Prototype PowerPlex® Y23 System: A concordance study.

The Prototype PowerPlex(®) Y23 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4. A total of 951 samples from six populations were typed to evaluate the kit and examine concordance for 17 of the loci that are in common with those that can be typed using the AmpFlSTR(®) Yfiler™ kit (Life Technologies, Carlsbad, CA). A total of 16,167 loci were analyzed for each multiplex, and overall concordance was observed. Because of different kit designs, and although concordant for the genetic type, discordant calls can occur due to a deletion at the DYS448 locus. Users should take into consideration such nomenclature anomalies when comparing Y STR profiles. This new kit allows a large battery of Y STR loci to be analyzed using the same basic technologies already employed in forensic laboratories.

Developmental validation of the PowerPlex® Y23 System: a single multiplex Y-STR analysis system for casework and database samples.

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 62.5 pg 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 62.5 pg of male DNA in a background of 400 ng of female DNA or 125 pg of male DNA mixed with 3000 ng of female DNA.

Developmental validation of the PowerPlex® 18D System, a rapid STR multiplex for analysis of reference samples.

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.

Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex ® ESX 17 and ESI 17 Systems.

The PowerPlex(®) ESX 17 and ESI 17 Systems for short tandem repeat (STR) amplification were developed by the Promega Corporation to meet the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group recommendations for increasing the number of STR loci included in the European Standard Set (ESS). The PowerPlex ESX 17 and ESI 17 Systems utilize different PCR primer combinations to co-amplify the following 17 loci: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, vWA, SE33, and the sex-typing locus amelogenin. A total of 1443 U.S. population samples were evaluated with pre-commercialization versions of both kits. Stutter and heterozygote peak height ratios have been used to characterize kit performance. Typing results have been used to estimate the match probabilities provided by the chosen loci as well as in concordance studies. Full concordance between the typing results for the two kits was observed in 99.994% (49,055 out of 49,062) STR allele calls compared. All genotyping discrepancies were confirmed by DNA sequence analysis. As a result of these comparisons, a second forward primer for the D22S1045 locus has been added to the PowerPlex ESX 17 System to address a primer binding site mutation and the D1S1656 locus reverse primer in the PowerPlex ESI 17 System was modified to eliminate an amplification-efficiency reducing primer dimer.

Developmental validation of the PowerPlex 16 HS System: an improved 16-locus fluorescent STR multiplex.

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.

Post-injection hybridization of complementary DNA strands on capillary electrophoresis platforms: a novel solution for dsDNA artifacts.

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.