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

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.

Quantifiler® Duo DNA Quantification Kit: A Guiding Tool for Short Tandem Repeat Genotyping of Forensic Samples

“…None of the other species investigated exhibited amplification signal except for chimpanzee, suggesting that the developed triplex assay is specific for human DNA and some higher primates. Cross reactivity of the human DNA quantification assays as well as STR profiling systems to higher primates is reported by many investigators [6][7][8][9][10][11].…”

A Novel Method for Rapid and Selective Extraction of Male DNA from Rape Kits using Alkaline Lysis and Pressure Cycling Technology (PCT)

“…It also aids in identifying unidentified remains by comparing mitochondrial DNA profile to any maternal relative [127].…”

“…They also help identify more than one male contributor to the sample. Though not as discriminatory as autosomal loci, these can be used to determine paternal lineage or identify victims in mass disasters by establishing match with the male relatives [95,127].…”

“…Similarly Ychromosome STR analysis plays a vital role in analyzing mixtures such as sexual assault samples where the male component may be masked by the overwhelming quantities of female tissue [127].…”

The Development of Direct Ultra-Fast PCR for Forensic Genotyping Using Short Channel Microfluidic Systems With Enhanced Sieving Matrices

“…Multi-locus probes and those capable of detecting autosomal and Y-specific probes for male DNA have added advantages and are capable of detecting low amounts of DNA. 68 The use of primatespecific Alu short interspersed nuclear elements (SINE) found throughout thousands of positions in the human genome allows for a high sensitivity method with a wide range (2pg-15ng) of detectable DNA. Compound repeats contain at least two simple repeats combined, for example AGAGACTAAGAGACTA.…”