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Method for detecting loss of heterozygous BRCA1/2 gene and primers adopted by method

A gene deletion and heterozygosity technology, applied in the field of molecular biology, can solve problems such as difficult to accurately detect heterozygosity loss and amplification bias

Active Publication Date: 2016-04-27
重庆精准医疗产业技术研究院有限公司
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Problems solved by technology

However, traditional high-throughput sequencing requires PCR amplification during library construction, which can lead to severe or slight amplification bias, making it difficult to accurately detect loss of heterozygosity

Method used

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  • Method for detecting loss of heterozygous BRCA1/2 gene and primers adopted by method
  • Method for detecting loss of heterozygous BRCA1/2 gene and primers adopted by method
  • Method for detecting loss of heterozygous BRCA1/2 gene and primers adopted by method

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Embodiment 1

[0048] Embodiment 1. Applying the present invention, the BRCA1 / 2 gene of a verified BRCA1 mutation breast cancer patient and a verified normal person were detected.

[0049] 1. Experimental methods and steps

[0050] 1. Obtaining BRCA1 / 2 positive samples: 2ml of blood was taken with EDTA anticoagulation from a breast cancer patient diagnosed in a certain hospital. Genomic DNA in blood was extracted with QiagenDNeasyBloodTissueKit kit (Germany Quick and Fine, Cat. No. 69506), and the Nanodrop concentration was 50ng / ul. At the same time, the sample has been verified by the MLPA method of Shanghai Jikang Biotechnology Co., Ltd. to only have the deletion of exon 11 of BRCA1.

[0051] 2. Obtain the sequence of each exon of the BRCA1 / 2 gene from the NCBI data, and then design primers (primer5 software can be used). The primers are shown in Table 1. The primers are synthesized by Yingwei Jieji (Shanghai) Trading Co., Ltd., and purified by HPLC .

[0052] 3. Amplify the genomic DNA...

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Abstract

The invention discloses primers for detecting loss of a heterozygous BRCA1 / 2 gene, wherein the primers are 370-400bp specific primers which are designed in accordance with 49 exon regions of the BRCA1 / 2 gene; each pair of the specific primers includes an overlay region which is at least 20bp; and at the 5' terminal of the specific primers, a universal sequence and five continuous N bases are designed, called tags. The invention also provides a method for detecting the loss of the heterozygous BRCA1 / 2 gene by virtue of the primers. Through a next generation sequencing technology, large-fragment deletion of the heterozygous BRCA1 / 2 gene can be detected, so that a high-risk group with the BRCA1 / 2 disease can be found out.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for detecting gene deletion mutations based on next-generation sequencing. Background technique [0002] Breast cancer is a cancer with a rapidly increasing incidence worldwide. In the World Geographic Atlas of Breast Cancer published by WHO, the number of incidences per 100,000 population is: Australia (83 / 100,000), the United States, Canada (82 / 100,000), and Europe (70 / 100,000). Followed by South American countries (65 / 100,000), Eastern Europe (49 / 100,000), North Africa, South Africa, Japan, Israel, and Arabia (28 / 100,000), while Central Africa and Asia had the lowest incidence (16-19 / 100,000). 100,000). [0003] Breast cancer accounts for the first incidence rate among female cancers, and accounts for more than 1 / 4 of all female cancers. One of every 7-9 females in Europe and the United States will develop breast cancer in their lifetime. Women who carr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 洪旭涛祁鸣
Owner 重庆精准医疗产业技术研究院有限公司
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