The objectives of the research presented in this report were to evaluate and develop methods that show promise for increasing the net yield of DNA and its purity through the following efforts: evaluation of DNA loss during its extraction and purification against standards; exploration of means to mitigate DNA loss and/or further damage to the molecules in the standards; and further improvement and/or development of novel methods to remove PCR inhibitors from DNA elutes and/or subdue their influences within the forensic workflow.
This final technical paper reports on a project that addressed the challenges of aged, degraded, and/or low copy number (LCN) DNA samples. The project was divided into four distinct phases that addressed specific issues relating to the outlined problems, and the paper includes cross references between the phases which are presented as sub-projects with their own sub-project reports that serve as a basic draft of a paper that will be submitted for peer-reviewed publication. Each sub-project is supported by figures and tables that are found at the end of each sub-project reports; supplemental files of raw data are provided as Excel files, and a single bibliography is provided at the end of the entire report. The authors note some project highlights, including: DNA extraction kit/methods evaluated in the project were associated with variable losses of concentrations of DNA standards and their inadvertent fragmentation; the authors measured abundant molecules “lost during a silica-based extraction; storage of DNA standards over the course of approximately 10 months at room temperature was met with substantial stability of quality and quantity of the DNA standards, and degradation was no more notable at room temperature than to storage of -80 degrees, Celsius; degradation of DNA by heat treatment is related positively to temperature exposed as well as its duration; increasing PCR extension times may subdue the influence of PCR inhibition; 500 cycle PCRs did not fail; and employing lower-than-standard denaturing temperature in PCR was a largely ineffective approach to studying degraded DNA.