Lee C K, Brown B G, Bombick B R, Doolittle D J
Laboratory of Environmental and Molecular Toxicology, Research and Development, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina 27102, USA.
Anal Biochem. 1995 May 1;227(1):156-61. doi: 10.1006/abio.1995.1265.
Among the currently available methods for detecting bulky hydrophobic compounds bound to DNA, the 32P-postlabeling is the most sensitive and most widely practiced. One of the key reaction steps of this assay is the polynucleotide kinase (PNK)-catalyzed incorporation of 32Pi into nucleotides. To ensure high sensitivity and reproducibility of the assay, it is imperative that the PNK reaction be conducted under optimal conditions. The PNK reaction is generally conducted at pH 9.6 in the 32P assay. We have investigated pH profiles of the PNK reactions using both normal (2'-deoxyadenosine 3'-monophosphate) and adducted (benzo[ alpha]pyrene- and cigarette smoke condensate-modified) nucleotides. The optimum pH range for the PNK reaction was between 7.4 and 8.4 with both types of nucleotides. Higher pH levels such as 9.4 and 9.6 were detrimental to the PNK reaction, yielding only about one-third or less of the labeling obtained at the optimum pH. Based on these results, the PNK reaction in the 32P-postlabeling assay should be conducted in the pH range between 7.8 and 8.0 to achieve quantitative labeling of adducted nucleotides.
