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

The goal of 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 we have 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. We 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 we 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.