Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation

A detection solution and chip technology, applied in the field of molecular biology, can solve the problems of pollution, poor timeliness, and false positive rate criticism, and achieve the effect of overcoming low sensitivity, improving detection accuracy, and good signal-to-noise ratio.

Active Publication Date: 2014-08-27
SUREXAM BIO TECH
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  • Application Information

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Problems solved by technology

The PCR-sequencing method has the advantage of being able to determine the range and type of mutations, and is currently a widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of clinical applications, especially for heterogeneity of tumor somatic mutations, low sensitivity will lead to a large number of missed detections
At the same time, the detection operation of the sequencing method is complicated and the timeliness is poor. For clinical detection that requires high timeliness and high sensitivity, the limitations of the sequencing method have long been highlighted
The real-time fluorescent quantitative PCR detection technology has high detection efficiency and strong timeliness, but its high false positive rate is also criticized by clinical applications.
Based on the problems of the above-mentioned detection technology, the detection method of "PIK3CA gene mutation detection probe, liquid chip and its detection method" (application number: 200810220266.1) successfully developed by the applicant in the early stage is simple to operate, and a large number of The interference caused by the wild-type sequence has good specificity, high sensitivity, and an accuracy of more than 99%. However, this method involves two rounds of PCR operations, which is easy to cause pollution, and the probes in the hybridization detection step are relatively close (only the mutation site is different). ), it is not convenient for parallel detection of multiple gene mutation sites

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  • Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation
  • Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation
  • Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation

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Experimental program
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Effect test

Embodiment 1PI

[0021] Embodiment 1 PIK3CA gene mutation detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Specific primer sequences were designed for five mutant types of PIK3CA gene Exon9: E542K, E545K, E545G, E545A, E545D, and two mutant types of Exon20: H1047R, H1047L.

[0024] The design points of ASPE primers for PIK3CA gene mutation detection are as follows:

[0025]ASPE primers consist of "Tag sequence + specific primer sequence". Among them, the 5' end is the Tag sequence designed according to the PIK3CA gene mutation detection. The designed Tag sequence can avoid the secondary structure that may be formed by the ASPE primer in the reaction system to the greatest extent, and the Tag sequence and the Tag sequence, the Tag sequence and the specific There is no cross-reaction between the primer sequences. The Tag sequence and the specific primer sequence form complete ASPE primers, and enable all ASPE primers to react synchronously in a uniform reaction system ...

Embodiment 2

[0052] Example 2 Detection of samples using PIK3CA gene mutation detection liquid chip

[0053] The formula of described various solutions is as follows:

[0054] 50mM MES buffer (pH5.0) formula (250ml):

[0055] Reagent

[0056] 2×Tm hybridization buffer

[0057] Reagent

[0058] Store at 4°C after filtration.

[0059] ExoSAP-IT kit was purchased from US USB Company.

[0060] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0061] 1. Sample DNA extraction:

[0062] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0063] 2. PCR amplification of samples to be tested

[0064] Using Primer5.0 to design primers, multiplex PCR amplifies the target sequence with detection sites in one step, and the product sizes are 141bp and 108bp, respectively. The primer sequences (SEQ NO.81-84) are shown in Table 7 above.

[0065] First prepare the PCR primer ...

Embodiment 3

[0117] The selection of wild-type and mutant-type specific primer sequences with different source sequences in embodiment 3

[0118] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0119] For the PIK3CA gene Exon9 and Exon20 mutation sites, the specific primer sequences of the 3' end of the wild-type and mutant ASPE primers were designed respectively, and the specific primer sequences for detecting Exon9 and Exon20 mutations were derived from the SEQ ID of SEQ.NO11-20 NO.21-SEQ ID NO.40, or SEQ ID NO.51-SEQ ID NO.70 derived from SEQ NO.41-50. The Tag sequences at the 5' ends of the wild-type and mutant ASPE primers are respectively selected from SEQ ID NO.1-10, and correspondingly, the anti-tag sequences coated on the microspheres that are complementary to the corresponding tag sequences are respectively selected from SEQ ID NO. .71-80. The specific design is shown in the following table (Table 12). The synthesis of...

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Abstract

The present invention provides a liquid chip for detecting PIK3CA gene mutations, which comprises wild-type and mutant ASPE primer pairs designed according to the mutational sites of PIK3CA gene; beads coded by different colors and coated with specific anti-tag sequences, wherein said anti-tag sequences are complementary with the tag sequences selected; and amplification primers for amplifying target sequences of PIK3CA gene which comprise the corresponding mutational sites of Exon 9 or Exon 20. The present invention also provides specific primers for detecting PIK3CA gene mutations.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a PIK3CA gene mutation detection liquid phase chip. Background technique [0002] PI3Ks (phosphatidylinositol3-kinases) are a group of protein multimers, which are divided into three classes (ClassI, II and III). Class I proteins may include two subunits: IA and IB. IA is a heterodimer composed of a p110 catalytic subunit and a p85 regulatory subunit. The PIK3CA gene encodes the p110α catalytic subunit of class IA. PI3Ks can be activated by growth factor receptor tyrosine kinases (RTKs). Activation of PI3K produces a variety of biological effects, including regulation of cell proliferation, survival, and cell cycle regulation. [0003] The site of PIK3CA point mutation is found in multiple exons, but mainly occurs in the kinase and helical domains. The most common mutations are E542K and E545K of exon 9 (Exon 9), and H1047R ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/6834
Inventor 许嘉森李国强余刚秦会娟
Owner SUREXAM BIO TECH
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