An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

• NCJ Number: 308316
• Journal: Forensic Science International - Genetics Volume: Online Dated: 2010
• Author(s): H. E. Lasalle; G. Duncan; B. McCord
• Date Published: 2010
• Grant Number(s): 2006-DN-BX-K006
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical)
• Award: An Investigation of the Effect of DNA Degradation and Inhibition on PCR Amplification of Single Source and Mixed Forensic Samples

Annotation

The authors of this paper explore two different, commercially available approaches to determine the relative quantities of autosomal Y chromosomal DNA using a real-time polymerase chain reaction.

Abstract

The goal of the research reported in this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler® Duo, utilizes a TaqMan® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches the authors utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. They also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However, the authors did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus, the choice of which system to use should depend upon the goals of the user. (Published Abstracts Provided)