Elsevier

Analytical Biochemistry

Volume 337, Issue 1, 1 February 2005, Pages 89-97
Analytical Biochemistry

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification

https://doi.org/10.1016/j.ab.2004.09.036Get rights and content

Abstract

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100–0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.

Section snippets

PCR primer and probe design

Oligonucleotide PCR primers and TaqMan-MGB probes were designed using Primer Express software (Applied Biosystems). Primers were purchased from Sigma–Genosys, and probes were purchased from Applied Biosystems (Table 1). Each primer pair was evaluated in our laboratory using standard agarose gel electrophoresis prior to the purchase of the probes.

The human nuclear DNA quantification assay is an intra-Alu-based design incorporating the 7-bp duplicated region characteristic of the Yb lineage of Alu

Results

Here, we report the development of a comprehensive set of human-specific, target-specific, multiplex PCR assays using TaqMan-MGB fluorescent-labeled probes, including the first reported triplex assay for the simultaneous detection and quantitation of human nDNA, mtDNA, and male Y DNA in a single reaction. The nuclear target in this multiplex quantification system is an intra-Alu PCR assay designed within the largely human-specific subfamily of AluYb8 elements (Fig. 1). There are about 1800

Discussion

In this study we have designed and evaluated a comprehensive set of human-specific multiplex PCR assays for the rapid quantitation of human nuclear, mitochondrial, and sex chromosome DNA. Forensic DNA testing typically involves analysis of autosomal STRs, Y-chromosome STRs, or mitochondrial DNA sequence determination at the hypervariable regions 1 and 2 (HV-1 and HV-2). The decision to implement one or more of these approaches is based on several factors, such as the type of evidence, case

Acknowledgments

This research was supported by the Louisiana Board of Regents Governor’s Biotechnology Initiative GBI (2002-005) (M.A.B.), National Science Foundation EPS-0346411 (M.A.B.), and the State of Louisiana Board of Regents Support Fund (M.A.B.). Benjamin Perodeau was supported by National Institutes of Health P20 RR16456 from the BRIN program of the National Center for Research Resources. Nadica Stoilova and Meredith Laborde were supported by a Howard Hughes Medical Institute grant through the

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