Developmental validation of an Improved Multiplex Assay for use in forensic casework: The simultaneous gender-typing and quantification of total human and male-only DNA within forensic samples

Scope and Method of Study. An end-point PCR-based multiplex assay was developmentally validated for use in forensic DNA casework that could simultaneously gender-type and quantify total human and male-only DNA. The developmental validation included the studies that are required by the Revised Validation Guidelines set forth by the Scientific Working Group on DNA Analysis Methods. DNA was extracted by an organic extraction method, or the Hair Extraction Kit. The samples were purified and de-salted using DNA IQ(TM) System with Differex(TM) or CentriconsRTM. The NIST SRM 2372 quantitation standards were purchased from the National Institute of Standards and Technology. Samples were amplified using an ABI 9700 thermal cycler and analyzed using an ABI Prism 310 Genetic Analyzer. The peak height and sizing of alleles was determined using the GeneScanRTM Analysis version 3.1.2 software and the alleles were labeled using a Genotyper RTM version 2.5.2 software macro developed for this research.;Findings and Conclusions. The Improved Multiplex Assay (IMA) has been developmentally validated as a valid quantitation method for use in criminal casework in CODIS participating forensic labs and meets or exceeds national standards. Based on a one to one relationship between the amount of amplified product from a known quantity of input DNA to a sample pre-amplification, a standard curve can be generated by serially diluting the NIST SRM 2372 Standards, amplifying, analyzing, generating a standard curve, and then comparing unknown samples to the standard curve to estimate the concentration of total human and male-only DNA in the unknown sample pre-amplification. The IMA is sensitive enough and can perform at a level that allows it to be used to screen evidence to determine what type of profiling test would provide optimal results. Because the IMA relies on the current equipment already used in DNA testing labs, it is cost effective to implement in an existing DNA testing lab. Any forensic laboratory can implement a low cost and DNA Advisory Board (DAB) and ASCLAD/LAB compliant DNA quantitation method based on this developmental validation.