WO2002101086A2 - Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante - Google Patents

Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante Download PDF

Info

Publication number
WO2002101086A2
WO2002101086A2 PCT/CA2002/000848 CA0200848W WO02101086A2 WO 2002101086 A2 WO2002101086 A2 WO 2002101086A2 CA 0200848 W CA0200848 W CA 0200848W WO 02101086 A2 WO02101086 A2 WO 02101086A2
Authority
WO
WIPO (PCT)
Prior art keywords
disease
mtdna
mutations
mitochondrial
mitochondrial dna
Prior art date
Application number
PCT/CA2002/000848
Other languages
English (en)
Other versions
WO2002101086A3 (fr
Inventor
Mark Birch-Machin
Gabriel D. Dakubo
Ryan Parr
Robert Thayer
Alioune Ngom
John Th'ng
Original Assignee
1304854 Ontario Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 1304854 Ontario Ltd. filed Critical 1304854 Ontario Ltd.
Priority to CA002450403A priority Critical patent/CA2450403A1/fr
Priority to JP2003503836A priority patent/JP2005506057A/ja
Priority to EP02737683A priority patent/EP1397508A2/fr
Publication of WO2002101086A2 publication Critical patent/WO2002101086A2/fr
Priority to US10/732,374 priority patent/US20050026167A1/en
Publication of WO2002101086A3 publication Critical patent/WO2002101086A3/fr
Priority to PCT/CA2004/002124 priority patent/WO2005056573A1/fr
Priority to US11/339,751 priority patent/US20070190534A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention is related to the field of mitochondrial genomics. In particular it is related to mutations in the mitochondrial genome and their utility as an indicator of the very genesis of disease.
  • the mitochondrial genome is a compact yet critical sequence of nucleic acid.
  • the mitochondrial genome codes for enzyme subunits necessary for cellular respiration.
  • Mitochondrial DNA, or "mtDNA” is a minuscule genome of nucleic acid at 16,569 base pairs (bp Anderson et al., 1981; Andrews et al., 1999) in contrast to the immense nuclear genome of 3.3 billion bp. Its genetic complement is astronomically smaller than that of its nuclear cell mate (0.0005%); however, communication or chemical signalling, routinely occur (Sherratt et al., 1997). Moreover, specific nuclear components are responsible for maintenance and integrity of mitochondrial sequence (Croteau et al., 1999).
  • mtDNA The essential role of mtDNA is the generation of the cellular fuel, adenosine triphosphate (ATP), which fires cellular metabolism.
  • ATP adenosine triphosphate
  • mitochondrial genome is dependent on seventy nuclear encoded proteins to accomplish the oxidation and reduction reactions necessary to this vital function, in addition to the thirteen polypeptides supplied by the mitochondrial genome (Leonard and Shapira, 1997).
  • Different tissues and organs depend on oxidative phosphorylation to a varied extent.
  • mutations in the mitochondrial genome are associated with a variety of chronic, degenerative diseases (Gattermann et al. 1995).
  • Diseases related to defective oxidative phosphorylation (OXPHOS) appear to be closely linked to mtDNA mutations (Byrne, 1992). Consequently as OXPHOS diminishes due to increased severity of mtDNA mutations, organ specific energetic thresholds are exceeded which give rise to a variety of clinical phenotypes.
  • 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
  • these 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).
  • specific base pair alterations, deletions, or combinations of are 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.
  • mtDNA is passed to offspring exclusively through the ovum, it is imperative to understand mitochondrial sequences through this means of inheritance.
  • the sequence of mtDNA varies widely between maternal lineages (Ward et al., 1991), hence mutations associated with disease must be clearly understood in comparison to this variation.
  • a specific T to C transition noted in the sequence of several individuals, associated with a specific cancer could in reality be natural variation in a maternal lineage widespread in a given particular geographical area or associated with ethnicity.
  • Native North Americans express an unusually high frequency of adult onset diabetes.
  • Lineage A is distinguished by a simple point mutation resulting in a Hae III site at bp 663 in the mitochondrial genome, yet there is no causative relationship between this mutation and the adult onset of diabetes.
  • sequence variation even within lineage clusters there is sequence variation.
  • a substantial mtDNA sequence database is a clear prerequisite to accurate forecasting of potential disease as a natural process, or through exposure to causative agents.
  • the entire molecule must be sequenced for its full information content.
  • the entire suite of 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) must be characterized as a whole over the entire mitochondrial genome. This ensures that all possible information available in the mitochondrial genome is captured.
  • the genome of cytoplasmic mitochondria (16,569bp) has been sequenced at an individual level, like its nuclear counte ⁇ art, the mitochondrial genome has not been sequenced at a population level for use as a diagnostic tool.
  • UV ultraviolet radiation
  • mitochondrial sequence loses integrity.
  • the 4977bp deletion increases in frequency with age (Fahn et al., 1996). Beginning at age 20, this deletion begins to occur in small numbers of mitochondria. By age 80, a substantial number of molecules have been deleted. This deletion characterizes the normal aging process, and as such serves as a biomarker for this process. Quantification of this aging process may allow medical or other interventions to slow the process.
  • NMSC Human non-melanoma skin cancer
  • BCC basal cell carcinoma
  • SCC squamous cell carcinoma
  • SCCs show loss of heterozygosity affecting several chromosomes which suggests the involvement of several tumour suppressor genes in their development.
  • AKs an equal or greater degree of genetic loss is observed in these precursor lesions compared to SCCs (Rehman et al. 1994; Rehman et al. 1996). This is important for the proposed invention because it suggests that other mechanisms, in addition to inactivation of tumour suppressor genes, are likely to be involved in the development of SCCs.
  • MtDNA as a molecular marker was used to study the relation between chronological aging and photo aging in human skin.
  • a 3 -primer quantitative PCR method was used to study the changes in the ratio of the 4977 bp-deleted to wild type mtDNA in relation to sun exposure and chronological age of human skin.
  • Deletions or mutations of mtDNA may therefore be useful as a marker of cumulative ultraviolet radiation exposure. Furthermore, a study using a South-Western Blot approach involving monoclonal antibodies against thymine dimers, provided direct evidence for the presence of UV- induced damage in purified mtDNA (Ray et al. 1998).
  • deletions are difficult to characterize. Long PCR is typically used which produces a ladder of deletions which then have to be characterized.
  • 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
  • Prostate cancer exhibits a wide variety of histological behaviour involving both erogenous 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).
  • Familial cancers refer to the incidences within a family, but are not inherited. This form accounts for up to 25% of prostate cancers (Walsh & Partin, 1997).
  • Hereditary refers to a subtype of prostate cancer with a Mendelian inheritance of a predisposing gene(s) and accounts for approximately 9% of reported cases. A positive family history of prostate cancer for this disease suggests that these predisposing gene(s) play an important role in prostate cancer development and progression.
  • Prostate cancer prognosis mainly depends on the tumour stage and grade at diagnosis.
  • Aging consists of an accumulation of changes with time both at the molecular and cellular levels; however, the specific molecular mechanisms underlying the aging process remain to be elucidated.
  • mitochondrial genomes in older subjects are compared to the genomes of younger subjects from the same maternal lineage.
  • One deletion associated with aging is known as the common deletion, or 4977-bp deletion. Aging research has been limited to this common deletion and polymo ⁇ hisms in the control region. For a clear understanding of these mutations, the entire genome must be analyzed. Other deletions are seen in Table 1 adapted from Wei, 1992.
  • Oxygen free radicals are a probable cause of this deletion, which increases in frequency with age.
  • Existing literature demonstrates a strong association between mtDNA (mtDNA) mutations, chronological age, and the overall aging process in postmitotic tissues such as muscle and brain; however, comparative maternal line studies are needed to discriminate between aging associated mutational events and those mutations without an aging association.
  • An object of the present invention is to provide a simple, straightforward system for monitoring the vast nuclear genome for early transitions associated with cancer, aging, and other human diseases with a DNA component.
  • a small biological sample which includes tissue or fluid samples such as urine, prostate fluid, skin cells, or saliva is taken from an individual. These samples are examined, using any suitable method including histological examination, to identify cells demonstrating disease mo ⁇ hology. Using any suitable method, including without limitation; laser capture, identified cells demonstrating disease mo ⁇ hology are recovered from the sample and the mtDNA therefrom is sequenced, followed by comparison to a database of known mitochondrial sequences associated with both health and disease.
  • the entire mitochondrial genome is sequenced at a population level to determine the variation of mtDNA sequences associated with disease.
  • the presence of mutation progression may signal the beginning and continuing development of disease.
  • Mutation load may also indicate progression or disease state.
  • mtDNA sequences from prostate massage fluid are compared to a mtDNA sequence database of normal, transitory, and metastatic mtDNA sequences clearly associated with prostate cancer.
  • This comparative data set is based on studies of maternal lines, and other normal maternal line variation present in the population stored in a maternal line database affording a lucid picture of mtDNA mutations clearly associated with disease, as opposed to variation present in mitochondrial lineages existing in the general population.
  • mtDNA sequences from suspected non-melanoma skin cancers are compared to a mtDNA sequence database of normal and mtDNA sequences clearly associated with non-melanoma skin cancer.
  • a method of detecting in a subject containing mtDNA the genesis or progression of disease comprising obtaining a biological sample from the subject, extracting DNA from the biological sample, and detecting the presence of mutations in the mtDNA.
