In the past 5 years, there has been a substantial increase in the use of Y-short tandem repeat loci (Y-STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFlSTR Yfiler PCR amplification kit simultaneously amplifies 17 Y-STR loci including the loci in the "European minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y-STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross-reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.
The human ecto-apyrase gene family consists of five reported members (CD39, CD39-L1, CD39-L2, CD39-L3, and CD39-L4). The family can be subdivided into two groups by conservation of proposed structural domains. The CD39, CD39-L1, and CD39-L3 genes all encode hydrophobic portions in their carboxy and amino termini, serving as transmembrane domains for CD39 and potentially for the other two members. CD39-L2 and CD39-L4 genes encode hydrophobic portions in their amino termini, suggesting that they might encode secreted apyrases. We demonstrate that the CD39-L4 gene encodes the first reported human secreted ecto-apyrase. COS-7 cells transfected with a CD39-L4 expression construct utilizing the naturally occurring leader peptide express recombinant protein outside of the cells. This expression can be blocked by brefeldin A, a chemical that inhibits a step in mammalian secretory pathways. We also demonstrate expression of CD39-L4 message in macrophages, suggesting that the protein is present in the circulation. Furthermore, we show that CD39-L4 is an E-type apyrase, is dependent on calcium and magnesium cations, and has high degree of specificity for NDPs over NTPs as enzymatic substrates. A potential physiological role in hemostasis and platelet aggregation is presented.
DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpF'STR Ò MiniFiler TM PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpF'STR Ò Identifiler Ò PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpF'STR Ò SGM Plus Ò kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI ⁄ National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpF'STR Ò Identifiler Ò and SGM Plus Ò kits. These data support that the MiniFiler TM kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.
The GlobalFiler™ PCR Amplification Kit is a single multiplex assay that amplifies a set of 24 markers, which encompass the European Standard Set and CODIS (Combined DNA Index System) recommended composite set of loci. In addition to more loci and a 6-dye chemistry format, the Master Mix has been formulated to allow higher sample loading volume for trace DNA samples. The GlobalFiler™ Kit has been optimized to deliver high performance on casework samples, while also delivering fast thermal cycling, with an amplification time of approximately 80 min. Here, we report the results of the developmental validation study which followed the SWGDAM (Scientific Working Group on DNA Analysis Methods) guidelines and includes data for PCR-based studies, sensitivity, species specificity, stability, precision, reproducibility and repeatability, concordance, stutter, DNA mixtures, and performance on mock casework samples. The results validate the multiplex design as well as demonstrate the kit’s robustness, reliability, and suitability as an assay for human identification with casework DNA samples.Electronic supplementary materialThe online version of this article (10.1007/s00414-018-1817-5) contains supplementary material, which is available to authorized users.
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