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Specific primer and liquid chip for detecting polymorphism of SLCO1B3 gene

A technology for gene polymorphism and detection solution, applied in the field of molecular biology, can solve problems such as inability to meet practical applications and cannot be used, and achieve the effects of good signal-to-noise ratio, consistent detection effect, 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 SLCO1B3 gene
  • Specific primer and liquid chip for detecting polymorphism of SLCO1B3 gene
  • Specific primer and liquid chip for detecting polymorphism of SLCO1B3 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 SLCO1B3 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 two common genotypes T182G and G77A of the SLCO1B3 gene, respectively. 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 SLCO1B3 gene (tag sequence + specific primer sequence)

[0031]

[0032]

[0033] 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.

[0034] 2. Microspheres coated with anti...

Embodiment 2

[0045] Example 2 Using the SLCO1B3 gene polymorphism detection liquid chip described in Example 1 to detect samples The formulations of the various solutions are as follows:

[0046] 50mM MES buffer (pH5.0) formula (250ml):

[0047] Reagent

[0048] ethanesulfonic acid)

[0049] 2×Tm hybridization buffer

[0050] Reagent

[0051] Store at 4°C after filtration.

[0052] ExoSAP-IT kit was purchased from US USB Company.

[0053] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0054] 1. Sample DNA extraction:

[0055] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0056] 2. PCR amplification of samples to be tested

[0057] Two pairs of primers were designed, and multiplex PCR amplified two target sequences containing two common genotypes T182G and G77A of the SLCO1B3 gene in one step. The product sizes were 315bp and 203bp, respective...

Embodiment 3

[0100] Example 3 Detection of the SNP site of the SLCO1B3 gene by the liquid chip of different ASPE primers

[0101] 1. Design of liquid phase chip preparation (selection of tag sequence and Anti-tag sequence)

[0102] Taking the T182G site mutation detection liquid chip of the SLCO1B3 gene as an example, the specific primer sequences at the 3' end of the ASPE primer were designed for the wild type and mutant type of T182G, and the tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1- SEQ ID NO.4, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.9-SEQ ID NO.12. 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.

[0103] Table 7 Design of liquid phase chip preparation

[0104]

[0...

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Abstract

The invention discloses a specific primer and liquid chip for detecting polymorphism of an SLCO1B3 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 polymorphic site of the target gene; the specific primer sequences are SEQ ID NO.5 and SEQ ID NO.6 aiming at the T182G site and / or SEQ ID NO.7 and SEQ ID NO.8 aiming at the G77A 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 SLCO1B3 gene polymorphism and a liquid phase chip. Background technique [0002] Organic anion-transporting polypeptide OATP1B3 (also known as organic anion-transporting polypeptide 8, OATP8, encoding gene SLCO1B3), OATP1B3 drug transporter plays a very important role in drug resistance and pharmacokinetics, it is related to drug tolerance, absorption , distribution, excretion, drug efficacy, and drug toxicity are closely related. Studies have shown that the polymorphism of SLCO1B3 gene is closely related to the activity of its encoded protein. [0003] At present, there are few methods for detecting and analyzing the polymorphism of SLCO1B3 gene, 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 chang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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