  • the step of detecting the presence of mutations is selected from the group consisting of sequencing the mtDNA, amplifying the mtDNA by PCR, South- Western blotting, denaturing HPLC, hybridization to microarrays, gene chips or biochips, molecular marker analysis or combinations thereof. Further, the mtDNA of the biological sample is compared to a database, the database containing data of mutations associated with the mtDNA sequences of non-disease and disease associated mitochondrial genomes.
  • a method of detecting in a human subject the presence of a disease comprising obtaining a biological sample from the human subject, extracting DNA from the biological sample, detecting mutations in the mitochondrial DNA of the biological sample, and comparing the mitochondrial DNA sequence of the biological sample to a database, the database containing data of mutations associated with the mitochondrial DNA sequences of non- disease and disease associated mitochondrial genomes Mutation rates of mitochondria DNA associated with a specific disease may be an important indicator of disease development and prognosis. This may allow specific identification of disease stage, improving disease definition resulting in better disease intervention and specific therapy application.
  • the invention may be used to monitor the progression of disease by watching important sites targeted by metastasis.
  • a method of determining a predisposition to a disease or disorder indicated by mutations in a mitochondrial DNA sequence comprising: obtaining a biological sample from the human subject, extracting DNA from the biological sample, detecting mutations in the mitochondrial DNA of the biological sample, and comparing the mitochondrial DNA sequence of the biological sample to a database, the database containing data of mutations associated with the mitochondrial DNA sequences of individuals who are predisposed to the disease or disorder, and individuals who are not predisposed to the disease or disorder.
  • a DNA microarray is used in determining the sequence of the mitochondrial DNA.
  • Other technologies can also be used. For example, direct sequencing of a subset, or the complete human genome, SNaP shotTM, SNP detection, real time PCR or other methods as is standard in the art.
  • a method for assessing the status of the aging process of a human subject comprising obtaining a biological sample from the human subject, extracting DNA from the biological sample, detecting mutations in the mitochondrial DNA of the biological sample, and comparing the mitochondrial DNA sequence of the biological sample to a database, the database containing data of mutations of TDNA associated with aging.
  • the step of detecting the presence of mutations in the mtDNA can be selected from: sequencing the mtDNA, amplifying mtDNA by PCR, Southern, Northern, Western, South- Western blot hybridizations, denaturing HPLC, hybridization to microarrays, biochips or gene chips, molecular marker analysis or a combination of any of the above.
  • a database containing a plurality of human mitochondrial DNA sequences, the mitochondrial DNA sequences selected from the group of normal control sequences associated with non- disease states, sequences associated with the presence of disease or sequences indicative of the predisposition to disease.
  • a kit for diagnosis of a disease comprising a disposable chip, microarray, means for holding the disposable chip, means for extraction of mitochondrial DNA and means for access to a database of mitochondrial DNA sequences.
  • a method of diagnosing a disease in a patient comprising hybridizing a nucleic acid sample obtained from mitochondrial DNA to an array comprising a solid substrate and a plurality of nucleic acid members, wherein each member is indicative of the presence of a disease, wherein each nucleic acid member has a unique position and is stably associated with the solid substrate, and wherein hybridization of said nucleic acid sample to one or more nucleic acid members comprising said array is indicative of the presence of prostate cancer.
  • kits for determining predisposition to a disease comprising a disposable chip, microarray, means for holding the disposable chip, means for extraction of DNA and means for access to a database of mitochondrial DNA sequences.
  • a method of determining a predisposition to or developing symptoms of a disease or disorder indicated by mutations in a mitochondrial DNA sequence comprising obtaining a biological sample from the human subject, extracting mitochondrial DNA from the biological sample, sequencing the mitochondrial DNA of the biological sample, and comparing the mitochondrial DNA sequence of the biological sample to a database, the database containing population-level data of mutations associated with the mtDNA sequences of non-disease and disease associated mitochondrial genomes.
  • a method of diagnosing non-melanoma skin cancer in a patient comprising: hybridizing a nucleic acid sample obtained from mitochondrial DNA to an array comprising a solid substrate and a plurality of nucleic acid members, wherein each member is indicative of non-melanoma cancer, wherein each nucleic acid member has a unique position and is stably associated with the solid substrate, and wherein hybridization of said nucleic acid sample to one or more nucleic acid members comprising said array is indicative of the presence of non- melanoma skin cancer.
  • non-specific mutations may reach a threshold effect beyond which cancer develops.
  • prostate cancer can also be diagnosed.
  • a method of detecting heteroplasmy in a subject containing mtDNA comprising obtaining a biological sample from the subject; extracting DNA from the biological sample; and performing denaturing HPLC on the sample.
  • a method of detecting mutations associated with disease in a subject containing mtDNA comprising: obtaining a biological sample from the subject, extracting DNA from the biological sample, detecting the presence of mutations in the mtDNA, and comparing the mtDNA of the biological sample to a database, the database containing data of common population variants in non-disease and disease associated mitochondrial genomes.
  • the method of the present invention can be used to diagnose diseases linked to mtDNA.
  • the method of the present invention provides for amplification of the mitochondrial genome of an individual from a biological sample, sequencing a portion of the mitochondrial genome, preferably the entire mitochondrial genome of the individual using any known means.
  • Denaturing high performance liquid chromatography (DHPLC) may also be used to rapidly screen many samples.
  • DHPLC can focus on hotspots of mutations. DHPLC is more sensitive than automated sequencing in terms of detecting mutations, 2% heteroplasmy, compared with 20-25% for ordinary sequencing. Methods for detecting lower levels of heteroplasmy ( ⁇ 2%) may also be developed.
  • the "presence" of a mutation in mtDNA includes heteroplasmic mutations and, therefore, it is contemplated that there may be additionally the presence of some normal mtDNA in a sample in which the mutated DNA is present.
  • actinic kerotoses means proposed precursor epidermal lesion of a squamous cell carcinoma.
  • aging refers to an accumulation of changes with time, both at the molecular and cellular levels.
  • alleles means one of several alternative forms of a given DNA sequence occupying a specific place on a chromosome.
  • attaching or “spotting” refers to a process of depositing a nucleic acid onto a solid substrate to form a nucleic acid array such that the nucleic acid is irreversibly bound to the solid substrate via covalent bonds, hydrogen bonds or ionic interactions.
  • basic cell carcinoma means a type of cancer of skin cells.
  • Boen's disease means in situ epidermal carcinoma.
  • diagnosis means using the presence or absence of a mutation or combination of mutations as a factor in disease diagnosis or management.
  • the detection of the mutation(s) can be a step in the disease state diagnosis.
  • disease includes a disorder or other abnormal physical state.
  • disease associated mitochondiral genomes means genomes containing mutations indicative or otherwise associated with a particular disease.
  • database means an electronic storage system (computer based using standard industry software) which will have the capacity to store and provide retrievable information that will enable researchers to rapidly determine the structure of the nucleotide sequences.
  • the database will also store descriptive information about those individuals who provide the biological samples. This descriptive information will include health status and other pertinent indices which may be correlated to the biological sample.
  • deletion means removal of a region of DNA from a contiguous sequence of nucleic acids, where once a deletion has occurred, the gap is repaired by rejoining of the ends. Deletions can range in size from one base to thousands of bases or larger.
  • duplications means when a specific sequence of DNA is copied and inserted behind or forward of the original copy one or more times or elsewhere in the genome.
  • heteroplasmy is defined by the ratio of mutant: to wild type mtDNA molecules, where 100% mutant mtDNA is termed "homoplasmic". Heteroplasmic mutations are those mutations which occur in some, but not all of the copies of the mitochondrial genome.
  • homoplasmy means all mitochondrial sequences are identical.
  • inversions refers to when a length of DNA is excised and reinserted in reverse orientation.
  • mitochondria which are inherited through the cytoplasm of the ovum.
  • mitochondrial DNA refers to the clonal sequence of mitochondrial DNA as passed down through successive generations from the mother.
  • mitochondria means a eukaryotic cytoplasmic organelle that generates ATP for cellular processes.
  • mutation encompasses any change in a DNA sequence from the wild type sequence, including without limitation point mutations, transitions, insertions, transversions, translocations, deletions, inversions, duplications, recombinations or combinations thereof.
  • mutant load refers to an increase in mutations in mtDNA which eventually leads to compromised function of the involved gene or the entire genome.
  • nucleic acid array refers to a plurality of unique nucleic acids attached to one surface of a solid support at a density exceeding 20 different nucleic acids/cm 2 wherein each of the nucleic acids is attached to the surface of the solid support in a non-identical preselected region.
  • the nucleic acid attached to the surface of the solid support is DNA.
  • the nucleic acid attached to the surface of the solid support is cDNA.
  • the nucleic acid attached to the surface of the solid support is cDNA synthesized by polymerase chain reaction (PCR).
  • a nucleic acid array comprises nucleic acids of at least 150 nucleotides in length.
  • a nucleic acid array comprises nucleic acids of less than 6,000 nucleotides in length. More preferably, a nucleic acid array comprises nucleic acids of less than 500 nucleotides in length.
