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Primer pair, probe and kit used for detecting polymorphism of MTHFR gene

A gene polymorphism and primer pair technology, applied in the field of molecular biology, can solve the problems of easy contamination of reaction products and high detection cost, and achieve the effects of shortening the time required for detection, avoiding pollution and reducing errors.

Active Publication Date: 2017-10-20
广州德臻生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Based on this, it is necessary to solve the problems of high detection cost and easy contamination of reaction products in the prior art. The purpose of the present invention is to provide a method for detecting MTHFR gene polymorphism that has low detection cost and can be detected by closing the cap of a single reaction tube. Primer pairs, probes and kits

Method used

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  • Primer pair, probe and kit used for detecting polymorphism of MTHFR gene
  • Primer pair, probe and kit used for detecting polymorphism of MTHFR gene
  • Primer pair, probe and kit used for detecting polymorphism of MTHFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Primer pair for detecting polymorphism of MTHFR gene

[0034] In this example, a primer pair was designed for the c.677 position of the MTHFR gene.

[0035] The primer pair was designed according to the rs1801133 sequence (SEQ ID No. 5), the sequence of the primer pair is as follows:

[0036] Forward primer (SEQ ID No. 1): 5'C (amino group) TTGAAGGAGAAGGT 3';

[0037] Reverse primer (SEQ ID No. 2): 5'C (amino group) AAAGCGGAAGAAT 3'.

[0038] In the process of preparing the above primer pair, the primer design software primer express of ABI was used to test the Tm values ​​of the base sequence before the amino group modification and the base sequence after the amino group modification. The results are as follows:

[0039] Before modification: the Tm value of the base sequence of the forward primer is 36.8°C, and the Tm value of the base sequence of the reverse primer is 39.4°C;

[0040] After modification: the Tm value of the base sequence of the forward primer is 59.3°C...

Embodiment 2

[0043] Example 2. Probes for detecting MTHFR gene polymorphism

[0044] In this example, a fluorescent probe was designed for the c.677 position of the MTHFR gene.

[0045] Design wild-type probes and mutant probes based on the rs1801133 sequence (SEQ ID No. 5) mutant fragment:

[0046] Wild type probe (SEQ ID No.3):

[0047] 5'FAM-CGGG (amino group) AGCCGATTTCAT-BHQ1 3', the fourth base (G) from the 5'end is modified with an amino group, and the seventh base from the 5'end is the SNP site.

[0048] Mutant probe (SEQ ID No. 4):

[0049] 5’Cy5-CGGGAGTCGA (amino group) TTTCATCATC-BHQ2 3’;

[0050] The tenth base (A) from the 5'end is modified with an amino group, and the seventh base from the 5'end is the SNP site.

[0051] In the above probe process, ABI's primer design software primer express was used to test the Tm values ​​of the base sequence before the amino group modification and the base sequence after the amino group modification. The results are as follows:

[0052] Before modifica...

Embodiment 3

[0055] Example 3. Kit for detecting MTHFR gene polymorphism

[0056] In this example, a kit containing the primer pair of Example 1 and the probe of Example 2 was designed for the c.677 position of the MTHFR gene.

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Abstract

The invention discloses a primer pair, a probe and a kit used for detecting polymorphism of an MTHFR gene. The kit comprises: the primer with the base sequences represented by SEQ ID No.1 and SEQ ID No.2, which is designed for the c.677 site of the MTHFR gene, wherein the first base at the 5' end of the base sequence represented by the SEQ ID No.1 has an amino group modification group, and the first base at the 5' end of the base sequence represented by the SEQ ID No.2 has an amino group modification group; and the probe with the base sequences represented by SEQ ID No.3 and SEQ ID No.4, which is designed for the c.677 site of the MTHFR gene, wherein one base of the base sequence represented by the SEQ ID No.3 has an amino group modification group, and one base of the base sequence represented by the SEQ ID No.4 has an amino group modification group. Compared with kits in the prior art, the kit disclosed in the invention has the advantages of remarkable shortening of the detection time, reduction of nonspecific amplification, detection of SNPs and point mutation, reduction of the design difficulty and the detection cost, no influences on the diagnosis precision, realization of mutation detection using a single reaction tube without cover opening, maximal avoiding of aerosol pollution, convenience in clinic use, and reduction of operation induced errors.

Description

Technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a primer pair, probe and kit for detecting MTHFR gene polymorphism. Background technique [0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. Fluorescence PCR (qPCR) detection is a real-time detection technology developed on the basis of PCR technology. It uses fluorescence signal accumulation to monitor the entire PCR process in real time, and finally quantitatively analyzes unknown templates through standard curves or detects target genes based on fluorescence signal accumulation. This technology has achieved a leap from qualitative to quantitative PCR. Compared with conventional PCR, it has the characteristics of stronger specificity, effective solution to PCR pollution problems, and high degree of automation. It has been widely used in the early 21st century. [0003] Although the PCR detection method ha...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6883C12Q2600/156C12Q2563/107
Inventor 金京勋崔红易其易梅仙
Owner 广州德臻生物技术有限公司
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