Simple, Rapid, and Accurate Quantitation of Human DNA

• NCJ Number: 215340
• Author(s): Eric Buel Ph.D.
• Date Published: January 2006
• Length: 101 pages
• Grant Number(s): 2000-IJ-CX-K012
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: Simple, Rapid, and Accurate Quantitation of Human DNA

Annotation

This final report presents research undertaken to develop a faster, easier, and more quantitative approach to predicting human DNA amounts from forensic casework samples.

Abstract

The research was undertaken in three stages. The first stage of research sought to: (1) develop a method to predict the amount of DNA obtained from an extracted sample; (2) validate the method for forensic use; and (3) determine if an automated system could be developed to perform the quantitation. Researchers used human specific PCR primers with a microplate format and reader to determine human specificity. The human specific sequences chosen for PCR analysis was the human multicopy Alu sequence. The final product was an Alu-based assay using PCR with SYBR Green and a florescence based plate reader to quantitate human DNA. The second stage of research sought to: (1) determine alternative approaches to current methods for the quantitation of human DNA; (2) evaluate each approach for ease of use, flexibility in analytical analysis, and ability to semi-automate the analysis; and (3) validate the method for forensic use. The SYBR Green, Alu-based assay was fully developed and validated and has been implemented for casework and distributed to forensic laboratories. The limited dynamic range of the SYBR Green assay led to the investigation of molecular beacon-type Eclipse probes, TaqMan probes, and LUX primers as possible improvements over SYBR Green. The Eclipse and TaqMan probes yielded similar results. A manuscript for the Eclipse method is presented at the end of Supplement 1. The third stage of research aimed to: (1) develop an assay to quantitate male and female DNA; (2) develop a fast and simple assay that detected alleles at one or two STR loci to be used as a quick sample screening method; (3) improve the original Alu PCR Real-time PCR assay; and (4) disseminate the methods to the forensic community. A TaqMan-based duplex Alu and DYZ5 sequence based assay was developed and validated. Figures, tables, references