  • the array comprises at least 500 different nucleic acids attached to one surface of the solid support.
  • the array comprises at least 10 different nucleic acids attached to one surface of the solid support.
  • the array comprises at least 10,000 different nucleic acids attached to one surface of the solid support.
  • nucleic acid as used herein, is interchangeable with the term "polynucleotide”.
  • nucleic acid target or "a target nucleic acid” is defined as a nucleic acid capable of binding to a nucleic acid member of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a nucleic acid target may include natural (i. e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
  • the bases in nucleic acid probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • nucleic acid targets may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • the nucleic acid targets are derived from human tissue or fluid extracts. More preferably, the nucleic acid targets are single- or double-stranded DNA, RNA, or DNA-RNA hybrids synthesized from human tissue of fluid extracts.
  • nucleus means the most conspicuous organelle in the eucaryotic cell, contains all of the chromasomal DNA.
  • PSA Test means prostate-specific antigen test; an antigen found in blood that may be indicative of cancer of the prostate.
  • point mutation means the change of a single nucleotide in DNA.
  • polymo ⁇ hism means sequence variation in a population of alleles or mtDNA genomes.
  • precursor lesions means a DNA mutation, or combinations thereof, indicating potential disease association.
  • predisposed to a disease or a “predisposition to a disease” means that individuals are at higher risk for developing the disease or disorder or are at higher risk for early onset of the disease or disorder than the average individual, due to the presence or absence of mutations which are associated with the disease or disorder.
  • preselected region refers to a localized area on a substrate which is, was, or is intended to be used for the deposit of a nucleic acid and is otherwise referred to herein in the alternative as a "selected region” or simply a "region.”
  • the preselected region may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
  • a preselected region is smaller than about 1 cm , more preferably less than 1 mm , still more preferably less than 0.5 mm , and in some embodiments about 0.125 to 0.5 mm .
  • “somatic mutation” means a change in DNA sequence after fertilization.
  • solid substrate or “solid support” refers to a material having a rigid or semi-rigid surface.
  • substrate and “support” are used interchangeable herein with the terms “solid substrate” and “solid support”.
  • the solid support may be biological, non-biological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc.
  • the substrate is a silicon or glass surface, (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene, polycarbonate, a charged membrane, such as nylon 66 or nitrocellulose, or combinations thereof.
  • the solid support is glass.
  • at least one surface of the substrate will be substantially flat.
  • the surface of the solid support will contain reactive groups, including, but not limited to, carboxyl, amino, hydroxyl, thiol, or the like.
  • the surface is optically transparent.
  • squamous cell carcinoma means a type of cancer of skin cells.
  • stably associated refers to a nucleic acid that is irreversibly bound to a solid substrate to form an array via covalent bonds, hydrogen bonds or ionic interactions such that the nucleic acid retains its unique preselected position relative to all other nucleic acids that are stably associated with an array, or to all other preselected regions on the solid substrate under conditions wherein an array is analyzed (i.e., hybridization and scanning).
  • a "statistically significant" number of mitochondrial DNA sequences is determined by or through the use of standard chi-square statistical algorithms using or determining observed versus expected scores.
  • transitions means substitution of like nitrogenous bases, pyrimidine to pyrimidine, purine to purine. A mutation in which one pyrimidine is substituted by the other, or in which one purine is substituted by the other.
  • conversions means substitution of unlike nitrogenous bases, purine to pyrimidine, pyrimidine to purine. A mutation in which a purine is substituted or replaced by a pyrimidine or vice versa.
  • mtDNA a less complex genome, is easily understood at an individual and population level, hence a large mtDNA database with normal and disease associated genomes renders individual diagnosis extremely accurate. Accordingly, variation, in relationship to disease, is understood.
  • mtDNA has a 10-fold higher mutation rate than nuclear DNA (Wallace 1992). Nuclear rearrangements, suggestive of preliminary disease, are rapidly communicated to mitochondria, where they appear as somatic mutations.
  • mtDNA has a maternal inheritance pattern, and is essentially clonal in that all mitochondria begin with the same mtDNA sequence, hence variation from this clonal condition is easily detected. Additionally, mtDNA does not show convincing evidence of recombination, thus any alterations in sequence are a somatic event. Any one mitochondrion harboring a mutation(s) is in a sense 'recessive' as a consequence of there being many mitochondrial genomes (2-10 copies) per mitochondrion, and many mitochondria per cell (500-2,000). Moreover, mitochondrial genomes can tolerate very high levels (up to 90%) of mitochondria with damaged genomes. This happens through complementation by the remaining wild type mtDNA (Chomyn et al. 1992).
  • mutated genomes have a replicative advantage over wild type genomes because they are usually smaller (Hayashi et al. 1991), hence there is clonal expansion of mutated mtDNA (Brierley et al. 1998), suggesting that unlike nuclear genes, there is little or no selection against cells harboring mtDNA mutations. Because of this elevated mutation rate, mutations and/or deletions that appear in mtDNA are maintained through the life span of the cell and may serve as a record of exposures to various mutagens. The integrity of mtDNA is maintained by nuclear repair mechanisms, and a defect at these loci has been suggested to result in an autosomal dominant disorder associated with multiple mitochondrial deletions (Zeviani et al. 1990). Consequently, mtDNA may function as an early warning sentinel of early nuclear events related to a variety of cancers or other diseases. Finally, the mitochondrial genome can be sequenced and monitored for mutations on an individual basis.
  • a system for early diagnosis of mtDNA changes in non-melanoma skin cancer (NMSC) and their precursor lesions indicative of solid tumour development is provided.
  • the particular changes, such as the common deletion and associated mutations, and the incidence of as yet uncharacterised deletions in mtDNA serve as reliable bio-markers of potential skin cancer.
  • the mutation finge ⁇ rint of the entire mtDNA genome in human NMSC and its precursor lesions is determined.
  • mtDNA changes are established as an early bio-marker of human skin cancer and its precursor lesions.
  • Denaturing HPLC can then be used to assess low levels of heteroplasmy at the sequences of interest. This approach can also provide an insight into the development of early changes in other human tumours.
  • a system for diagnosis of prostate cancer is provided.
  • Age related accumulation of mtDNA defects might predispose an individual to the appearance of certain clinical disorders such as prostate cancer which is prevalent in middle age and older men.
  • routine prostate cancer screening takes place through mitochondrial genome sequencing from prostate massage fluid.
  • the presence of epithelial cells transformed into cancer cells can be determined through amplification of mtDNA from prostate massage fluid, eclipsing current diagnostic techniques such as digital rectal examination and PSA.
  • the system and method of the present invention may be used to assess aging, based on the increasing frequency of mutations such as the "common deletion" of 4977-bp and other mutations of the mitochondrial genome (Liu et al. 1997).
  • This information in conjunction with health survey data, allows crucial statistical discrimination between separate causes resulting in the same mutation/deletion.
  • mtDNA is inherited exclusively through the ovum and is essentially clonal in nature (Van De Graaff & Fox, 1995). This permits carefully controlled studies of mutations/deletions within maternal lines through several generations to determine a reliable age related deletion frequency. This information may be used to develop treatment methods which slow the aging process.
  • Biological samples can be collected by any known means, whether for the pu ⁇ ose of constructing a mtDNA sequence database, or performing a diagnostic test on an individual.
  • Samples destined for database generation include, but are not limited to: tumour banks, maternal lineage studies involving affected and unaffected individuals from the same maternal lineage, as well as maternal lineage studies from groups or populations with high frequencies of specific disease such as, but not limited to: skin and prostate cancer, assessment of health status and aging.
  • FTA ® Gene Cards ® may be used to collect and archive biological samples.
  • Suitable samples include any tissue or body fluid derived from mesothelium, epithelium, or endothelium.
  • Such tissues and fluids include, but are not limited to blood, sputum, buccal cells, saliva, prostate massage fluid, sweat, bone, hair, lymph tissue, cervical smears, breast aspirate, fecal matter, ejaculate, menstrual flow and biopsy tissue.
  • skin cells or tissue, (from normal, NMSC and precursor lesions) is taken from skin biopsy or a routine suction blistering technique. Where a disease is suspected, primary care physicians, oncologists or other practitioners, may extract both normal and suspected disease tissue from the patient.
  • HE hematoxylin and eosin
  • MG methyl green
  • HE stains are graded by a pathologist for normal, precursor, and applicable grades of tumour progression.
  • Replicate MG slides are used for laser capture, according to manufacturers recommendations (Arcturus) of graded cells.
  • Extraction of DNA may take place using any method known in the art, followed by sequencing of the mitochondrial genome, as described in Current Protocols in Molecular Biology.
  • Polynucleotide sequences of the invention can be amplified by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • PCR requires the presence of a nucleic acid to be amplified, two single stranded oligonucleotide primers flanking the sequence to be amplified, a DNA polymerase, deoxyribonucleoside triphosphates, a buffer and salts.
  • the method of PCR is well known in the art. PCR is performed as described in Mullis and Faloona, 1987, Methods Enzvmol., 155: 335, herein inco ⁇ orated by reference.
