Multiplex compositions and methods for quantification of human nuclear DNA and human male DNA and detection of PCR inhibitors
AssigneeApplied Biosystems, LLC
InventorsJaiprakash G. Shewale, Manohar Furtado, Pius Brzoska, Maura Barbisin, Rixun Fang, Michael Malicdem, Cristin O'Shea
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Abstract
The invention relates to a method for simultaneous quantification of human nuclear DNA and human male DNA in a biological sample while also detecting the presence of PCR inhibitors in a single reaction. The multiplex quantification method also provides a ratio of human nuclear and male DNA present in a biological sample. Such sample characterization is useful for achieving efficient and accurate results in downstream molecular techniques such as genotyping.
Claims18
A method for simultaneously quantitating human nuclear DNA and human male DNA in a sample and ascertaining the accuracy of said quantitation, the method comprising:
- combining the sample under amplification conditions with primer pairs capable of specifically amplifying a 60-500 base pair sequence comprising human specific RPPH1, and a 60-500 base pair sequence comprising human male specific SRY loci;
- an internal plasmid control (IPC) comprising SEQ ID NO:1, and a primer pair capable of amplifying a 60-500 base pair sequence comprising SEQ ID NO:1;
- labeled probes for detecting the amplified RPPH1, SRY, and SEQ ID NO:1;
- amplifying the loci and plasmid sequence by a multiplex real-time polymerase chain reaction (PCR);
- and
- determining the amount of the amplified loci and plasmid sequence,
- wherein the amount of amplified loci provides a quantitative value of the amount of the amplified loci and the amount of the amplified plasmid sequence indicates the accuracy of said quantitation.
A method for simultaneously quantitating human nuclear DNA and human male DNA in a sample and ascertaining the accuracy of said quantitation, the method comprising:
- combining the sample under amplification conditions with primer pairs capable of specifically amplifying a 60-500 base pair sequence comprising human specific RPPH1, and a 60-500 base pair sequence comprising human male specific SRY loci;
- an internal plasmid control (IPC) comprising SEQ ID NO:2, and a primer pair capable of amplifying a 60-500 base pair sequence comprising SEQ ID NO:2;
- labeled probes for detecting the amplified RPPH1, SRY, and SEQ ID NO:2;
- amplifying the loci and plasmid sequence by a multiplex real-time polymerase chain reaction (PCR);
- and determining the amount of the amplified loci and plasmid sequence, wherein the amount of amplified loci provides a quantitative value of the amount of the amplified loci and the amount of the amplified plasmid sequence indicates the accuracy of said quantitation.
Dependencies are inferred from the claim text.