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Targeted capturing and sequencing kit for 33 lung cancer-related genes and application of targeted capturing and sequencing kit

A targeted capture and kit technology, applied in the field of genetic engineering, can solve the problems of incorrect sequencing results, high price, and unsatisfactory detection results in unknown regions, and achieve the goal of maximizing use, generating economic benefits, and reducing clinical testing costs Effect

Active Publication Date: 2018-05-04
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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  • Targeted capturing and sequencing kit for 33 lung cancer-related genes and application of targeted capturing and sequencing kit
  • Targeted capturing and sequencing kit for 33 lung cancer-related genes and application of targeted capturing and sequencing kit
  • Targeted capturing and sequencing kit for 33 lung cancer-related genes and application of targeted capturing and sequencing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Preparation of a probe kit for detecting lung cancer-related 33 gene profiles

[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 related to the pathogenesis of lung cancer, chemotherapy and radiotherapy, targeted drug resistance and metastasis in the Chinese population or their parallels , Adjacent pathway genes, determine 33 genes included in the kit (Table 1). Then find out the exact corresponding position coordinates on the chromosome and the position coordinates of the fusion area one by one.

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

[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

[0053] 2. Design 33 gene hybridization pr...

Embodiment 2

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

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

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

[0062] (2) Connect the DNA fragments with sticky ends to the Adapters to obtain the connection 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) Purifying the target fragment hybridized with the probe and then PCR amplifying it to obtain an amplified product;

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

[0066] (6) The library is sequenced through the Ion Proton second-generation...

Embodiment 3

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

[0127] 1. Verification Select 9 human lung adenocarcinoma tissue samples (from Guangdong Provincial People's Hospital) whose mutations have been detected by direct sequencing, and 200ng DNA is required for each sample. The operating 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 Ion Proton second-generation sequencing platform analysis in Example 2, except for the initial use The DNA samples are different.

[0128] 2. The kit of the present invention detects 9 cases of lung cancer tissue samples and compares the results 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 the direct sequencing method, and...

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Abstract

The invention discloses a targeted capturing and sequencing kit for 33 lung cancer-related genes. The kit can be used for high-throughput detection on multiple sites of the 33 lung cancer-related genes simultaneously. The detection efficiency can be significantly improved, and disadvantages of high price, long time consumption, complicated operation and the like of the traditional single-gene detection method are changed. The invention further discloses an application of the targeted capturing and sequencing kit for the 33 lung cancer-related genes in analysis of the 33 lung cancer-related genes based on an Ion Proton next-generation sequencing platform.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a lung cancer-related 33 gene targeted capture sequencing kit and its application. Background technique [0002] Molecular biology experiments have multiple technology platforms, such as quantitative PCR, FISH, immunohistochemistry, etc., which can only measure one or a limited number of gene mutations at a time, and use sequential detection methods for the same sample. The limitation of the traditional method is that since each test is limited to one gene, the sample size required increases with the number of genes tested. In the foreseeable future, it will be difficult to meet the needs of determining all target genes. The DNA second-generation sequencing method based on target region capture can detect dozens or even hundreds of genes in parallel at one time, including point mutations (SNP), insertion deletions (INDEL), DNA copy number changes (CNV), and fus...

Claims

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Application Information

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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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