcatcgcact aatttacact cacaacaccc taggctcact 11280aaacattcta ctactcactc tcactgccca agaactatca aactcctgag ccaacaactt 11340aatatgacta gcttacacaa tagcttttat agtaaagata cctctttacg gactccactt 11400atgactccct aaagcccatg tcgaagcccc catcgctggg tcaatagtac ttgccgcagt 11460actcttaaaa ctaggcggct atggtataat acgcctcaca ctcattctca accccctgac 11520aaaacacata gcctacccct tccttgtact atccctatga ggcataatta taacaagctc 11580catctgccta cgacaaacag acctaaaatc gctcattgca tactcttcaa tcagccacat 11640agccctcgta gtaacagcca ttctcatcca aaccccctga agcttcaccg gcgcagtcat 11700tctcataatc gcccacgggc ttacatcctc attactattc tgcctagcaa actcaaacta 11760cgaacgcact cacagtcgca tcataatcct ctctcaagga cttcaaactc tactcccact 11820aatagctttt tgatgacttc tagcaagcct cgctaacctc gccttacccc ccactattaa 11880cctactggga gaactctctg tgctagtaac cacgttctcc tgatcaaata tcactctcct 11940acttacagga ctcaacatac tagtcacagc cctatactcc ctctacatat ttaccacaac 12000acaatggggc tcactcaccc accacattaa caacataaaa ccctcattca cacgagaaaa 12060caccctcatg ttcatacacc tatcccccat tctcctccta tccctcaacc ccgacatcat 12120taccgggttt tcctcttgta aatatagttt aaccaaaaca tcagattgtg aatctgacaa 12180cagaggctta cgacccctta tttaccgaga aagctcacaa gaactgctaa ctcatgcccc 12240catgtctaac aacatggctt tctcaacttt taaaggataa cagctatcca ttggtcttag 12300gccccaaaaa ttttggtgca actccaaata aaagtaataa ccatgcacac tactataacc 12360accctaaccc tgacttccct aattcccccc atccttacca ccctcgttaa ccctaacaaa 12420aaaaactcat acccccatta tgtaaaatcc attgtcgcat ccacctttat tatcagtctc 12480ttccccacaa caatattcat gtgcctagac caagaagtta ttatctcgaa ctgacactga 12540gccacaaccc aaacaaccca gctctcccta agcttcaaac tagactactt ctccataata 12600ttcatccctg tagcattgtt cgttacatgg tccatcatag aattctcact gtgatatata 12660aactcagacc caaacattaa tcagttcttc aaatatctac tcatcttcct aattaccata 12720ctaatcttag ttaccgctaa caacctattc caactgttca tcggctgaga gggcgtagga 12780attatatcct tcttgctcat cagttgatga tacgcccgag cagatgccaa cacagcagcc 12840attcaagcaa tcctatacaa ccgtatcggc gatatcggtt tcatcctcgc cttagcatga 12900tttatcctac actccaactc atgagaccca caacaaatag cccttctaaa cgctaatcca 12960agcctcaccc cactactagg cctcctccta gcagcagcag gcaaatcagc ccaattaggt 13020ctccacccct gactcccctc agccatagaa ggccccaccc cagtctcagc cctactccac 13080tcaagcacta tagttgtagc aggaatcttc ttactcatcc gcttccaccc cctagcagaa 13140aatagcccac taatccaaac tctaacacta tgcttaggcg ctatcaccac tctgttcgca 13200gcagtctgcg cccttacaca aaatgacatc aaaaaaatcg tagccttctc cacttcaagt 13260caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 13320atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 13380aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 13440acttcaacct ccctcaccat tggcagccta gcattagcag gaataccttt cctcacaggt 13500ttctactcca aagaccacat catcgaaacc gcaaacatat catacacaaa cgcctgagcc 13560ctatctatta ctctcatcgc tacctccctg acaagcgcct atagcactcg aataattctt 13620ctcaccctaa caggtcaacc tcgcttcccc acccttacta acattaacga aaataacccc 13680accctactaa accccattaa acgcctggca gccggaagcc tattcgcagg atttctcatt 13740actaacaaca tttcccccgc atcccccttc caaacaacaa tccccctcta cctaaaactc 13800acagccctcg ctgtcacttt cctaggactt ctaacagccc tagacctcaa ctacctaacc 13860aacaaactta aaataaaatc cccactatgc acattttatt tctccaacat actcggattc 13920taccctagca tcacacaccg cacaatcccc tatctaggcc ttcttacgag ccaaaacctg 13980cccctactcc