EFFECT OF PH OF THE T4 POLYNUCLEOTIDE KINASE REACTION ON THE P-32 POSTLABELING ASSAY OF DNA-ADDUCTS

Among the currently available methods for detecting bulky hydrophobic compounds bound to DNA, the P-32-postlabeling is the most sensitive an d most widely practiced. One of the key reaction steps of this assay i s the polynucleotide kinase (PNK)-catalyzed incorporation of P-32(i) i nto nucleotides. To ensure high sensitivity and reproducibility of the assay, it is imperative that the PNK reaction be conducted under opti mal conditions. The PNK reaction is generally conducted at pH 9.6 in t he P-32 assay. We have investigated pH profiles of the PNK reactions u sing both normal (2'-deoxyadenosine 3'-monophosphate) and adducted (be nzo[a]pyrene- and cigarette smoke condensate-modified) nucleotides. Th e optimum pH range for the PNK reaction was between 7.4 and 8.4 with b oth types of nucleotides. Higher pH levels such as 9.4 and 9.6 were de trimental to the PNK reaction, yielding only about one-third or less o f the labeling obtained at the optimum pH. Based on these results, the PNK reaction in the P-32-postlabeling assay should be conducted in th e pH range between 7.8 and 8.0 to achieve quantitative labeling of add ucted nucleotides. (C) 1995 Academic Press, Inc.