This grant provided critical resources for the redesign of the STR assay and a mechanism for thoroughly testing and validating both the mitochondrial (mtDNA) and short tandem repeat (STR) assays. The project developed a 24-primer multiplexed assay that amplified human mtDNA hyper variable region 1 (HV1) coordinates 15924 to 16428 (primers span 158 to 16451) and HV2 coordinates 31 to 493 (primers span 5 to 603). The assay consists of eight triplexes PCR reactions that occupy one column of a 96-well plate. The assay produces a profile of base compositions anchored on revised Cambridge Reference Sequence (rCRS) coordinates. By anchoring on standardized coordinates, any base composition profile can be directly compared to any other profile produced in this assay, or to any mtDNA sequence that spans the full range of amplified coordinates for each base composition to be compared (the base composition that would be produced by any sequence that spans one of the primer pairs’ amplified coordinates can be predicted accurately). This grant facilitated close collaboration with forensic scientists from several laboratories, including the FBI DNA Unit II, AFDIL, and NIST. The project was divided into four aims, each designed to develop, validate, or automate a different aspect of the assay. These aims touched on various facets of development and/or performance validation of the automated mass spectrometry platform, the design and production of the assay kits, and the software used to interpret and report the results. 19 tables, 42 figures, 17 references, and appended GenBank accession numbers for human mtDNA genomes
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