Efficient and Effective SNP System for Analysis of Highly Degraded DNA Samples

Forensic DNA typing is a highly sensitive set of methodologies, such that a minute amount of DNA can be analyzed for a wide range of applications including identifying missing persons from degraded human remains and analysis of crime scene biological evidence. A single hair shaft can provide answers to support investigative leads. Because of this sensitivity, there has been an increased demand to analyze challenging (i.e., low quantity or low quality) samples. Analysis of touch samples, hairs, and human remains pushes the limits of current technologies, especially when samples are highly degraded. To date there has not been a technology that can accommodate such samples. Reverse complement polymerase chain reaction (RC-PCR) is an innovative, one-step, single-tube PCR target enrichment technology adapted for the amplification of highly degraded DNA and can be the solution to analyzing degraded samples. Based on work from a previously funded NIJ grant, a multiplex of 27 single nucleotide polymorphisms (SNPs) for human identification was developed. The SNPs are all contained within approximately 50 base long amplicons and the system has substantial sensitivity of detection, tested down to 60 picograms of input DNA. With the promising results from the initial study, the multiplex should be expanded to provide a high level of discrimination and developed with a concomitant robustness for forensic applications. The goals of this project are to expand the number of SNPs to 82 commonly used SNPs and enhance performance of the system through primer redesign, degenerate primers, and inhibition enhancers so that the most challenging of samples may be analyzed. The SNPs will be designed to reside within amplicons less than 100 bases in length. Increased number to the RC-PCR Human Identification panel will improve the overall power of discrimination. An enhanced robustness, demonstrated with standard validation testing, will enable the SNP panel to be applied to a variety of highly degraded and contaminated samples. The one-step, closed-tube process is conducive to automation, decreases the labor required to perform library preparation, and substantially reduces the probability of contamination due to less sample manipulation. The unique features of the RC-PCR technology lend itself to a seamless application to successfully target SNPs on highly degraded templates. Because of the reduced amplicon size and higher typing success, the RC-PCR method is anticipated to yield results with samples for which no or very limited data are obtained with standard DNA typing methods. 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