TOXI 108 |
| Many mutations occur as a result of DNA synthesis past the site of DNA damage by a replicative of DNA damage bypass polymerase. The frequency and type of mutations not only depend on the type of damage, but also on the sequence context, as revealed from analysis of mutation spectra of heterogeneous DNA sequences exposed to a mutagen. To best distinguish between the effects of DNA damage structure and sequence context, researchers have relied on using a pure DNA substrate containing a specific type of damage at a specific site. Unfortunately, this approach would require the synthesis of a multitude of substrates to determine the effect of flanking sequence, and goes at(4)n where n is the number of flanking bases to be examined. Herein we report a new approach for the study of mutations and their sequence context effects that we call SAMS for serial analysis of mutation spectra. That makes use of the methodology underlying serial analysis of gene expression (SAGE) to analyze the mutations that result from DNA synthesis past a DNA lesion embedded in a library of DNA sequences. In our example, we constructed a 45-mer template containing an abasic (ab) analog, flanked by a random library of nucleotides, NabN, and two EcoRI restriction sites to each side. This template was then used for primer extension by yeast pol eta, followed by separation of the primer extended product from the template, PCR amplification, EcoRI cleavage, ligation to form multimers, and then cloned into pZEro vector. The frequency of mutations resulting from the bypass of individual sequences was then determined by sequence analysis of the clones.(This work was supported by NIH CA40463.) |
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Poster Session and Awards
6:00 PM-10:00 PM, Tuesday, August 21, 2007 BCEC -- 204 A/B, Poster
Division of Chemical Toxicology |