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Nucleic Acids Research, 1994, Vol. 22, No. 12 2351-2359
© 1994

METHODS

Assessment of DNA damage and repair in specific genomic regions by quantitative immuno-coupled PCR

Mikhail F. Denissenko*, Sundaresan Venkatachalam, Edith F. Yamasaki and Altaf A. Wani

Department of Radiology and Biochemistry Program, The Ohio State University 103 Wiseman Hall, 400 W. 12th Avenue, Columbus, OH 43210, USA

*To whom correspondence should be addressed

Received February 18, 1994. Revised March 12, 1994. Accepted March 12, 1994.

Fine analysis of DNA damage and repair at the subgenomic level has indicated a microheterogeneity of DNA repair in mammalian cells, including human. In addition to the well established Southern hybridization-based approach to investigate gene-specific DNA damage and repair, alternative methods utilizing the sensitivity of PCR have been evaluated. The latter technique has relied on decreased PCR amplification due to damage in template DNA. We have developed a novel quantitative assay combining the selective recovery of DNA damage containing genomic fragments with the PCR amplification. DNA isolated from 7,8-dihydroxy-anti-9, 10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) treated human skin fibro-blasts was immunoprecipitated with polyclonal antibody BP-1. Recovered target sequences were amplified by PCR using primers encompassing a 149 bp target region around codon 12 of the H-ras protooncogene. Quantitative DNA damage specific response was observed with nanogram amounts of genomic DNA. This approach allowed analysis of the initial DNA damage at a level less than 1 anti-BPDE adduct per 6.4 kbp ras gene fragment. Repair proficient GM637 cells exposed to 2 µM anti-BPDE showed a faster removal of the adducts from the H-ras gene segment than from the genome overall. Gene-specific repair was not apparent in GM4429 xeroderma pigmentosum (complementation group A) cells. The established technique could be extended to the quantitative measurement of the repair of diverse DNA base lesions in any genomic region of known sequence.


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