  • PCR is performed using template DNA (at least lfg; more usefully, 1-
  • a typical reaction mixture includes: 2 ⁇ l of DNA, 25 pmol of oligonucleotide primer, 2.5 ⁇ l of 10X PCR buffer 1 (Perkin-El er, Foster City, CA), 0.4 ⁇ l of 1.25 ⁇ M dNTP, 0.15 ⁇ l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, CA) and deionized water to a total volume of 25 ⁇ l.
  • Mineral oil is overlaid and the PCR is performed using a programmable thermal cycler.
  • the length and temperature of each step of a PCR cycle, as well as the number of cycles, are adjusted according to the stringency requirements in effect.
  • Annealing temperature and timing are determined both by the efficiency with which a primer is expected to anneal to a template and the degree of mismatch that is to be tolerated.
  • the ability to optimize the stringency of primer annealing conditions is well within the knowledge of one of moderate skill in the art.
  • An annealing temperature of between 30°C and 72°C is used.
  • initial denaturation of the template molecules normally occurs at between 92°C and 99°C for 4 minutes, followed by 20-40 cycles consisting of denaturation (94-99°C for 15 seconds to 1 minute), annealing (temperature determined as discussed above; 1-2 minutes), and extension (72°C for 1 minute).
  • the final extension step is generally carried out for 4 minutes at 72°C, and may be followed by an indefinite (0-24 hour) step at 4°C.
  • DNA Sequencing Any known means to sequence the mitochondrial genome may be used.
  • mtDNA is amplified by PCR prior to sequencing.
  • PCR products can be sequenced directly or cloned into a vector which is then placed into a bacterial host. Examples of DNA sequencing methods are found in Brumley, R. L. Jr. and Smith, L.M., 1991, Rapid DNA sequencing by horizontal ultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 and Luckey, J.A., et al, 1993, High speed DNA sequencing by capillary gel electrophoresis, Methods Enzymol. 218: 154-172.
  • LX-PCR long extension PCR
  • a semi-quantitative PCR method (Corral-Debrinski et al 1991) can be used to estimate the proportion of the mtDNA 4977 deletion in the total mtDNA.
  • Southern Blot and probing technology labeled with isotopes or any other technique as is standard in the art may be used for deletion detection as well.
  • Sequencing of PCR products Any known means may be used to sequence the PCR products. Preferably, the entire DNA sequence is characterized by di-deoxy sequencing using ABI Big Dye TerminatorTM technology and a series of 72 overlapping primers each for heavy and light strands. Sequencing occurs on one, several, or a combination of ABI platforms such as the 310, 3100, or 3700. Sequencing reactions are performed according to manufacturer's recommendation.
  • DHPLC denaturing high performance liquid chromatography
  • Rapid screening for heteroplasmic mtDNA mutations is determined using the relatively new technique of denaturing high performance liquid chromatography (DHPLC) (Oefner & Underhill, 1998). This technique has recently been used to rapidly screen and identify whole mtDNA genomes for heteroplasmic point mutations down to levels ⁇ 5% (Van den Bosch et al. 2000).
  • DPLC denaturing high performance liquid chromatography
  • the DHPLC may be performed on the WAVETM DNA Fragment Analysis System (Transgenomic, Omaha, USA) which provides a fully automated screening procedure.
  • the same technology can be used to screen for mtDNA heteroplasmic mutations.
  • the entire mtDNA genome is amplified by PCR in 13 overlapping fragments using two different PCR conditions as described by van den Bosch et al. (2000).
  • the 1-2 kb PCR products are digested into fragments of 90-600bp and resolved at their optimal melting temperature. Mutations are represented as two peaks and mutations with low percentages, such as ⁇ 2% heteroplasmy as a 'shoulder' in the peak.
  • DNA sequencing can also take place using a microarray, as is known in the art (Chee et al. 1996).
  • Data obtained from the sequencing of the individual mitochondrial genome is compared to population level data.
  • the data is obtained through obtaining samples and sequencing mtDNA as described above.
  • the database contains information from maternal line studies.
  • the population level data is maintained in a database. Any suitable database can be used.
  • a multidimensional evaluation research database of clinical and biological data is used, which provides the bio-informatics infrastructure necessary for the collection, processing and dissemination of information amassed by the laboratories involved in this venture.
  • the database is a centralized electronic system which links networks resulting in a dynamic and powerful resource.
  • the database may be accessed through any known means, and preferably through a secure Internet pathway.
  • the database is developed using an e-commerce algorithm, built on a server and deployed using an application server which supports a high volume of concurrent users through optimized performance and scalability features.
  • a separate "web" server can provide the foundation of the web-site architecture since it can serve as the central point through which all content, applications, and transactions must flow before reaching users.
  • Data mining algorithms known in the art are used to discover patterns, clusters and models from data (SAS 2000). Moreover, intelligent algorithms and methods will be developed for: occurrence of mutation and mutation rates, patterns of mutations for disease detection, information retrieval, and other complex sequence analysis software.
  • the invention provides for nucleic acid members and probes that bind specifically to a target nucleic acid sequence.
  • the target nucleic acid sequence is a nucleic acid or a region of a nucleic acid that is to be detected, as indicative of disease such as prostate cancer, non-melanoma skin cancer and the like.
  • the target nucleic acid sequences to be analyzed using a microarray of the invention are preferably derived from human tissue or fluid samples.
  • the invention provides for target nucleic acid sequences comprising RNA or nucleic acid corresponding to RNA, (i.e., cDNA), or DNA.
  • Nucleic acid members are stably associated with a solid support to comprise an array according to the invention.
  • the nucleic acid members may be single or double stranded, and may be a PCR fragment amplified from cDNA.
  • the invention also provides for polynucleotide sequences comprising a probe.
  • probe refers to an oligonucleotide which forms a duplex structure with a sequence in the target nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the target region.
  • the probe may be labeled, according to methods known in the art.
  • a probe according to the invention may be single or double stranded.
  • the invention includes diagnostic devices such as biochips, gene chips or microarrays used to diagnose specific diseases or identify specific mutations. All sequenced mitochondrial genomes are assessed to create a consenus structure of the base pair arrangement and are assigned a prohibiting index for proportion of base pair deletions and mutations associated with a particular disease or disorder. The diagnostic arrangement is then used to create biochips, gene chips, or microarrays. Once sequences associated with particular diseases, disease states or disorders are identified, hybridization of mtDNA to an array of oligonucleotides can be used to identify particular mutations. Any known method of hybridization may be used. Preferably, an array is used, which has oligonucleotide probes matching the wild type or mutated region, and a control probe.
  • arrays such as microarrays or gene chips are suitable. These arrays contain thousands of matched and control pairs of probes on a slide or microchip, and are capable of sequencing the entire genome very quickly. Review articles describing the use of microarrays in genome and DNA sequence analysis is available at www.gene-chips.com.
  • Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides in a sample comprising one or more target nucleic acid sequences.
  • the arrays of the invention are useful for gene expression analysis, diagnosis of disease and prognosis of disease (e.g., monitoring a patient's response to therapy, drug screening, and the like).
  • any combination of the polynucleotide sequences of mtDNA indicative of disease, aging, or other health related mutations are used for the construction of a microarray.
  • the target nucleic acid samples to be analyzed using a microarray are derived from any human tissue or fluid which contains adequate amounts of mtDNA, as previously described, preferably prostate massage fluid, solid tumours, blood, or urine.
  • the target nucleic acid samples are contacted with polynucleotide members under hybridization conditions sufficient to produce a hybridization pattern of complementary nucleic acid members/target complexes.
  • the microarray comprises a plurality of unique polynucleotides attached to one surface of a solid support, wherein each of the polynucleotides is attached to the surface of the solid support in a non-identical preselected region.
  • Each associated sample on the array comprises a polynucleotide composition, of known identity, usually of known sequence, as described in greater detail below. Any conceivable substrate may be employed in the invention.
  • the array is constructed using any known means.
  • the nucleic acid members may be produced using established techniques such as polymerase chain reaction (PCR) and reverse transcription (RT). These methods are similar to those currently known in the art (see e.g. PCR Strategies, Michael A. Innis (Editor), et al. (1995) and PCR: Introduction to Biotechniques Series, C. R. Newton, A. Graham (1997)).
  • Amplified polynucleotides are purified by methods well known in the art (e.g., column purification).
  • a polynucleotide is considered pure when it has been isolated so as to be substantially free of primers and incomplete products produced during the synthesis of the desired polynucleotide.
  • a purified polynucleotide will also be substantially free of contaminants which may hinder or otherwise mask the binding activity of the molecule.
  • the polynucleotide compositions are stably associated with the surface of a solid support, wherein the support may be a flexible or rigid solid support.
  • any solid support to which a nucleic acid member may be attached may be used in the invention.
  • suitable solid support materials include, but are not limited to, silicates such as glass and silica gel, cellulose and nitrocellulose papers, nylon, polystyrene, polymethacrylate, latex, rubber, and fluorocarbon resins such as TEFLONTM.
  • the solid support material may be used in a wide variety of shapes including, but not limited to slides and beads.
