TY - JOUR
T1 - A modified quantitative polymerase chain reaction assay for measuring gene-specific repair of UV photoproducts in human cells
AU - McCarthy, M. J.
AU - Rosenblatt, J. I.
AU - Lloyd, R. S.
N1 - Funding Information:
The authors wish to acknowledge the following individuals that made contributions to this project. Drs. John A. Phillips III and Joy Cogan provided the primer sequencea nd conditions for the 2.7 kb hGH PCR. Dr. Veronica Maher at Michigan State Univer- sity provided the SL89 cell line which was karyotyped by Dr. Merlin Butler and Ms. Lora Hedges. We thank the Recombinant DNA Laboratory in the Scaly Center for Molecular Science for the synthesis of the oligonucleotides.T his researchw as supported by NIH CA60056, NIH ES06676 and American Cancer Society FRA 38 1.
PY - 1996/5/15
Y1 - 1996/5/15
N2 - 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.
AB - 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.
KW - Gene specific repair
KW - Human
KW - Polymerase chain reaction
KW - UV photoproduct
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U2 - 10.1016/0921-8777(95)00061-5
DO - 10.1016/0921-8777(95)00061-5
M3 - Article
C2 - 8632778
AN - SCOPUS:0029915021
SN - 0921-8777
VL - 363
SP - 57
EP - 66
JO - Mutation Research - DNA Repair
JF - Mutation Research - DNA Repair
IS - 1
ER -