Quadruplex real-time PCR for forensic DNA quantitation |
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Authors: | Martin R. Whittle Denilce R. Sumita |
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Affiliation: | Genomic Engenharia Molecular Ltda, Rua Itapeva 500/5AB, 01332-903 São Paulo, SP, Brazil |
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Abstract: | Forensic DNA quantitation is an important initial step preceding PCR amplification of the STR loci even though information concerning the quality of the DNA is not revealed. A quadruplex real-time PCR (qPCR) assay was developed to quantify four DNA targets: (1) the human RB1 gene in nuclear DNA, (2) the DAZ gene present on the human Y chromosome, (3) the ATPase8 gene present in human mitochondrial DNA and (4) an artificial internal positive control to reveal possible PCR inhibition. Primers labeled with four different fluorophores are used together with a single quencher using the antiprimer quenching-based qPCR method in one reaction, in which the resultant amplicons are less than 127 bp in size. Sensitivity was shown to be less than ten copies for all four targets in the absence of amplification inhibition. The amplification remained sensitive in the presence of an excess of non-human DNA. |
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Keywords: | Multiplex real-time PCR PCR inhibition Forensic DNA quantitation |
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