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Method and kit for detecting mutation of BRAF gene of human colorectal cancer

A technology for colorectal cancer and human detection, applied in the fields of biotechnology and medicine, can solve problems such as large limitations, and achieve the effects of simple operation, low detection cost, and accurate genotyping

Inactive Publication Date: 2012-07-18
苏州科贝生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Due to their own defects, the above detection methods have relatively large limitations in clinical application, and they are still only used as a detection method, not a finished kit.

Method used

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  • Method and kit for detecting mutation of BRAF gene of human colorectal cancer
  • Method and kit for detecting mutation of BRAF gene of human colorectal cancer
  • Method and kit for detecting mutation of BRAF gene of human colorectal cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Selection of samples and extraction of genomic DNA

[0058] The collected genomic DNA comes from whole blood, cells, fresh tissue, and paraffin-fixed tissue. The extraction method refers to the operation manual provided by the kit, and some optimizations have been made at the same time.

[0059] Whole blood genomic DNA of healthy people was used as the wild-type control of the experiment, and was extracted using the BloodGen Mini Kit of Kangwei Century;

[0060] Genomic DNA derived from the colo201 cell line was used as a control for the V600E homozygous mutant type (1799T>A) of exon 15 of the BRAF gene in the experiment,

[0061] The genomic DNA derived from the HT29 cell line was used as the control of the V600E heterozygous mutant type (1799T>A) of exon 15 of the BRAF gene in the experiment, and was extracted using the DNA FlexGenDNA Kit of Kangwei Century;

[0062] Both fresh tissue and paraffin tissue were derived from mice, and were used to evaluate th...

Embodiment 2

[0068] Example 2: Identification of mutation sites

[0069] HRM technology was used to detect the samples with known BRAF gene exon 15 V600E codon genotype.

[0070] 1. Specific primers and probes are as follows:

[0071] SEQ ID No: 3, Forward primer:

[0072] 5'-ACAACTGTTCAAACTGATGGGACC-3'

[0073] SEQ ID No: 4, Reverse primer:

[0074] 5'-TCCTTTACTTACTACACCTCAGATAT-3'

[0075] SEQ ID No: 2, specific probe: 5'-TCTAGCTACAGTGAAATCTCGAT-3'

[0076]2. Amplify a fragment near the V600E codon by PCR; prepare a mixture: add 1 μl of the genomic DNA solution prepared before, 2 μl of PCR buffer (10×), 1.6 μl of dNTP, 0.1 μl of Taq DNA polymerase, and 0.4 μl of forward primer and 0.08 μl of reverse primer, 2 μl of SYTO 9 fluorescent dye, and PCR-grade pure water to make the reaction volume 20 μl. Reaction at 95°C for 15 minutes, 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds, for 20 cycles, 82°C for 15 seconds, 60°C for 15 seconds, 72°C for 15 seconds, for 15 cycles...

Embodiment 3

[0080] Example 3: Kit Sensitivity Verification

[0081] The homozygous mutant colo201 genome and the wild-type genome extracted from whole blood were mixed in a certain proportion, so that the homozygous mutant genome accounted for 0.05% of the total DNA amount. The concentration of the above mixed genomic DNA and wild-type genomic DNA was adjusted to 10 ng per microliter.

[0082] 1. Specific primers and probes are as follows:

[0083] SEQ ID No: 3, Forward primer:

[0084] 5'-ACAACTGTTCAAACTGATGGGACC-3'

[0085] SEQ ID No: 4, Reverse primer:

[0086] 5'-TCCTTTACTTACTACACCTCAGATAT-3'

[0087] SEQ ID No: 2, specific probe: 5'-TCTAGCTACAGTGAAATCTCGAT-3'

[0088] 2. Amplify a fragment near the V600E codon by PCR; prepare a mixture: add 1 μl of the genomic DNA solution prepared before, 2 μl of PCR buffer (10×), 1.6 μl of dNTP, 0.1 μl of Taq DNA polymerase, and 0.4 μl of forward primer and 0.08 μl of reverse primer, 2 μl of SYTO 9 fluorescent dye, and PCR-grade pure water to...

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Abstract

The invention relates to a method and a kit for detecting gene mutation, in particular to a method and a kit for detecting the mutation of BRAF gene. The invention is characterized in that the kit comprises a specific probe used for carrying out genotyping on No. 15 exon codon V600E of the BRAF gene, wherein the specific probe of the No. 15 exon codon V600E comprises a nucleotide sequence of V600E codon. by the technology combining the conventional polymerase chain reaction (PCR) amplification with a Cold-PCR enrichment amplification product and a high resolution melting curve analysis technology, the kit provided by the invention can be used for completing the judgment on sample genotyping.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a method and kit for detecting BRAF gene mutation. Background technique [0002] Colon cancer is one of the common malignant tumors in humans. In recent years, studies have found that in addition to the classic "adenoma-carcinoma" pathway, there is also a "deformed crypt focus-hyperplastic polyp-serrated adenoma-carcinoma" pathway, that is, the serrated adenoma pathway. This pathway is regulated by the mitogen-activated protein kinases (mitogen-activated protein kinases, MAPK) / extracellular signal-regulated kinases (ERK) signaling pathway, and the BRAF gene is an important regulator. [0003] 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 As an amino acid protein kinase, BRAF protein is located at the entran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 姬云张泓侯青
Owner 苏州科贝生物技术有限公司
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