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Human MTHFR gene polymorphism detection kit

A detection kit and kit technology, applied in the biological field, can solve the problems of long operation time, unfavorable large-scale promotion, low cost, etc., and achieve the effect of stable typing detection

Inactive Publication Date: 2019-07-12
江苏正大天创生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the high-resolution melting curve method has special requirements for equipment, there are certain difficulties in clinical promotion; PCR-PFLP technology relies on restriction endonuclease digestion, electrophoresis analysis and other reasons, often resulting in misjudgment of results, which affects its detection accuracy In addition, its detection cycle is long and the throughput is low, and it is not suitable for rapid screening of a large number of people.
As the gold standard of genetic testing, DNA direct sequencing method is difficult to achieve large-scale promotion due to its low cost, long operation time and low sensitivity.
DNA chip technology has been widely used in SNP detection due to the advantages of high throughput, simple and fast operation, etc., but this technology is expensive, complicated, poor in repeatability, and low in sensitivity, which is not conducive to large-scale promotion

Method used

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  • Human MTHFR gene polymorphism detection kit
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  • Human MTHFR gene polymorphism detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. Primer and probe synthesis:

[0064] Design and synthesize 2 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; 2 sets of specific probes SEQ ID NO:7, SEQ ID NO:8; SEQ ID NO:9, SEQID NO:10, and label FAM, HEX, Cy3 and Cy5 fluorescent groups at the 5' end, and NFQ-MGB at the 3' end without luminescent quenching Killing group; 2 enrichment primers SEQ ID NO:14, SEQ ID NO:15. The primers and probes were prepared into 100 μM stock solutions for storage.

[0065] 1. Prepare the internal standard system: design and synthesize a pair of internal standard primers designed for the human genome, the primer pair sequences are SEQ ID NO: 11 and SEQ ID NO: 12; design and synthesize internal standard probes, the probes is SEQ ID NO:13. The primers and probes were prepared into 100 μM stock solutions for storage.

[0066] 2. Prepare other reagents: prepare PCR buffer, which contains 1.0mM MgCl2, dATP, dUTP, dGTP and dCTP each ...

Embodiment 2

[0072] Use the human MTHFR gene polymorphism detection kit prepared in Example 1 to detect the sample to be treated.

[0073] 1. Genomic DNA extraction from blood samples

[0074] Use the blood genome column extraction kit to extract the human blood genome according to the instructions. Use an ultraviolet spectrophotometer to detect the concentration of the obtained sample DNA solution, then dilute the sample DNA to 10ng / μl, take 2μl-5μl respectively and add them to the kit prepared in Example 1 and carry out the next step of PCR reaction.

[0075] 2. Take 2 μl of the DNA samples diluted in step 1 and add them to the reaction system of the kit of Example 1 of 23 μl in turn, and put them into a fluorescent quantitative PCR instrument, set the PCR reaction program as shown below and perform amplification reaction:

[0076] 50℃ for 2min, 95℃ for 10min;

[0077] 95℃ for 15s, 48℃~53℃ for 1min, 5 cycles;

[0078] 95°C for 15s, 60°C-62°C for 1min, 40 cycles; after each cycle, the...

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Abstract

The invention relates to a method for detecting gene polymorphism with labeled probes and specific primer sequences and a kit including specific probes and the specific primer sequences, and is suitable for biotechnology and medical fields. The kit provides the specific primer sequences and the labeled probes for simultaneous detection of polymorphism of C677T and A1298C loci of a human MTHFR gene. The kit contains a Taq enzyme, the specific primers, the specific probes and an internal standard system. The primers and kit provided by the invention can be used for simultaneous detection of thepolymorphism of the C677T and A1298C loci of the MTHFR gene, and has the advantages of high specificity, high sensitivity, simple and fast operation, high throughput, safety and objective result interpretation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a polymorphism detection kit of C677T site and A1298C site of human MTHFR gene and its preparation method and application. Background technique [0002] Folic acid is a water-soluble B vitamin (vitamin B9), which does not exist in nature and has no biological activity, but is a precursor of biologically active folate (folate), and its active form in the body is 5 -Methyltetrahydrofolate, which can transfer a one-carbon group (methyl or formyl) to deoxyuridine acid to make it into deoxythymidylate, and then synthesize DNA. It is an essential substance for tissue repair and an indispensable nutrient for embryonic development. In recent years, a large number of studies have confirmed that folic acid is an indispensable nutrient for fetal growth and development, and can help prevent neural tube defects, including very serious birth defects such as spina bifida and anencephaly. In addit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2537/143C12Q2563/107C12Q2531/113C12Q2545/101C12Q2547/00
Inventor 李思慧吴凯林伟强魏赵延
Owner 江苏正大天创生物工程有限公司
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