Analysis of 2-chloro-2'-deoxyadenosine incorporation into cellular DNA by quantitative polymerase chain reaction.
Thermus aquaticus (Taq) DNA polymerase elongation is blocked by several DNA adducts. This property has been exploited in polymerase chain reaction (PCR) methods to analyze cellular DNA damage and repair after exposure to damaging agents. Such methods have not been applied previously to detect nucleoside analog incorporation into cellular DNA. 2-Chloro-2'-deoxyadenosine (CldAdo), a deoxyadenosine analog, is clinically effective for hairy cell leukemia. CldAdo is taken up by cells, converted to the triphosphate, and incorporated into cellular DNA. Here, we measured by primer extension the ability of CldAMP residues in 98-base single-stranded DNA to block Taq elongation. In contrast to control DNA, no full-length 98-mers were produced on CldAMP-containing templates, and Taq polymerase was halted at the first CldAMP site. We then examined the possibility of using quantitative PCR to measure CldATP incorporation into the N-ras gene after incubation of cultured human leukemia cells with CldAdo or with cisplatin as a positive control for DNA damage. Treatment with either drug resulted in reduced amounts of amplified DNA product compared to untreated cells. CldAMP residues within cellular DNA inhibited PCR amplification in a dose-dependent manner; 100 nM CldAdo produced approximately 0.4 CldAMP sites within a 523-bp region of the N-ras sequence. Thus, PCR analysis with Taq polymerase provides a sensitive assessment of nucleoside analog incorporation after cellular exposure to antileukemic drugs.
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Department of Cellular and Molecular Pharmacology, Chicago Medical School, 3333 Green Bay Road, North Chicago, Illinois, 60064, USA.
Tibudan
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M
Hentosh
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D000970:Antineoplastic Agents / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D017338:Cladribine / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D017338:Cladribine; D004247:DNA; D019914:Taq Polymerase; D002945:Cisplatin