PCR-Based Assays for the Detection and Quantitation of DNA Damage and Repair
Exposure to genotoxic agents from both environmental and endogenous sources which result in damage to cellular DNA poses a significant health risk to the individual if such damage is left unrepaired. Several human diseases, including Cockayne’s syndrome and xeroderma pigmentosum, have been associated with defects in the repair of DNA damage (Friedberg et al., 1995). An overall decrease in DNA repair capacity is observed in the latter, whereas the former is associated with a defect in a transcription-coupling factor which facilitates rapid repair in the transcribed strand of an active gene. One general method for the analysis of gene- and strand-specific repair is based on Southern analysis of DNA strand breaks induced by a damage-specific endonuclease (Smith and Mellon, 1990; Bohr and Okumoto, 1988). This endonuclease-sensitive site (ESS) technique employs the use of T4 endonuclease V which cleaves DNA at pyrimidine dimers (Ganesan et al., 1980), eliminating its ability to hybridize to a radioactive probe on alkaline Southern analysis. Although this methodology has been pivotal in the elucidation of gene-specific repair in single-copy genes (Bohr et al., 1987; Mellon et al., 1987; Mellon and Hanawalt, 1989), there are certain limitations. It requires some information regarding restriction sequence information flanking the gene or genomic segment of interest, a lesion-specific endonuclease to incise near the damaged base, and perhaps most significant is the requirement for large quantities of DNA (5–10 μg) generally used in Southern assays.
KeywordsQuantitative Polymerase Chain Reaction Quantitative Condition Dhfr Gene Lesion Frequency Polymerase Chain Reaction Target
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