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Composition and kit for detecting c-MET gene exon 14 skip mutation

An exon and composition technology, applied in the field of molecular biology, can solve problems such as inability to achieve, and achieve the effects of short detection time, good specificity and simple operation

Inactive Publication Date: 2017-06-09
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, technologies such as direct sequencing are far from meeting the needs of their practical applications, and it is urgent to develop a highly specific and sensitive c-MET exon 14 read-skipping mutation detection composition to realize c-MET exon 14 read skipping mutation detection composition. 14 Highly sensitive detection of skipping mutations, thus providing a reference for clinical individualized drug guidance

Method used

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  • Composition and kit for detecting c-MET gene exon 14 skip mutation
  • Composition and kit for detecting c-MET gene exon 14 skip mutation
  • Composition and kit for detecting c-MET gene exon 14 skip mutation

Examples

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Embodiment 1

[0059] In this example, plasmids were used to detect the system of the present invention, and a mutant plasmid template (deleting c-MET exon 14) and a wild-type plasmid template (containing c-MET exon 14) constructed by genetic engineering were 1 each. Utilize the present invention to implement fluorescent PCR to detect c-MET exon 14 skipping mutation method as follows:

[0060] (1) Plasmid construction and processing:

[0061] According to the human c-MET published by COSMIC data is the wild-type gene sequence, the c-MET sequence missing exon 14 was used as the mutant sequence to construct mutant plasmids and wild-type plasmids, respectively. The two plasmids were respectively dissolved in ultrapure water, and the quality control was carried out with a micro-spectrophotometer NanoDrop 8000, and the concentration was determined, and then the plasmid concentration was adjusted to 0.01pg / μl, that is, 5000 copies / μl, and 2μl was used as a PCR template for PCR Amplify.

[0062] ...

Embodiment 2

[0074] The cell lines detected by the present invention include human lung squamous cell line H596 and human lung adenocarcinoma cell line Calu-3. Utilize the specific ARMS primers and probes of the present invention to implement fluorescent PCR detection of c-MET exon 14 skipping mutation method as follows:

[0075] (1) Sample processing and RNA extraction:

[0076] (a) Take the cultured cell lines H596 and Calu-3 cells about 3X10 6 , respectively add 1ml 1×PBS to wash, centrifuge at room temperature for 4min at 1000rpm, discard the supernatant, gently tap your fingers to break up the cell mass, add 350μl RLT Buffer, shake and mix well, add 350μl 70% ethanol, blow and mix with the tip of the pipette, and quickly dissolve Add 700μl of the mixture to the RNA Mini Spin column, centrifuge at 12000rpm for 15s at room temperature, and discard the filtrate;

[0077] (b) Add 350μl RW1 Buffer to the column, 12000rpm, centrifuge at room temperature for 15s, discard the filtrate, add ...

Embodiment 3

[0097] Using the present invention to detect clinical samples, 20 cases of clinical lung adenocarcinoma paraffin-embedded tissue (FFPE) samples to be tested were taken and sent to our company, including 10 males and 10 females, aged 48-77 years, with an average age of 62 years. Utilize the specific ARMS primers and probes of the present invention to implement fluorescent PCR detection of c-MET exon 14 skipping mutation method as follows:

[0098] (1) Sample processing and RNA extraction:

[0099] (a) Take the above-mentioned lung adenocarcinoma samples and slice them into 5um thick slices. Take 2 slices from each sample and quickly place them in a 1.5ml centrifuge tube, add 1ml xylene, vortex for 10s, and centrifuge at full speed for 2min at room temperature. Carefully suck up the supernatant with a pipette, be careful not to touch the precipitate, add 1ml of absolute ethanol, vortex, and centrifuge at full speed for 2min. Carefully suck off the supernatant, and use a smaller...

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Abstract

The invention discloses a composition and a kit for detecting human c-MET gene exon 14 skip mutation. The composition and the kit comprise the following 16 primers shown from SEQ.ID NO 01 to SEQ.ID NO 16, and probes. The primers and probes comprise positive specific amplification mutation target sequence ARMS primers and corresponding TAQMAN probes and reverse specific amplification mutation target sequence ARMS primers and corresponding TAQMAN probes, and the c-MET gene exon 14 skip mutation can be specifically detected. The composition and the kit for detecting human c-MET gene exon 14 skip mutation disclosed by the invention are high in specificity, high in sensitivity, capable of detecting mutation of 10 copies, convenient in detection, simple in operation, high in detection speed and wide in clinical application range.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a composition and a kit for detecting the skipping mutation of exon 14 of c-MET gene. Background technique [0002] The c-MET proto-oncogene is located on human chromosome 7, with a size of more than 120kb, including 21 exons and 20 introns. The c-MET proto-oncogene encodes a hepatocyte growth factor (HGF) receptor, which belongs to the receptor-type tyrosine protein kinase, and is expressed in both normal cells and tumor cells. Its continuous activation is the cause of tissue cell canceration or cancer cell An important cause of hyperproliferation. After c-MET binds to HGF, the tyrosine residues close to the four phosphorylation sites in the cell undergo spontaneous phosphorylation, activating important cell signals such as PI-3K, ERK1 / 2, PLC-Y, STAT and FAK Pathways that promote cell proliferation, proliferation, migration and invasion. In non-tumor cells, the c-MET / HGF sign...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2535/137C12Q2561/101
Inventor 孙青芝林敏张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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