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c-KIT gene mutation detection liquid-phase chip

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

Active Publication Date: 2013-04-24
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the c-KIT gene mutation detection products 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. Quantitative limitations, can not meet the needs of practical applications

Method used

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Embodiment 1

[0018] The c-KIT gene mutation detection liquid chip described in this embodiment mainly includes:

[0019] 1. ASPE Primers

[0020] Specific primer sequences were designed for the four common mutation sites AY502-503dup, V560G, T670I, and D816H / V of the c-KIT gene. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0021] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0022]

[0023]

[0024] 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.

[0025] 2. Microspheres coated with anti-tag sequences

[0...

Embodiment 2

[0037] Example 2 Detection of samples by c-KIT gene mutation detection liquid chip

[0038] The formula of described various solutions is as follows:

[0039] 50mM MES buffer (pH5.0) formulation (250mL):

[0040]

[0041] 2×Tm hybridization buffer:

[0042]

[0043] Store at 4°C after filtration.

[0044] ExoSAP-IT kit was purchased from US USB Company.

[0045] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0046] 1. Sample DNA extraction:

[0047] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0048] 2. PCR amplification of samples to be tested

[0049] Using Primer5.0 to design 3 pairs of primers, the c-KIT genes AY502-503dup, V560G, T670I, and D816H / V were amplified in one step by multiplex PCR, and the product sizes were 350bp, 309bp, and 301bp, respectively. 33) See Table 3 above.

[0050] First prepare the multiplex PCR primer working solution:...

Embodiment 3

[0099] The liquid phase chip of embodiment 3 different ASPE primers detects c-KIT gene mutation site

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

[0101]Taking the c-KIT gene AY502-503dup 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 AY502-503dup, and the Tag sequence at the 5' end of the ASPE primer was selected. From SEQ ID NO.1-SEQ ID NO.9, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.19-SEQ ID NO.27. 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]

[010...

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Abstract

The invention discloses a c-KIT gene mutation detection liquid-phase chip, which comprises tag sequence at 5' terminal and ASPE (Allele Specific Primer Extension) primers consisting of specific primers specific to mutational sites at 3' terminal. The specific primers consist of sequences shown in SEQ ID No.10 and SEQ ID No.11 specific to AY502-503dup mutational sites, sequences shown in SEQ ID No.12 and SEQ ID No.13 specific to V560G mutational site, sequences shown in SEQ ID No.14 and SEQ ID No.15 specific to T670I mutational site and / or sequences shown in SEQ ID No.16-SEQ ID No.18specific to D816H / V mutational site, the tag sequence which is selected from sequences shown in SEQ ID No.1-SEQ ID No.9, microballoons which have different color codings and are respectively enveloped with a specific anti-tag sequence and amplification primers. The coincidence ratio of the result of sequencing method with that of the c-KIT gene mutation detection liquid-phase chip reaches up to 100 percent. The c-KIT gene mutation detection liquid-phase chip can simultaneously detect a plurality of 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 c-KIT gene mutation detection liquid phase chip. Background technique [0002] Gastrointestinal stromal tumor (GIST) is a special tumor originating from the gastrointestinal tract. In recent years, with the in-depth research on the molecular pathology of GIST and the application of targeted drugs, GIST has attracted more and more attention. Surgical resection is still the only curative treatment for GIST, but the recurrence rate is extremely high. The molecular targeted drug imatinib mesylate (Gleevec) has achieved very good curative effect in the treatment of GIST and is the drug of choice for GIST patients today. [0003] The mechanism of action of imatinib is to inhibit the tyrosine kinase of platelet-derived growth factor (PDGFR) receptor and c-KIT receptor, thereby inhibiting the cell behavior mediated by PDGFR and c-KIT, and then i...

Claims

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Application Information

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