Multiplex PCR for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA
A comprehensive set of human specific, target specific, multiplex PCR assays for DNA quantitation is provided. Our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100 ng to 0.1 ng. Human-specificity was demonstrated by the accurate detection of 0.05% and 5% human DNA, respectively, from a complex source of starting templates. Target-specificity was confirmed by the lack of cross-amplification among targets. A high throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X chromosome based system. Background cross-amplification with DNA templates derived from fourteen other species was negligible aside from the male Y assay which produced spurious amplifications from other non-human primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.
1. A process for simultaneous quantitation of a human mitochondrial DNA and at least one of human nuclear DNA and human Y chromosome DNA in a sample, said process comprising the steps of:
performing multiplex polymerase chain reaction for simultaneously amplifying a region of the human mitochondrial DNA containing a human/chimp mismatch and a mitochondrial DNA/nuclear DNA mismatch by using a primer pair specific to human mitochondrial DNA, the region of the human mitochondrial DNA being not the hypervariable region, and at least one region of the human nuclear DNA containing a duplicated region of the Yb lineage of Alu subfamilies and an AluYb8 diagnostic base by using a primer pair specific to human nuclear DNA, and a region of the human Y chromosome DNA containing a 90 base pair sequence which is deleted on the human X-chromosome in an X-Y chromosome homologous region by using a primer pair specific to the human Y chromosome DNA in order to obtain an amplified products; and
quantitating the human mitochondrial DNA, and at least one of the human nuclear DNA and the human Y chromosome DNA by comparing the amplified products with a reference standard.
2. The process of claim 1 , wherein said at least one region includes both the human nuclear DNA and the human Y chromosome DNA.
3. The process of claim 1 , wherein said at least one region is the human nuclear DNA.
4. The process of claim 2 , wherein the primer pair amplifying the region of the human nuclear DNA comprises the following sequences:
5′ CTTGCAGTGAGCCGAGATT 3′;
(SEQ ID NO:13)
and
5′ GAGACGGAGTCTCGCTCTGTC 3′.
(SEQ ID NO:14)
5. The process of claim 2 , wherein the primer pair amplifying the region of the human Y chromosome DNA comprises the following sequences:
5′ CACCTACTGTGCCAGACAATGTG 3′;
(SEQ ID NO:16)
and
5′ CCCATGCCATGTTTGTCATACT 3′.
(SEQ ID NO:17)
6. The process of claim 3 , wherein the primer pair amplifying the region of the human mitochondrial DNA comprises the following sequences:
5′ AATATTAAACACAAACTACCACCTACCT 3′;
(SEQ ID NO:10)
and
5′ TGGTTCTCAGGGTTTGTTATAA 3′.
(SEQ ID NO:11)
7. The process of claim 1 , wherein the quantitation step comprises detecting the amplified products by using a quantitative PCR (qPCR) system.
8. The process of claim 1 , wherein the quantitation step further compnses using a nucleotide probe for the quantitation of the human nuclear DNA, the nucleotide probe containing the following sequence:
5′ ACTGCAGTCCGCAGTCCGGCCT 3′.
(SEQ ID NO:15)
9. The process of claim 1 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human nuclear DNA, the nucleotide probe containing the following sequence:
5′VIC ACTGCAGTCCGCAGTCCGGCCT3′ MGBNFQ
(5′VIC-SEQ ID NO:15-3′MGBNFQ).
10. The process of claim 2 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human Y chromosome DNA, the nucleotide probe containing the following sequence:
5′ TAGGCTCTAGGAATACAAAA 3′.
(SEQ ID NO:18)
11. The process of claim 2 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human Y chromosome DNA, the nucleotide probe containing the following sequence:
5′ NED TAGGCTCTAGGAATACAAAA 3′ MGBNFQ (5′ NED-SEQ
ID NO:18-3′ MGBNFQ).
12. The process of claim 3 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human mitochondrial DNA, the nucleotide probe containing the following sequence:
5′ CCTCACCAAAGCCCATA 3′.
(SEQ ID NO:12)
13. The process of claim 3 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human mitochondrial DNA, the nucleotide probe containing the following sequence:
5′ FAM CCTCACCAAAGCCCATA 3′MGBNFQ
(5′FAM-SEQ ID NO:12-3′MGBNFQ).
