Method of detecting or quantitatively determining mitochondrial DNA 3243 variation, and kit therefor

Abstract

A method for detecting a DNA having the mitochondrial DNA 3243 mutation is disclosed. Quantitative PCR is used with a primer having a nucleotide sequence complementary to the nucleotide sequence starting from the nucleotide number 243 in SEQ ID NO: 2 and having a length of 12 to 30 nucleotides. A method is also disclosed for detecting a DNA having the mitochondrial DNA 3243 mutation by using a nucleic acid probe which is end labeled with a fluorescent dye. The fluorescence of the fluorescent dye decreases upon hybridization. The nucleic acid probe has a nucleotide sequence complementary to the nucleotide sequence starting from nucleotide number 230 in the nucleotide sequence of SEQ ID NO: 2 and a length of 14 to 40 nucleotides. The 3′ end of the probe is labeled with the fluorescent dye.

What is claimed is: 
     
       1. A method for detecting a mutation comprising performing a melting curve analysis for a nucleic acid having a single nucleotide polymorphism site by using a nucleic acid probe having a 3′ terminal cytosine labeled with a fluorescent dye and measuring fluorescence of the fluorescent dye, and detecting the mutation on the basis of the result of the melting curve analysis, wherein the single nucleotide polymorphism is a mutation at the 3243rd position in a mitochondrial DNA, and the nucleic acid probe consists of the nucleotide sequence of SEQ ID NO: 21 or 22,
 wherein a region containing the single nucleotide polymorphism site in a nucleic acid contained in a sample is amplified to obtain the nucleic acid showing the single nucleotide polymorphism, and 
 wherein the amplification is performed by a method of using a DNA polymerase in the presence of a nucleic acid probe. 
 
     
     
       2. A method of determining the ratio of a mutant type deoxyribonucleic acid (DNA) of mitochondrial DNA 3243 mutation in a sample comprising:
 adding to the sample a labeled nucleic acid probe which hybridizes to the mutant type DNA, wherein the probe consists of the nucleotide sequence of SEQ ID NO: 21 or 22,   obtaining a melting curve for the probe to the DNA in the sample, and   comparing the melting curve for the probe to the DNA in the sample with at least one melting curve with a known mutant ratio, wherein the melting curve with a known mutant ratio indicates the mutant ratio in the sample.   
     
     
       3. The method according to claim 2, wherein the probe is labeled with a fluorescent dye, and the melting curve is obtained by detecting fluorescence of the fluorescent dyes. 
     
     
       4. The method according to claim 2, wherein the probe consists of the nucleotide sequence of SEQ ID NO: 21. 
     
     
       5. The method according to claim 2, wherein the probe consists of the nucleotide sequence of SEQ ID NO: 22. 
     
     
       6. The method according to claim 2, further comprising amplifying a region containing the mutation prior to obtaining the melting curve. 
     
     
       7. The method according to claim 6, wherein the amplification is performed in the presence of the probe. 
     
     
       8. The method according to claim 2, further comprising:
 adding the probe to a sample with a known mutant ratio,   obtaining a melting curve with a known mutant ratio.   
     
     
       9. The method according to claim 2, wherein the melting curve for the probe to the DNA in the sample is compared with two or more melting curves with known mutant ratios. 
     
     
       10. The method according to claim 2, wherein the probe consists of the nucleotide sequence of SEQ ID NO: 21 or 22 and is labeled with a fluorescent dye, and the melting curve for the probe to the DNA in the sample is compared with two or more melting curves with known mutant ratios. 
     
     
       11. A method for detecting a mutation comprising
 performing a melting curve analysis for a nucleic acid having a single nucleotide polymorphism site by using a nucleic acid probe having a 3′ terminal cytosine labeled with a fluorescent dye and   measuring fluorescence of the fluorescent dye, and   detecting the mutation on the basis of the result of the melting curve analysis, wherein the single nucleotide polymorphism is a mutation at the 3243rd position in a mitochondrial DNA, and the nucleic acid probe consists of the nucleotide sequence of SEQ ID NO: 21 or 22.   
     
     
       12. The method according to claim 11,
 wherein a region containing the single nucleotide polymorphism site in a nucleic acid contained in a sample is amplified to obtain the nucleic acid showing the single nucleotide polymorphism, and   wherein the amplification is performed by a method of using a DNA polymerase in the presence of a nucleic acid probe.