  • Slides provide several functional advantages and thus are a preferred form of solid support. Due to their flat surface, probe and hybridization reagents are minimized using glass slides. Slides also enable the targeted application of reagents, are easy to keep at a constant temperature, are easy to wash and facilitate the direct visualization of RNA and/or DNA immobilized on the solid support. Removal of RNA and/or DNA immobilized on the solid support is also facilitated using slides.
  • the particular material selected as the solid support is not essential to the invention, as long as it provides the described function. Normally, those who make or use the invention will select the best commercially available material based upon the economics of cost and availability, the expected application requirements of the final product, and the demands of the overall manufacturing process.
  • spotting Numerous methods are used for attachment of the nucleic acid members of the invention to the substrate (a process referred as spotting).
  • polynucleotides are attached using the techniques of, for example U.S. Pat. No. 5,807,522, which is inco ⁇ orated herein by reference for teaching methods of polymer attachment.
  • spotting is carried out using contact printing technology.
  • each composition will be sufficient to provide for adequate hybridization and detection of target polynucleotide sequences during the assay in which the array is employed.
  • the amount of each nucleic acid member stably associated with the solid support of the array is at least about 0.1 ng, preferably at least about 0.5 ng and more preferably at least about 1 ng, where the amount may be as high as 1000 ng or higher, but will usually not exceed about 20 ng.
  • the diameter of the "spot” will generally range from about 10 to 5,000 ⁇ m, usually from about 20 to 2,000 ⁇ m and more usually from about 50 to 1000 ⁇ m.
  • Control polynucleotides may be spotted on the array and used as target expression control polynucleotides and mismatch control nucleotides to monitor non-specific binding or cross-hybridization to a polynucleotide in the sample other than the target to which the probe is directed.
  • Mismatch probes thus indicate whether a hybridization is specific or not. For example, if the target is present the perfectly matched probes should be consistently brighter than the mismatched probes. In addition, if all central mismatches are present, the mismatch probes are used to detect a mutation.
  • the targets for the microarrays are derived from human fluid or tissue samples. It may be desirable to amplify the target nucleic acid sample prior to hybridization.
  • amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified polynucleotides.
  • Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction.
  • the high density array may then include probes specific to the internal standard for quantification of the amplified polynucleotide.
  • PCR Protocols A Guide to Methods and Applications, Innis et al., Academic Press, Inc. N.Y., (1990).
  • Other suitable amplification methods include, but are not limited to polymerase chain reaction (PCR) (Innis, et al., PCR Protocols. A guide to Methods and Application. Academic Press, Inc.
  • LCR ligase chain reaction
  • the invention provides for labeled target or labeled probe.
  • Any analytically detectable marker that is attached to or inco ⁇ orated into a molecule may be used in the invention.
  • An analytically detectable marker refers to any molecule, moiety or atom which is analytically detected and quantified.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 I, 35S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • Patents teaching the use of such labels include U.S.
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • the labels may be inco ⁇ orated by any of a number of means well known to those of skill in the art. However, in a preferred embodiment, the label is simultaneously inco ⁇ orated during the amplification step in the preparation of the sample polynucleotides.
  • PCR polymerase chain reaction
  • labeled primers or labeled nucleotides will provide a labeled amplification product.
  • transcription amplification as described above, using a labeled nucleotide (e.g. fluorescein- labeled UTP and/or CTP) inco ⁇ orates a label into the transcribed polynucleotides.
  • a label may be added directly to the original polynucleotide sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed.
  • Means of attaching labels to polynucleotides are well known to those of skill in the art and include, for example nick translation or end-labeling (e.g. with a labeled RNA) by kinasing of the polynucleotide and subsequent attachment (ligation) of a polynucleotide linker joining the sample polynucleotide to a label (e.g., a fluorophore).
  • the target will include one or more control molecules which hybridize to control probes on the microarray to normalize signals generated from the microarray.
  • Labeled normalization targets are polynucleotide sequences that are perfectly complementary to control oligonucleotides that are spotted onto the microarray as described above.
  • the signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, "reading" efficiency and other factors that may cause the signal of a perfect hybridization to vary between arrays.
  • Polynucleotide hybridization involves providing a denatured probe or target nucleic acid member and target polynucleotide under conditions where the probe or target nucleic acid member and its complementary target can form stable hybrid duplexes through complementary base pairing. The polynucleotides that do not form hybrid duplexes are then washed away leaving the hybridized polynucleotides to be detected, typically through detection of an attached detectable label. It is generally recognized that polynucleotides are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the polynucleotides.
  • hybrid duplexes e.g., DNA:DNA, RNA:RNA, RNA:DNA, cDNA:RNA and cDNA:DNA
  • hybrid duplexes e.g., DNA:DNA, RNA:RNA, RNA:DNA, cDNA:RNA and cDNA:DNA
  • specificity of hybridization is reduced at lower stringency.
  • higher stringency e.g., higher temperature or lower salt
  • Methods of optimizing hybridization conditions are well known to those of skill in the art (see, e.g., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Polynucleotide Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).
  • non-hybridized labeled or unlabeled polynucleotide is removed from the support surface, conveniently by washing, thereby generating a pattern of hybridized target polynucleotide on the substrate surface.
  • wash solutions are known to those of skill in the art and may be used.
  • the resultant hybridization patterns of labeled, hybridized oligonucleotides and/or polynucleotides may be visualized or detected in a variety of ways, with the particular manner of detection being chosen based on the particular label of the test polynucleotide, where representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement and the like.
  • the resultant hybridization pattern is detected.
  • the intensity or signal value of the label will be not only be detected but quantified, by which is meant that the signal from each spot of the hybridization will be measured and compared to a unit value corresponding to the signal emitted by a known number of end labeled target polynucleotides to obtain a count or absolute value of the copy number of each end-labeled target that is hybridized to a particular spot on the array in the hybridization pattern.
  • data analysis can include the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e., data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the test polynucleotides from the remaining data.
  • the resulting data is displayed as an image with the intensity in each region varying according to the binding affinity between associated oligonucleotides and/or polynucleotides and the test polynucleotides.
  • the hybridization pattern is used to determine quantitative information about the genetic profile of the labeled target polynucleotide sample that was contacted with the array to generate the hybridization pattern, as well as the physiological source from which the labeled target polynucleotide sample was derived.
  • genetic profile is meant information regarding the types of polynucleotides present in the sample, e.g. in terms of the types of genes to which they are complementary, as well as the copy number of each particular polynucleotide in the sample.
  • the invention provides for diagnostic tests for detecting diseases.
  • the invention also provides for prognostic tests for monitoring a patient's response to therapy.
  • the presence of disease or the patient's response to therapy is detected by obtaining a fluid or tissue sample from a patient.
  • a sample comprising nucleic acid is prepared from the fluid or tissue sample.
  • the nucleic acid extracted from the sample is hybridized to an array comprising a solid substrate and a plurality of nucleic acid members, wherein each member is indicative of the presence of disease or a predisposition to a disease or disorder.
  • hybridization of the sample comprising nucleic acid to one or more nucleic acid members on the array is indicative of disease, a predisposition to a disease or disorder, or in the case of a prognostic test, indicative of a patient's response to therapy.
  • Other utilities for the present invention such as that described above and in the following examples, will be readily apparent to those skilled in the art.
  • Example 1 Prostate Tumours Following acquisition of prostate fluid or surgery to remove prostate tumours, biopsy slides are prepared to identify transforming or cancerous cells.
  • Laser Capture Microdissection (LCM) microscopy is used to isolate cells that are either normal, benign, or malignant from the tissue section. Procurement of diseased cells of interest, such as precancerous cells or invading groups of cancer cells is possible from among the surrounding heterogeneous cells.
  • LCM Laser Capture Microdissection
  • DNA extraction from each of these cells was purified according to a modification of the protocol outlined by Arcturus Engineering Inc.
  • DNA was extracted from cells with a 50 ⁇ l volume of 1 mg/ml proteinase K (PK), in lOmM Tris pH 8.0, O.lmM EDTA pH 8.0, and 0.1% Tween 20, at 42°C overnight. Following incubation overnight at 42°C the tubes were removed from the incubation oven. The samples were microcentrifuged for 5 min at 640O ⁇ m(2000 x g). The CapSureTM was removed from the tube and discarded. The tube was incubated at 95°C for 10 minutes (PK is inactivated) and then cooled to room temperature. 5-50 ⁇ l of the sample was used for PCR amplification.
  • PK proteinase K
  • HV1 hypervariable region 1
  • HV2 hypervariable region 2
  • Example 2 Duplications in the non-coding region of mtDNA from sun-exposed skin DNA was extracted from tissue samples as described in Example 1 , with the use of
  • NCR non-coding region
