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Primer pair, kit and method for detecting breast cancer susceptibility gene variation based on long fragment PCR

A susceptibility gene, long-segment technology, used in biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc., to achieve the effect of promoting efficient completion

Pending Publication Date: 2022-03-11
奥明(杭州)基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ordinary PCR can only amplify fragments of 2-3kb, and it is difficult to effectively amplify long-segment genes above 5kb

Method used

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  • Primer pair, kit and method for detecting breast cancer susceptibility gene variation based on long fragment PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Step 1: DNA Extraction

[0033] Take 200 microliters of peripheral blood, and perform DNA extraction according to the instructions of the blood / cell / tissue genomic DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and extract DNA for subsequent experiments or store at -20°C.

[0034] Step 2: PCR amplification and detection

[0035] (1) The BRCA1 and BRCA2 upstream and downstream primer pairs shown in the nucleotide sequence SEQ ID 1-36 are pre-mixed according to the mixing ratio;

[0036] (2) In the PCR tube, prepare the following reaction system:

[0037] Total volume 50 μl, containing 1 μl Phanta Max Super-Fidelity DNA Polymerase, 25 μl 2×Phanta Max Buffer at 2 mmol / L, 1 μl dNTP Mix at 10 mmol / L, 1 Microliter 2% DMSO, 2 microliter upstream primers (SEQ ID 1 , SEQ ID 3 , SEQ ID5 , SEQ ID 7 , SEQ ID 9 , SEQ ID 11 , SEQ ID 13 at a concentration of 10 millimolar per liter , SEQ ID 15, SEQ ID 17, SEQ ID 19, SEQ ID 21, SEQ ID 23, SEQ...

Embodiment 2

[0055] Step 1: DNA Extraction

[0056] Take 200 microliters of peripheral blood, and perform DNA extraction according to the instructions of the blood / cell / tissue genomic DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and extract DNA for subsequent experiments or store at -20°C.

[0057] Step 2: PCR amplification and detection

[0058] (1) The BRCA1 and BRCA2 upstream and downstream primer pairs shown in the nucleotide sequence SEQ ID 01-36 are pre-mixed according to the mixing ratio;

[0059] (2) In the PCR tube, prepare the following reaction system:

[0060]Total volume 50 μl, containing 1 μl Phanta Max Super-Fidelity DNA Polymerase, 25 μl 2×Phanta Max Buffer at 2 mmol / L, 1 μl dNTP Mix at 10 mmol / L, 1 Microliter 2% DMSO, 2 microliter upstream primers (SEQ ID 1 , SEQ ID 3 , SEQ ID5 , SEQ ID 7 , SEQ ID 9 , SEQ ID 11 , SEQ ID 13 at a concentration of 10 millimolar per liter , SEQ ID 15, SEQ ID 17, SEQ ID 19, SEQ ID 21, SEQ ID 23, SEQ...

Embodiment 3

[0078] Step 1: DNA Extraction

[0079] Take an appropriate amount of tissue, and perform DNA extraction according to the instructions of the blood / cell / tissue genomic DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and extract DNA for subsequent experiments or store at -20°C.

[0080] Step 2: PCR amplification and detection

[0081] (1) The BRCA1 and BRCA2 upstream and downstream primer pairs shown in the nucleotide sequence SEQ ID 01-36 are pre-mixed according to the mixing ratio;

[0082] (2) In the PCR tube, prepare the following reaction system:

[0083] Total volume 50 μl, containing 1 μl Phanta Max Super-Fidelity DNA Polymerase, 25 μl 2×Phanta Max Buffer at 2 mmol / L, 1 μl dNTP Mix at 10 mmol / L, 1 Microliter 2% DMSO, 2 microliter upstream primers (SEQ ID 1 , SEQ ID 3 , SEQ ID5 , SEQ ID 7 , SEQ ID 9 , SEQ ID 11 , SEQ ID 13 at a concentration of 10 millimolar per liter , SEQ ID 15, SEQ ID 17, SEQ ID 19, SEQ ID 21, SEQ ID 23, SEQ ID...

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Abstract

The invention discloses a primer pair, a kit and a method for detecting breast cancer susceptibility gene variation based on long fragment PCR (Polymerase Chain Reaction). The specific breast cancer susceptibility gene variation long fragment PCR detection primer pair is designed, PCR reaction and gene sequencing are combined, rapid and low-cost detection of breast cancer susceptibility gene variation is achieved, the detection rate reaches up to 100%, the specificity is 100%, and the method can be used for clinical detection of breast cancer susceptibility gene variation.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a pair of primers for detecting mutations of breast cancer susceptibility genes BRCA1 and BRCA2 based on long-segment PCR, a kit and applications thereof. Background technique [0002] According to the latest global cancer burden data released by the International Agency for Research on Cancer of the World Health Organization in 2020, the number of new cases of breast cancer, known as the "pink killer", reached 2.26 million, surpassing the 2.2 million cases of lung cancer and becoming the number one in the world big cancer. In my country, the incidence of breast cancer is increasing year by year, and more than 300,000 women are diagnosed with breast cancer every year. In the eastern coastal areas and large cities with developed economies, the incidence of breast cancer has increased significantly; from the perspective of onset age, The age of onset of breast cancer in my c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/11
CPCC12Q1/6886C12Q1/686C12Q2600/156
Inventor 童云广任永丽邓贵
Owner 奥明(杭州)基因科技有限公司
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