Method and reagent kit for constructing BRCA 1/2 (breast cancer susceptible genes 1/2) detection libraries

A technology for gene detection and method construction, applied in chemical libraries, biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of increased library construction costs, instrument maintenance costs, manual operation costs, increased manual operations, and fragment loss , Interrupt the problem of large original sample volume and other problems, achieve the simple and fast library building steps, reduce the purification operation of magnetic beads, and comprehensively detect the variation types

Inactive Publication Date: 2017-07-28
BGI GENOMICS CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Library construction generally needs to be carried out in steps. After fragmentation by physical fragmentation method and conventional enzyme fragmentation method, it is necessary to further select fragments of appropriate size for screening by gel electrophoresis or magnetic beads, followed by end repair, addition of A, addition of adapters, etc. Steps, many steps and cumbersome, long operation time, easy to cause pollution
[0009] Fragmentation o

Method used

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  • Method and reagent kit for constructing BRCA 1/2 (breast cancer susceptible genes 1/2) detection libraries
  • Method and reagent kit for constructing BRCA 1/2 (breast cancer susceptible genes 1/2) detection libraries
  • Method and reagent kit for constructing BRCA 1/2 (breast cancer susceptible genes 1/2) detection libraries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] In this example, 8 samples were used for library construction, and the Nimblegen chip containing BRCA1 / 2 gene was used for hybrid capture and combined with the high-throughput sequencer BGISEQ-500 platform to detect three types of BRCA1 / 2 gene SNV, InDel and CNV mutations. The specific operation process is as follows:

[0082] 1. Sample Preparation

[0083] 1.1 Sample extraction and quantification: DNA extraction kits from QIAGEN were used to extract gDNA from blood samples and cell lines, and 1 μL was taken for Qubit HS quantification. If it exceeds 100ng / μL, the sample needs to be diluted and re-quantified, and if it is less than 15ng / μL, it is enriched by the method of purification and recovery with Ampure XP magnetic beads.

[0084] 1.2 Homogenization of samples: 200ng of each sample was diluted to 15μL with NF water (Nuclease-free water), and transferred to a new PCR tube for library construction.

[0085] 2. DNA Fragmentation and End Repair

[0086] 2.1 Reagent...

Embodiment 2

[0214] In this example, 8 samples were used for library construction, and BGI chips containing BRCA1 / 2 genes were used for hybrid capture and combined with the high-throughput sequencer BGISEQ-500 platform to detect three types of BRCA1 / 2 gene SNV, InDel and CNV mutations. The specific operation process is as follows:

[0215] Steps 1 to 5 are the same as in Example 1.

[0216] 6. Hybrid Capture

[0217] 6.1 Library mixing and purification before hybridization:

[0218] 6.1.1 Each group of 8 samples shall be mixed, and a total of 1-3 μg shall be rehydrated to 160 μL.

[0219] 6.1.2 Optional step: add 0.6 times (the range can be floated) XP magnetic beads, after resting for 5 minutes, centrifuge instantly. Place the centrifuge tube on the magnetic stand, wait for the supernatant to be clarified, and pipette the supernatant into a new centrifuge tube. Add 0.6 times (the range can be floated) XP magnetic beads again for purification, and 21 μL for recovery.

[0220] 6.1.3 Ta...

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Abstract

The invention discloses a method and a reagent kit for constructing BRCA 1/2 (breast cancer susceptible genes 1/2) detection libraries. The method includes carrying out fragmentation and tail-end repair on homogenized DNA (deoxyribonucleic acid) samples in DNA fragmentation and tail-end repair systems under the effects of interruption enzymes and tail-end repair enzymes; linking products obtained at the previous step with linker sequences under the effects of DNA ligase; utilizing linker linking products obtained at the previous step as templates and carrying out PCR (polymerase chain reaction) amplification by the aid of primers of linker sequences under the effects of DNA polymerases; hybridizing probe sequences with BRCA 1/2 and products obtained at the previous step to trap DNA fragments with the BRCA 1/2 and carrying out elution to obtain hybridization trapped products; carrying out cyclization on single strands of the hybridization trapped products under the effects of ligase to obtain the BRCA 1/2 detection libraries. The method and the reagent kit have the advantages that steps for constructing the libraries are simple, convenient and speedy, the cost can be effectively reduced, workload can be relieved, and variation types are comprehensive and accurate and are high in flux.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and kit for constructing a BRCA1 / 2 gene detection library. Background technique [0002] According to the 2015 cancer statistics released by the National Cancer Center in the "Journal of Cancer for Clinicians", the annual incidence of ovarian cancer in Chinese women is 52,100, and the annual death rate is 22,500, with a mortality rate of about 43%. It ranks first among the three major gynecological tumors . Breast cancer has an annual incidence of 272,000 cases and an annual death rate of 70,700 cases, with a mortality rate of about 26%. In addition, research by the American Cancer Society shows that the 5-year survival rate of patients with type I breast cancer is 88%, and the 5-year survival rate of type IV breast cancer is only 15%, which shows that the higher the grade, the lower the survival rate; The higher the survival rate. Therefore, it is of great sig...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/68
CPCC12Q1/6869C12Q1/6806C40B50/06C12Q2535/122C12Q2565/519C12Q2525/191C12Q2521/501C12Q2521/301
Inventor 李红梅叶明芝李世勇苏孟媛
Owner BGI GENOMICS CO LTD
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