Method for detecting polymorphism rs9005 of human stomach cancer susceptibility gene IL-1RN through MspI

A gene polymorphism, susceptibility gene technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of expensive instruments, poor specificity, long steps, etc., to achieve a wide range of applications, predict susceptibility , the effect of simple operation

Inactive Publication Date: 2016-05-11
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The Taqman fluorescent probe method requires expensive instruments and long steps, the cost is high, and it is difficult to be widely used in the laboratory
[0007] The disadvantage of ...

Method used

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  • Method for detecting polymorphism rs9005 of human stomach cancer susceptibility gene IL-1RN through MspI
  • Method for detecting polymorphism rs9005 of human stomach cancer susceptibility gene IL-1RN through MspI
  • Method for detecting polymorphism rs9005 of human stomach cancer susceptibility gene IL-1RN through MspI

Examples

Experimental program
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Embodiment 1

[0073] Example 1. Determination of human IL-1RNrs9005 polymorphism in human gastric cancer tissue samples

[0074] In a specific embodiment of the present invention, the specific steps for detecting the human IL-1RN gene polymorphism rs9005 are as follows:

[0075] (l) Obtain the surgically excised gastric cancer tissue, and use the phenol-chloroform method to extract the genomic DNA of the gastric cancer tissue as the DNA to be tested. The extraction steps are as follows:

[0076] 1) Thaw the gastric cancer tissue block, wash away blood stains with physiological saline, cut out 0.1g tissue for grinding, add 1ml sterile water, mix upside down, centrifuge at 10000rpm for 10min, discard the supernatant, repeat the above steps twice

[0077] 2) Add 200μl of DNA lysis buffer and 5μl of proteinase K to mix well, and digest in a water bath at 55°C overnight.

[0078] 3) After the digestion is completed, add an equal volume of phenol-chloroform mixture (1:1), shake vigorously to make it become...

Embodiment 2

[0088] Example 2. Determination of human IL-1RNrs9005 polymorphism in whole blood samples of human peripheral blood

[0089] The steps are basically the same as in Example 1, except that the following method is used to extract genomic DNA from human peripheral blood as the test DNA.

[0090] Follow the operating steps of the NEP004-1 Whole Blood Genomic DNA Extraction Kit to extract the genomic DNA of the blood sample to be tested. The specific steps are as follows:

[0091] l) After adding 300μl of blood cells to a 1.5ml centrifuge tube, then add 900μl of cell lysate to mix evenly. After placing it on ice for 10 minutes, centrifuge at 12000rpm in a centrifuge for 1min, discard the supernatant, and add another 900μl of cell lysate. After blowing up the precipitate and mixing well, repeat the above steps;

[0092] 2) Add 600μl of solutionB solution to the precipitate, gently blow up the precipitate with a pipette, add 10μl of proteinase K to mix well, place in a 70°C water bath for 10 ...

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Abstract

The invention discloses a method for detecting polymorphism rs9005 of a human stomach cancer susceptibility gene IL-1RN through MspI. A polymerase chain reaction-restrictive fragment length polymorphism method is adopted, target DNA fragments are amplified through a polymerase chain reaction, then digestion is conducted on the DNA fragments to be detected with restriction enzyme, the restriction enzyme identifies and cuts a specific sequence, then a product obtained after digestion is subjected to electrophoresis, then specific cutting loci of the sequence are analyzed through a restriction enzyme map, and difference of gene sequences of different sources is compared through fragment diversity. Briefly speaking, the corresponding target fragments are amplified through the PCR, then a restriction enzyme digestion reaction is conducted, and difference of the sequences is analyzed by observing the restrictive map after electrophoresis is conducted. The method is good in repeatability, easy to operate and low in cost, and the digestion result is easy to identify.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for detecting the IL-1RN polymorphism rs9005 of the human gastric cancer susceptibility gene by using MspI. Background technique [0002] Single nucleotide polymorphism (singlenucleotide polymorphism, SNP), mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of human heritable variation. It accounts for more than 90% of all known polymorphisms. SNPs are widespread in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that their total number can reach 3 million or more. CAPs (cleavedamplificationpolymorphismsequence-taggedsites) CAPs technology is also known as PCR-RFLP, restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) technology. The basic principle of PCR-RFLP is to use PCR to amplify the target DNA, and then the amplified ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/683C12Q1/6886C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 王凯娟宋春花侯瑞生薛云红高三友杨文杰陈晓霖徐娅娟范琦琪
Owner ZHENGZHOU UNIV
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