Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-04-14
2002-10-08
Myers, Carla J. (Department: 1655)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024310, C435S069100, C435S252300, C435S320100, C435S325000
Reexamination Certificate
active
06462190
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified mitochondrial polynucleotide sequences having age-dependent mutations, and the use of such polynucleotides, as well as the production and isolation of such polynucleotides. More particularly, methods and compositions useful in modulating and diagnosing age-related disorders are provided.
BACKGROUND
Mitochondria are organelles primarily responsible for synthesizing ATP (adenosine triphosphate), the chief store of chemical energy for eukaryotic cells. Oxidative phosphorylation is the process by which ATP is synthesized from ADP with the concomitant oxidation of a reduced substrate derived from the breakdown of sugars and fatty acids. The energy for ATP synthesis is provided by the flow of electrons from the reduced substrate along an electron transport system consisting of a series of electron carriers embedded in the inner mitochondrial membrane. The electron transport system catalyzes the transfer of electrons from reduced substrates to molecular oxygen, resulting in substrate oxidation and reduction of oxygen to water.
Mitochondria generate high levels of reactive oxygen species as by-products of the electron transport process and constitute the main source of free radical generation in eukaryotic cells. Free radicals are believed to act as DNA damaging agents and may be responsible for the high mutation rate of mitochondrial DNA (mtDNA). The metabolic importance of mitochondrial genome-encoded protein products is indicative of the significance of mtDNA-associated mutational events. Such events may be pathogenically involved in degenerative processes, Thus, there is a desire to isolate and identify mutated nucleic acid sequences associated with a mitochondrial dysfunction as it relates to the aging process.
SUMMARY OF THE INVENTION
The invention relates to the identification and quantification of genetic mutations in a mitochondrial control region that segregate with age-related disorders or are age dependent. The invention provides methods for detecting and quantifying such mutations as a diagnostic for an age related disorder, either before or after the onset of clinical symptoms. More specifically, the present invention provides a method for detecting the presence or risk of age-related disorders by obtaining a biological sample containing mitochondrial nucleic acid from a subject and determining the presence of at least one mutation in a mitochondrial DNA main control region including positions 1 and 660 of the Cambridge sequence (Anderson et al., Nature 250:57, 1981), which correlates with the age of a subject. The invention also involves isolated nucleic acid sequences that are useful in the above-mentioned diagnostics, namely those that correspond, or are complementary, to portions of the mitochondrial control region, and where the sequences contain mutations which correlate with age-related disorders.
The invention provides a method for determining the presence or risk of mitochondrial-associated disease or age dependent mutations in a subject by cleaving nucleic acids in a sample containing mitochondrial nucleic acids with one or more enzymes specific for nuclear nucleic acids in order to eliminate nuclear pseudogenes; amplifying a region of interest by contacting the sample with a pair of primers flanking the region of interest; an subjecting one or more amplified products to an analysis to determine a difference in the amplified product sequence compared to a control sequence, wherein a difference is indicative of a mitochondrial-associated disorder or age dependent mutation. In one embodiment, the method of analysis is by denaturing gradient gel electrophoresis (DGGE). In another embodiment the method of amplification and DGGE is repeated one or more times.
The invention also provides an isolated polynucleotide having a sequence as set forth in SEQ ID NO:1 and having one or more of the following mutations selected from the group consisting of T414G, A368G, a T insertion after position 383, T285C, A249G, T195C, T152C, T146C, variations in length or compositon of the homopolymer tract (HT) D310 at positions 303-315 or variations in length or composition of a CA repeat (positions 514-523), and any combination thereof.
The invention further provides a vector containing a polynucleotide sequence of the invention as well as host cells containing the vector.
The invention also provides a method for detecting an age-related disorder, comprising detecting a mutation in a control region of a mitochondrial polynucleotide sequence, wherein the wild type control region has a sequence as set forth in SEQ ID NO:1.
The invention provides a method for diagnosing a subject having or at risk of having an age-related disorder or mutation by determining the presence of at least one mutation in the nucleic acid sequence of a mitochondrial control region wherein the presence of at least one mutation correlates with risk of having an age-related disorder or mutation.
The invention also provides a method for detecting an age-related mutation of DNA by contacting a sample containing mitochondrial DNA with a nucleic acid probe under conditions that allow the probe and the mitochondrial DNA to hybridize; and detecting hybridization of the probe with the DNA, wherein hybridization of the probe to the DNA is indicative of an age-related mutation.
Also provided a kit useful for the detection of an age-related mutations of mitochondrial DNA comprising carrier means containing therein one or more containers wherein a first container contains a nucleic acid probe that hybridizes to a nucleic acid sequence as set forth in SEQ ID NO:1 wherein SEQ ID NO:1 has one or more of the mutations selected from the group consisting of T414G, A368G, a T insertion after position 383, T285C, A249G, T195C, T152C, T146C, variations in length or compositon of the homopolymer tract (HT) D310 at positions 303-315 or variations in length or composition of a CA repeat (positions 514-523), and combinations thereof
The invention further provides a method for determining the presence or risk of mitochondrial-associated disease or age dependent mutations in a subject by cleaving nucleic acids in a sample containing mitochondrial nucleic acids with one or more enzymes specific for nuclear nucleic acids in order to eliminate nuclear psudogenes; amplifying a region of interest by contacting the sample with a pair of primers flanking the region of interest; and subjecting one or more amplified products to an analysis to determine a difference in the amplified product sequence compared to a control sequence, wherein a difference is indicative of an mitochondrial-associated disorder or age dependent mutation.
REFERENCES:
patent: 6027883 (2000-02-01), Hernstadt et al.
patent: WO 98/23632 (1998-06-01), None
Anderson et al., “Sequence and organization of the human mitochondrial genome,”Nature, 290:457-465 (Apr. 9, 1981).
Fleming et al., “Age Dependent Changes in Mitochondria,”Symposium on Molecular Biology of Aging(1984:Brookhaven National Laboratory), pp. 143-156.
Fleming et al., “Is Cell Aging Caused by Respiration-Dependent Injury to the Mitochondrial Genome?”Gerontology, 28:44-53 (1982).
Jazin et al., “Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA,”Proc. Natl. Acad. Sci. USA, 93:12382-12387 (Oct. 1996).
Larsson et al., “Progressive Increase of the Mutated Mitochondrial DNA Fraction in Kearns-Sayre Syndrome,”Pediatric Research, 28(2):131-136 (1990).
Linnane et al., “Mitochondrial DNA Mutations as an Important Contributor to Ageing and Degenerative Diseases,”The Lancet, pp. 642-645 (Mar. 25, 1989).*
Linnane et al., “Mitochondrial Gene Mutation: The Ageing Process and Degenerative Diseases,”Biochemistry International, 22(6):1067-1076 (Dec. 1990).*
T. Ozzawa, “Mitochondrial Genome Mutation in Cell Death and Aging, ”Journal of Bioenergetics and Biomembranes, 31(4):377-390 (1999).*
Park and Ames, “7-Methylguanine adducts in DNA are normally present at high levels and increase on aging: Analysis by HPLC with elect
Attardi Giuseppe M.
Michikawa Yuichi
California Institute of Technology
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Johannsen Diana
Myers Carla J.
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