Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Human mthfr gene polymorphism detection kit and method based on taqman-mgb probe

A technology of gene polymorphism and detection kit, which is applied in the field of genetic detection, can solve problems such as prone to false positives, poor interpretation of results, and troublesome operation, so as to reduce the risk of false positives, facilitate high-throughput detection, The effect of improving the detection efficiency

Inactive Publication Date: 2016-03-30
HENAN UNIV OF SCI & TECH
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Advantages: accurate results, sequencing method is the gold standard for gene polymorphism site detection; Disadvantages: high cost, cumbersome operation, long test period
Advantages: low cost, intuitive results; Disadvantages: Some polymorphic sites do not have suitable restriction enzyme sites, and mutations need to be introduced manually. In addition, the operation is cumbersome and the test cycle is long
Disadvantages: PCR products need to be analyzed later, and the operation is cumbersome; in addition, the results are not easy to interpret, and false positives are prone to occur
Disadvantages: A sample needs to be divided into 2 tubes for PCR testing to detect the genotype of a SNP site, which is troublesome to operate
Advantages: One SNP site genotype only needs one tube of PCR to complete the detection; Disadvantages: It is not easy to interpret the difference in Tm value according to the HRM melting curve, and the difference in the peak diagram of the melting curve of different genotypes is not obvious
[0011] At present, the Taqman probe method has the following disadvantages: the results of different genotypes are not significantly different, and three genotype controls must be set up for each qPCR test. In addition, the fluorescence quenching of the TaqMan probe is not complete, and the background is high

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Human mthfr gene polymorphism detection kit and method based on taqman-mgb probe
  • Human mthfr gene polymorphism detection kit and method based on taqman-mgb probe
  • Human mthfr gene polymorphism detection kit and method based on taqman-mgb probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: PCR reaction solution configuration in the kit

[0063] Prepare PCR reaction solution for detecting C677T and A286C polymorphism sites of human MTHFR gene respectively. The PCR reaction solution contains primers and corresponding Taqman-MGB probes, Taq enzyme, dNTP mixture, MgCl2 solution, and fluorescent quantitative PCR reaction buffer solution, ddH2O.

[0064] The reaction system for each PCR amplification is 20ul, including 2.0μl of 10×PCR reaction buffer, 2.4μl of 25mMMgCl2 solution, 1.6μl of 2.5mM dNTP mixture, 0.1μl of 5U / ul Taq enzyme, 1μl of DNA template (about 50ng), 20uM 0.5 μl each of upstream and downstream primers, 0.5 μl each of 10uM mutant wild probe, ddH2O 12.7 μl

[0065] Primers and Taqman-MGB probes in the MTHFRC677T reaction solution:

[0066] Upstream primer MTHFR677F: 5'-TGTGCTGTGCTGTTGGAAGGTG-3';

[0067] Downstream primer MTHFR677R: 5'-TCAGAGCCCCCAAAGCAGAGGACTC-3';

[0068] Mutation probe MTHFR677T: 5'-FAM-TCATGGCATTTCTCA-NFQ; ...

Embodiment 2

[0075] Embodiment 2: the use of detection kit

[0076] 1. Extract DNA template

[0077] For human blood DNA extraction, Genomic DNA was extracted from blood using the Whole Blood DNA Extraction Kit from Tiangen Biochemical Biotechnology Company.

[0078] 2. PCR reaction system configuration

[0079] Use the PCR reaction solution configured in Example 1 for detection, one sample, and 2 tubes of PCR detection at the same time. Take 19ul of MTHFRC677TPCR reaction solution into one PCR reaction tube, take 19ul of MTHFRA286CPCR reaction solution into another PCR reaction tube, and add 1ul (10~100ng) of DNA extracted in the same step 1 to the two PCR reaction tubes respectively.

[0080] 3. Fluorescent PCR detection

[0081] Put the configured PCR system into a fluorescent PCR instrument for fluorescent PCR amplification detection; the reaction conditions are: denaturation and enzyme activation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, annealing at 62°C for 30 s...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the field of genetic gene detection, and in particular relates to a kit and a method for detecting the gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on a Taqman-MGB (Minor Groove Binder) probe. By adopting the kit and method, two SNP (single nucleotide polymorphism) sites namely C677t and A1298C can be detected at the same time, and the results are easy to judge and read. The MGB probe has a nonluminous 3'-end quenching group and a relatively low background, is more sensitive to detection of a single base mutation, and can judge and read different genotypes only according to a Ct value, and the result can be judged and read more easily compared with that of conventional methods of Taqman probe detection and HRM (high resolution melting) detection. The probe price is relatively low, the detection cost is relatively low, a polymorphism site only needs one-tube qPCR (quantitative polymerase chain reaction) detection, and the operation is simple. Moreover, the method disclosed by the invention does not need subsequent analysis of PCR products, so that while the detection cost is saved, the detection circle is greatly shortened, the detection efficiency is improved and the risk of false positive caused by PCR product pollution is reduced.

Description

technical field [0001] The invention belongs to the field of genetic detection, in particular to a human MTHFR gene polymorphism detection kit and method based on Taqman-MGB probes. Background technique [0002] 5,10-methylenetetrahydrofolatereductase (MTHFR) is a key enzyme in the folate metabolism system, which catalyzes the reduction of 5,10-methylenetetrahydrofolate to the methyl donor 5-methyltetrahydrofolate . On the one hand, 5-methyltetrahydrofolate can be used as an indirect donor of methyl groups to participate in the synthesis of purines and pyrimidines and the methylation of DNA, RNA, and proteins; on the other hand, under the catalysis of methionine synthase, Vitamin B12 is used as a coenzyme to re-methylate homocysteine ​​in the blood to produce methionine, thereby maintaining normal homocysteine ​​levels in the body. There are two common polymorphic sites in the MTHFR gene, C677T (SNPID: rs1801133) and A1296C (SNPID: rs1801131), among which the C677T site is...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 杨曦
Owner HENAN UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com