A MODIFIED QUANTITATIVE POLYMERASE CHAIN-REACTION ASSAY FOR MEASURINGGENE-SPECIFIC REPAIR OF UV PHOTOPRODUCTS IN HUMAN-CELLS

Methods for measuring the induction and repair of ultraviolet (UV) ind uced modifications in short DNA fragments are essential for the study of gene-specific DNA repair. Measurements in genomic fragments of 14 k ilobases (kb) or larger can be obtained using the enzyme-sensitive sit e (ESS) assay introduced by Hanawalt and Bohr (Bohr et al., 1985), Mor e recently, several assays based on variations of the polymerase chain reaction (PCR) technique have been developed which have increased sen sitivity (Govan et al., 1990; Kalinowski et al., 1992; Jennerwein and Eastman, 1991), even nucleotide resolution (Pfeifer et al., 1993). How ever, examination of these reports indicates that the PCR based DNA re pair assays lad; precision (Govan et al., 1990; Kalinowski et al., 199 2; Tornaletti and Pfeifer, 1991; Jennerwein and Eastman, 1991). We rep ort here, the development of a highly precise QPCR DNA repair assay. T he assay was used to measure the induction and repair of UV photoprodu cts in a 2.7 kb genomic fragment containing the human growth hormone ( hGH) gene in SL89 (wild-type) fibroblasts. The assay was exceedingly r eproducible 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) i nduction and repair.