Linear amplification mapping of polycyclic aromatic hydrocarbon-reactive sequences in H-ras gene

Dhrubajyoti Chakravarti, Ercole Cavalieri, Eleanor G Rogan

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

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 reactions would be expected to synthesize full-length single-stranded DNA, the synthesis is often prematurely terminated. We describe the optimization 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 amounts of proofreading DNA polymerases to a nick-translating polymerase is optimum for linear amplification. From these results, a linear amplification protocol was developed to map DNA synthesis-blocking sites generated by the reaction of (±) anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, 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 indicate that the central A of H-ras codon 61 (CAA) reacts with these polycyclic aromatic hydrocarbons.

Original languageEnglish (US)
Pages (from-to)529-539
Number of pages11
JournalDNA and Cell Biology
Volume17
Issue number6
DOIs
Publication statusPublished - Jan 1 1998

    Fingerprint

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this