LINEAR AMPLIFICATION MAPPING OF POLYCYCLIC AROMATIC HYDROCARBON-REACTIVE SEQUENCES IN H-RAS GENE

Linear amplification, or primer directed single-strand DNA synthesis, is commonly used in applications such as cycle sequencing and mapping replication block sites in DNA, Although linear amplification reaction s would be expected to synthesize full-length single-stranded DNA, the synthesis is often prematurely terminated. We describe the optimizati on of a linear amplification protocol for synthesizing a full-length ( 985-nt) single-stranded pBR322 segment, The enzyme activities of five DNA polymerases commonly used in PCR amplification, namely, AmpliTaq, Stoffel fragment, Tth, Pfu, and Vent, were tested either singly or in combination, The results indicate that the additive action of small am ounts of proofreading DNA polymerases to a nick-translating polymerase is optimum for linear amplification. From these results, a linear amp lification protocol was developed to map DNA synthesis-blocking sites generated by the reaction of(+/-) anti-benzo[a]pyrene-7,8-diol-9,10-ep oxide, or anti-or syn-dibenzo[a,l]pyrene-9,10-diol-11,12-epoxide with H-ras DNA surrounding the oncogenic codon 61 region. The results indic ate that the central A of H-ras codon 61 (CAA) reacts with these polyc yclic aromatic hydrocarbons.