Assay for quantitation of human DNA using
View Patent ↗An assay for determining presence of human DNA in a sample in which non-human DNA may also be present and for quantitating such human DNA. One embodiment uses intra-Alu based PCR and another uses inter-Alu based PCR. The assays are performed without unique, expensive equipment. The assays are based on detection of multiple-copy Alu elements recently integrated into the human genome that are largely absent from non-human primates and other mammals.
1. A process for quantitating a human DNA in a sample, said process comprising the steps of:
providing a sample to be analyzed;
amplifying predetermined genomic DNA of an Alu element subfamily by using primers, said Alu element subfamily being more enriched in the human genome than in any non-human primate genome, the amplification being intra-Alu polymerase chain reaction amplification, each of said primers including a subfamily-specific diagnostic mutation, said Alu element subfamily being Yb8 subfamily or Yd6 subfamily; and
measuring the amount of the human DNA by comparing the amplified DNA with a reference to quantitate the human DNA in the sample.
2. The process of claim 1 , wherein the amplification is a polymerase chain reaction with the primers containing the following sequences:
5′ CGAGGCGGGTGGATCATGAGGT 3′
(SEQ ID NO: 3)
and
5′ TCTGTCGCCCAGGCCGGACT 3′.
(SEQ ID NO: 4)
3. The process of claim 1 , wherein the amplification is a polymerase chain reaction with the primers containing the following sequences:
5′ GAGATCGAGACCACGGTGAAA 3′
(SEQ ID NO: 5)
and
5′ TTTGAGACGGAGTCTCGTT 3′.
(SEQ ID NO: 6)
4. The process of claim 1 , wherein the measurement step comprises the step of measuring the amount of the human DNA on an agarose gel stained with ethidium bromide.
5. The process of claim 1 , wherein the measurement step comprises the step of measuring the amount of the human DNA by using a qPCR system.
6. The process of claim 1 , wherein the measurement step comprises the step of measuring the amount of the human DNA by using TaqMan chemistry.
7. A process for quantitating a human DNA in a sample, said process comprising the steps of:
providing a sample to be analyzed;
amplifying predetermined genomic DNA of an Alu element subfamily by using primers, each of said primers including a subfamily-specific diagnostic mutation, a copy number of said predetermined genomic DNA in the human genome being higher than a copy number of said predetermined genomic DNA in any non-human primate genome, the amplification being intra-Alu polymerase chain reaction amplification, said Alu element subfamily being Yb8 subfamily or Yd6 subfamily; and
measuring the amount of the human DNA by comparing the amplified DNA with a reference.
8. A process for quantitating a human DNA in a sample, said process comprising the steps of:
providing a sample to be analyzed;
amplifying predetermined genomic DNA of an Alu element subfamily by using primers, said Alu element subfamily being Yd6 subfamily, said Alu element subfamily being more enriched in the human genome than in any non-human primate genome, the amplification being intra-Alu polymerase chain reaction amplification; and
measuring the amount of the human DNA by comparing the amplified DNA with a reference to quantitate the human DNA in the sample.
9. A process for quantitating a human DNA in a sample, said process comprising the steps of:
providing a sample to be analyzed;
amplifying predetermined genomic DNA of an Alu element subfamily by using primers, said Alu element subfamily being Yd6 subfamily, said predetermined genomic DNA including subfamily-specific diagnostic mutations, a copy number of said predetermined genomic DNA in the human genome being higher than a copy number of said predetermined genomic DNA in any non-human primate genome, the amplification being intra-Alu polymerase chain reaction amplification; and
measuring the amount of the human DNA by comparing the amplified DNA with a reference.