Enrichment and detection method of target gene fragment

A technology of target genes and fragments, applied in biochemical equipment and methods, microbial determination/testing, DNA preparation, etc.

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

[0013] Aiming at the above-mentioned technical problems, the present inventor has developed a method for capturing DNA sequences of specific genes based on hybridization selection through a large number of experiments. By using this method, specific target gene fragments enriched by thousands of times can be obtained. The enriched target gene Fragment samples can be selectively applied to various genetic detection technologies, especially the next generation sequencing technology can be used to detect gene mutations, deletions, additions, and transversions

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  • Enrichment and detection method of target gene fragment
  • Enrichment and detection method of target gene fragment
  • Enrichment and detection method of target gene fragment

Examples

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Comparison scheme
Effect test

Embodiment 1

[0144] Example 1: Using the method of the present invention to enrich and detect the KRAS gene in cells

[0145] In this example, the KRAS gene of the MDA-MB-231 cell line was enriched and detected.

[0146] 1. Prepare the DNA sample bank of the MDA-MB-231 cell line to be tested

[0147] 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")

[0148] 1.1 DNA extraction

[0149] Using Qiagen Blood&Tissue DNeasy kit (article number: 69506), whole genome DNA was extracted from the MDA-MB-231 cell line (human breast cancer cell line, from the ATCC cell bank, article number: HTB-26). Follow the instructions in the manual.

[0150] Use a spectrophotometer and a gel electrophoresis system to detect the quality and concentration of DNA. The 260nm absorbance of dsDNA is greater than 0.05, and the absorbance ratio of A260 / A280 is between 1.8 and 2.

[0151] 1.2 DNA frag...

Embodiment 2

[0229] Example 2: Using the method of the present invention to enrich and detect the KRAS gene in cells

[0230] In this example, the KRAS gene of the HCT-116 cell line was enriched and detected.

[0231] Using the probe library composed of SEQ ID NO. 1 to SEQ ID NO. 10 obtained in Example 1, the HCT-116 cell line (human colorectal cancer cell line, derived from ATCC cells) was detected by the same method as in Example 1. Library, catalog number: ATCC-CCL-247) KRAS gene, a single base mutation was found in the KRAS gene of HCT-116 cell line, which is 38G> A. See the result Figure 4 .

[0232] After DNA extraction, PCR amplification and Sanger sequencing, the above mutations have been verified.

Embodiment 3

[0233] Example 3: Use the method of the present invention to enrich and detect the EGFR gene in cells

[0234] This example detects the EGFR gene of the K-562 cell line.

[0235] 1. Prepare the DNA sample bank of the K-562 cell line to be tested

[0236] Except that the K-562 cell line (human chronic leukemia cell line, from the ATCC cell bank, catalog number: ATCC-CCL-243) is different, the experimental operation is the same as the first step of Example 1.

[0237] 2. Prepare DNA probe library for EGFR gene

[0238] Except for the difference of EGFR mRNA (NCBI accession number: NM_005228.3), the experimental operation is the same as the second step of Example 1. Through the search of the human gene bank, it is ensured that the selected probe has a small off-target rate. After screening, first of all, 85 probes met the standard. Through IDT DNA Technologies, each probe was synthesized separately and analyzed by mass spectrometry to ensure the quality, and there was Biotin at the 5'en...

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Abstract

The invention provides a method for enriching target gene fragments. The method comprises the following steps: (1) obtaining a partial or whole-length DNA (deoxyribonucleic acid) sample library containing a target gene; (2) obtaining a DNA probe library capable of hybridizing with the target gene; (3) hybridizing the DNA probe library and the DNA sample library; (4) separating a hybrid product obtained in the step (3), and releasing the target gene fragments subjected to hybridizing and enriching. According to the enrichment method, the invention further provides a method for detecting the gene structure mutation of the target gene. The method for detecting the gene structure mutation of the target gene comprises the following steps: (1) enriching the target gene fragments according to the method; (2) detecting the structure mutation of the target gene. The target gene fragments enriched by using the method can be used for next-generation sequencing technique to detect the gene structure mutation and comprise single-alkali base mutation, mRNA (Messenger RNA) absence 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 gene enrichment and extraction method, which can accurately enrich required gene fragments, and then can selectively detect gene structural mutations. Background technique [0002] DNA sequencing technology is undergoing radical changes. Its outstanding feature is the simultaneous observation and analysis of many sites (massive parallelism), thereby gradually realizing a substantial increase in sequencing throughput and the cost of sequencing each base in the original data. The sharp decline. Based on this, previously unattainable luxury activities (such as personal genetic sequencing, metagenomics research) have gradually become more and more feasible. Especially with the development of science, since traditional Sanger sequencing can no longer fully meet the needs of research, next-generation sequencing technology (Next-generation sequencing) ha...

Claims

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Application Information

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