Developmental validation of a real-time quantitative PCR assay for automated quantification of human DNA.
Our laboratory has developed an automated real-time quantitative PCR assay for detecting human DNA. The assay utilizes an in-house, custom-designed TaqMan-MGB sequence-specific probe (CFS-HumRT) and the ABD 7900HT SDS platform. Developmental validation has followed TWGDAM (1) guidelines and demonstrates that the assay is primate specific, is highly sensitive, yields consistent results, and works with human DNA extracted from a variety of body fluid stains. When operating within the dynamic range of the system using high-quality DNA samples. the technique yields similar quantification results to our current QuantiBlot assay with the added benefit of time saving through automation. Furthermore, the QPCR assay identifies how much amplifiable DNA is in a sample and thus has the potential to predict PCR success in downstream applications such as STR analysis.
Richard
Melanie L
ML
Biology Section, Centre of Forensic Sciences, Toronto, Ontario, Canada.
Frappier
Roger H
RH
Newman
Jonathan C
JC
D000818:Animals; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D005006:Ethnicity / Q000235:genetics; D005554:Forensic Medicine / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences; D015003:Yeasts / Q000235:genetics
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA