NCJ Number
226736
Journal
Problems of Forensic Sciences Volume: 76 Dated: 2008 Pages: 355-368
Date Published
2008
Length
14 pages
Annotation
This report describes the development of a novel method of simultaneous quantitative determination of human nuclear and male Y-chromosome DNA by real-time polymerase chain reaction (RT-PCR) assay.
Abstract
The authors have based their investigations on the method proposed by Walker et al., which consists of a simultaneous amplification of the Alu Yb8 and Y-DNA sequences by RT-PCR and allowing analysis of both autonomic DNA and Y-chromosome DNA. The work done by the current authors involved the development of a method for specific determination of total human DNA content (nDNA) and male DNA (Y-DNA) in a single multiplex RT-PCR (nYDNA) reaction. In order to determine these components, the authors used the Alu Yb8 sequence and the human Y chromosome-specific sequence. The method developed is highly sensitive and reliable. It detects DNA concentration at the level of a single haploid cell (nDNA) and is characterized by high species specificity, while allowing the determination of whether male Y-chromosome DNA is present in a given sample; and if so, in what amount. The investigations were performed using DNA quantitation standards and human and animal DNA samples. DNA was extracted by a non-enzymatic method, and DNA concentration was determined by fluorometry. Other descriptions of materials and methods address real-time PCR primers and probes, real-time PCR reagents, real-time PCR quantitation of DNA, determination of multiplex RT-PCR assay sensitivity, determination of the effect of animal DNA admixture on quantitation of nDNA and Y-DNA, and determination of degraded DNA concentration levels using a Quantifiler kit and a multiplex RT-PCR (nYDNA) reaction. 4 tables, 4 figures, and 13 references