Multiplex PCR for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA

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.

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.