Nucleic Acids Research, 1992, Vol. 20, No. 13 3485-3494
© 1992
MOLECULAR BIOLOGY |
Analysis of DNA damage and repair in murine leukemia L1210 cells using a quantitative polymerase chain reaction assay
1Departments of Pathology Burlington, VT 05405, USA 2Departments of Molecular Genetics and Microbiology Burlington, VT 05405, USA 3Departments of Biochemistry, University of Vermont Burlington, VT 05405, USA
*To whom correspondence should be addressed
Received January 28, 1992. Revised June 8, 1992. Accepted June 8, 1992.
The polymerase chain reaction (PCR) represents an alternative to the current methods for investigating DNA damage and repair in specific genomic segments. In theory, any DNA lesion which blocks Taq polymerase can be measured by this assay. We used quantitative PCR (QPCR) to determine the lesion frequencies produced by clsplatin and ultravioiet light (UV) in a 2.3 kilobase (kb) segment of mitochondrial DNA and a 2.6 kb segment of the DHFR gene in mouse leukemia L1210 cells. The frequency of UV-induced lesions increased linearly with dose, and was 0.58 lesions/10kb/10 J/m2 in the mitochondrial DNA, and 0.37 iesions/10kb/10J/m2 in the DHFR gene. With cisplatin, the lesion frequency also increased linearly with dose, and was 0.17 lesions/10kb/10µM in the DHFR gene, and 0.07 lesions/10kb/10µM in mitochondrial DNA. This result is contrary to that of Murata et al., 1990 (1), in which mitochondrial DNA received greater cisplatin damage than did nuclear DNA. Using PCR to measure the repair of UV-induced lesions in the DHFR gene segment, we observed that iess than 10% of the lesions were removed by 4 h, but over 70% of the lesions were removed by 8 h. Repair of 43% of UV-induced ieslons in mitochondrial DNA was also observed during a 24 h period.
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