A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells.

摘要

Methods for measuring the induction and repair of ultraviolet (UV) induced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 kilobases (kb) or larger can be obtained using the enzyme-sensitive site (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985). More recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sensitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). However, examination of these reports indicates that the PCR based DNA repair assays lack precision (Govan et al., 1990; Kalinowski et al., 1992; Tornaletti and Pfeifer, 1994; Jennerwein and Eastman, 1991). We report here, the development of a highly precise QPCR DNA repair assay. The assay was used to measure the induction and repair of UV photoproducts in a 2.7 kb genomic fragment containing the human growth hormone (hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly reproducible with an overall coefficient of variation from the mean of about 2.5%. This level of precision enabled the apparent simultaneous resolution of cyclobutane dimer (CPD) and (6-4) photoproduct (6-4PP) induction and repair.