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Simultaneous Determination of Total Human and Male DNA Using a Duplex Real‐Time PCR Assay
Author(s) -
Nicklas Janice A.,
Buel Eric
Publication year - 2006
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/j.1556-4029.2006.00211.x
Subject(s) - taqman , dna , primer (cosmetics) , microbiology and biotechnology , biology , duplex (building) , microsatellite , polymerase chain reaction , str analysis , real time polymerase chain reaction , genetics , gene , chemistry , allele , organic chemistry
A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan ™ technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 μL of 64 ng/μL to 0.5 pg/μL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.