Primer, method and kit for detecting mutation sites of human BRCA1 and BRCA2 gene all-coding sequences

A mutation site and gene encoding technology, applied in the field of gene mutation detection in biotechnology, can solve the problems of low sensitivity, long cycle and high cost, and achieve the effects of improving primer specificity, simple operation and cost saving.

Active Publication Date: 2019-03-01
杭州链康医学检验实验室有限公司
View PDF7 Cites 17 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a primer, method and kit for detecting mutation sites in the full coding sequence of BRCA1 and BRCA2 genes related to human genetic susceptibility based on multiplex PCR and high-throughput sequencing technology, aiming to solve the current There are problems such as high cost, many steps, low sensitivity, long cycle and easy pollution in the technology

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer, method and kit for detecting mutation sites of human BRCA1 and BRCA2 gene all-coding sequences
  • Primer, method and kit for detecting mutation sites of human BRCA1 and BRCA2 gene all-coding sequences
  • Primer, method and kit for detecting mutation sites of human BRCA1 and BRCA2 gene all-coding sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The performance verification of this kit is carried out by detecting the standard of known mutation type and frequency.

[0037] A. Standard DNA Quantification

[0038] Use Novagen human genomic DNA (Merck, Cat. No. 69237) as a standard DNA template, use Fluorescence Quantitative Instrument and Corresponding The quantification reagent (Invitrogen) was used for quantification, and the concentration was 100 ng / uL.

[0039] B. The multiplex PCR method amplifies the target region of the sample genome.

[0040]1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepare the PCR reaction system as shown in the table below, and be careful to avoid cross-contamination.

[0041] components

volume

2×PCR mix

6.25 μL

specific primer

2.0 μL

Universal Primer R5

0.5μL

Universal Primer R7

0.5μL

DNA template, 50ng

xμL

nuclease free water

3.25-x μL

total capacity

12.5μL ...

Embodiment 2

[0064] BRCA1 / 2 gene mutations were detected in 20 patients with breast tumors.

[0065] A. Genomic DNA extraction from whole blood of patients

[0066] Genomic DNA was extracted from the patient's peripheral blood samples using a blood genomic DNA extraction kit (Tiangen, Cat. No. DP318-02), and the operation was as described in the instructions of the extraction kit. use Fluorescence Quantitative Instrument and Corresponding Quantification reagent (Invitrogen) was used for quantification. The quantitative results are shown in Table 2.

[0067] Table 2. Sample quantification results

[0068]

[0069]

[0070] B. The multiplex PCR method amplifies the target region of the sample genome.

[0071] 1). Prepare a sterile, nuclease-free 200 μL PCR tube and place it on ice; prepare the PCR reaction system as shown in the table below, and be careful to avoid cross-contamination.

[0072] components

volume

2×PCR mix

6.25 μL

specific primer ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a primer, a method and a kit for detecting mutation sites of human BRCA1 and BRCA2 gene all-coding sequences. The primer comprises forward and reverse primers which can amplifyfor covering all the 24 coding zones of BRCA1 gene and of which base sequences are shown as SEQ ID NO:3-52. The primer further comprises forward and reverse primers which can amplify for covering allthe 27 exon coding zones of BRCA2 gene and of which base sequences are shown as SEQ ID NO: 53-134. A detection method based on NGS technique comprises the following steps: combining a specific primerwith a target template DNA sequence; adopting a universal primer for amplifying a to-be-detected sample target area; purifying a library by using a magnetic bead; performing high-throughput sequencing on the acquired library and analyzing mutation of BRCA1 and BRCA2 genes. The primer, method and kit disclosed by the invention can cover all the to-be-detected mutation sites; uniformity is high; experimental procedures can be simplified; detection sensitivity is promoted; a guidance is supplied for selective medication of patients suffering from familial breast cancer and ovarian cancer as wellas for genetic predisposition; morbidity risk is effectively reduced.

Description

technical field [0001] The invention relates to the field of gene mutation detection in biotechnology, and more specifically relates to a primer, method and kit for detecting mutation sites in the full coding sequences of human BRCA1 and BRCA2 genes. Background technique [0002] The high incidence of breast cancer has seriously threatened women's health and is one of the most common cancers in women. Breast cancer can be divided into two categories: sporadic and hereditary. At present, it is believed that most hereditary breast cancers are caused by gene mutations, among which BRCA1 and BRCA2 are the most direct breast cancer susceptibility genes discovered so far. Studies have shown that people with BRCA1 and BRCA2 gene mutations have a higher risk of developing breast cancer and recurrence than the general population. Women with BRCA1 and BRCA2 gene mutations have a lifetime risk of breast cancer as high as 87%. . Not only that, but the BRCA1 / 2 gene is also a susceptibi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156Y02A50/30
Inventor 郎秋蕾周小川李璐璐林彬方超张瑞静梁洪殷楠楠
Owner 杭州链康医学检验实验室有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap