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Breast cancer susceptibility gene detection kit

A technology for susceptibility genes and breast cancer, which is applied in the determination/inspection of microorganisms, nucleotide libraries, library creation, etc., which can solve the problem of high proportion of non-specific sequences, inappropriate optimization of annealing temperature and extension time, and impact on the target. Regional amplification efficiency and product quantity

Active Publication Date: 2021-04-02
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Similarly, inappropriate optimization of amplified fragments, primer design, annealing temperature, and extension time will lead to an increase in non-specific bands, and there may be more than a dozen pairs of primers mixed in one reaction, often resulting in reciprocal pairing or increased mismatched products , affect the amplification efficiency and product quantity of the target region, and ultimately lead to poor uniformity of sequencing data and a large proportion of non-specific sequences

Method used

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  • Breast cancer susceptibility gene detection kit
  • Breast cancer susceptibility gene detection kit
  • Breast cancer susceptibility gene detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0181] Example 1 Using the above primer sets 1-12, PCR amplification was performed using human genomic DNA as a template to obtain gene fragments of breast cancer susceptibility genes BRCA1 and BRCA2, and to construct a DNA library for next-generation sequencing

[0182] Preparation before experiment:

[0183] Prepare 30 μL of a human genomic DNA sample with a concentration of 20 ng / μL extracted from a case of blood cells, dilute the primers to a concentration of 10 μM with TE buffer (1×) pH 8.0, and thaw the PCR reagent on ice for use.

[0184] Experimental steps:

[0185] 1) Take several 0.2mL EP tubes, and configure a PCR system in each tube, the system is as follows:

[0186] 0.6 μL each of upstream primer and downstream primer (respectively constituting the above primer sets 1-12), 0.2 μL Taq enzyme, 2.5 μL 10×PCR buffer, 4 μL dNTP, 1 μL template, add ddH 2 O to 25 μL. In addition, a negative control was made without adding template. After the preparation is complete,...

Embodiment 2

[0198] Example 2 Using 216 pairs of primer sets (including the above primer sets 1-12), using the WaferGen platform to perform PCR amplification and library construction at the nanoliter level

[0199] In this embodiment, Barcode Primer, TE master mix and Universal Outer Primer were purchased from WaferGen Company.

[0200] 1. Preparation before the experiment:

[0201] 1) Start-up preparations for the WaferGen platform sampler, including checking the helium pressure, confirming the internal humidity of the system, checking the pressure bottle, waste liquid tank, lotion bottle, running the warm up program and tip clean program when starting up;

[0202] 2) Prepare test consumables such as 96-well plate, 384-well plate, 1.5mL EP tube;

[0203] 3) Prepare 85% ethanol, pre-equilibrate the magnetic beads, and melt the library preparation reagent on ice for later use;

[0204] 2. Test sample

[0205] A total of 23 cases of blood extraction samples and 1 case of negative control ...

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Abstract

The invention provides a breast cancer susceptibility gene detection reagent kit. The breast cancer susceptibility gene detection reagent kit comprises 12 primer groups for carrying out PCR (polymerase chain reaction) amplification on mutation hotspots of breast cancer susceptibility genes BRCA1 and BRCA2. The breast cancer susceptibility gene detection reagent kit has the advantages that the breast cancer susceptibility genes are basically identical in PCR amplification efficiency, accordingly, PCR amplification products can be used for second-generation sequencing on DNA (deoxyribonucleic acid) fragments with sequencing requirements, and ultimately obtained sequencing data for the mutation hotspots are excellent in uniformity.

Description

technical field [0001] Specifically, the present invention relates to a next-generation sequencing DNA library construction method and a detection kit for detecting mutations in breast cancer susceptibility genes BRCA1 and BRCA2. Background technique [0002] Breast cancer is a malignant tumor that seriously threatens women's health, and its mortality rate is second only to lung cancer. In many large and medium-sized cities in my country, breast cancer has accounted for the first cause of death from malignant tumors in women, seriously threatening women's health. [0003] Early diagnosis and early detection, the cure of breast cancer condition is quite optimistic. Only 20% of breast cancer patients diagnosed at stage IV can survive beyond 5 years, while 100% of patients diagnosed at stage I survive beyond 5 years. Therefore, the early diagnosis of breast cancer is an urgent problem to be solved. [0004] BRCA (breast cancer susceptibility gene) is a susceptibility gene of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C40B50/06C40B40/06
CPCC12Q1/6886C12Q2600/156C12Q2600/16C40B40/06C40B50/06
Inventor 王秀莉王旺玄兆伶李大为梁峻彬陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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