Quantifying and Qualifying the Influence of Standard Laboratory Procedures on Aged, Degraded, and/or Low Copy Number DNA

As submitted by the proposer: The analysis of aged, degraded, and/or LCN DNA is complicated largely by: (1) the presence of modern contaminating DNA; (2) the co-extraction of impurities (PCR inhibitors) along with DNA; and (3) the degree of damage accumulated by endogenous DNA postmortem or from the time of deposition of the biological material. These problems make the analysis of DNA from aged, degraded, and/or LCN sources far more challenging than working with modern DNA. Objectives of this research are to evaluate and develop methods that show promise to increase the net yield of DNA and its purity. These include: (1) evaluating DNA loss during its extraction and purification against standards (phase I); (2) exploring means to mitigate DNA loss and/or further damage to the molecules in the standards (phases I & II); and (3) further improving and/or development of novel methods to remove PCR inhibitors from DNA elutes and/or subdue their influences within the forensic workflow (Phase III). Moreover, there are still a number of poorly understood aspects of how aged, degraded, and/or LCN DNA "behaves" during routine laboratory methods and, critically, whether some or all of those behaviors truly differ from that of modern contaminating DNA. There are hypotheses that newly observed damage patterns from ancient & endogenous DNA molecules are consistent and, thus, predictable. If these hypotheses are accurate, presumably there would be measurably different expectations for the behavior of - and damage accrued in - modern exogenous contaminating DNA due to the reduced time since creation and deposition of the biological material. To be clear, if the damage patterns observed in contaminating DNA mirror those routinely observed in authentic aged and degraded DNA, then damage patterns cannot be a used as a reliable means by which to discriminate between contaminating and endogenous DNA. These ideas will be evaluated by experiments described in phases II & IV. This proposed research employs state-of-the-art technology for the quantification and/or qualification of DNA using a Fragment Analyzer, CFX96 Touch Deep Well Real-Time PCR Detection System, and an Illumina HiSeq. The combination of these technologies will permit for unique evaluation of the above outlined issues and hypotheses in novel ways. The observations made during this project will directly benefit forensic genetics, criminal justice, as well as the fields of ancient DNA (aDNA) and paleogenomics. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). ca/ncf