tcctagacct aacctgacta gaaaagctat tacctaaaac aatttcacag 14040caccaaatct ccacctccat catcacctca acccaaaaag gcataattaa actttacttc 14100ctctctttct tcttcccact catcctaacc ctactcctaa tcacataacc tattcccccg 14160agcaatctca attacaatat atacaccaac aaacaatgtt caaccagtaa ctactactaa 14220tcaacgccca taatcataca aagcccccgc accaatagga tcctcccgaa tcaaccctga 14280cccctctcct tcataaatta ttcagcttcc tacactatta aagtttacca caaccaccac 14340cccatcatac tctttcaccc acagcaccaa tcctacctcc atcgctaacc ccactaaaac 14400actcaccaag acctcaaccc ctgaccccca tgcctcagga tactcctcaa tagccatcgc 14460tgtagtatat ccaaagacaa ccatcattcc ccctaaataa attaaaaaaa ctattaaacc 14520catataacct cccccaaaat tcagaataat aacacacccg accacaccgc taacaatcaa 14580tactaaaccc ccataaatag gagaaggctt agaagaaaac cccacaaacc ccattactaa 14640acccacactc aacagaaaca aagcatacat cattattctc gcacggacta caaccacgac 14700caatgatatg aaaaaccatc gttgtatttc aactacaaga acaccaatga ccccaatacg 14760caaaactaac cccctaataa aattaattaa ccactcattc atcgacctcc ccaccccatc 14820caacatctcc gcatgatgaa acttcggctc actccttggc gcctgcctga tcctccaaat 14880caccacagga ctattcctag ccatgcacta ctcaccagac gcctcaaccg ccttttcatc 14940aatcgcccac atcactcgag acgtaaatta tggctgaatc atccgctacc ttcacgccaa 15000tggcgcctca atattcttta tctgcctctt cctacacatc gggcgaggcc tatattacgg 15060atcatttctc tactcagaaa cctgaaacat cggcattatc ctcctgcttg caactatagc 15120aacagccttc ataggctatg tcctcccgtg aggccaaata tcattctgag gggccacagt 15180aattacaaac ttactatccg ccatcccata cattgggaca gacctagttc aatgaatctg 15240aggaggctac tcagtagaca gtcccaccct cacacgattc tttacctttc acttcatctt 15300gcccttcatt attgcagccc tagcaacact ccacctccta ttcttgcacg aaacgggatc 15360aaacaacccc ctaggaatca cctcccattc cgataaaatc accttccacc cttactacac 15420aatcaaagac gccctcggct tacttctctt ccttctctcc ttaatgacat taacactatt 15480ctcaccagac ctcctaggcg acccagacaa ttatacccta gccaacccct taaacacccc 15540tccccacatc aagcccgaat gatatttcct attcgcctac acaattctcc gatccgtccc 15600taacaaacta ggaggcgtcc ttgccctatt actatccatc ctcatcctag caataatccc 15660catcctccat atatccaaac aacaaagcat aatatttcgc ccactaagcc aatcacttta 15720ttgactccta gccgcagacc tcctcattct aacctgaatc ggaggacaac cagtaagcta 15780cccttttacc atcattggac aagtagcatc cgtactatac ttcacaacaa tcctaatcct 15840aataccaact atctccctaa ttgaaaacaa aatactcaaa tgggcctgtc cttgtagtat 15900aaactaatac accagtcttg taaaccggag atgaaaacct ttttccaagg acaaatcaga 15960gaaaaagtct ttaactccac cattagcacc caaagctaag attctaattt aaactattct 16020ctgttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca 16080accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat 16140acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16200caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc 16260cctcacccac taggatacca acaaacctac ccacccttaa cagtacatag tacataaagc 16320catttaccgt acatagcaca ttacagtcaa atcccttctc gtccccatgg atgacccccc 16380tcagataggg gtcccttgac caccatcctc cgtgaaatca atatcccgca caagagtgct 16440actctcctcg ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16500ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct taaataagac 16560atcacgatg 16569922DNAArtificial Sequencesynthetic primer forward 3.4 kb deleted sequence 9cccactcatc acctaaacct ac 221020DNAArtificial Sequencesynthetic primer reverse 3.4 kb deleted sequence 10ggtaggagtc aggtagttag 20

