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Method for identifying polymorphisms of rs6470502 of human LINCRNA-CCAT1 gene by using BstUI

A technology of LINCRNA-CCAT1 and gene polymorphism, which is applied to biochemical equipment and methods, and the measurement/testing of microorganisms, can solve the problems of difficulty in popularization, expensive restriction endonucleases, and high experimental costs, and achieve repeatable results. Good performance, low cost and simple operation

Inactive Publication Date: 2017-11-21
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, when identifying human LINCRNA-CCAT1 gene polymorphism rs6470502, the restriction endonucleases commonly used 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 polymorphisms of rs6470502 of human LINCRNA-CCAT1 gene by using BstUI
  • Method for identifying polymorphisms of rs6470502 of human LINCRNA-CCAT1 gene by using BstUI
  • Method for identifying polymorphisms of rs6470502 of human LINCRNA-CCAT1 gene by using BstUI

Examples

Experimental program
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Embodiment 1

[0076] Example 1. Determination of Human LINCRNA-CCAT1rs6470502 Polymorphism in Human Gastric Cancer Tissue Specimens

[0077] In a specific embodiment of the present invention, the specific steps of detecting human LINCRNA-CCAT1 gene polymorphism rs6470502 are as follows:

[0078] (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:

[0079] 1) Thaw the gastric cancer tissue block, wash away blood stains with normal saline, cut 0.1g of tissue for grinding, add 1ml of sterilized water, mix by inverting, centrifuge at 10000rpm for 10min, discard the supernatant, repeat the above steps twice

[0080] 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.

[0081] 3) After the digestion is completed, add an equal volume of phenol-chloroform mixture (1:1) and shake vigo...

Embodiment 2

[0091] Example 2. Determination of Human LINCRNA-CCAT1rs6470502 Polymorphism in Human Peripheral Blood Whole Blood Specimen

[0092] 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.

[0093] 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:

[0094] l) Add 300µl of blood cells to a 1.5ml centrifuge tube, then add 900µl of cell lysate and mix well, place on ice for 10 minutes, centrifuge at 12000rpm for 1min in a centrifuge, discard the supernatant, add 900µl of cell lysate again, and use a gun After blowing up the precipitate and mixing it, repeat the above steps;

[0095] 2) Add 600µl solutionB solution to the precipitate, gently blow up the precipitate with a pipette gun, add 10µl proteinase K and mix well, place in a 70°C water bath fo...

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Abstract

The invention discloses a method for identifying the rs6470502 polymorphism of the human LINCRNA-CCAT1 gene by using BstUI, comprising the following steps: providing human genome DNA to be tested; providing upstream primers and downstream primers for amplifying the sequence near the rs6470502 site of the human LINCRNA-CCAT1 gene polymorphism Primers, using the extracted human genome DNA to be tested as a template, perform PCR amplification to obtain amplified products; use restriction endonucleases to digest the obtained amplified products to obtain corresponding digested products; digest the digested products 3% agarose gel was used for electrophoresis to determine the genotypes of LINCRNA‑CCAT1 gene polymorphism rs6470502. The invention provides a method and a detection kit for detecting polymorphism rs6470502 of the human gastric cancer susceptibility gene LINCRNA-CCAT1 with simple operation, low cost and wide application range.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for identifying rs6470502 polymorphism of human LINCRNA-CCAT1 gene by using BstUI. Background technique [0002] Single nucleotide polymorphism (single nucleotide 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. Accounting 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. The basic principle of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology is to use PCR to amplify the target DNA, and then the amplified product is digested with specific endonuclease and cut into fragments of different sizes, which are directly electrophore...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/683
Inventor 王凯娟董凯艳杨倩高甚方王轩智张叶闫雅丽范琦琪
Owner ZHENGZHOU UNIV