Probe, method and reagent for detecting EGFR (epidermal growth factor receptors)/KRAS/BRAF gene mutation sites on basis of single-molecule targeted sequencing technologies and application of probe, method and reagent

A targeted sequencing and technical detection technology, applied in the field of molecular biology, can solve the problems of cumbersome operation, complicated library construction process, and long time, and achieve the effect of simplifying the preparation steps

Inactive Publication Date: 2016-06-08
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although high-throughput sequencing (NGS) has high sensitivity and can detect multiple mutation sites at the same time, its library construction process is complex, cumbersome, time-consuming, and expensive

Method used

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  • Probe, method and reagent for detecting EGFR (epidermal growth factor receptors)/KRAS/BRAF gene mutation sites on basis of single-molecule targeted sequencing technologies and application of probe, method and reagent
  • Probe, method and reagent for detecting EGFR (epidermal growth factor receptors)/KRAS/BRAF gene mutation sites on basis of single-molecule targeted sequencing technologies and application of probe, method and reagent
  • Probe, method and reagent for detecting EGFR (epidermal growth factor receptors)/KRAS/BRAF gene mutation sites on basis of single-molecule targeted sequencing technologies and application of probe, method and reagent

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment

[0129] Material and Reagent Description:

[0130] Samples to be tested: from Shenzhen Second People's Hospital. Unless otherwise specified, the reagents used in the examples of the present invention are commercially available, and the databases used in the examples of the present invention are all public online databases.

[0131] 20xSSC: Dissolve 175.3g NaCl and 88.2g sodium citrate in 800ml water, add a few drops of 10mol / l NaOH solution to adjust the pH value to 7.0, add water to make up to 1L, aliquot and autoclave. SSC is the most standard blotting and molecular hybridization treatment solution in molecular biology. 20*SSC is a common concentrated buffer used for denaturation and cleaning of hybridization experiments.

[0132] The reagents used in the examples of the invention are all commercially available, and the databases used in the examples of the invention are all public online databases.

[0133] Specifically, multiple PCR primers of the present invention are as...

Embodiment 1

[0143] Example 1 Method for preparing samples for single-molecule targeted sequencing of PCR products

[0144] The embodiment of the present invention provides a sample preparation method for single-molecule targeted sequencing, including the following steps:

[0145]1. Multiplex PCR

[0146] The tissues containing cancer cells were obtained from the hospital, and the genomic DNA was extracted using the QIAamp DNA Mini Kit (51304) kit, and the concentration and purity of the DNA were determined with Nanodrop2000 (Thermo), and then the genomic DNA was preserved.

[0147] The 14 pairs of primers in Table 2 were used for multiplex PCR, and the multiplex PCR system and PCR parameters were as follows:

[0148] Reaction system: Refer to QIAGEN's MultiplexPCR Kit (Product No.: 206143) for configuration.

[0149]

[0150] Loop parameters:

[0151]

[0152] Finally, a 30ul PCR product was obtained by purification with a Qiagen purification kit.

[0153] 2. Measure the concent...

Embodiment 2

[0165] Example 2 Preparation of samples for single-molecule targeted sequencing of gDNA

[0166] The embodiment of the present invention provides a sample preparation method for single-molecule targeted sequencing, including the following steps:

[0167] 1. Obtain tissues containing cancer cells from the hospital, use the QIAamp DNA Mini Kit (51304) kit to extract genomic DNA, and use Nanodrop2000 (Thermo) to measure the concentration and purity of DNA, and then save the genomic DNA.

[0168] DNA fragmentation

[0169] Add the following components to a sterile PCR tube

[0170] DNA (5ng-3μg)

1~16μL

10×Fragmentase Reaction Buffer v2

2μL

Sterile Water

variable

dsDNA Fragmentase (Cat. No. M0348S)

2μL

Total

20 μL

[0171] Incubate at 37°C for 30min, then place at 70°C for 10min.

[0172] 2. Electrophoresis judgment

[0173] Take 2-3ul of the reaction product for electrophoresis, use a gel imager to take pictures and...

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Abstract

The invention provides a probe, a method and a reagent for detecting EGFR (epidermal growth factor receptor) / KRAS / BRAF gene mutation sites on the basis of single-molecule targeted sequencing technologies and application of the probe, the method and the reagent, and further provides a method for preparing single-molecule targeted sequencing samples and application of the method. The probe comprises a probe body or a plurality of probe bodies for detecting a gene or a plurality of genes among EGFR, KRAS and BRAF genes. Nucleotide sequences of the probe include a sequence or a plurality of sequences shown as SEQ ID NO:1-SEQ ID NO:16. The probe, the method, the reagent and the application have the advantages that the probe can be used for efficiently detecting the diversity of the EGFR / KRAS / BRAF gene mutation sites and can cover exons NO.18-21 of EGFR, exons NO.2-3 of KRAS and exons NO.15 of BRAF.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a probe, method, reagent and application for detecting EGFR / KRAS / BRAF gene mutation sites based on single-molecule targeted sequencing technology, and a non-invasive detection gene mutation site Methods. Background technique [0002] Epidermal growth factor receptor (EGFR) is the expression product of the proto-oncogene C-erbB-1 (HER-1) located on the short arm of chromosome 7, and is one of the four members of the epidermal growth factor receptor family (HER). One of the members, the HER family plays an important regulatory role in cell physiological processes. Mutations in the EGFR tyrosine kinase region mainly occur in exons 18-21, and mutations in exons 19 and 21 account for 90% of the total mutations. Abnormality of EGFR signaling is the cause of many tumors. Studies have shown that EGFR is expressed in a variety of tumors, such as colorectal cancer, breast cancer, pancre...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6806C12Q1/6874C12Q2537/143C12Q2525/10
Inventor 葛良进高雁李改玲邓力蔚吴增丁蔡金森纪道锐黄天逊
Owner GENEMIND BIOSCIENCES CO LTD
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