Validation of half-reaction amplification using Promega PowerPlex 16.

DNA amplification is a fundamental yet costly process used in DNA analysis. This study evaluated half-reaction amplification (12.5, 12, and 13 microL) using the Promega Powerplex 16 Kit with the hope of reducing sample analysis costs by half.

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency.

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.

Comparison of seven protocols to identify fecal contamination sources using Escherichia coli.

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly.

Effect of DNA damage on PCR amplification efficiency with the relative threshold cycle method.

Polymerase stop assays used to quantify DNA damage assume that single lesions are sufficient to block polymerase progression. To test the effect of specific lesions on PCR amplification efficiency, we amplified synthetic 90 base oligonucleotides containing normal or modified DNA bases using real-time PCR and determined the relative threshold cycle amplification efficiency of each template. We found that while the amplification efficiencies of templates containing a single 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were not significantly perturbed, the presence of a single 8-oxo-7,8-dihydro-2'-deoxyadenosine, abasic site, or a cis-syn thymidine dimer dramatically reduced amplification efficiency.

Extraction and analysis of human nuclear and mitochondrial DNA from electron beam irradiated envelopes.

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.