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Method for identifying polymorphism of human breast cancer genes BRCA1 rs8176318 by aid of NsiI

A gene polymorphism, breast cancer technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high experimental cost, difficult to popularize and use, and expensive restriction endonucleases, etc., to achieve results. Accurate and reliable, easy to distinguish, overcoming the effect of limited choice

Inactive Publication Date: 2016-06-22
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the restriction endonucleases often used to identify the human BRCA1 gene polymorphism rs8176318 are expensive (refer to Table 1 below for the reference price of some endonucleases), and the experimental cost is high, so it is difficult to be widely used in the laboratory

Method used

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  • Method for identifying polymorphism of human breast cancer genes BRCA1 rs8176318 by aid of NsiI
  • Method for identifying polymorphism of human breast cancer genes BRCA1 rs8176318 by aid of NsiI
  • Method for identifying polymorphism of human breast cancer genes BRCA1 rs8176318 by aid of NsiI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Determination of Human BRCA1rs8176318 Polymorphism in Whole Blood Specimen of Human Peripheral Blood

[0056] 1 Materials and methods

[0057] 1.1 Main instruments and reagents

[0058] Instruments: BCD-228CH Refrigerator (Xinfei Electric), HH-2 Digital Display Constant Temperature Water Bath (Huafon Instruments), SmartGe Gel Imager (Beijing Saizhi Venture), GT9612 Gradient PCR Instrument (Biteke Biology), WD900SL23- 2 models of microwave ovens (Galanz Electric Appliances), DG-300C electrophoresis apparatus (Dingguo Changsheng Biology), etc.

[0059] Reagents: NEP004-1 DNA Extraction Kit (Dingguo Changsheng Biology), 50bpDNALadder (Laifeng Biology), 2×TaqPCRMix (Laifeng Biology), NsiI (Thermo), Agarose (SIGMA), etc.

[0060] 1.2 Primer design

[0061] In the dbSNP database of NCBI, the nucleotide sequence of BRCA1rs8176318 was searched. In the primer design software PrimerPremier6.0, parameters such as the length of the primer and the length of the target ...

Embodiment 2

[0086] Example 2. Determination of Human BRCA1rs8176318 Polymorphism in Human Breast Cancer Tissue Specimens

[0087] The steps are basically the same as in Example 1, except that the following method is used to extract DNA from breast cancer tissue samples as the DNA to be tested.

[0088] The breast cancer tissue was excised with a mobile phone, and the genomic DNA of the breast cancer tissue was extracted by the phenol-chloroform method as the DNA to be tested.

[0089] 1) Thaw the breast cancer tissue block, wash away the blood stains with normal saline, cut about 0.1g of the tissue and grind it, add 1ml of sterilized water, mix by inversion, centrifuge at 10,000 rpm for 10 minutes, and discard the supernatant. There should be sediment at the bottom of the tube. repeat twice

[0090] 2) Add 200ul of DNA lysate, mix well with 5ul of proteinase K, and digest at 55 overnight.

[0091] 3) After the digestion is completed, add an equal volume of phenol-chloroform mixture (1:...

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Abstract

The invention discloses a method for using NsiI to identify the rs8176318 polymorphism of human breast cancer BRCA1 gene. By creating an enzyme cutting site method and applying primer 3' end mismatch technology, genotype identification is carried out after PCR products are subjected to enzyme digestion and electrophoresis , thereby providing a method and a detection kit for detecting human BRCA1rs8176318 polymorphism with simple operation, low cost and wide application range. The present invention improves the traditional PCR-RFLP method, overcomes the shortcomings of the traditional PCR-RFLP method, such as small selection of endonucleases, high price, and high experiment cost. Inexpensive, and at the same time, the purity of the PCR product is high, and the digestion results are easy to distinguish. The detection method is verified by sequencing, and its results are accurate and reliable.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for identifying rs8176318 polymorphism of human breast cancer BRCA1 gene by using NsiI. 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, including the form of substitution / insertion and deletion of editing. SNP is the most common type of human heritable variation, and it has been widely used in genetic linkage analysis / association analysis of simple and complex diseases and the location of disease susceptibility genes, guiding the cloning of susceptibility genes. The more common single base The detection methods of polymorphic sites include polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), three-primer allelic amplification (TP-PCR) and sequencing. Although these methods have their adva...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2521/301C12Q2565/125
Inventor 宋春花赵艳艳蔡建有薛云红闫芮曹晶晶郭巧云
Owner ZHENGZHOU UNIV
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