An Analysis of Single and Multi-Copy Methods for DNA Analysis by Real-Time Polymerase Chain Reaction

• NCJ Number: 255288
• Date Published: June 2011
• Length: 9 pages
• Author(s): Heather E. LaSalle; George Duncan; Bruce McCord
• Agencies: NIJ-Sponsored
• Publication Type: Research (Applied/Empirical), Report (Study/Research), Report (Grant Sponsored), Program/Project Description
• Grant Number(s): 2006-DN-BX-K006
• Award: An Investigation of the Effect of DNA Degradation and Inhibition on PCR Amplification of Single Source and Mixed Forensic Samples

Annotation

The goal of this project was to examine and compare two commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR.

Abstract

One approach, Quantifiler Duo, uses a TaqMan assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY , uses a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches, the study used 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. The study also examined data from both systems, using casework samples. The results indicate that both systems produced linear estimates for DNA quantity over a broad range of input DNA; however, researchers observed some apparent copy number effects when comparing the three NIST standards, which was attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy, and the multi-copy approach produced better sensitivity. Thus, the choice of which system to use should depend upon the goals of the user. 26 references (publisher abstract modified)