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Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection

A technology of gene polymorphism and detection kit, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of unsatisfactory detection results of gene polymorphism and achieve true results Credible and stable typing detection, simple and objective results interpretation method

Inactive Publication Date: 2015-10-28
WUHAN YZY MEDICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art, and provide specific primer sequences and kits for simultaneous detection of human MTHFR and MTRR gene polymorphisms, so as to solve the problem of low sample size in the existing gene polymorphism detection technology Unsatisfactory detection effect of genetic polymorphism

Method used

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  • Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection
  • Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection
  • Specific sequence specific primers-polymerase chain reaction primers and kit for human MTHFR and MTRR gene polymorphism detection

Examples

Experimental program
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Effect test

Embodiment 1

[0068] The preparation of the MTHFR and MTRR gene polymorphism detection kit of the present invention includes the following steps:

[0069] 1. Synthesis of primers and probes:

[0070] Design and synthesis of 3 sets of specific primers SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3; SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO: 9; 3 sets of specific probes, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 12, SEQ ID NO: 13; SEQ ID NO: 14, SEQ ID NO: 15, and labeled FAM fluorophore at the 5' end of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, and labeled the NFQ-MGB non-luminescence quenching group at the 3' end, at SEQ ID NO: 15 The 5' end of NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15 was labeled with a VIC fluorophore, and the 3' end was labeled with a NFQ-MGB non-luminescence quenching group. Primers and probes were prepared as 100 μM stock solutions for storage.

[0071] 2. Prepare an internal standard system: design and synthesize a pair of internal stand...

Embodiment 2

[0084] Use the human MTHFR and MTRR gene polymorphism detection kits prepared in Example 1 to detect the samples on the other side.

[0085] In this example, 16 anticoagulated whole blood samples with folic acid metabolism disorder were collected, and genomic DNA was extracted from them, and the human MTHFR and MTRR gene polymorphism detection kit obtained in Example 1 was used to detect the MTHFR and MTRR of the samples to be tested. Gene polymorphisms.

[0086] 1. Genomic DNA extraction from blood samples

[0087] Take 300 μl of whole blood, add 900 μl of cell lysate CL, invert and mix, let stand for 5 min, centrifuge at 10,000 rpm (11,500 × g) for 1 min, aspirate the supernatant, leave the cell pellet, add to the cell pellet collected by centrifugation 200 μl Buffer GS, shake until thoroughly mixed. Add 20 μl of Proteinase K solution and mix. Add 200 μl of buffer GB, invert and mix thoroughly, place at 56°C for 10 minutes, invert and mix several times during this time, a...

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Abstract

The present invention relates to the fields of biotechnology and medicine, and provides specific sequence specific primers-polymerase chain reaction primer sequences and a kit for simultaneously detecting human MTHFR gene polymorphism and human MTRR gene polymorphism, wherein the kit contains the specific primers, specific probes, an internal standard system, Taq enzyme and UNG enzyme. According to the present invention, the primers and the kit have advantages of strong specificity, high sensitivity, simple and rapid operation, high-throughput, safety, objective result interpretation, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to primers, probes and kits for detecting human MTHFR and MTRR gene polymorphisms. Background technique [0002] Folic acid (Folic Acid) refers to a class of water-soluble B vitamins with related biological activities. Its active form in the body is 5-methyltetrahydrofolate, which is an essential substance for cell growth and reproduction, and participates in many important metabolic reactions. As a carrier of one-carbon units in the body, folic acid participates in the synthesis and transformation of purines, thymine, nucleotides and other substances, and at the same time affects the production of phospholipids, creatine, and nerve mediators, and promotes the development of nerve cells and brain cells. Folic acid deficiency can lead to hyperhomocysteinemia, causing multi-system damage in the body, which is neonatal neural tube defects, cancer, chronic atrophic gastritis, colitis, live...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2600/166
Inventor 周鹏飞蔡从利张喆叶婷张浩
Owner WUHAN YZY MEDICAL SCI & TECH
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