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ABCB1 gene polymorphism detection specific primers and liquid chip

A gene polymorphism and detection solution technology, applied in the field of molecular biology, can solve problems such as unusable and unsatisfactory for practical applications, and achieve consistent detection results, improved detection accuracy, and low cross-reaction rate

Inactive Publication Date: 2013-02-06
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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  • ABCB1 gene polymorphism detection specific primers and liquid chip
  • ABCB1 gene polymorphism detection specific primers and liquid chip
  • ABCB1 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 ABCB1 gene polymorphism detection liquid chip mainly includes:

[0027] 1. ASPE Primers

[0028] Specific primer sequences were designed for the wild-type and mutant types of the three common genotypes T61C, T101G / A and C81T of the ABCB1 gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0029] The ASPE primer sequence (Tag sequence+specific primer sequence) of table 2ABCB1 gene

[0030]

[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-t...

Embodiment 3

[0099] The detection of the polymorphic site of ABCB1 gene by the liquid chip of embodiment 3 different ASPE primers

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

[0101] Taking the ABCB1 gene T61C 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 T61C, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1 -SEQ ID NO.7, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.15-SEQ ID NO.21. 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.

[0102] Table 8 Design of liquid phase chip preparation

[0103]

[01...

Embodiment 4

[0111] The selection of embodiment 4ABCB1 gene SNP detection specific primer sequence

[0112] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0113] Taking the SNP site detection liquid chip of ABCB1 gene T101G / A as an example, using the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, design ASPE primers for the wild type and mutant type of T101G / A respectively The specific primer sequences at the 3' end include the preferred specific primer sequences and 2 alternative specific primer sequences in Example 1 of the present invention, as shown in Table 10. in, Inner bases are polymorphic sites.

[0114] Table 10 specific primer sequence

[0115]

[0116] Taking the polymorphic site detection liquid chip of ABCB1 gene T101G / A as an example, different specific primer sequences were selected for T101G / A, and the Tag sequence at the 5' end of the ASPE...

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Abstract

The invention discloses ABCB1 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of 5' terminal tag sequence and 3' terminal specific primer sequences aiming at target gene mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.8 and SEQ ID NO.9 aiming at T61C site; SEQ ID NO.10 and SEQ ID NO.11 and / or SEQ ID NO.12 aiming at T101G / A site; and / or SEQ ID NO.13 and SEQ ID NO.14 aiming at C81T 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 ABCB1 gene polymorphism and a liquid phase chip. Background technique [0002] There are many mechanisms of tumor multidrug resistance, among which intracellular drug efflux mediated by members of ABC transporter superfamily (ATP-binding cassette transporter superfamily) plays an important role in MDR, and it is also one of the hot research topics at home and abroad. one. [0003] Multidrug resistance gene ABCB1 (multidrug resistance 1, or MDR1), also known as multidrug resistance-related protein (multidrug resistance protein, MRP1), is the coding gene of P-glycoprotein (P-glycoprotein, P-gp). P-glycoprotein, the expression product of multidrug resistance gene MDR1, is an ATP-dependent membrane transport protein, which can excrete drugs and many other related molecules from cells and participate in many norma...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森刘志明
Owner SUREXAM BIO TECH
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