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MEK1 gene mutation detection specificity primer and liquid chip thereof

A detection solution and specificity technology, which is applied in the field of molecular biology, can solve the problems of not being able to meet the practical application and use, and achieve the effects of improving detection accuracy, consistent detection effect, and low cross-reaction rate

Active Publication Date: 2013-03-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • MEK1 gene mutation detection specificity primer and liquid chip thereof
  • MEK1 gene mutation detection specificity primer and liquid chip thereof
  • MEK1 gene mutation detection specificity primer and liquid chip thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 The MEK1 gene mutation detection liquid chip mainly includes:

[0020] 1. ASPE Primers

[0021] Specific primer sequences were designed for the wild-type and mutant types of four common genotypes Q56P, K57N, D67N and P124L of the MEK1 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0022] Table 1 ASPE primer sequence of MEK1 gene (tag sequence + specific primer sequence)

[0023]

[0024]

[0025] Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0026] 2. Microspheres coated with anti-tag seque...

Embodiment 2

[0037] Example 2 Detection of samples using the MEK1 gene mutation detection liquid chip described in Example 1

[0038] The formula of described various solutions is as follows:

[0039] 50mM MES buffer (pH5.0) formula (250ml):

[0040]

[0041]

[0042] 2×Tm hybridization buffer

[0043] Reagent

[0044] Store at 4°C after filtration.

[0045] ExoSAP-IT kit was purchased from US USB Company.

[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] 1. Sample DNA extraction:

[0048] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0049] 2. PCR amplification of samples to be tested

[0050] Two pairs of primers were designed, and two target sequences containing Q56P, K57N, D67N and P124L were amplified in one step by multiplex PCR, among which, Q56P, K57N and D67N were located in the same amplification product. The sizes of the products ...

Embodiment 3

[0084] Example 3 Detection of MEK1 gene mutation site by liquid chip with different ASPE primers

[0085] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0086] Taking the MEK1 gene Q56P and D67N site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of Q56P and D67N, and the tag sequence of the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.8, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. The specific design is shown in the following table (Table 6). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0087] Table 6 Design of liquid phase chip preparation

[0088]

[0089] ...

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Abstract

The invention discloses an MEK1 gene mutation detection specificity primer and a liquid chip thereof. The liquid chip mainly comprises: an ASPE primer composed of a 5'-terminal tag sequence and 3'-terminal specificity primer sequences focused on target gene mutation sites, wherein the specificity primer sequences comprise SEQ ID NO.9 and SEQ ID NO.10 focused on a Q56P site, SEQ ID NO.11 and SEQ ID NO.12 focused on a K57N site, SEQ ID NO.13 and SEQ ID NO.14 focused on a D67N site, and / or SEQ ID NO.15 and SEQ ID NO.16 focused on a P124L; a microsphere coated by an anti-tag sequence; and an amplimer. The consistency between the detection result of the detection liquid chip provided by the invention and the detection result of a sequencing method is high to 100%, and the wild-type and mutant parallel detection of a plurality of mutation sites is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a MEK1 gene mutation detection specific primer and a liquid phase chip. Background technique [0002] The MEK1 gene is involved in a large number of life processes and plays an important role in embryonic development. Studies have shown that mutations of the MEK1 gene occur frequently in tumors, and the research hotspots generally focus on four sites: Q56P, K57N, D67N and P124L. [0003] At present, there are few methods for detecting and analyzing MEK1 gene mutations, mainly including direct sequencing and PCR-RFLP analysis, among which the most commonly used method is PCR-RFLP analysis. The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森刘志明
Owner SUREXAM BIO TECH
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