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PDGFRA gene mutation detection liquid-phase chip

A detection solution and chip technology, applied in the field of molecular biology, can solve the problems of high false positive rate, easy contamination of samples, high price, etc., and achieve the effects of avoiding cross-reaction, strong scalability, and improved sensitivity

Active Publication Date: 2011-03-09
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection products of PDGFRA polymorphism mainly include quantitative PCR, direct sequencing, pyrosequencing pyrosequencing technology, DHPLC, etc., which have disadvantages such as low sensitivity, easy sample contamination, high false positive rate, and high price. limitations and cannot meet the needs of practical applications

Method used

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  • PDGFRA gene mutation detection liquid-phase chip
  • PDGFRA gene mutation detection liquid-phase chip
  • PDGFRA gene mutation detection liquid-phase chip

Examples

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Embodiment 1

[0017] Embodiment 1 PDGFRA gene mutation detection liquid chip mainly includes:

[0018] 1. ASPE Primers

[0019] Specific primer sequences were designed for four common mutations V561D, DIMH 842-845 deletion mutation, IMHD 843-846 deletion mutation, and D842V of the PDGFRA gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0020] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0021]

[0022]

[0023] 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 100pmol / mL stock solution with 10mmol / LTris Buffer.

[0024] 2. Microspheres coated with anti-tag s...

Embodiment 3

[0102] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of PDGFRA gene mutation site

[0103] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0104] Taking the PDGFRA gene locus V561D 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 V561D, 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 NO.21. 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.

[0105] Table 7 Design of liquid phase chip preparation

[0106]

[0...

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Abstract

The invention discloses a PDGFRA gene mutation detection liquid-phase chip, which comprises tag sequence at 5' terminal and ASPE (Allele Specific Primer Extension) primers specific to mutational sites at 3' terminal. The ASPE primers consist of sequences shown in SEQ ID No.8-SEQ ID No.9 specific to V561D sites, sequences shown in SEQ ID No.10-SEQ ID No.12 specific to DIMH 842-845 and / or IMHD 843-846 deletion mutational sites, and / or sequences shown in SEQ ID No.13-SEQ ID No.14 specific to D842V site, the tag sequence which is selected from sequences shown in SEQ ID No.1- SEQ ID No.7, color coding microballoons which are respectively enveloped with a specific anti-tag sequence and amplification primers. The coincidence ratio with a sequencing method of the PDGFRA gene mutation detection liquid-phase chip reaches up to 100 percent. The PDGFRA gene mutation detection liquid-phase chip can simultaneously detect aiming at a plurality of deletion mutational sites with excellent signal to noise ratio.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a PDGFRA gene mutation detection liquid phase chip. Background technique [0002] Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract, and its incidence is increasing year by year. Imatinib mesylate (Gleevec) is a molecularly targeted drug for the treatment of unresectable or metastatic malignant gastrointestinal stromal tumors, and has achieved very good results in the treatment of GIST. The mechanism of action of imatinib is to inhibit the tyrosine kinase of the platelet-derived growth factor (PDGFR) receptor, thereby inhibiting the cell behavior induced by PDGFR. In vitro experiments show that imatinib can inhibit the proliferation of GIST cells and induce their apoptosis. Death. Although the curative effect of imatinib is very good, the problem of drug resistance inevitably arises. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森余刚
Owner SUREXAM BIO TECH
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