Method for identifying polymorphism of human breast cancer genes RAD51 rs7180135 by aid of BccI
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A breast cancer, polymorphism technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of difficult universal use, expensive restriction endonucleases, and high experimental costs, and achieves simple operation, Low cost, predicts the effect of susceptibility
Inactive Publication Date: 2016-06-22
ZHENGZHOU UNIV
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At present, the restriction endonucleases often used to identify the human RAD51 gene polymorphism rs45549040 are expensive (for the reference price of some endonucleases, please refer to Table 1 below), the cost of the experiment is high, and it is difficult to be widely used in the laboratory
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Embodiment 1
[0075] Example 1. Determination of human RAD51rs7180135 polymorphism in human breast cancer tissue samples.
[0076] In a specific embodiment of the present invention, the specific steps for detecting the human RAD51 gene polymorphism rs7180135 are as follows:
[0077] (l) Obtain surgically removed 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:
[0078] 1) Thaw the gastric cancer tissue block, wash away blood stains with normal saline, cut out 0.1g of tissue for grinding, add 1ml of sterilized water, mix upside down, centrifuge at 10000rpm for 10min, discard the supernatant, repeat the above steps twice
[0079] 2) Add 200 μl of DNA lysate and 5 μl of proteinase K, mix well, and digest overnight in a water bath at 55°C.
[0080] 3) After the digestion is completed, add an equal volume of phenol-chloroform mixture (1:1) and shake vigorously to make...
Embodiment 2
[0090] Example 2. Determination of Human RAD51rs7180135 Polymorphism in Whole Blood Specimen of Human Peripheral Blood
[0091] 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 DNA to be tested.
[0092] Follow the operation steps of 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:
[0093] l) After adding 300 μl of blood cells into a 1.5ml centrifuge tube, add 900 μl of cell lysate and mix evenly. After placing it on ice for 10 minutes, centrifuge at 12,000 rpm for 1 minute in a centrifuge, discard the supernatant, add 900 μl of cell lysate again, and use a gun After blowing up the precipitate and mixing it, repeat the above steps;
[0094] 2) Add 600 μl solution B solution to the precipitate, gently blow up the precipitate with a pipette gun, add 10 μl proteinase K and mix well, pla...
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Abstract
The invention discloses a method for identifying the polymorphism of human breast cancer genes RAD51 rs7180135 by the aid of BccI. The method is implemented by the aid of polymerase chain reaction-restriction fragment length polymorphism processes, and includes amplifying target DNA (deoxyribonucleic acid) fragments by means of polymerase chain reaction; digesting to-be-detected DNA fragments by the aid of restriction incision enzymes; identifying and cutting specific sequences by the aid of the restriction incision enzymes; carrying out electrophoresis on digested products; analyzing specific cleavage sites of the sequences of the fragments by the aid of restriction maps; comparing the differences of the gene sequences from different sources by the aid of the diversity of the fragments. The method has the advantages that briefly speaking, the corresponding target fragments are amplified by means of PCR (polymerase chain reaction) at first, then digestion reaction is carried out by the aid of the restriction incision enzymes, the restriction maps are observed and compared after electrophoresis is carried out, and accordingly the difference between the sequences can be analyzed; the method is good in repeatability, easy to implement and low in cost, and digestion results are easy to identify; the method and detection reagent kits which are easy to implement, low in cost and wide in application range can be provided for detecting the polymorphism of the human breast cancer susceptible genes RAD51 rs7180135.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for identifying rs7180135 polymorphism of human breast cancer RAD51 gene by using BccI. 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 heritable variation in humans. Accounts for more than 90% of all known polymorphisms. SNPs exist widely in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. CAPs (cleaved amplification polymorphism sequence-tagged sites) CAPs technology is also called 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 product is digest...
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