ABCB2 gene polymorphism detection specific primers and liquid chip

A gene polymorphism and detection solution technology, which is applied in the field of molecular biology, can solve problems such as unusable and unsatisfactory for practical applications, and achieve consistent detection results, simple steps, and good detection specificity

Inactive Publication Date: 2014-08-27
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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  • ABCB2 gene polymorphism detection specific primers and liquid chip
  • ABCB2 gene polymorphism detection specific primers and liquid chip
  • ABCB2 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 ABCC2 gene polymorphism detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Specific primer sequences were designed for wild type and mutant types of six common genotypes of ABCC2 gene, G142A, G88A, G154A, C86T, C150T and A92T. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 1ABCC2 gene

[0025]

[0026]

[0027] 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.

[0028] 2. Microspheres coated with a...

Embodiment 3

[0095] The liquid phase chip of embodiment 3 different ASPE primers detects the polymorphic site of ABCC2 gene

[0096] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0097] Taking the ABCC2 gene G142A and G154A 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 C298T and T102G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.12, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. The specific design is shown in the following table (Table 8). 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.

[0098] Table 8 Design of liquid phase chip preparation

...

Embodiment 4

[0109] The selection of embodiment 4ABCC2 gene polymorphism detection specific primer sequence

[0110] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0111] Taking the ABCC2 gene G88A and C150T site mutation detection liquid chip as an example, using the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, ASPE primers were designed for the wild type and mutant type of G88A and C150T respectively3 The specific primer sequences at the 'end include the preferred specific primer sequences and two alternative specific primer sequences in Example 1 of the present invention, as shown in Table 11. in, Inner bases are polymorphic sites.

[0112] Table 11 specific primer sequence

[0113]

[0114]

[0115] Taking the ABCC2 gene G88A and C150T site mutation detection liquid chip as an example, using the forward or reverse complementary sequence of the targ...

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Abstract

The invention discloses ABCB2 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of tag sequence and specific primer sequences aiming at mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.13 and SEQ ID NO.14 aiming at G142A site; SEQ ID NO.15 and SEQ ID NO.16 aiming at G88A site; SEQ ID NO.17 and SEQ ID NO.18 aiming at G154A site; SEQ ID NO.19 and SEQ ID NO.20 aiming at C86T site; SEQ ID NO.21 and SEQ ID NO.22 aiming at C150T site; and / or SEQ ID NO.23 and SEQ ID NO.24 aiming at A92T site. The matching rate of the detection result obtained by using the liquid chip provided by the invention and a result obtained by using a sequencing method is 100%. Therefore, wild-type and mutant-type parallel detections of a plurality of mutation sites are realized.

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 ABCC2 gene polymorphism and a liquid phase chip. Background technique [0002] Multidrug resistance-related protein ABCC2 (MRP2) is widely distributed in the small intestine, liver and kidney of mammals, and is specifically located on the apical membrane of polar cells. At present, there are many methods for detecting and analyzing the polymorphism of the ABCC2 gene, such as: direct sequencing, Real time-PCR, PCR-RFLP analysis, etc., 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 restriction endonuclease. The size of the fragment is observed b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森邹凤文
Owner SUREXAM BIO TECH
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