DNA probe library for hybridizing with kras gene and method for enriching kras gene fragments using the same

A DNA probe, gene fragment technology, applied in the field of KRAS gene enrichment and extraction

Active Publication Date: 2015-09-30
GENESEEQ TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Aiming at the above-mentioned technical problems, the present inventor has developed a method for capturing the KRAS gene sequence based on hybridization selection through a large number of experiments. By using this method, KRAS gene enriched by thousands of times can be obtained. The enriched KRAS gene sample can be selected It can be applied to various genetic detection technologies, especially the detection of gene mutations, deletions, additions, and transversions using next-generation sequencing technology

Method used

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  • DNA probe library for hybridizing with kras gene and method for enriching kras gene fragments using the same
  • DNA probe library for hybridizing with kras gene and method for enriching kras gene fragments using the same
  • DNA probe library for hybridizing with kras gene and method for enriching kras gene fragments using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1: Enrichment and detection of KRAS gene in MDA-MB-231 cell line

[0149] (1) Prepare the DNA sample bank of the MDA-MB-231 cell line to be tested

[0150] 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")

[0151] 1.1 DNA extraction

[0152] 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.

[0153] 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.

[0154] 1.2 DNA Fragmentation

[0155] Dilute 3 µg of high-quality genomic DNA to 120 µl with low TE buffer. According to...

Embodiment 2

[0233] Example 2: Enrichment and detection of KRAS gene in HCT-116 cell line

[0234] Using the probe library consisting of SEQ ID NO.1 to SEQ ID NO.10 obtained in Example 1, the same method as in Example 1 was used to detect the HCT-116 cell line (human colorectal cancer cell line, from ATCC cells Library, product number: ATCC-CCL-247) of the KRAS gene, a single base mutation was found in the KRAS gene of the HCT-116 cell line, which was 38G>A. see results Figure 4 .

[0235] After DNA extraction, PCR amplification and Sanger sequencing, the above mutations have been verified.

[0236]

[0237]

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Abstract

The invention provides a DNA (Deoxyribonucleic Acid) probe library used for hybridization with a KRAS gene. The DNA probe library comprises one or more DNA probes which can be hybridized with the KRAS gene, wherein the DNA probes include the following sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10. The invention further provides a method for enriching the KRAS gene segments by adopting the DNA probe library. Based on this, the invention further provides a method for detecting gene structure mutations of the KRAS gene. The method disclosed by the invention can be adopted to obtain the KRAS gene segments by enriching in thousands of times, and the KRAS gene segments can be used for a next generation sequencing technology to detect the gene structure mutations including single base mutation, mRNA deletion 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 KRAS gene enrichment and extraction method. The method can accurately enrich the KRAS gene, and then can selectively detect the structural mutation of the KRAS gene. 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 (Next-genera...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B40/06C12N15/10C12Q1/68
Inventor 邵阳
Owner GENESEEQ TECH INC
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