Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of high false positive rate, easy sample contamination, low sensitivity, etc., and achieve the effect of good detection specificity, consistent detection effect and simple steps

Active Publication Date: 2012-07-11
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the products for detecting STK39 are generally PCR-based RT-PCR, fluorescent quantitative PCR, traditional solid-phase chip technology, direct sequencing, etc., each of which has shortcomings such as low sensitivity, easy sample contamination, and high false positive rate. The limitation of flux cannot meet the needs of practical applications

Method used

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  • Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene
  • Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene
  • Specific primers and liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of STK39 (Serine/Threonine Kinase) gene

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1 STK39 gene SNP detection liquid chip mainly includes:

[0020] 1. ASPE Primers

[0021] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes T107C, G232A and A190C of the STK39 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0022] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 STK395 gene

[0023]

[0024] 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.

[0025] 2. Microspheres coated with anti-tag sequences

[00...

Embodiment 2

[0036] Example 2 Detection of samples using STK39 gene detection liquid chip

[0037] The formula of described various solutions is as follows:

[0038] 50mM MES buffer (pH5.0) formulation (250mL):

[0039]

[0040] 2×Tm hybridization buffer

[0041] Reagent

[0042] Store at 4°C after filtration.

[0043] ExoSAP-IT kit was purchased from US USB Company.

[0044] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0045] 1. Sample DNA extraction

[0046] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0047] 2. PCR amplification of samples to be tested

[0048] Three pairs of primers were designed, and multiplex PCR was used to amplify three target sequences of three common genotypes T107C, G232A and A190C containing STK39 gene in one step, and the sequence fragment sizes were 467bp, 400bp and 467bp, respectively. The primer sequences (SEQID NO.19-24)...

Embodiment 3

[0107] The liquid phase chip of embodiment 3 different ASPE primers to the detection of STK39 gene SNP site

[0108] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0109] Taking the STK39 gene T107C 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 T107C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 - SEQ ID NO.6, correspondingly, the anti-tag sequence coated on the microspheres and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). 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.

[0110] Table 7 Design of liquid phase chip preparation

[0111]

[0112] ...

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Abstract

The invention discloses specific primers and a liquid phase chip for SNP (Single Nucleotide Polymorphism) detection of a STK39 (Serine / Threonine Kinase) gene. The liquid phase chip mainly comprises an ASPE (Allele Specific Primer Extension) primer composed of a tag sequence at a 5' side and specific primers aiming at target gene mutation at a 3' side. The specific primers are respectively: SEQ ID NO.7 and SEQ ID NO.8 aiming at the T107C SNP site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the G232A SNP site, and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at the A190C SNP site; a microsphere coated with an anti-tag sequence; and an amplification primer. The coincidence rate of the detection result of the liquid phase chip for SNP detection of the STK39 gene provided by the invention and a sequencing method is as high as 100%. The prepared liquid phase chip has an excellent signal-noise ratio and can realize parallel detection of multiple polymorphism sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a STK39 gene SNP detection specific primer and a liquid phase chip. Background technique [0002] The serine / threonine kinase gene (serine / threonine kinase, STK39) is located on human chromosome 2, including 18 exons, with a total length of about 300kb. [0003] The main biochemical function of STK39 is to encode a specific protein SPAK, which is involved in regulating the process of renal excretion of salt. The researchers conducted functional studies of STK39 at the cellular level and found that STK39 interacts with WNK kinases and cation-chloride cotransporters. WNK kinases can activate the protein SPAK encoded by STK39, which can further interact with cation-chloride cotransporters to control NaCl transport in osmolarity regulation and renal NaCl secretion. If there is a mutation in STK39, the body's ability to excrete salt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森秦会娟郭婧朱泽尧
Owner SUREXAM BIO TECH
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