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Method and kit for constructing multi-gene mutation sequencing library of lung cancer driving genes

A technology for driving genes and sequencing libraries, applied in the field of biomedicine, can solve the problems of high cost, cumbersome and complicated operation, and long time.

Pending Publication Date: 2021-04-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the common sequencing library construction method currently on the market is the hybrid capture method, which requires multiple steps of fragmentation, end repair, adapter addition, purification, amplification and purification, hybridization, capture, PCR amplification and purification, and the total time-consuming needs to exceed 24 hours; and in addition to the PCR system and purified magnetic beads, a variety of enzymes and buffers involved in end repair, ligation reaction, hybridization reaction and multi-step purification are required, and the operation is cumbersome, time-consuming and costly

Method used

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  • Method and kit for constructing multi-gene mutation sequencing library of lung cancer driving genes
  • Method and kit for constructing multi-gene mutation sequencing library of lung cancer driving genes
  • Method and kit for constructing multi-gene mutation sequencing library of lung cancer driving genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] A kit for detecting multi-gene somatic mutation and gene fusion of lung cancer, the kit includes reverse transcription buffer, targeted amplification buffer, library amplification buffer, forward library amplification primer A5I, reverse library amplification Add primer A7I, purified magnetic beads, eluent, quality control.

[0076] Reverse transcription buffer, including reverse transcriptase, dNTP, buffer, random primer or Oligo(dT) primer, RNase inhibitor, used to reverse transcribe sample RNA into cDNA.

[0077] Targeted amplification buffer, including high-fidelity DNA polymerase, PCR buffer, dNTP mixture, and targeted amplification primers, are used to amplify the target mutation region in the nucleic acid to be tested. The target amplification primers (SEQ ID NO.1-SEQ ID NO.89), each primer structure includes a universal primer sequence and a specific primer sequence, and the amplification region at least covers all target gene mutation sites to be detected.

[...

Embodiment 2

[0094] Sample detection was performed using the kit described in Example 1.

[0095] 1. Sample DNA and RNA extraction:

[0096] Fresh tissue or cells, paraffin tissue samples, etc. were used as samples. DNA and RNA were extracted using a commercial company’s DNA / RNA extraction kit according to the instructions, and the concentration was measured with a Qubit4 fluorometer.

[0097] The loading amount of total RNA should not be lower than 200ng, and the loading amount of total DNA should not be lower than 50ng.

[0098] 2. Reverse transcription reaction: For each sample to be tested, configure the reaction system proportionally in a 0.2 μl PCR tube as shown in Table 3:

[0099] Table 3 reverse transcription system

[0100] Reagent name The actual amount sample total RNA 200-1000ng reverse transcription buffer 2μl nuclease free water Make up to 10 μL

[0101] After the system configuration is completed, use a pipette to mix gently by pipett...

Embodiment 3

[0132] Example 3 Effects of different sample nucleic acid concentrations on library construction

[0133]Select 5 examples of samples with different mutation types verified by Sanger sequencing, use the kit of Example 1, and according to the method of Example 2, add 10ng, 30ng, 50ng, 100ng, and 200ng sample DNA to carry out library construction, the results show (as shown in Table 10), when the nucleic acid concentration of the sample for constructing the library is as low as 10 ng, the corresponding mutation can still be detected correctly using the library construction kit and construction method provided by the present invention, and the results of the library construction using different initial amounts of DNA are measured. The mutation rate is close.

[0134] Table 10 Test results of sample library construction with different nucleic acid concentrations

[0135]

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Abstract

The invention provides a method and a kit for constructing a multi-gene mutation sequencing library of lung cancer driving genes. According to primers for amplifying the lung cancer driving genes, amplification products of the primers cover hotspot mutation sites and gene fusion types of the multiple lung cancer driving genes such as EGFR, ALK, ROS1, KRAS, BRAF, PIK3CA, HER2, RET, MET, NRAS, NTRK1-3 and MAP2K1, and aiming at the NTRK1-3 gene fusion, no second-generation sequencing library covering the fusion type exists in China at present. The primers of the kit provided by the invention can effectively capture the multi-gene sequences of the lung cancer driving genes, and the multi-gene mutation sequencing library of the lung cancer driving genes can be constructed only by two-step PCR amplification and purification with a multiplex PCR method, so that the amplification and detection of NTRK1-3 gene fusion are realized for the first time.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method and a kit for constructing a multi-gene mutation sequencing library for driving genes of lung cancer. Background technique [0002] There are about 781,000 cases of lung cancer in my country each year, and about 626,000 cases of death. It is currently the cancer with the highest morbidity and mortality in my country. Based on histopathological results, lung cancer is divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), of which non-small cell lung cancer accounts for about 80%. In recent years, with the continuous development of molecular diagnostic techniques and the emergence of new targeted drugs, the treatment of non-small cell lung cancer has entered the era of targeted therapy. [0003] Non-small cell lung cancer driver genes include EGFR, ALK, ROS1, KRAS, BRAF, PIK3CA, HER2, RET, MET, NRAS, NTRK1-3, MAP2K1 and other g...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11C40B50/06
CPCC12Q1/6858C40B50/06C12Q2535/122C12Q2531/113
Inventor 曾杰彭璨璨吴诗扬刘志明
Owner SUREXAM BIO TECH
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