NCJ Number
217651
Journal
Journal of Forensic Sciences Volume: 52 Issue: 2 Dated: March 2007 Pages: 364-370
Date Published
2007
Length
7 pages
Annotation
This paper reports on the development and validation of an improved assay method called human DNA quantification (H-Quant), which can be used for quantitation of human DNA in forensic samples by using the 7500 real-time PCR System (Applied Biosystems, Foster City, CA).
Abstract
The system's sensitivity was sufficient for quantification of as little as 7.6 pg/ml of human DNA. The data indicate that the H-Quant system is valid and reliable for quantitation of human DNA in forensic DNA analysis. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/ml of human DNA. The amplicon generated in the H-Quant assay is 216bp, which is within the same range of the common amplifiable short tandem repeat amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. 1 table, 6 figures, and 22 references