14. A process for simultaneous quantitation of human nuclear DNA, human mitochondrial DNA and human Y chromosome DNA in a sample, said process comprising the steps of:
performing multiplex polymerase chain reaction to simultaneously amplify a region of the human nuclear DNA containing a human nuclear specific sequence by using a first primer pair specific to the human nuclear specific sequence, a region of the human mitochondrial DNA containing a human mitochondria specific sequence by using a second primer pair specific to the human mitochondria specific sequence, and a region of the human Y chromosome DNA containing a human Y chromosome specific sequence by using a third primer pair specific to the human Y chromosome specific sequence, the amplified region of the human nuclear DNA containing a duplicated region of the Yb lineage of Alu subfamilies and an AluYb8 diagnostic base at the 3′ end of a forward primer of the first primer pair, the amplified region of the human mitochondrial DNA containing a human/chimp mismatch and a mitochondrial DNA/nuclear DNA mismatch, the region of the human mitochondrial DNA being not the hypervariable region, the amplified region of the human Y chromosome DNA containing a 90-bp sequence which is deleted on the X chromosome in an X-Y homologous region;
measuring the amplified products from the human nuclear DNA, the human mitochondrial DNA and the human Y chromosome DNA by using a first probe for the human nuclear DNA, a second probe for the human mitochondrial DNA, and a third probe for the human Y chromosome DNA, respectively; and
quantitating the human nuclear DNA, the human mitochondrial DNA and the human Y chromosome DNA by comparing the amplified products with a reference standard.
15. The process of claim 14 , wherein the first primer pair amplifying the human nuclear DNA comprises the following sequences:
5′ CTTGCAGTGAGCCGAGATT 3′;
(SEQ ID NO:13)
and
5′ GAGACGGAGTCTCGCTCTGTC 3′.
(SEQ ID NO:14)
16. The process of claim 14 , wherein the first probe for the human nuclear DNA comprises the following sequence:
5′ ACTGCAGTCCGCAGTCCGGCCT 3′.
(SEQ ID NO:15)
17. The process of claim 14 , wherein the first probe for the human nuclear DNA comprises the following sequence:
5′VIC ACTGCAGTCCGCAGTCCGGCCT3′ MGBNFQ
(5′VIC-SEQ ID NO:15-3′MGBNFQ).
18. The process of claim 14 , wherein the second primer pair amplifying the human mitochondrial DNA comprises the following sequences:
5′ AATATTAAACACAAACTACCACCTACCT 3′;
(SEQ ID NO:10)
and
5′ TGGTTCTCAGGGTTTGTTATAA 3′.
(SEQ ID NO:11)
19. The process of claim 14 , wherein the second probe for the human mitochondrial DNA comprises the following sequence:
5′ CCTCACCAAAGCCCATA 3′.
(SEQ ID NO:12)
20. The process of claim 14 , wherein the second probe for the human mitochondrial DNA comprises the following sequence:
5′ FAM CCTCACCAAAGCCCATA 3′MGBNFQ
(5′ FAM-SEQ ID NO:12-3′MGBNFQ.
21. The process of claim 14 , wherein the third primer pair amplifying the human Y chromosome DNA comprises the following sequences:
5′ CACCTACTGTGCCAGACAATGTG 3′;
(SEQ ID NO:16)
and
5′ CCCATGCCATGTTTGTCATACT 3′.
(SEQ ID NO:17)
22. The process of claim 14 , wherein the third probe for the human Y chromosome DNA comprises the following sequence:
5′ TAGGCTCTAGGAATACAAAA 3′.
(SEQ ID NO:18)
23. The process of claim 14 , wherein the third probe for the human Y chromosome DNA comprises the following sequence:
5′ NED TAGGCTCTAGGAATACAAAA 3′ MGBNFQ (5′ NED-SEQ
ID NO:18 3′ MGBNFQ).
24. A process for simultaneously quantitating human X and Y chromosome DNA in a sample, said process comprising the steps of:
performing duplex polymerase chain reaction to amplify a region in SEQ ID NO: 9 of human Y chromosome DNA containing a 90 base pair sequence which starts from position 85 on SEQ ID NO.9 and which is deleted on SEQ ID NO: 8 of the human X-chromosome in an X-Y chromosome homologous region by using a third primer pair specific to the human Y chromosome DNA and to amplify a region in SEQ ID NO: 8 of the human X chromosome DNA in the X-Y chromosome homologous region by using a fourth primer pair; and
quantitating the human Y chromosome DNA and the human X chromosome DNA by comparing the amplified products with a reference standard.
