Method for testing SNP combining restrictive zyme cutting method and mass spectrometry

A restriction enzyme and mass spectrometry detection technology, which is applied in biochemical equipment and methods, measuring devices, microbial determination/inspection, etc., can solve the problems of insufficient detection resolution and slow pre-processing speed, so as to improve detection efficiency, Save detection time and achieve high resolution

Inactive Publication Date: 2008-08-27
BIOYONG TECH
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  • Abstract
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Problems solved by technology

[0012] The present invention is a corresponding solution to the problems of slow pre-processing speed or i

Method used

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  • Method for testing SNP combining restrictive zyme cutting method and mass spectrometry
  • Method for testing SNP combining restrictive zyme cutting method and mass spectrometry
  • Method for testing SNP combining restrictive zyme cutting method and mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Design and amplify the primer of 669 position SNPs of hepatitis B virus P gene

[0049] Hepatitis B virus (HBV) infection is a serious worldwide health problem, and nearly one million people die from various diseases caused by HBV infection every year. However, for the diagnosed hepatitis B patients, there are multiple SNP mutations in the hepatitis B virus genome in different individuals, which leads to the resistance of patients with the same disease to the same drug. If the SNP markers in hepatitis B patients are studied, the root cause of the differences in the sensitivity of different individuals to the same drug will be revealed, which will help researchers to screen more suitable drugs.

[0050] Among them, the 669th nucleotide of the P gene (NCBI Gene ID: 944565) encoded by the hepatitis B virus genome undergoes a C→A mutation, which changes the encoded amino acid from leucine to methionine (L180M), thereby producing the antiviral drug Lamy Fuding...

Embodiment 2

[0055] Embodiment 2: Amplify the 669 SNP fragments comprising the P gene of hepatitis B virus

[0056] The size of the PCR reaction fragment of the two sets of primers is 55 nucleotides, and contains a 10-nucleotide restriction endonuclease recognition sequence. The PCR reaction system and reaction parameters of two groups of primers are also consistent, and the PCR reaction system is: 10ul system, wherein hepatitis B virus genomic DNA 1.5ul (initial concentration 10ng / ul), each 1ul of forward primer and reverse primer (initial concentration 5pmol / ul). ul), dNTPs 0.8ul (initial concentration 2.5mM each), buffer 1ul (initial concentration 1x, containing Mg 2+ ), HSTaq enzyme 0.05ul (initial concentration 5U / ul), sterile deionized water 4.65ul; the cycle parameters of the PCR reaction are: 95°C for 5min, then cycle 30 times at 95°C for 20s, 56°C for 20s, and 72°C for 20s, Then 72°C for 7min. After the PCR reaction, the copy number of the nucleotide sequence with the SNP site i...

Embodiment 3

[0057] Example 3: Digestion of amplified products with specific restriction enzymes

[0058] After the PCR reaction, a thorough restriction endonuclease reaction was carried out by corresponding restriction endonucleases, and the reaction system and reaction parameters were slightly different among the products of each group of primers.

[0059] PCR product of the first group of primers: 20ul system, wherein PCR product 10ul, enzyme buffer 2ul (initial concentration 10x), MlyI enzyme 0.4ul (initial concentration 10U / ul), sterile deionized water 7.6ul; enzyme digestion reaction The parameters are: 37°C for 40 minutes, 95°C for 20 minutes, and ice-water bath for 20 minutes.

[0060] The PCR product of the second group of primers: 20ul system, wherein PCR product 10ul, enzyme buffer 2ul (initial concentration 10x), HgaI enzyme 2ul (initial concentration 2U / ul), sterile deionized water 6ul; Enzyme digestion reaction parameters are : 37°C for 40 minutes, 65°C for 20 minutes.

[0...

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Abstract

The invention provides a novel method for checking the polymorphism of mononucleotide by a combination of a special restriction endonuclease and a mass spectrometric method. Key points of the invention are as follows: a primer taking part in the PCR reaction contains recognition sites and endonuclease sites of different positions of the special restriction endonuclease, and PCR products undergo the restriction endonuclease digestion, which generates a single chain nucleotide segment containing SNP sites; after processes of purification and point target, etc., a sample which can be directly used for the substrate auxiliary laser analytic ionization flight time mass spectrometric detection is prepared. Compared with the prior art, the novel method has the advantages of short time, low cost, high resolution and convenient and easy operation.

Description

technical field [0001] The present invention relates to mass spectrometry analysis of organic biomolecules, especially nucleic acid molecules, in genomics and molecular biology, and specifically provides a method for combining restriction enzyme digestion and mass spectrometry to detect single nucleotide polymorphisms, more specifically Specifically, a method for the detection of genomic single nucleotide polymorphisms in matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is provided. technical background [0002] Human genetic information is stored in the genome. In 2002, the Human Genome Project, an international cooperation project, was finally completed, drawing a fine map of the human genome structure and providing a reference sequence for related research. The human genome sequence consists of a total of 3 billion loci, and studies have shown that the genome difference between any two individuals is about 0.1%, that is, a differ...

Claims

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Application Information

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IPC IPC(8): G01N27/64C12Q1/68
Inventor 赵洪斌马庆伟于中连吕芳吕萍萍
Owner BIOYONG TECH
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