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Gene library constructing method for hereditary gastrointestinal tumor and kit

A technology for gastrointestinal tumors and gene libraries, which is applied in the field of genetic library construction methods and kits for hereditary gastrointestinal tumors, and can solve problems such as complicated processes and insufficient DNA quantity

Inactive Publication Date: 2019-03-15
ANNGEEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the genomic DNA fragmentation method is used in the sample library preparation process, there will be subsequent steps such as DNA end repair and A-tail addition. Not only is the process complicated, but the requirement for genomic DNA is more than 100ng. The sample will have insufficient amount of DNA extracted
In addition, DNA fragmentation requires the help of a DNA fragmentation instrument. After DNA fragmentation, it is necessary to use magnetic beads to enrich DNA fragments, which requires additional operation and cost for instrument maintenance.

Method used

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  • Gene library constructing method for hereditary gastrointestinal tumor and kit
  • Gene library constructing method for hereditary gastrointestinal tumor and kit
  • Gene library constructing method for hereditary gastrointestinal tumor and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Genomic DNA extracted from 10 peripheral blood samples was used for library construction, using exon regions encoding amino acids of 7 genes including APC, CDH1, MSH2, MLH1, MSH6, PMS2, and EPCAM, and 20 bases in each of the upstream and downstream exons The base region is used as the primer set of the target region for the amplification reaction of the target region, combined with the high-throughput sequencing platform Ion GeneStudioS5 for sequencing, and for the samples to be detected, single-base mutation (SNP) and small fragment insertion-deletion (InDel) detection and analysis . The specific operation process is as follows:

[0096] (1) Nucleic acid extraction and quality inspection: Genomic DNA extracted from peripheral blood samples is required to meet certain quality control standards after quality inspection: DNA concentration: ≥1ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230 >2; DNA total input amount: ≥2ng;

[0097] (1) Amplification of the target region:...

Embodiment 2

[0109] Genomic DNA extracted from 10 formalin-fixed paraffin-embedded samples was used for library construction, using exon regions and exon encoding amino acids of seven genes including APC, CDH1, MSH2, MLH1, MSH6, PMS2, and EPCAM Each 20-base region upstream and downstream of the target region is used as a primer set for the target region to amplify the target region, combined with the high-throughput sequencing platform Ion Torrent PGM for sequencing, and for the samples to be detected, single-base mutations (SNPs) and small fragment insertions are performed. Deletion (InDel) detection and analysis. The specific operation process is as follows:

[0110] (1) Nucleic acid extraction and quality inspection: Genomic DNA extracted from peripheral blood samples is required to meet certain quality control standards after quality inspection: DNA concentration: ≥1ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230 >2; DNA total input amount: ≥2ng;

[0111] (2) Amplification of the targe...

Embodiment 3

[0125] Genomic DNA extracted from 10 buccal swab samples was used for library construction, using exon regions encoding amino acids of 7 genes including APC, CDH1, MSH2, MLH1, MSH6, PMS2, and EPCAM, and 20 upstream and downstream exons each The base region is used as the primer set of the target region to carry out the amplification reaction of the target region, combined with the high-throughput sequencing platform IonTorrent PGM for sequencing, and for the samples to be detected, single-base mutation (SNP), small fragment insertion-deletion (InDel) detection and analyze. The specific operation process is as follows:

[0126] (3) Nucleic acid extraction and quality inspection: Genomic DNA extracted from peripheral blood samples is required to meet certain quality control standards after quality inspection: DNA concentration: ≥1ng / μL; DNA purity: OD260 / 280 1.8-2.0, OD260 / 230 >2; DNA total input amount: ≥2ng;

[0127] (4) Amplification of the target region: use the above (1) ...

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Abstract

The invention discloses a gene library constructing method for a hereditary gastrointestinal tumor, and relates to mutation of 7 genes, namely APC, CDH1, MSH2, MLH1, MSH6, PMS2 and EPCAM. Target regions are exon regions of encoding amino acids of the 7 genes and respective 20 base regions at the upstream and the downstream of exons. In order to ensure complete coverage of the target regions, a primer group necessary for PCR amplification is separated into two independent primer groups, and the target regions are amplified respectively. Then, sequencing tag connection, library elution and re-amplification are performed on a PCR product, and purification is performed after the amplification to obtain a sequencing library. The library constructing method is simple and rapid in steps, the costof library construction is effectively reduced and genes related to the gastrointestinal tumor are covered. Through combination with a high-throughput sequencing instrument, mutation of the related genes can be quickly and accurately obtained, so that the gene library constructing method is of great significance for the hereditary gastrointestinal tumor.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a gene library construction method and kit for hereditary digestive tract tumors. Background technique [0002] Gastrointestinal cancer is a malignant tumor with high incidence worldwide. Among malignant tumors, most of gastric cancer and colorectal cancer are sporadic, but about 10% of gastric cancer patients have familial aggregation, and about 5-10% of intestinal cancers have genetic related factors. Hereditary diffuse gastric cancer and hereditary nonpolyposis colorectal cancer (HNPCC) are two hereditary gastrointestinal tumors with relatively clear genetic factors. [0003] In hereditary diffuse gastric cancer families, the incidence of tumors such as lobular breast cancer and colon cancer also increased significantly. Some early-onset (age <50 years old) diffuse gastric cancers with no clear family history have CDH1 gene mutations. For CDH1 gene mutation carrie...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6869C12Q1/6886C40B50/06
CPCC12N15/1013C12Q1/6869C12Q1/6886C40B50/06C12Q2600/156C12Q2535/122
Inventor 周洋杨颖扶媛媛曹彦东
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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