Margaret C. Kline

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.

Results from the NIST 2004 DNA Quantitation Study

For optimal DNA short tandem repeat (STR) typing results, the DNA concentration ([DNA]) of the sample must be accurately determined prior to the polymerase chain reaction (PCR) amplification step in the typing process. In early 2004, the National Institute of Standards and Technology (NIST) conducted an interlaboratory study to help assess the accuracy of DNA quantitation in forensic DNA laboratories. This study was designed with four primary purposes: (1) to examine concentration effects and to probe performance at the lower DNA concentration levels that are frequently seen in forensic casework; (2) to examine consistency with various methodologies across multiple laboratories; (3) to examine single versus multiple source samples; and (4) to study DNA stability over time and through shipping in two types of storage tubes. Eight DNA samples of [DNA] from 0.05 ng/microL to 1.5 ng/microL were distributed. A total of 287 independent data sets were returned from 80 participants. Results were reported for 19 different DNA quantitation methodologies. Approximately 65% of the data were obtained using traditional slot blot hybridization methods; 21% were obtained using newly available quantitative real-time PCR (Q-PCR) techniques. Information from this interlaboratory study is guiding development of a future NIST Standard Reference Material for Human DNA Quantitation, SRM 2372.

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues.

Chromosomal Duplications Along the Y-Chromosome and their Potential Impact on Y-STR Interpretation

Y-chromosome short tandem repeat (Y-STR) markers are being used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA as is present in many sexual assault samples. Usually single copy Y-STR loci produce a single amplicon in single source samples, and thus the observation of multiple peaks at such a locus could suggest to an analyst that a mixture of more than one male contributor is present in the tested sample. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation. Reasons for the presence of duplications at multiple loci within a single sample are explored in the context of Y-STR marker location along the chromosome. True male-male mixtures commonly exhibit more than one locus-specific PCR product across multiple Y-STR loci that are not adjacent to one another on the Y-chromosome. In addition, duplicated loci typically possess alleles that differ by only a single repeat unit and possess similar peak heights.

Electrophoretic separations of polymerase chain reaction-amplified DNA fragments in DNA typing using a capillary electrophoresis- laser induced fluorescence system

Abstract Analysis of polymerase chain reaction (PCR)-amplified DNA fragments for human identification requires high-resolution separation and efficient detection of amplified alleles. Capillary electrophoresis (CE) with its speed, automation, high resolution and efficiency shows promise for analysing the amplified DNA fragments. CE with UV detection, however, suffers from lack of detector sensitivity owing to the limited detection path length of the capillary. By the use of intercalating dyes (TOTO and YOYO) a laser-induced fluorescence (LIF) detection system can provide much greater sensitivity for detecting DNA fragments. Femtogram quantities of dsDNA (φX174 HaeIII restriction digest mixture) per nanoliter of injected volume have been detected. Application of CE-LIF to analysis of PCR-amplified DNA fragments from three different genetic loci (apolipoprotein B, VNTR locus D1S80, mitochondrial DNA) is shown here. Further, the resolving power of a polymer-network capillary separation system is compared to that of a capillary-gel separation system.

Match criteria for human cell line authentication: Where do we draw the line?†‡

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross‐contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN‐0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines—duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50–79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Analysis of mutations in father–son pairs with 17 Y-STR loci

We have examined 389 father/son sample pairs from U.S. Caucasians, African Americans, Hispanics and Asians using the 17 Y-STR loci in the Yfilertrade mark kit and observed a total of 24 differences between father and son. Thirteen mutations resulted in the gain of a repeat in the son and 11 resulted in a loss of a repeat. All samples resulted in single repeat mutations except one sample which contained a two repeat loss at Y-GATA-H4. Furthermore, two different sample pairs were found to have two mutations. An African American sample pair had a mutation at DYS458 and a second at DYS635 and an Asian sample pair had mutations at DYS439 and Y-GATA-H4.

Standard Reference Material 2366 for Measurement of Human Cytomegalovirus DNA

Human cytomegalovirus (CMV), classified as human herpesvirus 5, is ubiquitous in human populations. Infection generally causes little illness in healthy individuals, but can cause life-threatening disease in those who are immunocompromised or in newborns through complications arising from congenital CMV infection. An important aspect in diagnosis and treatment is to track circulating viral load with molecular methods, particularly with quantitative PCR. Standardization is vital, because of interlaboratory variability (due in part to the variety of assays and calibrants). Toward that end, the U.S. National Institute of Standards and Technology produced a Standard Reference Material 2366 appropriate for establishing metrological traceability of assay calibrants. This standard is composed of CMV DNA (Towne(Δ147) bacterial artificial chromosome DNA). Regions of the CMV DNA that are commonly used as targets for PCR assays were sequenced. Digital PCR was used to quantify the DNA, with concentration expressed as copies per microliter. The materials were tested for homogeneity and stability. An interlaboratory study was conducted by Quality Control for Molecular Diagnostics (Glasgow, UK), in which one component of SRM 2366 was included for analysis by participants in a CMV external quality assessment and proficiency testing program.

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.

NIST mixed stain studies #1 and #2: interlaboratory comparison of DNA quantification practice and short tandem repeat multiplex performance with multiple-source samples.

The Mixed Stain Study 1 (MSS1, Apr.-Nov. 1997) and Mixed Stain Study 2 (MSS2, Jan.-May 1999) evaluated multiplexed short-tandem repeat (STR) DNA typing systems with samples containing DNA from more than one source. These interlaboratory challenge studies evaluated forensic STR measurement, interpretation, and reporting practice using well-characterized samples of very different analytical difficulty. None of the relatively few errors reported in either exercise resulted in a false identification of a reference source; several errors in evaluating the unknown source in three-source samples would hinder matching the profile in any archival database. None of the measurement anomalies reported is associated with any particular STR multiplex; all DNA amplification anomalies are associated with inefficient DNA extraction, inaccurate DNA quantitation, and/or analytical threshold policies.