Gene library construction method of hereditary gynecologic tumors and kit

A technology for gynecological tumors and gene libraries, which is applied in chemical libraries, biochemical equipment and methods, and microbial measurement/testing, and can solve the problems of insufficient DNA and complicated processes

Inactive Publication Date: 2019-04-26
ANNGEEN BIOTECHNOLOGY CO LTD
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  • 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 construction method of hereditary gynecologic tumors and kit
  • Gene library construction method of hereditary gynecologic tumors and kit
  • Gene library construction method of hereditary gynecologic tumors and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Genomic DNA extracted from 20 peripheral blood samples was used for library construction, using ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PIK3CA, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53, MSH2, MLH1, MSH6, PMS2, EPCAM, a total of 20 genes encoding amino acid exon regions and 20 base regions upstream and downstream of the exons are used as the primer set of the target region for the amplification reaction of the target region, combined with the high-throughput sequencing platform IonGeneStudio S5 Sequencing is performed, and single base mutation (SNP) and small fragment insertion-deletion (InDel) detection and analysis are performed on the samples to be tested. The specific operation process is as follows:

[0109] (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 / 23...

Embodiment 2

[0122] Genomic DNA extracted from 20 formalin-fixed paraffin-embedded samples was used for library construction, including ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PIK3CA, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53, MSH2, MLH1, MSH6, PMS2, EPCAM, a total of 20 genes encoding amino acid exon regions and 20 base regions upstream and downstream of the exons are used as the primer set for the target region to amplify the target region, and combined with high The throughput sequencing platform Ion Torrent PGM performs sequencing, and performs single base mutation (SNP) and small fragment insertion-deletion (InDel) detection and analysis on the samples to be tested. The specific operation process is as follows:

[0123] (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; D...

Embodiment 3

[0139] Genomic DNA extracted from 20 buccal swab samples was used for library construction, including ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, NBN, PIK3CA, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53, MSH2, MLH1 , MSH6, PMS2, EPCAM, a total of 20 gene coding amino acid exon regions and 20 base regions upstream and downstream of the exons are used as primer sets for the target region to amplify the target region, and combined with the high-throughput sequencing platform IonTorrent PGM is used for sequencing, and single base mutation (SNP) and small fragment insertion-deletion (InDel) detection and analysis are performed on the samples to be tested. The specific operation process is as follows:

[0140] (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 amou...

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Abstract

The invention discloses a gene library construction method of hereditary gynecologic tumors and a kit and relates to mutation of 20 genes including ATM (ATM serine / threonine kinase), BARD1 (BRCA1 associated RING domain 1), BRCA1 (BRCA1, DNA repair associated), BRCA2 (BRCA2, DNA repair associated), BRIP1 (BRCA1 interacting protein C-terminal helicase 1), CDH1 (cadherin 1), CHEK2 (checkpoint kinase2), NBN (nibrin), PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha), PALB2 (partner and localizer of BRCA2), PTEN (phosphatase and tensin homolog), RAD51C (RAD51 paralogC), RAD51D (RAD51 paralog D), STK11 (serine / threonine kinase 11), TP53 (tumor protein p53), MSH2 (mutS homolog 2), MLH1 (mutL homolog 1), MSH6 (mutS homolog 6), PMS2 (PMS1 homolog 2, mismatch repair system component) and EPCAM (epithelial cell adhesion molecule). Target regions comprise exon regions of coding amino acids of the above 20 genes and 20 basic group regions at each of the upstream anddownstream of each exon. In order to ensure that all the target regions are covered, a primer group necessary for a PCR (Polymerase Chain Reaction) amplification process is separated into two independent primer groups for carrying out target region amplification respectively. Then a PCR product is subjected to sequencing label connection, library elution and reamplification and purification afteramplification to obtain a library for sequencing. The library construction method disclosed by the invention is simple and rapid in steps; the library construction cost is effectively reduced; and thelibrary covers related genes of the hereditary gynecologic tumors. A high-throughput sequencing instrument is combined and related gene variation can be rapidly and accurately obtained; and the genelibrary construction method has important significance on the hereditary gynecologic tumors.

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 gynecological tumors. Background technique [0002] Associated syndromes in hereditary gynecologic cancers are characterized by an increased risk of associated tumors and are common in the following patients: hereditary ovarian cancer, breast cancer syndrome, colorectal cancer, pancreatic cancer, melanoma, diffuse gastric cancer, endometrium cancer etc. The common feature of hereditary gynecological tumors is a high genetic risk, not only limited to women, but also increases the risk of prostate cancer in men. For patients, it is impossible to determine whether it is a genetic disease by relying only on the current routine pathological detection method. In this case, the detection of genes related to hereditary gynecological tumors shows great advantages, and provides a basis for genetic detection to determine whe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6886C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6886C40B50/06C12Q2531/113C12Q2521/501C12Q2523/308C12Q2535/122
Inventor 曹彦东周洋扶媛媛杨颖
Owner ANNGEEN BIOTECHNOLOGY CO LTD
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