Multiplex compositions and methods for quantification of human nuclear DNA and human male DNA and detection of PCR inhibitors
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.
1. 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.
2. The method of claim 1 , wherein the human RPPH1 locus has the sequence SEQ ID NO:3.
3. The method of claim 2 , wherein the primers capable of amplifying human RPPH1 locus have the sequence SEQ ID NO:5 and SEQ ID NO:6, respectively.
4. The method of claim 1 , wherein the human SRY locus has the sequence SEQ ID NO: 4.
5. The method of claim 4 , wherein the primers capable of amplifying human SRY locus have the sequence SEQ ID NO:7 and SEQ ID NO:8, respectively.
6. The method of claim 1 , wherein the primers capable of amplifying SEQ ID NO:1 have the sequence SEQ ID NO:11 and SEQ ID NO:12, respectively.
7. The method of claim 1 , wherein the labeled probes are labeled with a first, second, and third fluorescent moiety that produces a detectable signal upon release of the labeled probe from being bound to its target DNA or plasmid, and the released probes are detected by real-time measurement of an amount of fluorescent emission; wherein the amount of fluorescent emission of each released labeled probe from being bound to the amplified loci provides a quantitative value of the amount of the amplified loci, and the amount of fluorescent emission detected by the release of the labeled probe to the plasmid sequence indicates the accuracy of said quantitation, the quantity of the released labeled probe to the plasmid sequence correlating inversely with the presence of PCR inhibitors.
8. The method of claim 1 , wherein the labeled probes have the sequence SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 13.
9. The method of claim 8 , wherein the RPPH1 probe is labeled with FAM; the SRY probe is labeled with VIC, and the SEQ ID NO:13 probe is labeled with NED.
10. 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.
11. The method of claim 10 , wherein the human RPPH1 locus has the sequence SEQ ID NO:3.
12. The method of claim 11 , wherein the specific primers capable of amplifying human RPPH1 locus have the sequence SEQ ID NO:5 and SEQ ID NO: 6, respectively.
13. The method of claim 10 , wherein the human SRY locus has the sequence SEQ ID NO: 4.
14. The method of claim 13 , wherein the specific primers capable of amplifying human SRY locus have the sequence SEQ ID NO:7 and SEQ ID NO: 8, respectively.
15. The method of claim 10 , wherein the labeled probes are labeled with a first, second, and third fluorescent moiety that produces a detectable signal upon release of the labeled probe from being bound to its target DNA or plasmid, and the released probes are detected by real time measurement of an amount of fluorescent emission; wherein the amount of fluorescent emission of each released labeled probe from being bound to the amplified loci provides a quantitative value of the amount of the amplified loci, and the amount of fluorescent emission detected by the release of the labeled probe to the plasmid sequence indicates the accuracy of said quantitation, the quantity of the released labeled probe to the plasmid sequence correlating inversely with the presence of PCR inhibitors.
16. The method of claim 10 , wherein the RPPH1 probe has the sequence SEQ ID NO: 9, the SRY probe has the sequence SEQ ID NO: 10, and the SEQ ID NO:2 probe has the sequence SEQ ID NO: 13.
17. The method of claim 16 , wherein the RPPH1 probe is labeled with FAM; the SRY probe is labeled with VIC, and the SEQ ID NO:2 probe is labeled with NED.
18. The method of claim 10 , wherein the labeled probes have the sequence SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 13.