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Method for detecting primer elongation reaction, method and apparatus for distinguishing kinds of basic groups

A technology of base types and detection methods, which can be used in measuring devices, biochemical equipment and methods, and microbial determination/inspection, etc., and can solve problems such as danger

Inactive Publication Date: 2004-06-02
PANASONIC CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in this method concentrated sulfuric acid is used, so it is very dangerous

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  • Method for detecting primer elongation reaction, method and apparatus for distinguishing kinds of basic groups
  • Method for detecting primer elongation reaction, method and apparatus for distinguishing kinds of basic groups
  • Method for detecting primer elongation reaction, method and apparatus for distinguishing kinds of basic groups

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Experimental program
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Embodiment approach 1

[0104] In this embodiment, a method for discriminating the base type of the SNP site of the target DNA in the sample will be described. A method of using four kinds of dNTPs by primer extension reaction (for example, PCR method, ICAN method, LCR method, SDA method, LAMP method, etc. amplification reaction) will be described with reference to FIG. 1 and FIG. 2 . FIG. 1 and FIG. 2 are process schematic diagrams of the method for discriminating the base type of the SNP site of the target DNA in the sample according to the present embodiment.

[0105] In the method of this embodiment, primers (hereinafter referred to as primers) that bind substantially complementary to the nucleotide sequence containing the SNP site of the target DNA and differ according to the elongation reaction of the base species of the SNP site of the target DNA are used. "typing primer"). In addition, in this embodiment, the base at the SNP site in the single-stranded target DNA that acts as the typing prim...

Embodiment approach 2

[0182] In this embodiment, a method for determining whether or not a sample contains DNA having a specific base sequence, that is, a method for detecting DNA having a specific base sequence will be described. The method (for example, PCR method, ICAN method, LCR method, SDA method, LAMP method, etc. amplification reaction) by primer extension reaction using four kinds of dNTPs will be described in detail with reference to FIG. 10 . Fig. 10 is a schematic diagram showing the steps of the method for determining whether or not DNA having a specific base sequence is contained in the sample according to the present embodiment.

[0183] In the method of this embodiment, a primer having a nucleotide sequence capable of complementary binding to DNA having a specific nucleotide sequence is used.

[0184] First, in the process shown in Fig. 10(a), primer 101 containing a base sequence capable of complementary binding to DNA having a specific base sequence, DNA polymerase, and four kinds...

Embodiment 1

[0217] In this example, the detection of λDNA in the sample was performed (for the complete base sequence of λDNA, refer to Accession Nos. V00636, J02459, M17233, and X00906 of the Gen Bank database).

[0218] First, a sample solution A in which λDNA (manufactured by Takara Shuzo Co., Ltd.) was dissolved in distilled water at a concentration of 10 ng / µL and a sample solution B consisting of distilled water alone were prepared. In addition, as shown in FIG. 14( a ), prepare two kinds of primers C and D obtained by completely hybridizing the specific nucleotide sequence of λDNA in distilled water respectively. Primer solutions E and F (both 20 μM) were prepared.

[0219] TaKaRa La Taq (5 U / μL, manufactured by Takara Shuzo Co., Ltd.), TaKaRa La Taq’s special buffer 2×GC buffer I (made by Takara Shuzo Co., Ltd.), dNTP mixture ( Each concentration was 2.5 mM, manufactured by Takara Shuzo Co., Ltd.), and primer solutions E and F were prepared to prepare PCR reaction solutions G and ...

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Abstract

Convenient techniques for discriminating the base type in a base sequence of a nucleic acid are provided. The technique includes the step (a) of preparing a sample solution containing a nucleic acid, a primer having a base sequence that includes a complementary binding region which complementarily binds to the nucleic acid, and a nucleotide; the step (b) of allowing the sample solution to stand under a condition to cause an extension reaction of the primer, and producing pyrophosphate when the extension reaction is caused; the step (c) of bringing the sample solution into contact with the front face of a H<+> hardly permeable membrane having H<+>-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face; the step (d) of measuring the H<+> concentration of at least either one of the solution at the front face side of the H<+> hardly permeable membrane or the solution at the back face side of the H<+> hardly permeable membrane, in a state where the H<+>-pyrophosphatase is immersed in the solution; the step (e) of detecting the extension reaction on the basis of the result of measurement in the step (d) ; and the step (f) of discriminating the base type in the base sequence of the nucleic acid on the basis of the result of detection in the step (e).

Description

[0001] technology area [0002] The present invention relates to a method for detecting an elongation reaction for detecting an elongation reaction of a primer, a method for discriminating base species and base types in a nucleic acid base sequence, and a method for discriminating base species and base types in a nucleic acid base sequence A device, a pyrophosphate detection device, a nucleic acid detection method, and a sample solution introduction chip. Background technique [0003] prior art 1 [0004] A technique for investigating the presence or absence of a nucleic acid having a specific base sequence is a very important technique. For example, it is an essential technology in the diagnosis of genetic diseases, the detection of food contamination caused by bacteria and viruses, and the detection of human infection by bacteria and viruses. [0005] It is known that genetic diseases such as severe combined immunodeficiency syndrome and familial hypercholesterolemia are c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/25C12Q1/68G01N33/00
CPCC12Q1/6869C12Q1/6858C12Q2565/301C12Q2521/543
Inventor 夜久英信行政哲男冈弘章
Owner PANASONIC CORP