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.