No abstract

DNA damage blocks DNA polymerase progression and increases miscoding. In this study we assessed the effects of specific lesions on Taq DNA polymerase fidelity and amplification efficiency. In the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), Taq DNA polymerase inserted dCMP and to a lesser extent dAMP. 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) instructed the incorporation of dTMP and caused a pronounced n-1 deletion not observed in other systems. The presence of an abasic lesion led to dAMP incorporation and n-1 deletions. In addition, we introduce the Mean Modified Efficiency (MME) as a more precise method for determining PCR amplification efficiency of damaged templates. Using this method, we were able to quantify reductions in amplification efficiency of templates containing 8-oxodG (single or multiple), 8-oxodA, or abasic sites. Because the MME method can detect small reductions in amplification efficiency, it may be useful in comparing the extent of damage in environmentally degraded or archival DNA specimens.

The United States Postal Service is considering methods such as electron beam irradiation to neutralize biological agents sent through the mail. While this is proven to reduce/eliminate pathogenic organisms, it may also degrade human genomic DNA and therefore hinder the ability to garner forensically informative genetic profiles. To determine the effects of electron beam irradiation on DNA typing, 16 white, standard letter-sized envelopes were licked. Half of the envelopes served as nonirradiated controls while the other half underwent irradiation at dosages sufficient to kill anthrax spores (29.3 and 51.6 kGy). Total cellular DNA was extracted from all envelopes; nuclear short tandem repeat loci, as well as the hypervariable region I from mitochondrial DNA, were amplified by means of the polymerase chain reaction. Short tandem repeat profiles and mitochondrial DNA sequence haplotypes were acquired on an ABI Prism® 310 Genetic Analyzer platform. Analysis of data from irradiated samples revealed evidence of DNA degradation; however, the ability to construct full genetic profiles from both nuclear and mitochondrial DNA remained largely unaffected. The use of the polymerase chain reaction, coupled with florescent fragment analysis and mitochondrial DNA sequencing, should be considered to profile biological material from evidence enduring irradiation to inactivate infectious agents.