Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-15
2003-08-12
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024330, C436S504000
Reexamination Certificate
active
06605433
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention is related to the field of environmental toxicology, in particular to methods for measuring the effects of environmental toxins.
BACKGROUND OF THE INVENTION
The human mitochondrial (mt) genome is small (16.5 kb) and encodes 13 respiratory chain subunits, 22 tRNAs and two rRNAs. Mitochondrial DNA is present at extremely high levels (10
3
-10
4
copies per cell) and the vast majority of these copies are identical (homoplasmic) at birth (1). Expression of the entire complement of mt genes is required to maintain proper function of the organelle, suggesting that even slight alterations in DNA sequences could have profound effects (2). It is generally accepted that mtDNA mutations are generated endogenously during oxidative phosphorylation via pathways involving reactive oxygen species (ROS), but they can also be generated by external carcinogens or environmental toxins. These mutations may accumulate partially because mitochondria lack protective histones and highly efficient DNA repair mechanisms as seen in the nucleus (3). Recently several mtDNA mutations were found specifically in human colorectal cancer (4).
SUMMARY OF THE INVENTION
It is an object of the present invention to provide methods of monitoring exposure of a person to an environmental pollutant.
It is another object of the present invention to provide a kit for monitoring exposure of a person to environmental pollutants.
It is an object of the invention to provide methods to aid in the detection of cancer or metastasis.
It is an object of the invention to provide probes and primers for detecting mitochondrial mutations.
It is an object of the invention to provide a method to aid in detecting the presence of tumor cells in a patient.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided for monitoring exposure of a person to an environmental pollutant. The presence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is determined at two or more time points. The amounts of mutations in mtDNA at different time points are compared. The amount of mutations correlates with amount of exposure to the environmental pollutant.
According to another embodiment another method is provided for monitoring exposure of a person to an environmental pollutant. The prevalence of one or more mutations in mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant is measured. A measured prevalence of one or more mutations in mtDNA of greater than 1% indicates clonal expansion of cells which harbor the one or more mutations in the person.
According to still another embodiment of the invention a method is provided for monitoring exposure of a person to an environmental pollutant. One or more mutations in a D-loop of mitochondrial DNA (mtDNA) in a body fluid of a person exposed to an environmental pollutant are measured. The number of mutations in mtDNA correlates with exposure to the environmental pollutant.
According to yet another embodiment of the invention a kit is provided. The kit comprises one or more primers which hybridize to a mitochondrial D-loop for making a primer extension product. In addition, the kit contains written material identifying mutations which are found in the D-loop as a result of exposure to one or more environmental pollutants.
According to another embodiment of the invention an oligonucleotide probe is provided. The probe comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The probe can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T→C at nucleotide 114; &Dgr;C at nucleotide 302; C→A at nucleotide 386; insert T at nucleotide 16189; A→C at nucleotide 16265; A→T at nucleotide 16532; C→T at nucleotide 150; T→C at nucleotide 195; &Dgr;C at nucleotide 302; C→A at nucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide 16519; G→A at nucleotide 16380; G→A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; T→C at nucleotide 16172; C→T at nucleotide 16292; A→G at nucleotide 16300; A→G at nucleotide 10792; C→T at nucleotide 10793; C→T at nucleotide 10822; A→G at nucleotide 10978; A→G at nucleotide 11065; G→A at nucleotide 11518; C→T at nucleotide 12049; T→C at nucleotide 10966; G→A at nucleotide 11150; G→A at nucleotide 2056; T→C at nucleotide 2445; T→C at nucleotide 2664; T→C at nucleotide 10071; T→C at nucleotide 10321; T→C at nucleotide 12519; &Dgr; 7 amino acids at nucleotide 15642; G→A at nucleotide 5521; G→A at nucleotide 12345; T→C substitution at position 710; T→C substitution at position 1738; T→C substitution at position 3308; G→A substitution at position 8009; G→A substitution at position 14985; T→C substitution at position 15572; G→A substitution at position 9949; T→C substitution at position 10563; G→A substitution at position 6264; A insertion at position 12418; T→C substitution at position 1967; T→A substitution at position 2299; and G→A at nucleotide 3054.
According to another aspect of the invention an oligonucleotide primer is provided. It comprises a sequence of at least 10 contiguous nucleotides of a human mitochondrial genome. The primer can optionally contain at least 12, 14, 16, 18, 20, 22, 24, 26, or 30 such contiguous nucleotides. The oligonucleotide comprises a mutation selected from the group consisting of: a mutation selected from the group consisting of: T→C at nucleotide 114; &Dgr;C at nucleotide 302; C→A at nucleotide 386; insert T at nucleotide 16189; A→C at nucleotide 16265; A→T at nucleotide 16532; C→T at nucleotide 150; T→C at nucleotide 195; &Dgr;C at nucleotide 302; C→A at nucleotide 16183; C→T at nucleotide 16187; T→C at nucleotide 16519; G→A at nucleotide 16380; G→A at nucleotide 75; insert C at nucleotide 302; insert CG at nucleotide 514; T→C at nucleotide 16172; C→T at nucleotide 16292; A→G at nucleotide 16300; A→G at nucleotide 10792; C→T at nucleotide 10793; C→T at nucleotide 10822; A→G at nucleotide 10978; A→G at nucleotide 11065; G→A at nucleotide 11518; C→T at nucleotide 12049; T→C at nucleotide 10966; G→A at nucleotide 11150; G→A at nucleotide 2056; T→C at nucleotide 2445; T→C at nucleotide 2664; T→C at nucleotide 10071; T→C at nucleotide 10321; T→C at nucleotide 12519; &Dgr; 7 amino acids at nucleotide 15642; G→A at nucleotide 5521; G→A at nucleotide 12345; T→C substitution at position 710; T→C substitution at position 1738; T→C substitution at position 3308; G→A substitution at position 8009; G→A substitution at position 14985; T→C substitution at position 15572; G→A substitution at position 9949; T→C substitution at position 10563; G→A substitution at position 6264; A insertion at position 12418; T→C substitution at position 1967; T→A substitution at position 2299; and G→A at nucleotide 3054.
Another aspect of the invention is a method to aid in detecting the presence of tumor cells in a patient. The presence of a single basepair mutation is detected in a mitochondrial genome of a cell sample of a patient. The mutation is found in a tumor of the patient but not in normal tissue of the patient. The tumor is not a colorectal tumor. The patient is identified as having a tumor if one or more single basepair mutations are determined in the mitochondrial genome of the cell sample of the patient.
Yet another embodiment of the invention is provided by another method to
Fliss Makiko
Jen Jin
Kinzler Kenneth W.
Polyak Komelia
Sidransky David
Banner & Witcoff , Ltd.
Fredman Jeffrey
The Johns Hopkins University
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