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 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.
Chakravarti
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Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, 68198-6805, USA.
Cavalieri
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Rogan
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EG
D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D017931:DNA Primers; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005784:Gene Amplification; D011905:Genes, ras; D008969:Molecular Sequence Data; D010957:Plasmids; D011084:Polycyclic Aromatic Hydrocarbons; D015183:Restriction Mapping
D003062:Codon; D017931:DNA Primers; D004277:DNA, Single-Stranded; D011084:Polycyclic Aromatic Hydrocarbons; D004259:DNA-Directed DNA Polymerase