25. The process of claim 24 , wherein the third primer pair amplifying the human Y chromosome comprises the following sequences:
5′ CACCTACTGTGCCAGACAATGTG 3′;
(SEQ ID NO:16)
and
5′ CCCATGCCATGTTTGTCATACT 3′.
(SEQ ID NO:17)
26. The process of claim 24 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human Y chromosome DNA, the nucleotide probe containing the following sequence:
5′ TAGGCTCTAGGAATACAAAA 3′.
(SEQ ID NO:18)
27. The process of claim 24 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human Y chromosome DNA, the nucleotide probe containing the following sequence:
5′ NED TAGGCTCTAGGAATACAAAA 3′ MGBNFQ
(5′ NED-SEQ ID NO:18-3′MGBNFQ).
28. The process of claim 24 , wherein the fourth primer pair amplifying the human X chromosome comprises the following sequences:
5′ CCACGAACTTTAATTAGTCACCTACTGT 3′;
(SEQ ID NO:19)
and
5′ ATTCCTCTCTCCATTATGTTCAATTACA 3′.
(SEQ ID NO:20)
29. The process of claim 24 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human X chromosome DNA, the nucleotide probe containing the following sequence:
5′ CAGACAATGTGATAAATG 3′.
(SEQ ID NO:21)
30. The process of claim 24 , wherein the quantitation step comprises using a nucleotide probe for the quantitation of the human X chromosome DNA, the nucleotide probe containing the following sequence:
5′ VIC CAGACAATGTGATAAATG 3′ MGBNFQ (5′ VIC-SEQ ID
NO:21-3′ MGBNFQ).
31. The process of claim 1 , wherein said at least one region is the human Y chromosome DNA.
32. The process of claim 31 , wherein the primer pair amplifying the region of the human Y chromosome DNA comprises the following sequences:
5′ CACCTACTGTGCCAGACAATGTG 3′;
(SEQ ID NO:16)
and
5′ CCCATGCCATGTTTGTCATACT 3′.
(SEQ ID NO:17)
33. The process of claim 32 , wherein the primer pair amplifying the region of the human mitochondrial DNA comprises the following sequences:
5′ AATATTAAACACAAACTACCACCTACCT 3′;
(SEQ ID NO:10)
and
5′ TGGTTCTCAGGGTTTGTTATAA 3′.
(SEQ ID NO:11)
34. The process of claim 31 , wherein the primer pair amplifying the region of the human mitochondrial DNA comprises the following sequences:
5′AATATTAAACACAAACTACCACCTACCT 3′(SEQ ID NO: 10); and
5′TGGTTCTCAGGGTTTGTTATAA 3′(SEQ ID NO: 11).
35. The process of claim 3 , wherein the primer pair amplifying the region of the human nuclear DNA comprises the following sequences:
5′ CTTGCAGTGAGCCGAGATT 3′;
(SEQ ID NO:13)
and
5′ GAGACGGAGTCTCGCTCTGTC 3′.
(SEQ ID NO:14)
36. The process of claim 35 , wherein the primer pair amplifying the region of the human mitochondrial DNA comprises the following sequences:
5′ AATATTAAACACAAACTACCACCTACCT 3′;
(SEQ ID NO:10)
and
5′ TGGTTCTCAGGGTTTGTTATAA 3′.
(SEQ ID NO:11)
37. The process of claim 2 , wherein the primer pair amplifying the region of the human mitochondrial DNA comprises the following sequences:
5′AATATTAAACACAAACTACCACCTACCT 3′(SEQ ID NO: 10); and
5′TGGTTCTCAGGGTTTGTTATAA 3′(SEQ ID NO: 11);
the primer pair amplifying the region of the human nuclear DNA comprises the following sequences:
5′ CTTGCAGTGAGCCGAGATT 3′;
(SEQ ID NO:13)
and
5′ GAGACGGAGTCTCGCTCTGTC 3′.
(SEQ ID NO:14)
the primer pair amplifying the region of the human Y chromosome DNA comprises the following sequences:
5′ CACCTACTGTGCCAGACAATGTG 3′;
(SEQ ID NO:16)
and
5′ CCCATGCCATGTTTGTCATACT 3′.
(SEQ ID NO:17)