Anonymous liquid blood samples with self-identified ethnicities were purchased from Interstate Blood Bank (Memphis, TN) and Millennium Biotech, Inc. (Ft. Lauderdale, FL) and extracted using a modified salt out procedure (1). The extracted DNA was then quantified using UV spectrophotometry at 260 nm and a PicoGreen assay (2). A 150-µL aliquot of the extracted DNA solution was directly quantified in a Cary 100 double-beam spectrophotometer (Varian Analytical Instruments, Walnut Creek, CA). Low volume micro-cuvettes allowed for accurate absorbance measurements (A = 0.2 to 0.6) without prior dilution of the stock extracted DNA. Sample concentrations were adjusted to 1 ng/µL for typing purposes using the PicoGreen assay values. Fifteen autosomal STR markers (the 13 CODIS core loci and D19S433 and D2S1338) were typed along with amelogenin using the Applied Biosystems AmpF1STR® Identifiler™ kit (3). PCR amplification was carried out on a GeneAmp® 9700 (Applied Biosystems) using 1 ng of DNA according to kit protocols (3) with the exception of reduced volume reactions (5 µL instead of 25 µL) and reduced cycles (26 instead of 28). Amplification products were diluted 1:15 in HiDi™ formamide and GS500-LIZ internal size standard (Applied Biosystems) and analyzed on the 16-capillary ABI Prism® 3100 Genetic Analyzer without prior denaturation of samples. POP™-6 (Applied Biosystems) rather than POP™-4 was utilized for higher resolution separations on a 36 cm array. Samples were injected

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/µL to 1.5 ng/µL 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 Mixed Stain Study 3 (MSS3) interlaboratory challenge exercise evaluated the 2001 performance of STR multiplex DNA typing systems using a set of seven DNA extracts of designed concentration and composition. This initial report focuses on the linkages connecting the measurement of the concentration of DNA ([DNA]) to the observed STR multiplex signal intensities. There is a causal relationship between [DNA] measurement accuracy and the efficiency of STR multiplex analysis. There are no intrinsic measurement performance differences among the [DNA] measurement technologies reported. However, there are large differences in the efficiencies of amplification, separation, and detection among participants using the same nominal measurement systems.

In collaboration with the Armed Forces Institute of Pathology's Department of Defense DNA Registry, the National Institute of Standards and Technology recently evaluated the performance of a short tandem repeat multiplex with dried whole blood stains on four different commercially available identification card matrixes. DNA from 70 stains that had been stored for 19 months at ambient temperature was extracted or directly amplified and then processed using routine methods. All four storage media provided fully typeable (qualitatively identical) samples. After standardization, the average among-locus fluorescence intensity (electropherographic peak height or area) provided a suitable metric for quantitative analysis of the relative amounts of amplifiable DNA in an archived sample. The amounts of DNA in Chelex extracts from stains on two untreated high-purity cotton linter pulp papers and a paper treated with a DNA-binding coating were essentially identical. Average intensities for the aqueous extracts from a paper treated with a DNA-releasing coating were somewhat lower but also somewhat less variable than for the Chelex extracts. Average intensities of directly amplified punches of the DNA-binding paper were much larger but somewhat more variable than the Chelex extracts. Approximately 25% of the observed variation among the intensity measurements is shared among the four media and thus can be attributed to intrinsic variation in white blood count among the donors. All of the evaluated media adequately "bank" forensically useful DNA in well-dried whole blood stains for at least 19 months at ambient temperature.

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.