qPCR Genotype Determination and Mixture Detection Using High Resolution Melting Curve Analysis of STR Loci

From submitted abstract (truncated to less than 400 words): In this proposal, the qPCR quantitation step has been chosen for incorporation of a meltcurve based mixture detection assay. This step has been chosen because of the ease and level of multiplexing that qPCR affords and because it is early enough in the workflow that changes to the downstream procedures could be made to improve the success rate of initial STR amplification of the samples. Further, by incorporating new targets into an existing quantitation assay (Qiagen Investigator™ Quantiplex kit and Life Technologies Quantifiler® DUO), this additional information could be obtained without any additional analytical steps or procedures. We propose to develop such an assay by simultaneously targeting two STR loci during the qPCR quantitation reaction. Melt curve analysis would immediately follow the traditional thermalcycling steps and would be used to presumptively identify a genotype group (“geno-group”) for the purposes of determining if a sample is from a single contritubor or from a mixture. Data groupings would also indicate likely number of contributors, if a mixture is detected.Melt curve analysis will be facilitated by the incorporation of recently developed intercalating dyes that have been shown to not interfere with existing qPCR kit chemistry. Two qPCR platforms will be used for this study. The Qiagen Rotor-Gene® Q is a qPCR instrument that is ideal for such an assay in that it allows for higher resolution melt curve analysis and has expanded fluorescent detection capabilities. In addition, the assay will be evaluated on the ABI 7500 qPCR instrument as it is the most common platform validated for use in forensic DNA laboratories. This instrument has a “dissociation” function to allow for standard melt-curve analysis. The STR loci selected for use in this study have been chosen based on their discriminatory power as well as their ability to generate amplicons whose melt patterns do not overlap in size range. Incorporation of two STR loci will improve the chances of successfully detecting a two or more person mixture and will improve the accuracy of any exclusionary genotyping data that could be obtained from single-source samples. The availability of this type of screening assay would allow for better decisions to be made early on in the analytical workflow which should lead to a concomitant reduction in sample retest rates, consumable costs, and hands-on examiner time. This project contains a research and/or development component, as defined in applicable law. ca/ncf