Abstract
Forensic analysts routinely encounter samples containing mixtures of DNA from male and female contributors and PCR inhibitors due to exposure to environmental insults. In order to select the appropriate STR analysis methodology for such samples and obtain optimal results at first pass, it is desirable to determine the relative quantities of male and female DNA and to detect the presence of PCR inhibitors at an early stage in the sample processing workflow. Here we describe a multiplex real-time PCR assay that can provide the desired information in a single reaction. Briefly, the simultaneous quantification of human and human male DNA is achieved by measuring the RPPH1 human target and the SRY male-specific target. At the same time a synthetic sequence is co-amplified as an Internal PCR Control (IPC) to detect the presence of PCR inhibitors. The assay has a good dynamic range (0.023–50 ng/μL) and can detect 25 pg/μL of human male DNA in the presence of ten thousand-fold excess of human female DNA. In addition, the ability of the assay to predict PCR inhibition was demonstrated by shifted IPC C T values in the presence of increasing quantities of hematin. All the real-time PCR results showed a good correlation with the downstream STR profiles obtained from a large set of various sample types therefore demonstrating that this assay can be considered a guiding tool to predict the performance of the STR genotyping kits with forensic samples.
Highlights
Quantification of human Deoxyribo Nucleic Acid (DNA) plays a central role in forensic short tandem repeat (STR) profiling for many reasons
We developed a multiplex assay for simultaneous amplification of the human autosomal target ribonuclease P RNA component H1 (RPPH1; Chromosome: 14; Location: 14q11.2; GeneID: 85495), the human male-specific Y-chromosome target sex determining region Y (SRY; Chromosome: Y; Location: Yp11.3; GeneID: 6736), and a synthetic oligonucleotide sequence embedded in a plasmid as an internal PCR control (IPC)
Measurement of the extent of amplification of RPPH1, SRY, and IPC enables the quantification of total human DNA, quantification of human male DNA and indication of the presence of inhibitors of PCR, respectively
Summary
Quantification of human DNA plays a central role in forensic short tandem repeat (STR) profiling for many reasons. Forensic evidence samples routinely contain DNA from non-human species, are contaminated with compounds that inhibit PCR, are mixtures from female and male contributors in various proportions, and have been exposed to environmental insults, leading to DNA degradation. All these factors make STR analyses more challenging. Detection of the minor male component in sexual assault samples during the quantification step can be valuable in determining the need to followup with Y-STR analysis. Detection of both total human and human male components in a single reaction reduces sample consumption. Detection of potential PCR inhibitors is useful in directing further sample analysis towards re-extraction of nucleic acid or the use of more robust kits like AmpFlSTR® MiniFilerTM or Identifiler® Plus PCR DNA Amplification kit
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