Method for testing SNP using restriction enzymes double zyme cutting

A technology of restriction endonuclease and restriction enzyme, which is applied in the field of detection of SNP by restriction endonuclease double digestion, can solve the problems of insufficient detection resolution and slow pre-processing speed, so as to improve detection efficiency and save Detection time, effect of high versatility

Inactive Publication Date: 2008-08-27
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The present invention is a corresponding solution to the problems of slow pre-processing speed or insufficient detection resolution in previous methods for detecting genomic SNP sites based on MALDI-TOF MS

Method used

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  • Method for testing SNP using restriction enzymes double zyme cutting
  • Method for testing SNP using restriction enzymes double zyme cutting
  • Method for testing SNP using restriction enzymes double zyme cutting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: Design and amplify the primer of 669 position SNPs of hepatitis B virus P gene

[0046] 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.

[0047]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

[0051] Embodiment 2: Amplify the 669 SNP fragments comprising the P gene of hepatitis B virus

[0052] The size of the PCR reaction fragment is 55 nucleotides, including a 10-nucleotide restriction endonuclease recognition sequence. The PCR reaction system is: 10ul system, including 1.5ul of hepatitis B virus genomic DNA (initial concentration 10ng / ul), 1ul each of forward primer and reverse primer (initial concentration 5pmol / ul), dNTPs 0.8ul (each initial concentration of 2.5mM) , buffer solution 1ul (initial concentration 1x, containing Mg 2+ ), HS Taq 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 , and then 72°C for 7min. After the PCR reaction, the copy number of the nucleotide sequence with the SNP site in the hepatitis B virus genome DNA is amplified. After the completion of the reaction, the PCR product can b...

Embodiment 3

[0053] Example 3: Digestion of amplified products with specific restriction enzymes

[0054] After the PCR reaction, carry out a thorough restriction reaction with corresponding restriction endonucleases. If other restriction endonucleases are used, the reaction system and reaction parameters are slightly different due to the selected restriction endonucleases.

[0055] The first enzyme digestion is a 20ul system, including 10ul of PCR product, 2ul of enzyme buffer (initial concentration 10x), 1ul of HgaI enzyme (initial concentration of 2U / ul), 7ul of sterile deionized water; the enzyme digestion reaction parameters are: 37 ℃ for 40min, 65℃ for 20min; the second digestion is a 30ul system, including 20ul of the first digestion product, 3ul of enzyme buffer (initial concentration 10x), 1.5ul of BsmFI enzyme (initial concentration of 2U / ul), sterile Deionized water 5.5ul; enzyme digestion reaction parameters: 65°C for 40min, 95°C for 20min, ice-water bath for 20min.

[0056] A...

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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: positive and negative primers taking part in the PCR reaction contain two special restriction endonuclease sites and recognition sites, and the recognition sites and the endonuclease sites are different in position. PCR products undergo the restriction endonuclease digestion twice, 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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Patent Type & Authority Applications(China)
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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