  • Primers pairs C/D and E/F are 'back to back' at the site of two separate sets of direct repeats in the non-coding region. As a result they only generate a product if a duplication is present at these points. Products generated are 260 bp and/or less common 200bp variant.
  • Modified PCR conditions are: lOOng total cellular DNA, 200 ⁇ M dNTPs, 2.5 U HotStarTaq polymerase and PCR buffer (Qiagen, Uk), 25 pmoles of primers: one cycle of 94°C for 4 minutes, 36 cycles of 94 °C x 1 minute, 55°C x 1 minute, 72°C x 1 minute and one cycle of 72°C x 7 minutes.
  • Example 3 Mutation fingerprint of mtDNA in human NMSC and its precursor lesions
  • DNA was extracted from human skin tissue samples as described in Example 1, with the use of DNeasyTM by Qiagen Using specific primers, mtDNA is amplified by PCR and following DNA sample preparation (Qiagen), mutations are identified by automated sequencing (PE Applied Biosystems) using BigDyeTM Terminator Cycle sequencing. This methodology is described in Healy et al. 2000; Harding et al. 2000. The entire 16,569bp human mitochondrial genome is sequenced using established PCR primer pairs, which are known not to amplify pseudogenes, or other nuclear loci. Any putative DNA changes are confirmed by comparison to the revised "Cambridge" human mtDNA reference (Andrews et al. 1999).
  • the sequences obtained from the tumour mtDNA are first compared for known polymo ⁇ hisms (Andrews et al. 1999; MITOMAP) and then compared with the mtDNA sequence from the normal perilesional skin to identify genuine somatic mutations.
  • DHPLC is performed on the WAVETM DNA Fragment Analysis System (Transgenomic, Omaha, USA) which provides a fully automated screening procedure. The same technology is used to screen for heteroplasmic mutations in the skin tumour mtDNA.
  • DNA length mutations i.e. tandem duplications
  • NCR non- coding region
  • Example 4 deletion spectrum of the entire mitochondrial genome in human NMSC and its precursor lesions
  • MtDNA damage in squamous cell carcinomas (SCCS), Basal cell carcinomas (BCCS) and putative precursor lesions such as Bowen's disease and actinic keratoses (As) was compared to adjacent perilesional skin taken from different sun-exposed body sites.
  • a long-extension PCR technique (LX-PCR) (Ray et al. 1998) was used to amplify the entire mitochondrial genome in order to determine the whole deletion spectrum of mtDNA.
  • LX-PCR Long-extension PCR technique
  • DNA is extracted by use of a commercial kit (Qiangen) according to the manufacturer's recommendations.
  • the entire mitochondrial genome is amplified in two separate reactions using the Expand TM Long Template PCR SystemTM (Boehringer Manheim, Switzerland).
  • the PCR primers used are those described by Kleinle et al. (1997) covering the following regions of the Cambridge sequence (Andrews et al. 1999): DIA(nucleotides (nt) 336-363), DIB (nt 282-255), OLA (nt 5756-5781), and OLB (nt 5745- 5781). These large products eliminate amplification of nuclear pseudogenes.
  • the sequences of the primers are as follows:
  • DIAF (336-363) 5' AACACATCTCTGCCAAACCCCAAAAACA 3' OLBR: (5745-5721) 5' CCGGCGGCGGGAGAAGTAGATTGAA 3' OLAF: (5756-5781) 5' GGGAGAAGCCCCGGCAGGTTTGAAGC 3' DIBR: (282-255) 5' ATGATGTCTGTGTGGAAAGTGGCTGTGC 3'
  • Amplifications are performed in 50 microlitre reactions containing 16 pmol of each primer, 500 ⁇ mol dNTPs, 10 x PCR buffer with 22.5mM MgCl 2 and detergents(kit), 0.75 ⁇ l of enzyme (3.5 x 10 3 units/ml) and 50-200ng of total DNA.
  • One reaction generates 1 l,095bp segments of the genome, while another results in 5,409bp lengths (e.g. Kleinle et al, 1997).
  • the PCR amplification conditions consists of a denaturing stage at 93°C for 1 min 30s, followed by 10 cycles of 93°C for 30s, 60°C for 30s and 68°C for 12 min, followed by a further 20 cycles of the same profile with an additional 5 s added to the elongation time every cycle. There is a final cycle of 93°C for 30s, 60°C for 30s and an elongation time of 68°C for 26 minutes. To ensure reproducibility, a known amount of DNA is separated on a 1% agarose gel and only samples which have at least the same amount of DNA are included in the analysis.
  • temporal maternal line comparisons i.e. great-grandchild through great- grand parents
  • the entire sequence of mtDNA extracted from a given tissue is rapidly, and accurately sequenced, in order to definitively state the arrangement of nucleotide base pairs for that specific molecule and possible changes through time.
  • These characterizations are compared to health status, aging indicators and between specific maternal lines, within larger populations.
  • This combined information allows crucial statistical discrimination between separate causes resulting in the same mutation/deletion and establishes that the mtDNA sequences, used as a bio-marker, has the required index of specificity and sensitivity in order to establish its validity.
  • the proportions of base pair deletions and mutations are compared for consistency in various tissues across the 4 maternal generations.
  • Skeletal muscle or leukocytes are obtained from a patient. DNA is extracted as set out in Example 1. The following primers were used:
  • Amplifications were performed in 50 microlitre reactions containing 2.0 ⁇ mol of each primer, 250 ⁇ mol dNTPs, 10 x PCR buffer(Thermopol Reaction Buffer), bovine serum albumin, 0.5units Deep vent polymerase and 50-200ng of total DNA.
  • the PCR amplification conditions consists of a denaturing stage at 95°C for 5 min (hot start), followed by 30 cycles of 94°C for 30s, 60°C for 60s and 72°C for 30s with a final extension at 72°C for 10 min.
  • Gel electrophoresis was performed on a 2% agarose gel at 125 volts for 60 min, stained with ethidium bromide, and visualized under UV light. To ensure reproducibility, a known amount of DNA was separated on a 2% agarose gel and only samples which have the same amount of DNA were included in the analysis.
  • the incidence of the common deletion is determined in a quantitative manner by a 3-primer PCR method which detects levels greater than 1-5% or a dilution PCR method which detects levels less than 1 % down to 10 "4 %.
  • Samples are obtained and DNA extracted as described in Example 1.
  • a 3-primer PCR procedure is used (as described in Birch-Machin et al 1998).
  • Primers A, and C correspond to heavy strand positions 13720-13705 and 9028-9008 respectively (Anderson et al., 1981); primer B corresponds to light strand positions 8273- 8289.
  • Primer C maps to a mtDNA region within the common deletion, whereas primers A and B flank the deleted region. Therefore primers B and C only amplify wt-mtDNAs and primers A and B only amplify deleted mtDNAs (the distance between the two primers in the absence of the deletion, approximately 5.5kb, is too long to be amplified under our PCR conditions as described below).
  • the PCR reaction mixture (25 ⁇ l total volume) contained lOOng total cellular DNA, 200 ⁇ M dNTPs, lOmM Tris-HCl (pH 8.8), 50mM KC1, 1.5mM Mg Cl 2 , 0.1% Triton X-100, 2.5U Taq DNA polymerase (BioTaq, BiolineUK Limited, London), 25 pmoles of primers A and B, 6.25 pmoles of primer C and 3 ⁇ Ci of [ ⁇ - 32 P]-dATP.
  • PCR conditions were 25 cycles of 94°C at 1 minute, 55°C at 1 minute, 72°C at 2 minutes including a final extension of 15 minutes at 72°C. These PCR products were then electrophoresed through a 6% nondenaturing polyacrylamide gel and the radioactive PCR fragments were quantified by phophorimage analysis using the ImageQuantTM software (Molecular Dynamics, Chesham UK).
  • Example 7 Serial Dilution PCR method to quantitatively detect low levels ( ⁇ 1%) of the common mtDNA deletion
  • a semi-quantitative PCR method (Corral-Debrinski et al 1991) is used to estimate the proportion of the common deletion in the total mtDNA extracted from the tissue/cell samples.
  • Biological samples are obtained and DNA extracted as described in Example 1.
  • the DNA sample is initially linearised using the restriction enzyme Bam HI (l ⁇ l enzyme and l ⁇ l of commercially supplied buffer) at 37°C for 90 minutes.
  • Serial dilutions are performed in two-fold steps (for total mtDNA there was an initial 10-fold dilution) and PCR performed for each dilution (l ⁇ l) using the following primers:
  • the reaction conditions are as follows:
  • the PCR productes are visualised on a UV transilluminator (TMW-20, Flowgen Ltd., Lichfield, UK) and a digital image of the gel obtained using image acquisition apparatus (Alpha Imager 2000, Alpha Innotech Co ⁇ oration, supplied by Flowgen Ltd., Lichfield, UK).
  • image acquisition apparatus Alpha Imager 2000, Alpha Innotech Co ⁇ oration, supplied by Flowgen Ltd., Lichfield, UK.
  • the associated image analysis software (Alpha Ease v3.3, Alpha Innotech Co ⁇ .) allows the calculation of the integrated optical density (IOD) for each PCR product in a dilution series.
  • the band where an IOD value of zero is obtained for both total mtDNA and deleted mtDNA and the corresponding dilution values are used to calculate the percentage of common deletion in the sample thus:
  • %common deletion total mtDNA dilution factor (I0D Zero) x 100 common deletion dilution factor (I0D Zero)
  • Example 8 Denaturing high performance liquid chromatography (DHPLC )
  • the 1 -2 kb PCR products are digested into fragments of 90-600bp and resolved at their optimal melting temperature. Mutations are represented as two peaks and mutations with low percentages, such as ⁇ 2% heteroplasmy as a "shoulder" in the peak.
  • DHPLC is performed with a mobile phase consisting of two eluents (pH 7.0).
  • Buffer A contains triethylammonium acetate (TEAA), which interacts with both the negatively charged phosphate groups on the DNA as well as the surface of the column.
  • TEAA triethylammonium acetate
  • Buffer B contains TEAA with 25% of the denaturing agent acetonitrile. Fragments were eluted with a linear acetonifrile gradient at a constant flow rate. Increasing the concentration of acetonifrile will denature the fragments. Table 3 below is an example of a standard method for DHPLC of a PCR reaction generated using the WAVEMAKER software (Transgenomics) according to manufacturer's instructions.
  • Birch-Machin MA et al., Methods in Toxicology, Volume 2, 51-69,1993
  • Birch-Machin MA et al., J.Invest.Dermatol, 1 10:149-152 1998
  • Birch-Machin MA Online Conference Report (Sunburnt DNA), International Congress of Biochemistry and Molecular Biology, New Scientist, 2000(a)
  • Croteau DL Stierum RH, Bohr VA, Mutat Res 434(3):137-148, 1999
  • Fahn H Wang L, Hseith R, Chang S, Kao S, Huang M, and Wei Y. American Journal of Respiratory Critical Care Medicine, 154: 1141-1145, 1996 Fahn HJ, Wang LS, Kao SH, Chang SC, Huang MH, Wei YH., Am. J Respir. Cell. Mol Biol, 19(6): 901 -9, 1998
  • Healy ter 1 The Human Melanocortin 1 Receptor Gene. RD (ed)). Humana Press Inc. New Jersey, USA,
  • MITOMAP A human mt genome database (www.gen.emorv.edu/mitomap.html " )
  • Woodwell DA National Ambulatory Medical Care Survey: 1997 Summary. Advance data from vital and health statistics; no. 305. Hyattsville, Maryland: National Center for Health Statistics. 1999

