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DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same

A technology of DNA probes and gene fragments, applied in the field of enrichment and extraction of BRAF gene fragments

Active Publication Date: 2014-03-26
GENESEEQ TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Aiming at the above-mentioned technical problems, the present inventor has developed a method for capturing BRAF gene fragment sequences based on hybridization selection through a large number of experiments. By using this method, BRAF gene fragments enriched by thousands or even tens of thousands of times can be obtained. The collected BRAF gene fragment samples can be selectively applied to various genetic detection technologies, especially the next generation sequencing technology can be applied to the detection of gene mutations, deletions, additions, and transversions

Method used

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  • DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same
  • DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same
  • DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene, and method for enriching BRAF gene segments by adopting same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Example 1: Enrichment and detection of BRAF gene in MDA-MB-231 cell line

[0147] 1. Prepare the DNA sample bank of the MDA-MB-231 cell line to be tested

[0148] 1. Extract the whole genome DNA of the MDA-MB-231 cell line, and then fragment it (the DNA sample bank obtained in this way is called "DNA sample bank derived from the whole genome")

[0149] 1.1 DNA extraction

[0150] Whole genome DNA was extracted from MDA-MB-231 cell line (human breast cancer cell line, from ATCC cell bank, catalog number: HTB-26) using Qiagen Blood&Tissue DNeasy Kit (Cat. No.: 69506). Operate according to the instructions in the manual.

[0151] Use spectrophotometer and gel electrophoresis system to detect the quality and concentration of DNA. The 260nm absorbance of dsDNA is greater than 0.05, and the absorbance A260 / A280 ratio between 1.8 and 2 is qualified.

[0152] 1.2 DNA Fragmentation

[0153] Dilute 3 µg of high-quality genomic DNA to 120 µl with low TE buffer. According t...

Embodiment 2

[0231] Example 2: Enrichment and detection of BRAF gene in HT-29 cell line

[0232] Utilize the probe library that is made up of SEQ ID NO.1 to SEQ ID NO.6 that obtains in embodiment 1, adopt and the method identical in embodiment 1 to detect HT-29 cell line (human colon cancer cell line, from ATCC cell line library, product number: ATCC-HTB-38) of the BRAF gene, a single base mutation was found in the BRAF gene of the HT-29 cell line, which was 1799T>A.

[0233] After DNA extraction, PCR amplification and Sanger sequencing, the above mutations have been verified.

[0234]

[0235]

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Abstract

The invention provides a DNA (Deoxyribonucleic Acid) probe library hybridized with BRAF (v-Raf murine sarcoma viral oncogene homolog B1) gene. The DNA probe library comprises one or more DNA probes which can be hybridized with the BRAF gene, each DNA probe comprises the following sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, OR SEQ ID NO.6. The invention also provides a method for enriching BRAF gene segments by adopting the same. Based on this, the invention further provides a method for detecting the gene mutation of the BRAF gene. The BRAF gene segments can be enriched by thousands of times through adopting the method, the BRAF gene segments can be used for next-generation sequencing technology for detecting gene structure mutation including single base mutation, mRNA deficiency or increase, mRNA structure transversion and mRNA splicing change.

Description

technical field [0001] The present invention relates to the field of gene detection. Specifically, the present invention relates to a method for enriching and extracting BRAF gene fragments. The method can accurately enrich BRAF gene fragments, and then selectively detect BRAF gene structural mutations. . Background technique [0002] DNA sequencing technology is undergoing drastic changes. Its outstanding feature is the observation and analysis of many sites at the same time (massively parallel), so as to gradually achieve a substantial increase in sequencing throughput. The cost of sequencing each base in the original data a sharp decline. Based on this, previously unattainable luxury activities (such as personal gene sequencing, metagenomics research) have gradually become more and more feasible. Especially with the development of science, because the traditional Sanger sequencing can no longer fully meet the needs of research, next-generation sequencing technology (Nex...

Claims

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Application Information

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IPC IPC(8): C12N15/11C40B40/06C12N15/10C12Q1/68
Inventor 邵阳
Owner GENESEEQ TECH INC
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