Typing Highly Degraded DNA Using Circularized Molecules and Target Enrichment

Forensic genetic typing of severely compromised DNA samples is one of the leading challenges faced by forensic DNA analysts. These degraded or damaged biological samples are difficult, if not impossible to test due to their highly fragmented nature. It is essential that techniques be developed to overcome the molecular limitations of highly damaged and degraded DNA based on use of nuclear DNA markers. This proposal defines two novel approaches that, in theory, can circularize short (i.e., highly degraded) DNA fragments that will serve as templates for target enrichment by capture and whole genome amplification (WGA) of human identity single nucleotide polymorphisms (SNPs). These proposed methods combine several established research techniques such as the circularization of genomic template DNA, bait capture, WGA via rolling circle amplification (RCA), and massively parallel sequencing (MPS) into an innovative approach to overcome the restrictions of typing highly compromised DNA. Preparation of the circular molecules, the basis of the proposed approaches, will focus on using Circligase TM II (Epicentre Biotechnologies; Madison, WI) and molecular inversion probes (MIPs). Both techniques will be investigated and compared employing a detailed list of assessment criteria. I hypothesize that through this combined methodology, sufficient genetic material will be generated to yield a genetic profile suitable for human identification. Our goal in this study is to create an approach that would allow for genetic typing of DNA from challenged biological samples and to provide forensic DNA analysts with the means to be less constrained by the limits of damaged and degraded DNA. This development would address an essential need that has always hampered forensic genetic investigation. The proposed research has the potential for significant advancement in the field of forensic genetics while simultaneously providing a substantial benefit to society by aiding in the identification of missing persons, contributing to the solving of criminal cases, and even addressing the analysis of historical remains. ca/ncf