Targeted trapping and sequencing kit for lung cancer-related 285 genes and application of targeted trapping and sequencing kit

A targeted capture and kit technology, applied in the field of kits, can solve the problems of wrong sequencing results, high price, unsatisfactory detection results in unknown regions, etc., achieve high throughput, meet maximum use, and reduce clinical testing costs Effect

Inactive Publication Date: 2018-06-12
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 trapping and sequencing kit for lung cancer-related 285 genes and application of targeted trapping and sequencing kit
  • Targeted trapping and sequencing kit for lung cancer-related 285 genes and application of targeted trapping and sequencing kit
  • Targeted trapping and sequencing kit for lung cancer-related 285 genes and application of targeted trapping and sequencing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Preparation of Lung Cancer Related 285 Gene Profile Detection Probe Kit

[0052] 1. Identify lung cancer-related driver genes or targeted drug resistance genes: Search the PUBMED database through the Internet, check the latest literature and unpublished data of the research team, and make statistics on the pathogenesis, chemoradiotherapy, targeted drug resistance and metastasis of lung cancer in the Chinese population. Related target genes or their parallel, adjacent pathway genes, identified 285 genes included in the kit (Table 2).

[0053] Table 2

[0054] JAK2

ABL2

ERCC3

TSHR

USP9X

SUFU

SOX2

RHBDF2

BRCA2

PTPRD

CDC73

LRP1B

DICER1

KDM6A

SMC3

EIF4A2

SRSF2

RB1

CDKN2A

ELF3

ACVR2A

AKT1

GATA1

FGFR2

AXIN1

BUB1B

ERCC5

FANCG

MDM4

NFE2L2

STK11

KDM5C

PDYN

TSC2

LTK

SMARCB1

PAX5

H3F3A

PMS1

GNA11 ...

Embodiment 2

[0058] Detection using the probe designed in Example 1 includes the following steps: fragmenting the genomic DNA to obtain DNA fragments; connecting the DNA fragments with sticky ends to Adapters to obtain ligated products; using the specific The ligation product is hybridized and captured by the sex probe to obtain the target fragment; the target fragment hybridized with the probe is purified, then PCR amplified, separated and purified, and the obtained amplified product constitutes the high-throughput sequencing library. The library was sequenced by the IonProton next-generation sequencing platform, and all the variations of the 285 genes detected in the samples were determined by analysis. The more specific steps are as follows (the operation of the kit is carried out according to the instructions):

[0059] 1. Sequencing library construction

[0060] (1) Prepare sample DNA (concentration greater than 20ng / μl, total amount greater than 200ng).

[0061] (2) Fragmentation o...

Embodiment 3

[0117] Detection of lung cancer patient tissue samples using the kit of Example 1

[0118] 1. Validation Select 13 human lung adenocarcinoma tissue samples (from Guangdong Provincial People's Hospital) whose mutations have been detected by direct sequencing. Each sample requires 200ng of DNA for detection. The operation steps and conditions of the kit of the present invention are the same as the specific steps in Example 2.

[0119] 2. 13 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 in these 13 tissue samples was completely consistent with the detection results of the direct sequencing method.

[0120] The verification result of the test kit of the present invention compared with the direct sequencing method of table 6

[0121]

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Abstract

The invention discloses a targeted trapping and sequencing kit for 285 lung cancer-related genes and application of the targeted trapping and sequencing kit. The kit is based on hybrid trapping of a probe, and can be used for preparing a high-throughput library of the lung cancer-related 285 genes and simultaneously detecting all variation loci on exons of the 285 genes, so that the disadvantagesof high cost, high time consumption, tedious operation and the like of conventional single-gene detection are overcome. Detection of a plurality of genes with a single small sample is implemented in the small sample for aspiration biopsy of lung cancer, and the clinical practice requirements of maximally using the small sample and obtaining a maximum amount of information are met. The kit is basedon an Ion Proton platform, has high accuracy and sensitivity, and has obvious advantages compared with a Sanger sequencing method, and a detection result in a residual DNA (deoxyribonucleic acid) sample of a paraffin sample is stable.

Description

Technical field [0001] The invention relates to a kit, in particular to a lung cancer-related 285 gene targeted capture sequencing kit and its application. Background technique [0002] Molecular biology tests have a variety of technical platforms, such as quantitative PCR, FISH, immunohistochemistry, etc., which can only detect one or a limited number of genetic variants at a time, and sequential detection methods are used for the same sample. A 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 demand for measuring 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, indels, DNA copy number changes, fusion rearrangements and other variant forms, thus detecting all genes at once. A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2565/519C12Q2531/113C12Q2525/191C12Q2535/122
Inventor 张绪超陈宇吴一龙
Owner GUANGDONG GENERAL HOSPITAL
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