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Currently, the amplification step of most forensic DNA profiling systems takes 3-4 h to complete. A decrease in the amplification time would allow for increased laboratory throughput, which may help reduce backlog when is due to limited cycling capacity. By using the SpeedSTAR™ HS Polymerase (Takara Bio, Otsu, Japan) in combination with the Veriti® (Applied Biosystems, Foster City, CA, USA) rapid thermal cycler, the amplification time for the PowerPlex® 16 HS (Promega, Madison, WI, USA) kit was reduced by 66 % (1 h). The sensitivity of this fast method was comparable to the standard system (0.13 ng). Although this rapid protocol showed an increase in average stutter ratios (2.8 %) and a decrease in average peak height ratios (7 %) across all loci when compared with standard conditions, it was able to consistently generate reliable DNA profiles. The results of this study indicate that the rapid protocol could be implemented in forensic laboratories with an optimal range of 0.25-2 ng of input DNA using appropriate analytical interpretation guidelines. [ABSTRACT FROM AUTHOR]

The Taiwanese aborigines have been regarded as the source populations for the Austronesian expansion that populated Oceania to the east and Madagascar off Africa to the West. Although a number of genetic studies have been performed on some of these important tribes, the scope of the investigations has been limited, varying in the specific populations examined as well as the maker systems employed. This has made direct comparison among studies difficult. In an attempt to alleviate this lacuna, we investigate, for the first time, the genetic diversity of all nine major Taiwanese aboriginal tribes (Ami, Atayal, Bunun, Rukai, Paiwan, Saisat, Puyuma, Tsou and Yami) utilizing a new generation multiplex Y-STR system that allows for the genotyping of 23 loci from a single amplification reaction. This comprehensive approach examining 293 individuals from all nine main tribes with the same battery of forensic markers provides for the much-needed equivalent data essential for comparative analyses. Our results have uncovered that these nine major aboriginal populations exhibit limited intrapopulation genetic diversity and are highly heterogeneous from each other, possibly the result of endogamy, isolation, drift and/or unique ancestral populations. Specifically, genetic diversity, discrimination capacity, fraction of unique haplotypes and the most frequent haplotypes differ among the nine tribes, with the Tsou possessing the lowest values for the first three of these parameters. The phylogenetic analyses performed indicate that the genetic diversity among all nine tribes is greater than the diversity observed among the worldwide reference populations examined, indicating an extreme case of genetic heterogeneity among these tribes that have lived as close neighbors for thousands of years confined to the limited geographical area of an island. [ABSTRACT FROM AUTHOR]

The PowerPlex® 21 System is a STR multiplex that has been optimized for casework samples while still being capable of database workflows including direct amplification. The loci included in the multiplex offer increasing overlap with core loci used in different countries and regions throughout the world. The PowerPlex® 21 System contains D1S1656, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, Amelogenin, CSF1PO, FGA, Penta D, Penta E, TH01, TPOX, and vWA. These loci represent all 13 core CODIS loci in addition to loci commonly used in Asia and Europe. A developmental validation study was completed to document performance capabilities and limitations of the PowerPlex® 21 System. Data from this validation work served as the basis for the following conclusions: genotyping of single-source samples was reliable across a range of template DNA concentrations with >95% alleles called at 50pg. Direct amplification of samples from FTA® storage cards was successfully performed using the reagents provided with the system and modified cycling protocols provided in the technical manual. Mixture analysis showed that over 95% of minor alleles were detected at 1:9 ratios. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, DNA polymerase, magnesium, and Master Mix were shown to be optimal and able to withstand moderate variations without affecting system performance. Reproducible results were generated by different users at different sites. Finally, concordance studies showed consistent results when comparing the PowerPlex® 21 System with other commercially available STR-genotyping systems. [ABSTRACT FROM AUTHOR]

Abstract: Analysis for short tandem repeat (STR) loci is widely performed in forensic laboratories for human identification that utilizes commercially available kits that employ fluorescently labeled primers and capillary electrophoresis. With only a few exceptions, the sequences of the primers included in a kit are not disclosed by the kit manufacturers. Therefore, we developed a simple method to determine the 5′ end of the binding site of the primers included in commercial kits. Our method requires only custom primers and human genome sequence data and routinely used equipment and consumables. One or two custom primers are added to the PCR reaction mixture containing kit primers and input human DNA prior to amplification, and PCR products are separated by capillary electrophoresis after amplification. With this method we can determine which primer of the pair is fluorescently labeled and the 5′ end of the binding site of primers based on the changes in an electropherogram that are caused by the addition of the custom primer(s), and the human genome sequence data. This method is also useful for the determination of the shortest possible lengths of labeled kit primers. [Copyright &y& Elsevier]

Abstract: The PowerPlex® Y23 System combines the seventeen Y-STR loci in current commercially available Y-STR kits (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and Y-GATA-H4) with six new highly discriminating Y-STR loci (DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643). These six new loci have higher gene diversities than most of the loci in other commercial Y-STR analysis kits, allowing for further distinction between unrelated male individuals. In addition, the inclusion of two rapidly mutating Y-STR loci may allow for the discrimination of related individuals. The PowerPlex® Y23 System is designed to amplify DNA from purified extracts as well as direct amplification from substrates used to collect database samples (e.g. swabs and storage cards). Validation of the PowerPlex® Y23 System includes all of the studies required by the FBI and SWGDAM. The results demonstrate that the PowerPlex® Y23 System is a robust and reliable amplification kit capable of overcoming high concentrations of commonly encountered inhibitors such as hematin, humic acid, and tannic acid. Full profiles are consistently detected with 62.5pg of male DNA, even in the presence of excessive amounts of female DNA, establishing the PowerPlex® Y23 System as a sensitive method for Y-STR testing. Complete Y-STR profiles are detected from mixed samples with 62.5pg of male DNA in a background of 400ng of female DNA or 125pg of male DNA mixed with 3000ng of female DNA. [Copyright &y& Elsevier]

