Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard

Biochemical Science Division, National Institute of Standards and Technology, 100 Bureau Drive, MS 8311, Gaithersburg, MD 20899-8311, USA.
Analytical and Bioanalytical Chemistry (Impact Factor: 3.44). 05/2009; 394(4):1183-92. DOI: 10.1007/s00216-009-2782-0
Source: PubMed


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.
NIST Standard Reference Material (SRM) 2372 Human Quantitation Standard

  • Source
    • "In this study, the clearly traceable E. coli BL21 from ATCC sources were selected as the source material for the E. coli DNA reference. For preparing this reference, WHO guidelines for standard materials were referred to and strictly followed [3], [18], [19], [20]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: This collaborative study developed the first national Escherichia coli (E. coli) DNA reference standard for standardizing quantitative residual DNA assay methods, fluorescence dye (PicoGreen) and quantitative PCR (q-PCR), which were widely employed to measure residual DNA contents of prokaryotic-derived recombinant products. High purity of E. coli strain BL21 was extracted by the cetyl triethyl ammonium bromide (CTAB)/phenol chloroform method, analyzed by UV-visible spectrophotometry and electrophoresis, diluted with tris-EDTA (TE) buffer and manually dispensed. Then, with a cooperative calibration among six laboratories, including five manufacturers and one national control laboratory, the concentration of E. coli DNA standard solution was determined as 96.2 μg/mL (95% C.I: 95.5-96.9 μg/mL, CV 3.4%). The candidate showed excellent stability both from accelerated degradation study and real time stability study. The applicability study showed that the E. coli DNA reference could reach the sensitivity of 0.781 ng/mL and 1 fg/μL, respectively, in fluorescent dye and q-PCR assay, and also had good linearity and precision. The consistency of the reference could meet the requirements of the national reference standard. As a conclusion, the candidate material was suitable to serve as a China national standard for E. coli residual DNA determination. The successful establishment of the E. coli DNA standard will facilitate the standardization of quantitative methods for testing residual host cell DNA.
    Preview · Article · Sep 2013 · PLoS ONE
  • [Show abstract] [Hide abstract]
    ABSTRACT: With the advent of forensic DNA profiling in the mid-1980s, this technology has had a positive impact on the criminal justice system, helping to convict the guilty and exonerate the innocent. The field has evolved from focusing on multilocus markers throughout the nuclear DNA genome to the use of autosomal short tandem repeat (STR) markers. Other marker systems such as mitochondrial DNA and Y-chromosomal STR testing have also found an important niche for the identification of missing persons and historical investigations. Given the importance of forensic DNA testing, it is critical that laboratories include proper controls and validated procedures for making quality measurements. In the United States, the National Institute of Standards and Technology (NIST) developed several standard reference materials (SRMs) to meet the needs of the forensic DNA community. Here, we discuss a brief history of forensic DNA testing and the development of NIST SRMs and educational resources for the field over the last 20years. KeywordsNational Institute of Standards and Technology (USA)–Standard reference materials–Short tandem repeat–DNA quantification–STRBase–Forensic DNA testing
    No preview · Article · Jun 2011 · Accreditation and Quality Assurance
  • [Show abstract] [Hide abstract]
    ABSTRACT: Eine erfolgreiche „Multiplex-short-tandem-repeat“(Multiplex-STR)-Typisierung wird durch den optimalen Einsatz an „Template“-DNA erleichtert. Die DNA-Konzentration kann mithilfe verschiedener Methoden bestimmt werden. In der vorgestellten Studie wurden die Fluorometrie mit PicoGreen (PG-F), die Nano-UV-Absorptionsspektroskopie (nUV-S) und die „Real-time polymerase chain reaction“ (qPCR) hinsichtlich ihrer Sensitivität verglichen. Außerdem wurde das Messverhalten der einzelnen Methoden bei der Quantifizierung von DNA-Lösungen untersucht, die mit UV-Licht bestrahlt oder mit im forensischen Bereich typischen Zusatzstoffen verunreinigt worden waren. Die qPCR und die PG-F zeigten die höchste Sensitivität und ein sehr robustes Messverhalten bei Verunreinigungen. Die UV-Licht-Bestrahlung der DNA beeinflusste dagegen das Messergebnis dieser Methoden deutlich, während die nUV-S hier nur geringe Messwertveränderungen aufwies.
    No preview · Article · Aug 2012 · Rechtsmedizin
Show more