Dennis J. Reeder

STRBase: a short tandem repeat DNA database for the human identity testing community

The National Institute of Standards and Technology (NIST) has compiled and maintained a Short Tandem Repeat DNA Internet Database (http://www.cstl.nist.gov/biotech/++ +strbase/) since 1997 commonly referred to as STRBase. This database is an information resource for the forensic DNA typing community with details on commonly used short tandem repeat (STR) DNA markers. STRBase consolidates and organizes the abundant literature on this subject to facilitate on-going efforts in DNA typing. Observed alleles and annotated sequence for each STR locus are described along with a review of STR analysis technologies. Additionally, commercially available STR multiplex kits are described, published polymerase chain reaction (PCR) primer sequences are reported, and validation studies conducted by a number of forensic laboratories are listed. To supplement the technical information, addresses for scientists and hyperlinks to organizations working in this area are available, along with the comprehensive reference list of over 1300 publications on STRs used for DNA typing purposes.

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit.

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.

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.

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.

A low-cost, high-throughput, automated single nucleotide polymorphism assay for forensic human DNA applications

Single nucleotide polymorphism (SNP) analysis of human DNA for the purpose of identification has some promising attributes. The question of approach is critical to the eventual adoption of this technology. The use of a low-volume open array platform was tested with a small selected set of eight SNP primers that have a low F(ST) (the proportion of the total genetic variance contained in a subpopulation [S subscript] relative to the total genetic variance [T subscript]) in human populations. Because multiple SNPs must be interrogated, issues concerning DNA concentration, total DNA, and whole genome amplification were investigated. Excellent correlations were obtained for seven of the eight SNP assays on a set of DNA samples of known configuration over a broad concentration range spanning 25-150ng/microl in blind studies. These seven SNP assays were then applied to 39 DNA samples in a population from southern India. These SNPs were sufficient to individualize each member of this sample population. In a paternity study, these same SNPs showed clear parental relationships. For low amounts of genomic DNA, the use of a commercially available whole genome amplification kit showed promise for genotyping sub-nanogram samples. Discrimination against nonhuman DNA was also demonstrated successfully. Because of the very low quantities of reagents used in the assay, the cost per test becomes reasonably inexpensive. Overall, using commercially available SNP assays, the OpenArray platform showed excellent promise as a highly automated, low-volume, high-throughput system for SNP analysis with potential applications to relevant forensic analyses such as identification and paternity.

Cloning and Expression of the Gene for a Novel Protein from Mycobacterium smegmatis with Functional Similarity to Eukaryotic Calmodulin

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.

Interlaboratory Comparison of Autoradiographic DNA Profiling Measurements: Precision and Concordance

Knowledge of the expected uncertainty in restriction fragment length polymorphism (RFLP) measurements is required for confident exchange of such data among different laboratories. The total measurement uncertainty among all Technical Working Group for DNA Analysis Methods laboratories has previously been characterized and found to be acceptably small. Casework cell line control measurements provided by six Royal Canadian Mounted Police (RCMP) and 30 U.S. commercial, local, state, and Federal forensic laboratories enable quantitative determination of the within-laboratory precision and among-laboratory concordance components of measurement uncertainty typical of both sets of laboratories. Measurement precision is the same in the two countries for DNA fragments of size 1000 base pairs (bp) to 10,000 bp. However, the measurement concordance among the RCMP laboratories is clearly superior to that within the U.S. forensic community. This result is attributable to the use of a single analytical protocol in all RCMP laboratories. Concordance among U.S. laboratories cannot be improved through simple mathematical adjustments. Community-wide efforts focused on improved concordance may be the most efficient mechanism for further reduction of among-laboratory RFLP measurement uncertainty, should the resources required to fully evaluate potential cross-jurisdictional matches become burdensome as the number of RFLP profiles on record increases.

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Abstract We describe a manual fluorometric method for the quantitation in protein solutions of total free amino compounds, expressed as norleucine. A trichloroacetic acid deproteinization step is employed, and o-phthalaldehyde, buffered with phosphate at pH 9,2, is used as the fluorogenic reagent. The method is linear, reproducible, and rapid. Recoveries of amino acids added to serum are quantitative. Sensitivity is in the picomole range. Results on unselected patient sera are diseussed.

Analysis and configuration assignments of the amino acids in a pyoverdine-type siderophore by reversed-phase high-performance liquid chromatography

The amino acid composition of the siderophore pyoverdine Pf244 was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) of phenylthiocarbamyl derivatives of the acid-hydrolyzed pyoverdine. The siderophore contains an acid-labile amino acid, Nδ-hydroxyornithine, and an amino acid previously unknown in naturally occurring systems, l-threo-β-hydroxyhistidine. Amino acid chirality assignments were determined by RP-HPLC of the β-d-glucopyranosyl isothiocyanate tetraacetate derivatives. Reactions with amino acid oxidases established the l-configuration of threo-β-hydroxyhistidine.

Graphical Tools for RFLP Measurement Quality Assurance: Single-Locus Charts

Forensic restriction fragment length polymorphism analyses typically provide two results for each sample, one result for each unique allele, at each genetic locus probed. In collaboration with the member laboratories of the Technical Working Group for DNA Analysis Methods, we have developed graphical techniques that compactly summarize even large numbers of such paired measurements. This paper provides a detailed description of the basic tool, a modified bivariate control chart for data from one sample at one locus. We demonstrate how various modifications and combinations of these “single-locus charts” can be used for within- and among-laboratory quality assurance activities.