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.     

Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA

National Institute of Standards and Technology SRM 2372 human DNA quantitation standard has been produced to support the need for a human-specific DNA quantitation standard in forensic casework and calibration of new quantitative polymerase chain reaction (qPCR) assays. The conventional DNA concentration has been assigned with one of the U.S. National Reference UV/Visible Spectrophotometers, assuming an absorbance of 1.0 at 260 nm equals 50 ng/μL of double stranded DNA. In addition, an interlaboratory study has been conducted, to verify that the SRM 2372 materials perform well in currently used DNA quantitation assays by the forensic DNA community. Each unit of SRM 2372 consists of three well-characterized DNA extracts. Component A is a single-source human male material derived from blood. Component B is a multiple-source human female material derived from blood. Component C was purchased as a purified unsheared genomic human DNA (Sigma-Aldrich Co., St. Louis, MO) obtained as a lyophilized human genomic extract and has both male and female donors. SRM 2372 is intended to enable the comparison of DNA concentration measurements across time and place. Manufacturers can use SRM 2372 to validate the values assigned to their own reference materials. Individual forensic laboratories can use SRM 2372 to validate DNA quantitation methods and to verify the assigned concentration of in-house or commercial DNA calibration standards.     

Associating gunpowder and residues from commercial ammunition using compositional analysis

Qualitatively identifying and quantitatively determining the additives in smokeless gunpowder to calculate a numerical propellant to stabilizer (P/S) ratio is a new approach to associate handgun-fired organic gunshot residues (OGSR) with unfired powder. In past work, the P/S values of handgun OGSR and cartridges loaded with known gunpowders were evaluated. In this study, gunpowder and residue samples were obtained from seven boxes of commercial 38 caliber ammunition with the goals of associating cartridges within a box and matching residues to unfired powders, based on the P/S value and the qualitative identity of the additives. Gunpowder samples from four of the seven boxes of ammunition could be easily differentiated. When visual comparisons of the cartridge powders were considered in addition to composition, powder samples from all seven boxes of ammunition could be reliably differentiated. Handgun OGSR was also collected and evaluated in bulk as well as for individual particles. In some cases, residues could be reliably differentiated based on P/S and additive identity. It was instructive to evaluate the composition of individual unfired gunpowder and OGSR particles. We determined that both the numerical centroid and dispersity of the P/S measurements provide information for associations and exclusions. Associating measurements from residue particles with those of residue samples collected from a test firing of the same weapon and ammunition appears to be a useful approach to account for any changes in composition that occur during the firing process.     

RFLP band size standards: NIST standard reference material 2390

The procedural standard for DNA profiling developed by the U.S. advisory board on DNA quality assurance methods mandates annual confirmation of forensic DNA measurement systems against an appropriate reference material supplied by or traceable to the National Institute of Standards and Technology (NIST). NIST Standard Reference Material (SRM) 2390 is a suitable and appropriate standard for HaeIII restriction enzyme-based restriction fragment length polymorphism (RFLP) profiling systems. Originally issued in 1992, an among-laboratory SRM 2390 recertification study was initiated in 1997. Using data provided by the 20 state, local, or commercial forensic laboratory participants, quantitative band sizes values (expected mean values and associated bivariate tolerance intervals) are established for two different-source DNAs (female cell line K562 and healthy male "TAW") for genetic loci D1S7, D2S44, D4S139, D5S110, D1OS28, and D17S79. Methods for validating an RFLP measurement system, validating a control material or other secondary standard, and for tracing a particular set of RFLP measurements to NIST SRM 2390 are described in detail.     

Investigating the effect of changing ammunition on the composition of organic additives in gunshot residue (OGSR)

The measurement of the organic additives in smokeless gunpowder is an attractive approach for the detection of handgun use because it provides compositional information that can help associate residues and unfired gunpowder. We investigate several factors that will be required to advance the characterization of organic gunshot residue (OGSR) as a useful forensic tool, including evaluating residue contamination from previously fired ammunition, particle-to-particle compositional variability, and compositional features resulting from the type of firing primer used. Using ammunition loaded with known smokeless powders containing different stabilizers, a sequence of shots was fired from a .357 magnum revolver, and the muzzle exit residues were collected. Compositional analysis of the residues, both in bulk and as single particles, showed only a trace of the previously fired powder in the first shot and none in subsequent shots. In an additional experiment testing conventional leaded and the new lead-free firing primers, the OGSR composition was found not to depend on the primer type.     

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.     

The single most polymorphic STR Locus: SE33 performance in U.S. populations

The STR locus SE33 (ACTBP2) located on chromosome 6 (6q14) is arguably the most polymorphic marker examined thus far by the forensic community with a heterozygosity of >0.95 in some populations. Three different primer sets were utilized in this study in order to assess the possibilities of primer binding site mutations. Population variation was measured in 460 U.S. Caucasian, 445 African American, 336 Hispanic, and 202 Asian samples along with mutation rates from almost 400 father–son pairs. In addition, the 10 genomic DNA components in NIST Standard Reference Material SRM 2391b were sequenced and found to exhibit a variety of additional base changes, insertions, and deletions outside of the SE33 repeat region.     

Mitochondrial DNA typing screens with control region and coding region SNPs

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to be recovered from highly degraded specimens. A major disadvantage to traditional forensic mtDNA analysis is that it is time-consuming and labor-intensive to generate and review the 610 nucleotides of sequence information commonly targeted in hypervariable regions I and II (HVI and HVII) of the control region. In addition, common haplotypes exist in HVI/HVII mtDNA sequences that can reduce the ability to differentiate two unrelated samples. In this report we describe the utility of two newly available screening assays for rapid exclusion of non-matching samples. The LINEAR ARRAY mtDNA HVI/HVII Region-Sequencing Typing Kit (Roche Applied Science, Indianapolis, IN) was used to type 666 individuals from U.S. Caucasian, African American, and Hispanic groups. Processing of the LINEAR ARRAY probe panels "mito strips" was automated on a ProfiBlot workstation. Observable variation in 666 individuals is reported and frequencies of the mitotypes within and between populations are presented. Samples exhibiting the most common Caucasian mitotype were subdivided with a multiplexed amplification and detection assay using eleven single nucleotide polymorphisms in the mitochondrial genome. These types of screening assays should enable more rapid evaluation of forensic casework samples such that only samples not excluded would be subjected to further characterization through full HVI/HVII mtDNA sequence analysis.