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Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof

A detection method and mutation detection technology, applied in DNA/RNA fragmentation, microbial determination/inspection, biochemical equipment and methods, etc. Improve detection accuracy, simple steps, and avoid interference

Active Publication Date: 2009-07-22
广州益善医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 the most widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of practical 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 also 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 a sensitivity of 1%-2%, high detection efficiency and strong timeliness, but its high false positive rate is also criticized by practical applications.

Method used

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  • Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof
  • Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof
  • Detecting probe and liquid phase chip for BRAF gene mutation and detecting method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Preparation of a Liquid Chip for BRAF Gene Exon 15 Mutation Detection

[0034] 1. Probe sequence design and microsphere coating

[0035] Specific oligonucleotide probes were designed for the wild-type and mutant sequences of exon 15 of BRAF. When the oligonucleotide probe sequence is synthesized, add a spacer sequence of 5-30 T (usually 10 T, the effect is consistent with 5-30 T). The specific steps of coating each microsphere are as follows (according to conventional method):

[0036] (1) Take the microsphere mother solution (purchased from Luminex Company) and vortex to form a microsphere suspension;

[0037] (2) Take out 8ul microsphere mother solution, containing 0.8×10 5 —1.2×10 5 microspheres into a 0.5ml centrifuge tube;

[0038] (3) Centrifuge at 10,000rpm for 3min, discard the supernatant;

[0039] (4) Add 10ul coupling solution (pH4.5) and mix well;

[0040] (5) Add 2 μl of 2 pmol / ul probe working solution;

[0041] (6) Add 2.5ul of EDC (1-ethy...

Embodiment 2

[0052] Example 2 Detection of Clinical Samples Using BRAF Gene Mutation Detection Liquid Chip

[0053] 1. Preparation of samples to be tested

[0054] Nucleic acid was extracted according to the instructions of the AxyPrep extraction kit, and 10 cases of DNA samples to be tested were obtained.

[0055] 2. PCR amplification and enzyme digestion enrichment of the samples to be tested:

[0056] (1) The first round of PCR amplification

[0057] PCR reaction system:

[0058] reaction system per reaction (μl) Sterilized ddH2O 28.8

[0059] 5×Buffer 10 2.5mM dNTP mix 2 MgCl 2 25mM 5 Primer F: B15F (10uM) 1 Primer R: B15R1 (10uM) 1 Taq enzyme (5U / ul) 0.2 template DNA 2 total capacity 50

[0060] PCR reaction conditions:

[0061]

[0062] (2) PCR product TspRI digestion:

[0063] The reaction system is as follows:

[0064] Sample system (incubated at 60°C for 2 hours) per reaction (μl...

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Abstract

The invention discloses a nucleic acid probe, a liquid chip and a detection method that are used for the mutation detection of a BRAF gene exon 15. The liquid chip that is used for the mutation detection of the BRAF gene exon 15 comprises microspheres and a primer; wherein, the microspheres are coated with wild and mutant probes which are decorated with amino groups and aim at the BRAF gene exon 15; and the primer is used for augmenting a target sequence that is enriched in mutant sites needing to be detected by the BRAF gene exon 15 and has a biotin marker in the tail end. The detection method that is provided by the invention has quickness, accuracy, simple and convenient operation, and greatly improves the detection efficiency.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection probe for BRAF gene mutation, a liquid phase chip and a detection method thereof. Background technique [0002] The full name of the BRAF gene is v-raf oncogenic homologue B1, located on human chromosome 7q34, and its functional coding region consists of 2510 base pairs, encoding the serine threonine in the MAPK pathway An amino acid protein kinase that transduces signals from RAS to MEK1 / 2, thereby participating in the regulation of cell growth, proliferation, and apoptosis. It is now generally accepted that the T1799A mutation on exon 15 of the BRAF gene causes the valine in the 600 residue of the BRAF protein product to be replaced by glutamic acid (V600E), thereby continuously activating the BRAF kinase, resulting in the continuous activation of the MAPK pathway , unlimited cell division and proliferation. . The frequency of other sites of BRAF gene mut...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q2600/156C12Q1/6883C12Q1/6886
Inventor 许嘉森朱泽尧陈玲
Owner 广州益善医学检验所有限公司
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