Method to detect DNA damage and measure DNA repair rate

Details Method to detect DNA damage and measure DNA repair rate Method to detect DNA damage and measure DNA repair rate

1997-07-31

1999-11-23

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 536 2433, C12Q 168, C12P 1934, C07H 2100

Patent

active

059898160

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the field of molecular biology and biochemistry. More specifically, it relates to detecting and quantitating DNA damage in animals and in cell culture, for monitoring the dynamics of DNA damage including rate of occurrence and remediation by DNA repair enzymes, and for detecting and assessing genotoxic risk in the environment.


BACKGROUND

Organisms are being constantly bombarded by endogenous and exogenous genotoxic agents which injure their DNA. This DNA damage, if left unrepaired, can lead to cell death, somatic mutations or cellular transformation, which at the organismal level can result in cancer, aging, and decreased immunocompetency. There is now a large amount of evidence which indicates that the repair of specific DNA lesions does not occur equally throughout the entire genome. For example, it has been shown that the DNA of actively transcribed regions is repaired more efficiently than DNA of inactive regions. This gene-specific DNA repair is due mainly to the rapid repair of the transcribed strand of actively expressed genes (Mellon et al., 1987; Mellon and Hanawalt, 1989; Smerdon and Thoma, 1990), and as a consequence can lead to the accumulation of mutations in the more slowly repaired non-transcribed strand (Chandrasekhar and Van Houten, 1994).
Preferential repair of DNA damage in actively transcribed genes was initially identified using a Southern hybridization-based assay employing T4 endonuclease V, which specifically cleaves DNA at pyrimidine dimer sites (Bohr and Okumoto, 1988). While this methodology has proven useful in the identification and study of gene-specific repair, it has the serious disadvantage of requiring a lesion-specific endonuclease to incise the DNA near the damaged nucleotide. Other disadvantages including: interest.
Another approach to determining DNA damage and repair in specific genomic DNA regions is the Taq enzyme and polymerase chain reaction (PCR) method of Govan et al., (Govan et al., 1990). This method is based on the fact that many DNA lesions can block Taq DNA polymerase, which causes in a decrease in amplification product. The method of Govan et al., however, was not very powerful because of the small size limitation on the DNA segments being amplified--less than 450 bp. Subsequently, the sensitivity such assays was increased to DNA segments of about 2 to 3 kb (Jennerwein and Eastman, 1991; Kalinowski et al., 1992). Presently the detection of DNA damage in specific gene sequences of bacterial and mammalian cells is limited to the quantitation of amplification products ranging in size from 334 bp to 3.2 kb.
Current PCR assay methods are not sufficient to detect DNA damage after exposure to biologically and environmentally relevant doses of genotoxic agents, including ionizing radiation. While the current PCR-based assay for the detection of DNA damage appears to be useful at high doses of damaging agent, it suffers from lack of sensitivity at biologically relevant doses, and therefore can not be used reliably for accurate DNA damage detection and determining DNA repair rates in humans. This is a major problem as the art currently stands. To meet these needs, the sensitivity of a DNA damage detection assay must be increased from a current detection limit of about one lesion/10.sup.4 nucleotides to one lesion/10.sup.5 nucleotides.


DEFINITIONS
complementary DNA strands which are detected after the amplification process. replication of the DNA sequence is interrupted at the damage site. damages DNA. These include compounds that arise in the cell naturally, or are directly derive from the environment, or indirectly from exposure to a agent in the environment (such as another compound or ionizing radiation). May also be called a mutagen. (>5,000 base pair) DNA templates to yield substantially only amplification products representing the intact (undamaged) complementary strands of the starting template sample. primer site on each of its complementary DNA strands separated by at least 5,000 base pairs on the i

REFERENCES:
patent: 5891682 (1999-04-01), Yoshida et al.
Casou et al. Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts Nucleic Acids Research vol. 20(23), pp. 6363-6368, 1992.
Piette, J. Biological consequences associated with DNA oxidation mediated by singlet oxygen J. Photochem, Photobiol. B: Biol., vol. 11, pp. 241-260, 1991.
Kalinowski et al. Analysis of DNA damage and repair in murine leukemina L1210 cells using a quantitative polymerase chain reaction assay, Nucleic Acid Research, vol. 20 (13), pp. 3485-3494, 1992.

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