Human Genomic DNA Quantitation System, H-Quant: Development and Validation for Use in Forensic Casework

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Abstract:

ABSTRACT:

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. 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/L of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

Keywords: Alu repeats; AluYb8; DNA quantitation; DNA typing; SYBR® green I; forensic science; human DNA; human identification; polymerase chain reaction; real-time PCR

Document Type: Research Article

DOI: http://dx.doi.org/10.1111/j.1556-4029.2006.00369.x

Affiliations: 1: ReliaGene Technologies, Inc., 5525 Mounes St. Suite 101, New Orleans, LA 70123. 2: Department of Biological Sciences, Biological Computation, and Visualization Center, Center for BioModular Multi-Scale Systems, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA 70803.

Publication date: March 1, 2007

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