Abstract

L'examen de mutations dans l'ensemble du génome mitochondrial sert de système diagnostique pour des maladies telles que le cancer de la prostate et le cancer de la peau sans mélanomes. Des mutations et des réarrangements caractéristiques tels que des mutations ponctuelles (transitions, transversions), délétions, inversions, duplications, recombinaisons, insertions ou des combinaisons de celles-ci dans le génome mitochondrial servent d'indicateurs précoces du cancer de la prostate et du cancer de la peau sans mélanomes. Par ailleurs, 4977bp ou « délétion commune » ainsi que d'autres mutations et/ou délétions associées servent de mesure du vieillissement.
PCT/CA2002/000848 2001-06-11 2002-06-10 Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante WO2002101086A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002450403A CA2450403A1 (fr) 2001-06-11 2002-06-10 Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante
JP2003503836A JP2005506057A (ja) 2001-06-11 2002-06-10 健康科学のための診断ツールとしての全ミトコンドリアゲノム配列
EP02737683A EP1397508A2 (fr) 2001-06-11 2002-06-10 Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante
US10/732,374 US20050026167A1 (en) 2001-06-11 2003-12-11 Complete mitochondrial genome sequences as a diagnostic tool for the health sciences
PCT/CA2004/002124 WO2005056573A1 (fr) 2002-06-10 2004-12-13 Sequences completes du genome mitochondrial utilisees comme outil de diagnostic pour des sciences de la sante
US11/339,751 US20070190534A1 (en) 2001-06-11 2006-01-26 Mitochondrial sites and genes associated with prostate cancer

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US29734001P 2001-06-11 2001-06-11
US60/297,340 2001-06-11
CA002450004A CA2450004A1 (fr) 2001-06-11 2003-12-18 Sequences completes du genome mitochondrial comme outil diagnostic pour les sciences de la sante

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/732,374 Continuation-In-Part US20050026167A1 (en) 2001-06-11 2003-12-11 Complete mitochondrial genome sequences as a diagnostic tool for the health sciences

Publications (2)

Publication Number Publication Date
WO2002101086A2 true WO2002101086A2 (fr) 2002-12-19
WO2002101086A3 WO2002101086A3 (fr) 2003-12-11

Family

ID=34808883

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2002/000848 WO2002101086A2 (fr) 2001-06-11 2002-06-10 Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante

Country Status (4)

Country Link
EP (1) EP1397508A2 (fr)
JP (1) JP2005506057A (fr)
CA (2) CA2450403A1 (fr)
WO (1) WO2002101086A2 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078661A1 (fr) * 2002-03-15 2003-09-25 Ulf Gyllensten Methode et trousse utiles pour detecter les mutations dans de l'adn mitochondrial
WO2005056573A1 (fr) * 2002-06-10 2005-06-23 1304854 Ontario Ltd. Sequences completes du genome mitochondrial utilisees comme outil de diagnostic pour des sciences de la sante
WO2006111029A1 (fr) * 2005-04-18 2006-10-26 Genesis Genomics Inc. Rearrangements et mutations mitochondriales utilises en tant qu'outil de diagnostic pour la detection de l'exposition au soleil, le cancer de la prostate et d'autres cancers
JP2010539908A (ja) * 2007-09-26 2010-12-24 ミトミクス インコーポレーテッド 癌の検出に使用するための３．４ｋｂのミトコンドリアＤＮＡ欠失
AU2008324675B2 (en) * 2007-11-09 2015-04-23 Mdna Life Sciences Inc. Mitochondrial DNA deletion between about residues 12317-16254 for use in the detection of cancer
US10308987B2 (en) 2005-04-18 2019-06-04 Mdna Life Sciences Inc. 3.4 kb mitochondrial DNA deletion for use in the detection of cancer
WO2019241273A1 (fr) * 2018-06-11 2019-12-19 The Broad Institute, Inc. Suivi de lignée à l'aide de mutations du génome mitochondrial et de la génomique cellulaire unique
US10813972B2 (en) 2013-02-06 2020-10-27 Malaysian Palm Oil Board Treatment of DNA damage and mitochondrial dysfunction using palm fruit juice

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2719718C (fr) * 2008-03-28 2019-07-02 Mitomics, Inc. Adn mitochondrial aberrant, produits de transcription de fusion associes et sondes d'hybridation pour celui-ci
EP2411522B1 (fr) * 2009-03-27 2017-09-06 MDNA Life Sciences Inc. Adn mitochondrial aberrant, transcrits de fusion et produits de traduction associes et sondes d'hybridation complementaires
JP6645737B2 (ja) * 2012-09-05 2020-02-14 漂太 高松 自己免疫疾患のバイオマーカー

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068923A2 (fr) * 2000-03-15 2001-09-20 The Johns Hopkins University Dosimetre a mitochondries

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001068923A2 (fr) * 2000-03-15 2001-09-20 The Johns Hopkins University Dosimetre a mitochondries

