A lung cancer-related 33 gene targeted capture sequencing kit and its application

A targeted capture and kit technology, applied in the field of genetic engineering, can solve the problems of wrong sequencing results, high price, unsatisfactory detection of unknown regions, etc.

Active Publication Date: 2021-07-20
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because this method is based on multiplex PCR amplification technology, it is easy to introduce amplification variation and cause errors in sequencing results. It is developed abroad, and the technical information is confidential. Therefore, it is of great significance to develop more reliable and effective multi-gene simultaneous detection methods

Method used

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  • A lung cancer-related 33 gene targeted capture sequencing kit and its application
  • A lung cancer-related 33 gene targeted capture sequencing kit and its application
  • A lung cancer-related 33 gene targeted capture sequencing kit and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Preparation of Lung Cancer Related 33 Gene Profile Detection Probe Kit

[0050] 1. Identify lung cancer-related driver genes or targeted drug resistance genes: Search the PUBMED database through the Internet, check the latest literature, and count the targeted genes or their parallels related to the pathogenesis, chemoradiotherapy, targeted drug resistance, and metastasis of lung cancer in the Chinese population. , adjacent pathway genes, and identified 33 genes included in the kit (Table 1). Then find out its exact corresponding position coordinates on the chromosome and the position coordinates of the fusion region one by one.

[0051] Table 1 List of 33 genes related to targeting kits

[0052] EGFR ALK KRAS ROS1 RET MET BRAF ERBB2 HRAS DDR2 NTRK1 NTRK2 NTRK3 FGFR1 FGFR2 FGFR3 ERBB3 STK11 TSC1 TSC2 mTOR PTEN PIK3CA AKT1 AKT2 AKT3 CDKN2A CDK4 RB1 CCND1 CCNE1 TP53 NF1 ...

Embodiment 2

[0059] Example 2 Application of the kit in the analysis of lung cancer-related 33 genes based on the Ion Proton next-generation sequencing platform

[0060] The above-mentioned kit is applied in the analysis of 33 genes related to lung cancer based on the Ion Proton next-generation sequencing platform, and specifically includes the following steps:

[0061] (1) Fragmentation of genomic DNA to obtain DNA fragments;

[0062] (2) linking the DNA fragments with sticky ends to Adapters to obtain a ligated product;

[0063] (3) using the specific probe described in Example 1 to hybridize and capture the ligation product to obtain the target fragment;

[0064] (4) the probe hybridization target fragment is purified and then PCR amplified, so as to obtain the amplified product;

[0065] (5) separating and purifying the amplification product, which constitutes the high-throughput sequencing library;

[0066] (6) The library is sequenced by the Ion Proton next-generation sequencing p...

Embodiment 3

[0126] Example 3 Using the kit of Example 1 to detect tissue samples of lung cancer patients

[0127] 1. Validation Select 9 human lung adenocarcinoma tissue samples (from Guangdong Provincial People's Hospital) whose mutations have been detected by direct sequencing method, and each sample requires 200ng of DNA for detection. The operation steps and conditions of the kit of the present invention are basically the same as the application steps of the lung cancer-related 33-gene targeted capture sequencing kit in Example 1 based on the analysis of the Ion Proton next-generation sequencing platform in Example 2, and the difference is only in the initial use different DNA samples.

[0128] 2. 9 cases of lung cancer tissue samples were detected by the kit of the present invention, and compared with the detection results of the direct sequencing method (see Table 6). Using the kit of the present invention, the detection results of 8 tissue samples were completely consistent with t...

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Abstract

The invention discloses a lung cancer-related 33 gene targeted capture sequencing kit, which can simultaneously perform high-throughput detection on multiple loci of the lung cancer-related 33 gene, can significantly improve detection efficiency, and is expensive to change traditional single-gene detection methods , time-consuming, cumbersome operation and other disadvantages, the present invention also discloses the application of the lung cancer-related 33 gene targeted capture sequencing kit in the analysis of lung cancer-related 33 genes based on the Ion Proton next-generation sequencing platform.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a lung cancer-related 33 gene targeted capture sequencing kit and application thereof. Background technique [0002] Molecular biology tests have a variety of technical platforms, such as quantitative PCR, FISH, immunohistochemistry, etc., only one or a limited number of gene variants can be measured at a time, and the same sample is detected by sequential detection. The limitation of traditional methods is that, since each assay is limited to one gene, the required sample size increases with the number of genes tested. In the foreseeable future, it will be difficult to meet the need to measure all target genes. The next-generation DNA sequencing method based on the capture of the target region can detect dozens or even hundreds of genes in parallel, including point mutations (SNPs), insertions and deletions (INDELs), DNA copy number changes (CNVs), fusio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/156C12Q2535/122
Inventor 张绪超陈宇吴一龙
Owner GUANGDONG GENERAL HOSPITAL
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