Martin Buoncristiani¹, Jeanette Wallin², Michi Lee¹, John Tonkyn¹ and Steve Lee^1
1 California Department of Justice DNA Laboratory, Berkeley, CA
² Perkin-Elmer, Applied Biosystems, Foster City, CA

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AmpFlSTR Blue is a short tandem repeat (STR) triplex kit containing the loci D3S1358, vWA and FGA. These tetranucleotide STR loci have a combined power of discrimination of approximately 0.9998. The kit primers are labeled with NHS-ester dye FAM, which emits at 535 nm (blue). To investigate the forensic conditions under which a reliable result may be obtained using the AmpFlSTR Blue kit, studies were performed as outlined by the TWGDAM committee. The results of several of these studies will be presented. These include Standard Specimen, Consistency, Reproducibility, Population, Environmental, Matrix, Single versus Multiple Locus Amplification, Differential Amplification, Mixed Specimen, Nonprobative Evidence, Nonhuman and Inheritance.

In general, expected types were observed in all cases. Environmental studies examined blood and semen under a variety of conditions. Highly degraded samples demonstrated that FGA was the first locus to drop out, followed by vWA, and then D3S1358. This is the expected pattern according to base pair size (FGA>vWA>D3S1358). Inhibition studies included samples deposited on a variety of substrates and samples amplified in the presence of purified hematin, the presumed inhibitor coextracted from blood-containing samples. While all samples extracted from substrates amplified with AmpFlSTR Blue, locus dropout was observed in amplifications with high concentrations of hematin. FGA was the first locus to dropout, followed by D3S1358, and then by vWA. Single versus multiple locus amplifications revealed no benefit in single-plexing, even in the presence of the inhibitor hematin. Differential amplification experiments included degraded and low template DNA, along with DNA in the presence of the inhibitor hematin and variation in amplification annealing and denaturation temperatures. All samples showed balanced peak heights except two in which some degree of differential amplification was observed at either vWA or FGA in the presence of high levels of hematin. Mixture studies included the examination of defined ratios of mixed genomic DNA, characterization of stutter bands and peak height ratios to assist in recognition and interpretation, and nonprobative sexual assault case evidence evaluation.