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BURGART L J ET AL: "SOMATIC MITOCHONDRIAL MUTATION IN GASTRIC CANCER" AMERICAN JOURNAL OF PATHOLOGY, PHILADELPHIA, PA, US, vol. 147, October 1995 (1995-10), pages 1105-1111, XP000863263 ISSN: 0002-9440 *
CORTOPASSI G ET AL: "MODELLING THE EFFECTS OF AGE-RELATED MTDNA MUTATION ACCUMULATION;COMPLEX 1 DEFICIENCY, SUPEROXIDE AND CELL DEATH" BIOCHIMICA ET BIOPHYSICA ACTA, AMSTERDAM, NL, vol. 1271, 24 May 1995 (1995-05-24), pages 171-176, XP000944048 ISSN: 0006-3002 cited in the application *
ESHAGHIAN ALEX ET AL: "Alterations of mitochondrial DNA in aging skin and in non-melanoma skin cancers." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 43, March 2002 (2002-03), pages 304-305, XP001153120 93rd Annual Meeting of the American Association for Cancer Research;San Francisco, California, USA; April 06-10, 2002, March, 2002 ISSN: 0197-016X *
FLISS MAKIKO S ET AL: "Facile detection of mitochondrial DNA mutations in tumors and bodily fluids." SCIENCE (WASHINGTON D C), vol. 287, no. 5460, 17 March 2000 (2000-03-17), pages 2017-2019, XP002244376 ISSN: 0036-8075 cited in the application *
JERONIMO C ET AL: "Mitochondrial mutations in early stage prostate cancer and bodily fluids." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 42, March 2001 (2001-03), page 63 XP001153112 92nd Annual Meeting of the American Association for Cancer Research;New Orleans, LA, USA; March 24-28, 2001, March, 2001 ISSN: 0197-016X *
JESSIE BENJAMIN C ET AL: "Accumulation of mitochondrial DNA deletions in the aging prostate." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 42, March 2001 (2001-03), pages 862-863, XP001153110 92nd Annual Meeting of the American Association for Cancer Research;New Orleans, LA, USA; March 24-28, 2001, March, 2001 ISSN: 0197-016X *
JESSIE BENJAMIN C ET AL: "Accumulation of mitochondrial DNA deletions in the malignant prostate of patients of different ages." EXPERIMENTAL GERONTOLOGY, vol. 37, no. 1, December 2001 (2001-12), pages 169-174, XP002244377 ISSN: 0531-5565 *
PARRELLA PAOLA ET AL: "Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates." CANCER RESEARCH, vol. 61, no. 20, 15 October 2001 (2001-10-15), pages 7623-7626, XP002244378 ISSN: 0008-5472 *
PENTA JS ET AL.: "Mitochondrial DNA in human malignancy" REVIEWS IN MUTATION RESEARCH, vol. 488, 2001, pages 119-133, XP002244375 cited in the application *
PETROS JOHN ANTHONY ET AL: "Mitochondrial DNA point mutations are common in prostate cancer and enhance malignant phenotype." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 42, March 2001 (2001-03), page 517 XP001153111 92nd Annual Meeting of the American Association for Cancer Research;New Orleans, LA, USA; March 24-28, 2001, March, 2001 ISSN: 0197-016X *
THAYER ROBERT ET AL: "Mitochondrial DNA mutations and/or deletions in prostate cancers." PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL, vol. 42, March 2001 (2001-03), pages 532-533, XP001153105 92nd Annual Meeting of the American Association for Cancer Research;New Orleans, LA, USA; March 24-28, 2001, March, 2001 ISSN: 0197-016X *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078661A1 (fr) * 2002-03-15 2003-09-25 Ulf Gyllensten Methode et trousse utiles pour detecter les mutations dans de l'adn mitochondrial
WO2005056573A1 (fr) * 2002-06-10 2005-06-23 1304854 Ontario Ltd. Sequences completes du genome mitochondrial utilisees comme outil de diagnostic pour des sciences de la sante
JP2012179053A (ja) * 2005-04-18 2012-09-20 Mitomics Inc 日光曝露、前立腺癌および他の癌の検出用の診断ツールとしての、ミトコンドリアの突然変異および再配置
CN101248180B (zh) * 2005-04-18 2013-09-25 米托米克斯公司 作为检测日光照射、前列腺癌和其它癌症的诊断工具的线粒体突变及重排
JP2008536496A (ja) * 2005-04-18 2008-09-11 ジェネシス ジェノミクス インコーポレーテッド 日光曝露、前立腺癌および他の癌の検出用の診断ツールとしての、ミトコンドリアの突然変異および再配置
EP1877559A4 (fr) * 2005-04-18 2009-06-10 Genesis Genomics Inc Réarrangements et mutations mitochondriales utilisés en tant qu'outil de diagnostic pour la détection de l'exposition au soleil, le cancer de la prostate et d'autres cancers
US11111546B2 (en) 2005-04-18 2021-09-07 Mdna Life Sciences, Inc. 3.4 KB mitochondrial DNA deletion for use in the detection of cancer
EP2339032A1 (fr) * 2005-04-18 2011-06-29 Mitomics Inc. Mutations mitochondriales et réagencements en tant qu'outil de diagnostic pour la détection de l'exposition au soleil, cancer de la prostate et autres cancers
AU2006238390B2 (en) * 2005-04-18 2011-07-28 Mitomics Inc. Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
US8008008B2 (en) 2005-04-18 2011-08-30 Mitomics Inc. Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
WO2006111029A1 (fr) * 2005-04-18 2006-10-26 Genesis Genomics Inc. Rearrangements et mutations mitochondriales utilises en tant qu'outil de diagnostic pour la detection de l'exposition au soleil, le cancer de la prostate et d'autres cancers
EP1877559A1 (fr) * 2005-04-18 2008-01-16 Genesis Genomics Inc. Réarrangements et mutations mitochondriales utilisés en tant qu'outil de diagnostic pour la détection de l'exposition au soleil, le cancer de la prostate et d'autres cancers
KR101363032B1 (ko) 2005-04-18 2014-02-13 미토믹스 인크. 일광 노출, 전립선암 및 다른 암의 검출용 진단 도구로서미토콘드리아 돌연변이 및 전위
US10907213B2 (en) 2005-04-18 2021-02-02 Mdna Life Sciences Inc. Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
US9745632B2 (en) 2005-04-18 2017-08-29 Mdna Life Sciences Inc. Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
US10308987B2 (en) 2005-04-18 2019-06-04 Mdna Life Sciences Inc. 3.4 kb mitochondrial DNA deletion for use in the detection of cancer
JP2010539908A (ja) * 2007-09-26 2010-12-24 ミトミクス インコーポレーテッド 癌の検出に使用するための３．４ｋｂのミトコンドリアＤＮＡ欠失
US10400290B2 (en) 2007-11-09 2019-09-03 Mdna Life Sciences, Inc. Mitochondrial DNA deletion between about residues 12317-16254 for use in the detection of cancer
AU2008324675B2 (en) * 2007-11-09 2015-04-23 Mdna Life Sciences Inc. Mitochondrial DNA deletion between about residues 12317-16254 for use in the detection of cancer
US10813972B2 (en) 2013-02-06 2020-10-27 Malaysian Palm Oil Board Treatment of DNA damage and mitochondrial dysfunction using palm fruit juice
WO2019241273A1 (fr) * 2018-06-11 2019-12-19 The Broad Institute, Inc. Suivi de lignée à l'aide de mutations du génome mitochondrial et de la génomique cellulaire unique

Also Published As

Publication number Publication date
EP1397508A2 (fr) 2004-03-17
CA2450004A1 (fr) 2005-06-18
CA2450403A1 (fr) 2002-12-19
WO2002101086A3 (fr) 2003-12-11
JP2005506057A (ja) 2005-03-03

Similar Documents

Publication Publication Date Title
US10907213B2 (en) Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
US20070190534A1 (en) Mitochondrial sites and genes associated with prostate cancer
JP2007502113A (ja) エピジェネティク・マーカーを使用して組織または細胞タイプを鑑別するための方法および組成物
JP2007504827A (ja) １３ｑ１４染色体変化の検出
US20050026167A1 (en) Complete mitochondrial genome sequences as a diagnostic tool for the health sciences
WO2002101086A2 (fr) Sequences completes du genome mitochondrial servant d'outil diagnostique dans les sciences de la sante
Yeung et al. Impact of copy neutral loss of heterozygosity and total genome aberrations on survival in myelodysplastic syndrome
EP1694695A1 (fr) Sequences completes du genome mitochondrial utilisees comme outil de diagnostic pour des sciences de la sante
AU2002312671A1 (en) Complete mitochondrial genome sequences as a diagnostic tool for the health sciences
AU2011242123B2 (en) Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
Roses Molecular genetics of myotonic and Duchenne muscular dystrophies
Zeehaida et al. MICROSPORIDIA INFECTION: A SERIES OF CASE REPORT
Malik Gene mapping in syndactyly families

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 530082

Country of ref document: NZ

Ref document number: 2003503836

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 10732374

Country of ref document: US

Ref document number: 2002312671

Country of ref document: AU

Ref document number: 2450403

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2002737683

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2002737683

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 2002737683

Country of ref document: EP