Abstract: As short tandem repeat markers remain the foundation of human identification throughout the world, new STR multiplexes require rigorous testing to ensure the assays are sufficiently robust and reliable for genotyping purposes. The PowerPlex® 18D System was created for the direct amplification of buccal and blood samples from FTA® storage cards and reliably accommodates other sample materials. The PowerPlex® 18D System allows simultaneous amplification of the 13 CODIS loci and amelogenin along with four additional loci: Penta E, Penta D, D2S1338, and D19S433. To demonstrate suitability for human identification testing, the PowerPlex® 18D System was tested for sensitivity, concordance, inhibitor tolerance, and performance with thermal cycling and reaction condition variation following SWGDAM developmental validation guidelines. Given these results, PowerPlex® 18D System can confidently be used for forensic and human identification testing. [Copyright &y& Elsevier]

Abstract: In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex® ESI 16 (European Standard Investigator 16) and the PowerPlex® ESI 17 Systems. The PowerPlex® ESI 16 System combines the 11 loci compatible with the UK National DNA Database®, contained within the AmpFlSTR® SGM Plus® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR® SGM Plus® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex® ESI 17 System amplifies the same loci as the PowerPlex® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR® SGM Plus® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors. [Copyright &y& Elsevier]

Abstract: STR multiplexes remain the cornerstone of genotyping forensic samples. The PowerPlex® 16 HS System contains the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex-determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. The PowerPlex® 16 HS System is an updated version of the PowerPlex 16® System; while the primers and dyes remain unchanged, it introduces an enhanced buffer system that includes hot-start Taq DNA polymerase and ensures robust performance. Due to the modification of the reaction mix, a multi-laboratory developmental validation study was completed to document performance capabilities and limitations for the revised assay. Data within this validation was generated by eight laboratories and served as the basis for the following conclusions: genotyping of single-source samples was consistent across a large range of template DNA concentrations with most laboratories obtaining complete profiles at 62.5pg. Mixture analyses showed that over 90% of minor alleles were detected at 1:9 ratios. Optimum amplification cycle number was ultimately dependent on the sensitivity of the detection instrument and could be adjusted to accommodate a range of DNA template concentrations. Reaction conditions including volume and annealing temperature as well as the concentrations of primers, Taq DNA polymerase, and magnesium were shown to be optimal and able to withstand moderate variations without affecting multiplexed STR amplification. Finally, data from non-probative samples and concordance studies showed consistent results when comparing the PowerPlex® 16 HS System with the PowerPlex® 16 System as well as other commercially available systems. [Copyright &y& Elsevier]

Abstract: Several laboratories have reported the occurrence of a split or n −1 peak at the vWA locus in PowerPlex® 16 and PowerPlex® ES amplification products separated on 4- and 16-capillary electrophoresis instruments. The root cause of this artifact is post-PCR reannealing of the unlabeled, unincorporated vWA primer to the 3′-end of the tetramethylrhodamine (TMR)-labeled strand of the vWA amplicon. This reannealing occurs in the capillary post-electrokinetic injection. The split peak is eliminated by incorporation into the loading cocktail of a sacrificial hybridization sequence (SHS) oligonucleotide that is complementary to the vWA primer. The SHS preferentially anneals to the primer instead of the TMR-labeled strand of the vWA amplicon. In addition, the n −10/n −18 artifact that may be seen at the vWA locus was determined to be due to double-stranded amplicon formed post-electrokinetic injection into the capillary. This was also eliminated by adding in two Complementary Oligo Targets (COT1 and COT2) in addition to the SHS oligonucleotide into the loading cocktail. These three oligonucleotides are complementary to the 33 bases at the 5′-end of the unlabeled vWA amplicon strand and the 60 bases at its 3′-end and therefore compete for hybridization to the TMR-labeled amplicon strand. Incorporation of these three oligonucleotides in the Internal Lane Standard 600 (ILS600) eliminate both the split peak and n −10/n −18 artifact in PowerPlex® 16 and PowerPlex® ES amplification products without affecting sizing of alleles at the vWA locus or any locus in the PowerPlex® 16, PowerPlex® Y, PowerPlex® ES, AmpFlSTR® Profiler Plus® ID, AmpFlSTR® Cofiler®, and AmpFlSTR® SGM Plus® kits. [Copyright &y& Elsevier]

Abstract: With over 6 million profiles currently stored in European databases and the number expected to increase with cross-border data sharing, the likelihood of random matches will undoubtedly increase as well. To improve the overall power of discrimination as well as provide standardization across Europe, the ENFSI and EDNAP committees have made a recommendation to extend the current European Standard Set (ESS) for STR systems. The five-color PowerPlex® ESX and ESI Systems allow co-amplification and detection of the current commonly tested loci, plus the five new recommended loci. These kits will be offered in multiple formats, including one to detect SE33, to accommodate various requirements or preferences. Additionally, the kits have increased tolerance to common inhibitors and increased sensitivity to obtain full profiles from low-level DNA, and are robust enough to genotype degraded DNA samples through the use of mini STR loci. In this manuscript we present an overview of these systems and data on performance, including sensitivity and resistance to inhibitors. The PowerPlex® ESX and ESI Systems are useful tools in database sharing and standardization throughout Europe. [Copyright &y& Elsevier]