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
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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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