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Quantitation of DNA for Forensic DNA Typing by qPCR (quantitative PCR): Singleplex and Multiple Modes for Nuclear and Mitochondrial Genomes, and the Y Chromosome

NCJ Number
Date Published
89 pages
This report presents three quantitation assays developed to improve the accuracy and precision of DNA quantification, as well as tools to increase the effectiveness of the analysis of complex forensic samples.
The quantitation of DNA plays a central role in all areas and applications of forensic DNA analysis. The careful evaluation of the quantity of DNA extracted from biological samples is an imperative for DNA typing via PCR. However, the quantitation methods commonly employed in forensics are often less accurate, time consuming, and require the consumption of a substantial percentage of what may be a precious sample. In this report, supported by the U.S. Department of Justice, National Institute of Justice, the use of real-time quantitative PCR (qPCR), a method commonly employed for a variety of molecular diagnostic and biotechnology procedures, was proposed for the quantification of DNA in forensic samples. The intent is to develop qPCR assays to quantify the human nuclear genome, the human mitochondrial genome, and the human Y-chromosome, and to multiplex these assays in order to quantify more than one genome (or chromosome) in a single reaction. Three quantitation assays were developed based on real-time quantitative PCR (qPCR) methods and include: (1) a duplex qPCR assay that quantifies simultaneously the human nuclear and mitochondrial genomes; (2) a triplex assay for assessing the quantity and quality of human nuclear DNA; and (3) several singleplex assays to quantify human male (Y-chromosomal) DNA. All assays (tests) were developed utilizing 5’-hydrolysis detection chemistries for data collection on the ABI 7000 instrument. References

Date Published: January 1, 2005