Robert S. McLaren

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(®) 21 System.

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 50 pg. 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.

Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant¨r) system

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assays specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

A rotationally-driven polyethylene terephthalate microdevice with integrated reagent mixing for multiplexed PCR amplification of DNA

We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for integrated on-chip reagent mobilization, mixing, and PCR amplification for genetic analysis of short tandem repeats (STR). Fluid flow, including reagent mobilization and mixing, is achieved by centrifugal force, eliminating the need for bulky instrumentation. The use of a passive valve also eliminates the need for extra hardware and simplifies the chip and the device design. A custom-built system is capable of thermocycling through a dual Peltier clamping system, as well as variable rate spinning with a DC motor. A multiplex PCR amplification of alleles associated with 18 genomic loci was successfully performed on-chip, followed by capillary electrophoretic separation, which showed efficient amplification of DNA from multiple sources. The genetic profiles generated were 100% concordant with those obtained using conventional PCR methods. The resultant system represents a novel microfluidic PCR amplification platform that uses inexpensive PCR microdevices that are simple to fabricate, yet effective for complex, multiplexed PCR.

Human cell line authentication: the critical first step in any project using human cell lines.

Short tandem repeat (STR) typing is a standard procedure used in many laboratories for the authentication of human cell lines. This technology, which is based on the informativeness of known polymorphism of numerous loci to uniquely identify a human cell line, has allowed for direct-amplification of human DNA stored on FTA(®) paper. We describe an application of this technology to create a unique STR profile by direct amplification of HCT 116 (ATCC(®) CCL-247™) cell line DNA, a cell line commonly used in colon research. The ability to perform direct-amplification of DNA opens up the possibility of using FTA(®) paper as a way to maintain long-term storage of DNA samples from a cell line and other human tissues, such as buccal cells.