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Method for identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI

A technology for gene polymorphism and human identification, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of difficulty in popularization and use, expensive restriction endonucleases, and high experimental costs, so as to achieve easy identification, Overcoming the effect of small choice and accurate and reliable results

Inactive Publication Date: 2016-06-08
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the restriction endonucleases often used to identify the human HOTAIR gene polymorphism rs920778 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

Method used

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  • Method for identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI
  • Method for identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI
  • Method for identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Example 1. Determination of Human HOTAIRrs920778 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), MspI (Thermo), Agarose (SIGMA), etc.

[0060] 1.2 Primer design

[0061] In the dbSNP database of NCBI, the nucleotide sequence of HOTAIRrs920778 was searched. In the primer design software PrimerPremier6.0, parameters such as the length of the primer and the length of the amplifi...

Embodiment 2

[0086] Example 2. Determination of Human HOTAIRrs920778 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 identifying human HOTAIR (HOX antisense intergenic RNA) gene polymorphism rs920778 by using MspI. Genome DNA of a sample is extracted; a forward primer and a reverse primer for amplifying a sequence nearby a human HOTAIR gene polymorphism rs920778 site are provided, to-be-detected human genome DNA is used as a template, a PCR (polymerase chain reaction) amplification reaction is performed, and an amplification product is obtained; the amplification product is subjected to enzyme digestion by using a restriction endonuclease, and a corresponding enzyme digestion product is obtained; the enzyme digestion product is subjected to electrophoresis by the aid of 3% agarose gel so as to judge genotypes of HOTAIR gene polymorphism rs920778. According to the established detection method for the HOTAIR rs920778 polymorphism, endonuclease is very cheap, meanwhile, the PCR product is higher in purity, and the enzyme digestion result is easy to distinguish. The detection method is accurate and reliable in result through sequencing validation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for identifying human HOTAIR gene polymorphism rs920778 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, 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 advantages, they also...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/683C12Q1/6886C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 宋春花王凯娟李丽蔡建有闫芮曹祯王静娴王艳娜
Owner ZHENGZHOU UNIV
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