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PCR primer and method for detecting genotype of SNP locus of MTRR gene

A genotyping and genotyping technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low efficiency and incompleteness of PCR products, and achieve high accuracy and accuracy , The effect of fast typing speed

Active Publication Date: 2018-07-06
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Aiming at the deficiencies in the prior art, the present invention improves the existing method for detecting the rs1801394 site of the MTRR gene using the restriction endonuclease NdeI, so that the PCR product has an internal control enzyme cleavage site as the quality of enzyme cleavage. Control, it can overcome the relatively low efficiency of fast restriction endonuclease NdeI digestion of PCR products, which is easy to cause incomplete digestion. It can identify the situation of incomplete digestion according to the electrophoretic pattern of the digestion product, and in the enzyme digestion In the case of incompleteness, the background interference of PCR products and intermediate products of enzyme digestion can be eliminated, and the genotype can be judged according to the status of the characteristic bands of each genotype, ensuring the accuracy of genotype discrimination

Method used

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  • PCR primer and method for detecting genotype of SNP locus of MTRR gene
  • PCR primer and method for detecting genotype of SNP locus of MTRR gene
  • PCR primer and method for detecting genotype of SNP locus of MTRR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Design of primers for detecting genotyping of MTRR gene rs1801394 polymorphism site

[0057] In this embodiment, a pair of primers are designed to obtain the sequence of 1000bp before and after the polymorphic site of MTRR gene rs1801394 (see figure 2 ), wherein the base sequence of the 6121-7260th position of the MTRR gene is shown in SEQ ID NO: 16, and specific typing primers are designed for the SNP site of rs1801394 according to the sequence. Among them, the 3' end of the forward primer is adjacent to the polymorphic site, and the penultimate 3rd base is C, so as to realize the introduction of a recognition sequence through PCR amplification C ATRTG, where C Introduced by a mismatched base, the corresponding sequence of the wild-type allele A is C ATATG, which can be recognized and cleaved by NdeI or isoschizase; the corresponding sequence of the mutant allele G is CATGTG cannot be recognized and cleaved by restriction endonuclease NdeI or isozyzyme, s...

Embodiment 2

[0064] Example 2 A method for detecting the genotype of the rs1801394 polymorphic site of the MTRR gene

[0065] 1) Genomic DNA extraction

[0066] Genomic DNA of human oral epithelial cells was extracted by using Tiangen's buccal swab genomic DNA extraction kit according to the instructions. The DNA source is not limited thereto, and any somatic cell-derived DNA is acceptable.

[0067] 2) PCR amplification of the target fragment

[0068] PCR reaction system:

[0069]

[0070]

[0071] PCR reaction conditions:

[0072]

[0073] PCR products were identified by 1.5% agarose gel electrophoresis.

[0074] 3) Restriction typing of PCR products

[0075] The restriction endonuclease used in this example is Thermo Scientific fast restriction endonuclease NdeI.

[0076] The total volume of the enzyme digestion system is 30 μL, and the specific formula is as follows:

[0077]

[0078] The amount (X μL) of the PCR product can be adjusted depending on the concentration ...

Embodiment 3

[0096] Example 3 A method for detecting the genotype of the rs1801394 polymorphic site of the MTRR gene

[0097] 1) Genomic DNA extraction

[0098] Genomic DNA is extracted from the subject's cells.

[0099] 2) PCR amplification of the target fragment

[0100] The forward primer F and the reverse primer R designed in the present invention are used for PCR amplification to obtain PCR primers.

[0101] 3) Restriction typing of PCR products

[0102] The PCR product was digested with restriction endonuclease NdeI. Agarose gel electrophoresis of PCR amplification products Figure 4 , Lanes 1-10 are PCR products, all of which are 478bp in size, and the size of the target band is consistent.

[0103] One of the electrophoresis patterns of the PCR product digested with restriction endonuclease NdeI Figure 5 , P is the PCR product with a size of 478bp; lanes 1-7 are PCR product digestion bands of 7 different samples, among which 1, 2, 4, and 5 are completely digested, and the ge...

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Abstract

The invention discloses a method and a kit for detecting a genotype of rs1801394 polymorphic locus of an MTRR gene. According to the method, the genotype of the rs1801394 locus of the MTRR gene is detected by a PCR-RFLP method; a PCR product carries an internal control restriction enzyme cutting site of NdeI or an isoschizomer thereof; a rapid restriction endonuclease is used for digestion of thePCR product to obtain a restriction map for genotyping, so that a typing time can be shortened; meanwhile, results can be comprehensively judged according to residual PCR products, residual digestionintermediate products and characteristic strips of each genotype sample after complete digestion so as to avoid genotype misjudgment caused by incomplete digestion of traditional methods.

Description

technical field [0001] The invention belongs to the field of molecular detection, in particular to a PCR primer and a method for detecting genotyping of an MTRR gene SNP site (ie, rs1801394 polymorphic site). Background technique [0002] Folic acid has important physiological functions for the human body, but the human body cannot synthesize folic acid itself and must be ingested from food. Insufficient folic acid supplementation or poor utilization ability will cause folic acid deficiency, lead to hyperhomocysteinemia, increase the risk of atherosclerosis, thrombosis and hypertension; folic acid deficiency can also cause DNA hypomethylation, Increase the risk of some cancers; lack of folic acid in pregnant women can lead to birth defects, and pregnant women themselves will also have many adverse symptoms, but excessive folic acid supplementation can also cause various side effects. [0003] Methionine synthase reductase, namely 5-methyltetrahydrofolate-homocysteine ​​meth...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 何震宇顾取良
Owner GUANGDONG PHARMA UNIV
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