DNA Quantitation Methods: Evaluation and Comparison

Participants: Margaret C. Kline, Amy E. Decker, and Peter M. Vallone

Project Timeframe: 2004 to present

Purpose: Real-time quantitative PCR (qPCR) has generated a great deal of interest in the forensic DNA typing community in the past several years as this technique can rapidly detect low levels of DNA with minimum hands-on time and minor sample consumption. The ability to utilize human specific assays in U.S. crime laboratories is important in order to meet federally mandated requirements (DAB/FBI Standard 9.3) to assess the quantity of human DNA in casework samples, particularly where bacterial contamination may exist. A number of published qPCR assays have been evaluated at NIST.

Progress: We have observed variability in commercial standards which will affect the relative concentration of DNA measurements and have noted that assay optimization is important to generate consistent results. Candidate materials have been selected for production of a quantitative reference material for human DNA. The three components present in SRM 2372 have been tested with a variety of qPCR kits and assays.

Publications or Presentations Resulting From This Project:

Kline, M.C., Vallone, P.M., Decker, A.E., Redman, J.W., Duewer, D.L., Butler, J.M. (2005) Testing candidate DNA quantitation standards with several real-time quantitative PCR methods. Proceedings of the 16^th International Symposium on Human Identification. http://www.promega.com/geneticidproc/ussymp16proc/abstracts/kline.pdf.

Margaret Kline talk at the 16th International Symposium on Human Identification (Grapevine, TX), September 29, 2005, "Testing Candidate DNA Quantitation Standards with Several Real-Time Quantitative PCR Methods" [.pdf]

Peter Vallone talk at 58th Annual Meeting of the American Academy of Forensic Sciences (Seattle, WA), February 23, 2006, "Examining Candidate DNA Quantitation Standards with Real-Time Quantitative PCR Assays" [.pdf]

Margaret Kline presentation at the NIJ DNA Grantees meeting (Crystal City, VA), June 26, 2006, "Progress Toward SRM 2372: Human DNA Quantitation" [.pdf]

Poster describing some NIST work regarding issues behind real-time qPCR for human DNA quantitation

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Last updated: 06/20/2007

Disclaimer: This project was supported by National Institute of Justice Grant Number 2003-IJ-R-029, which is an interagency agreement between NIJ and the NIST Office of Law Enforcement Standards, awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the US Department of Justice. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible.  In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose.