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EGFR gene extron 20 mutational detecting probe, liquid phase chip and detecting method thereof

A mutation detection and liquid phase chip technology, applied in the biological field, can solve the problems of hindering mutation gene detection and interference, and achieve the effects of simple steps, convenient sampling and good specificity

Active Publication Date: 2009-06-24
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, due to the heterogeneity of the tumor, there may be only a very small amount of mutated genes in the cell-free nucleic acid, but a large number of wild-type genes, mixing a large number of wild-type genes will hinder the detection of mutated genes, so how to exclude the wild-type sequence pair Detecting interference is the key issue to address first

Method used

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  • EGFR gene extron 20 mutational detecting probe, liquid phase chip and detecting method thereof
  • EGFR gene extron 20 mutational detecting probe, liquid phase chip and detecting method thereof
  • EGFR gene extron 20 mutational detecting probe, liquid phase chip and detecting method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Preparation of EGFR gene exon 20 mutation detection liquid chip kit

[0043] 1. Probe sequence design and microsphere coating

[0044] For the wild-type and mutant sequences of EGFR exon 20, design specific oligonucleotide probes, including base sequences of SEQ.ID NO: 1-6, for exon 20 wild-type probes, and SEQ.ID NO: 7-12, for exon 20 mutant probes, wherein, SEQ.ID NO: 4-6 is the reverse complementary sequence of SEQ.ID NO: 1-3, SEQ ID NO : 10-12 are the reverse complementary sequences of SEQ ID NO: 7-9, respectively.

[0045] The specific steps of each microsphere coating are as follows:

[0046] (1) Take the microsphere mother solution (purchased from Luminex Company) and vortex to form a microsphere suspension;

[0047] (2) Take out 8ul microsphere mother solution, containing 0.8×10 5 —1.2×10 5 microspheres into a 0.5ml centrifuge tube;

[0048] (3) Centrifuge at 10,000rpm for 3min, discard the supernatant;

[0049] (4) Add 10ul coupling solution (p...

Embodiment 2

[0061] Example 2 Detection of Lung Cancer Serum Samples Using EGFR Gene Mutation Detection Liquid Chip

[0062] 1. Preparation of samples to be tested (plasma, serum and pleural effusion supernatant free nucleic acid can be extracted according to the following methods):

[0063] Refer to the instructions of AxyPrep Whole Blood Genome Small Extraction Kit, the detailed steps are as follows:

[0064] (1) Take about 2.5ml of anticoagulated venous blood or pleural effusion from the patient, centrifuge at 3000rpm for 15 minutes, take 300μl of supernatant and add it to a 1.5ml clean and sterile centrifuge tube;

[0065] (2) Add 500 μl of AP1 buffer solution to the centrifuge tube, vortex and mix well;

[0066] (3) Add 100 μl of AP2 buffer, vortex and mix well;

[0067] (4) Centrifuge at 12,000 rpm for 10 minutes at room temperature

[0068] (5) Draw the supernatant and add it to the adsorption column AxyPrep placed on the 2ml collection tube, cover the lid,

[0069] Centrifuge a...

Embodiment 3

[0108] Example 3 Detection of Lung Cancer Tissue Samples Using the EGFR Gene Exon 20 Mutation Detection Liquid Chip in Example 1

[0109] Get the lung cancer tissue samples of patient No. 1-20 used in the above-mentioned embodiment 2 for detection, and the specific process is as follows:

[0110] 1. Preparation of samples to be tested

[0111] Extraction of DNA from lung cancer tissue samples: take 5-50 mg of tissue samples after lung cancer surgery or biopsy, grind them, and wash twice with PBS solution with pH 7.4; the washed tissue samples are resuspended in 1ml of digestive solution (50mmol / L Tris, 1mmo / L Na 2 EDTA, 0.5% Tween-20, 200ug / ml proteinase K 200, pH 8.5), digest in 55°C water bath for 1 hour, inactivate proteinase K in 99°C water bath for 15 minutes; centrifuge at 12000 rpm for 10 minutes; take the supernatant and pass through phenol - Chloroform-isoamyl alcohol extraction and ethanol precipitation to obtain DNA samples for PCR reactions. DNA can also be ext...

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Abstract

The invention discloses a probe for detecting EGFR gene exon 20 mutation, a liquidoid chip and a detection method thereof. The liquidoid chip of the EGFR gene exon 20 mutation comprises a microsphere enveloped by the probe, the microsphere enveloped by a wild amido modification probe with base sequence selected from any of SEQ ID NO: 1-SEQ ID NO: 6 aiming at exon 20 and the microsphere enveloped by a mutant amido modification probe with the base sequence selected from any of SEQ ID NO: 7-SEQ ID NO: 12 aiming at exon 20. A space arm is used for connecting the base sequence and the amido of each probe. Every microsphere has different color codes. The liquidoid chip of the EGFR gene exon 20 mutation also comprises a primer used for amplifying a target sequence with exon 20 mutant site. The method provided by the invention is fast and exact, is simple for operating and improves the detection efficiency greatly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection probe for EGFR gene exon 20 mutation, a liquid phase chip and a detection method thereof. Background technique [0002] Epidermal growth factor receptor (EGFR) is a multifunctional glycoprotein widely distributed on the cell membranes of various human tissues. It has tyrosine kinase activity and is one of the four members of the ErbB family, so it is also known as HER1 or ErbB1. After being activated by ligands, EGFR initiates intracellular signal transduction, and through the cascade reaction of adapter proteins and enzymes in the cytoplasm, it regulates the transcription of transcription factor-activated genes and guides cell migration, adhesion, proliferation, differentiation and apoptosis. Studies have shown that there is high or abnormal expression of EGFR in many solid tumors, which provides a theoretical basis and experimental basis for EGFR-targeted t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森吴诗扬郭元杰陈玲
Owner SUREXAM BIO TECH
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