Premium
Development of a Human‐Specific Real‐Time PCR Assay for the Simultaneous Quantitation of Total Genomic and Male DNA *
Author(s) -
Horsman Katie M.,
Hickey Jeffrey A.,
Cotton Robin W.,
Landers James P.,
Maddox Lewis O.
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.1556-4029.2006.00183.x
Subject(s) - genomic dna , taqman , genbank , primer (cosmetics) , real time polymerase chain reaction , microbiology and biotechnology , dna , biology , nuclease , primer dimer , southern blot , polymerase chain reaction , genetics , computational biology , gene , chemistry , multiplex polymerase chain reaction , organic chemistry
A duplex real‐time quantitative PCR assay was developed for forensic DNA analysis, which provides simultaneous quantitation of total genomic human DNA and human male DNA. The assay utilizes two spectrally resolved fluorogenic probes in a 5′ nuclease (TaqMan™) assay. Within the range of organisms empirically tested and based upon theoretical specificity using National Center for Biotechnology Information GenBank sequences, primer and probe sequences were shown to be human specific, and the Y‐chromosome probe, male‐specific. A mixture‐challenge study resulted in accurate quantitation of 25 pg male DNA in a mixture of up to 1:5000 (male:female DNA). Additional experimental results include comparisons with the slot blot method and commercial real‐time PCR kits. The assay developed addresses the shortcomings of the traditional slot blot method as well as the commercial real‐time PCR kits. This method is shown to be specific, relatively simple, rapid, has low limits of detection, and consumes limited sample in addition to reporting both the male and total genomic DNA concentrations present.