Claims

1. A method of detecting a cancer in an individual, the method comprising: a) obtaining a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome, wherein the step of quantifying comprises contacting the sample with a pair of amplification primers and amplifying a target region of mtDNA that is indicative of the deletion, wherein a first primer of the pair of primers is adapted to bind to a region of the mtDNA comprising a splice joining opposite ends of the mtDNA sequence after removal of the deletion; c) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA wherein the reference sample is from a known non-cancerous tissue or body fluid or from a known cancerous tissue or body fluid; wherein if the reference sample is from a known non-cancerous tissue or body fluid, then an elevated level of the deletion in the biological sample compared to the non-cancerous reference sample is indicative of cancer and if the reference sample is from a known cancerous tissue or body fluid sample then an equivalent or elevated level of the deletion in the biological sample compared to the cancerous reference sample is indicative of cancer.

2. The method of claim 1 wherein the deletion has a nucleic acid sequence set forth in SEQ ID NO: 1.

3. (canceled)

4. The method of claim 1, wherein the first primer has the nucleic acid sequence set forth in SEQ ID NO: 2.

5. The method of claim 1 wherein the step of quantifying is conducted using real-time PCR.

6. The method of claim 1 wherein the cancer is prostate cancer.

7. The method of claim 1 wherein the cancer is breast cancer.

8. The method of claim 1 wherein the biological sample is a body tissue or body fluid.

9. The method of claim 8 wherein the biological sample is breast tissue, prostate tissue, prostate massage fluid, or urine.

10. (canceled)

11. A method of monitoring an individual for the development of a cancer comprising; a) obtaining a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome, wherein the step of quantifying comprises contacting the sample with a pair of amplification primers and amplifying a target region of mtDNA that is indicative of the deletion, wherein a first primer of the pair of primers is adapted to bind to a region of the mtDNA comprising a splice joining opposite ends of the mtDNA sequence after removal of the deletion; c) repeating steps a) to b) over a duration of time; and d) wherein an increasing level of the deletion over the duration of time is indicative of cancer.

12. The method of claim 11 wherein the deletion has a nucleic acid sequence set forth in SEQ ID NO: 1.

13. The method of claim 11 further comprising at least one step selected from the group consisting of: (a) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and (b) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid.

14. (canceled)

15. The method of claim 11 wherein the step of quantifying is conducted using real-time PCR.

16. The method of claim 11, wherein the first primer has the nucleic acid sequence set forth in SEQ ID NO: 2.

17. The method of claim 11 wherein the cancer is prostate cancer.

18. The method of claim 11 wherein the cancer is breast cancer.

19. The method of claim 11 wherein the biological sample is a body tissue or body fluid.

20. The method of claim 19 wherein the biological sample is breast tissue, prostate tissue, prostate massage fluid, or urine.

21-35. (canceled)