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Method of detecting gene mutation

a gene mutation and gene technology, applied in the field of gene mutation detection methods, can solve the problems of complex procedures, high price, specialized instruments, and cannot be easily performed in clinical laboratories, and achieve the effect of simple and rapid detection of gene mutations

Inactive Publication Date: 2006-06-15
MATSUBARA YOICHI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] An object of the present invention is to provide a method of simply and quickly detecting a gene mutation.
[0007] The present inventors have used to achieve the present invention the findings that the use of certain primers and probes under a certain condition enables both amplification and hybridization of nucleic acids in one reaction system, and also enables a easy detection of a hybrid formed by the hybridization.
[0011] (2) The method according to item (1), wherein the mutation site is a point mutation, and the reaction solution for effecting the DNA amplification further contains an unlabeled oligonucleotide having a base sequence different in a single base at the position of the point mutation from the base sequence of the labeled hybridization probe, in an amount sufficient to enhance the specificity of hybridization of the amplified DNA to the hybridization probe.

Problems solved by technology

These methods require complicated procedures including hybridization or electrophoresis after DNA amplification.
However, these methods require high-priced, specialized instruments and cannot be easily performed at clinical laboratories.
Alternatively, the SSCP method, chemical cleavage method, and DHPLC method are widely used for screening of gene mutations, and are highly effective for broad screening of unknown gene mutations; but are inadequate to reliable detection of a known mutation.
In addition, the detection of a point mutation by the use of the sequencing method requires complicated procedures and high expenses, and is of undeniably too much quality for the detection of a known mutation.
At present, all of these methods described above involve special examinations performed at gene research laboratories and find a great difficulty in quick performance in clinical settings (or at bedside).

Method used

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  • Method of detecting gene mutation
  • Method of detecting gene mutation
  • Method of detecting gene mutation

Examples

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example 1

Detection of Mutation g727t in Glycogenosis Type Ia

(1) Reaction System and Experimental Procedure

[0076] For detecting g727t mutation in glycogenosis Type Ia, primers listed in Table 1 were prepared on the basis of known base sequences around the mutation site.

TABLE 1Primers and probes for detection of g727t mutationin glycogenosis type IaPCR forward primer (G6P-E5-1F-Dig):5′-Dig-CCCAAATCCTTCCTATCTCTCACAG-3′(SEQ ID NO: 1)PCR reverse primer (G6P-E5-1R(20)):5′-TGCTGGAGTTGAGAGCCAGC-3′(SEQ ID NO: 2)

[0077] For examining the effect of chain lengths of probes, oligonucleotides listed in Table 2 were prepared as hybridization probes and competing probes.

TABLE 2(I) Biotin-labeled oligonucleotide for detectionof normal base sequence:17 mer:5′-AAGCTGAACAGGAAGAA-Biotin-3′(SEQ ID NO: 3)15 mer:5′-AGCTGAACAGGAAGA-Biotin-3′(SEQ ID NO: 4)13 mer:5′-GCTGAACAGGAAG-Biotin-3′(SEQ ID NO: 5)11 mer:5′-CTGAACAGGAA-Biotin-3′(SEQ ID NO: 6)(II) Unlabeled competing oligonucleotide for de-tection of normal...

example 2

[0088] Detection of Mutation a985g of Medium-Chain Acyl-CoA Dehydrogenase Deficiency, Mutation g1691t of GLDC Gene in Hyperglycinemia, Mutation g681a of Drug-Metabolizing Enzyme Gene CYP2C19, and Point Mutation of Glu487Lys of Aldehyde Dehydrogenase 2 Polymorphism

[0089] The detection method of the present invention was carried out to detect a point mutation, including mutation a985g of medium-chain acyl-CoA dehydrogenase deficiency, mutation g1691t of GLDC gene in hyperglycinemia, mutation g681a of drug-metabolizing enzyme gene CYP2C19, and point mutation of Glu487Lys of aldehyde dehydrogenase 2 polymorphism.

[0090] The PCR primers for amplifying base sequences containing the respective point mutation sites were adjusted in chain length so as to carry out PCR reactions with setting of an annealing temperature of 55° C. In addition, the hybridization probes were designed to have Tm values in the range of 35 to 40° C. As a result, the chain lengths thereof were 10 mers to 15 mers. Th...

example 3

[0095] Detection of Delta F508 Deletion Mutation in Cystic Fibrosis Transmembrane Regulator Protein Gene, 1277insTATC Insertion Mutation in HEXA Gene of Tay-Sachs Disease, 5382insC Insertion Mutation in BRCA1 Gene of Breast Cancer, 6174delT Deletion Mutation in BRCA2 Gene of Breast Cancer, and G1691A Point Mutation in Blood Coagulation Factor V Gene of Thrombosis

[0096] The detection method of the present invention was carried out to detect a mutation, including deltaF508 deletion mutation in the gene of cystic fibrosis transmembrane regulator protein; 1277insTATC insertion mutation in HEXA gene of Tay-Sachs disease; 5382insC insertion mutation in BRCA1 gene of breast cancer; 6174delT deletion mutation in BRCA2 gene of breast cancer; and G1691A point mutation in Blood Coagulation Factor V gene of thrombosis.

[0097] The PCR primers for amplifying base sequences containing the respective mutation sites were adjusted in chain length so as to carry out PCR reactions with setting of an a...

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Abstract

DNA amplification and hybridization are successively carried out in a reaction system containing primers for the DNA amplification and hybridization probes, followed by detecting the hybrid in the reaction solution by affinity chromatography, wherein at least one of the primers to be used in the DNA amplification is labeled with a first labeling agent so that the amplified DNA will be labeled with the first labeling agent, a hybridization probe is labeled with a second labeling agent and contained in a reaction solution for effecting the DNA amplification, the base sequence of the hybridization probe is designed not to inhibit the DNA amplification, and a hybrid is detected by affinity chromatography with the use of the first and second labeling agents.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of detecting a base sequence, and more particularly to a method of detecting a base sequence containing a mutation site such as a point mutation, thereby detecting a gene mutation. BACKGROUND ART [0002] There exist a number of gene polymorphisms on the genome, which have been considered to be deeply associated with susceptibility to diseases, individual variations in drug metabolism, and the like. The detection of the gene polymorphism is indispensable for so-called tailor-made medicine and becomes one of the most important subjects on the clinical applications of genomic science. Among others, much interest is lately focused on SNP (single nucleotide polymorphism; gene polymorphism caused by substitution of a single base) as a marker of the gene polymorphism, on which huge research funds have been spent on a global basis. On the other hand, data on gene mutations associated with various genetic diseases has been accum...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34C07H21/04C12NC12N15/09G01N33/50G01N33/53G01N33/566G01N33/58
CPCC12Q1/6827C12Q2535/131G01N33/5308G01N33/58
Inventor MATSUBARA, YOICHIKURE, SHIGEO
Owner MATSUBARA YOICHI
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