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Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene

A gene polymorphism and detection solution technology, applied in the field of molecular biology, can solve the problems of unsatisfactory practical application and unusability, and achieve the effects of avoiding uncertain factors, consistent detection results, and low cross-reaction rate

Inactive Publication Date: 2013-03-06
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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  • Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene
  • Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene
  • Specific primer and liquid chip for detecting polymorphism of SLC22A6 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 SLC22A6 gene polymorphism detection liquid chip mainly includes:

[0028] 1. ASPE Primers

[0029] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes G129A, A109T and G108C of the SLC22A6 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0030] Table 1 ASPE primer sequence of SLC22A6 gene (tag sequence + specific primer sequence)

[0031]

[0032] 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.

[0033] 2. Microspheres coated with anti-tag sequenc...

Embodiment 2

[0046] Example 2 Detection of samples using the SLC22A6 gene polymorphism detection liquid chip described in Example 1

[0047] The formula of described various solutions is as follows:

[0048] 50mM MES buffer (pH5.0) formula (250ml):

[0049]

[0050] 2×Tm hybridization buffer

[0051] Reagent

[0052] Store at 4°C after filtration.

[0053] ExoSAP-IT kit was purchased from US USB Company.

[0054] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0055] 1. Sample DNA extraction:

[0056] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0057] 2. PCR amplification of samples to be tested

[0058] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes G129A, A109T and G108C of the SLC22A6 gene in one step. The product sizes were 356bp, 267bp and 239bp, respectively. ) are shown in T...

Embodiment 3

[0099] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of SLC22A6 gene

[0100] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0101] Taking the SLC22A6 gene G129A 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 G129A, and the tag sequence of 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 microsphere 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.

[0102] Table 7 Design of liquid phase chip preparation

[0103]

[0104] ...

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Abstract

The invention discloses a liquid chip and specific primer for detecting polymorphism of an SLC22A6 gene. The liquid chip mainly comprises ASPE (allele specific primer extension) primers, microspheres and amplification primers, wherein each ASPE primer is formed by tag sequences at the 5' terminal and specific primer sequences which are arranged at the 3' terminal and aim at the mutation site of the target gene; the specific primer sequences are SEQ ID NO.7 and SEQ ID NO.8 aiming at the G129A site, SEQ ID NO.9 and SEQ ID NO.10 aiming at the A109T site and / or SEQ ID NO.11 and SEQ ID NO.12 aiming at the G108C site; and the microspheres are enveloped by anti-tag sequences. The detecting liquid chip provided by the invention has the advantages that the consistency of the detection result and the sequencing method is as high as 100%, thus parallel detection of wild types and mutant types of a plurality of mutation sites is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for detecting SLC22A6 gene polymorphism and a liquid phase chip. Background technique [0002] SLC22A6 gene (solute carrier family 22 member 6) encodes organic anion transporter 1 (organic anion transporter1, OAT1) protein, which belongs to the solute carrier superfamily and is an important membrane transporter in animals and humans, widely distributed in the gastrointestinal tract , liver, kidney, blood-brain barrier, etc., mediate the transcellular transport of exogenous substances. At present, there are few methods for detecting and analyzing the SLC22A6 gene polymorphism, 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 ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森郭靖
Owner SUREXAM BIO TECH
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