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Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene

A technology of epidermal growth factor and deletion mutation, applied in the field of molecular biology, can solve the problems of shortened time for results, high price, and limitations

Inactive Publication Date: 2011-10-19
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, DNA sequencing is still the most widely used method for detecting EGFR gene mutations, but it needs to obtain tumor tissues, separate tumor cells, extract nucleic acids, and perform sequencing, which takes a long time, is expensive, and requires strict material and technical requirements. Clinical application is still limited to a certain extent
There are laboratories in the United States that specifically provide EGFR gene mutation testing. Although it avoids operational restrictions and shortens the time for results, it is expensive
At present, the human kits used by domestic clinical units are mainly imported from abroad, which are expensive and not suitable for domestic use

Method used

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  • Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene
  • Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene
  • Method for detecting exon 19 deletion mutation and exon 21 point mutation of epidermal growth factor receptor gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1 Primer and probe design

[0067] 1. Materials

[0068] Restriction endonucleases Msel and Mscl were purchased from NEB Company in the UK, and 2×Universe Taqman Mix was purchased from ABI Company in the United States (this Mix already included PCR buffer, dNTP, Mg 2+ , all provided by the supplier), the primers and probes used were synthesized by Shanghai Sangong, and the Tiangen tissue sample genomic DNA extraction kit was purchased from Beijing Tianhe Biotechnology Co., Ltd. The instrument used was ABI7300 fluorescent quantitative PCR instrument from ABI Company of the United States.

[0069] 2. Primer and probe sequence design

[0070] 1. Enrichment primer design

[0071] Primers that can amplify a section of 400-500bp were designed respectively, and the relevant parameters were: Tm value 65.0°C-68.0°C, GC value 40.0%-65.0%, primer size 23±3bp. The designed enrichment primer sequences are as follows:

[0072] E746_A750del upstream primer: 5'-CTTCCAAA...

Embodiment 2

[0086] Example 2 Using this method to detect E746_A750del and L858R mutations

[0087] The mutation-enriched ARMS fluorescent quantitative PCR detection method of the present invention integrates enzyme digestion enrichment PCR technology and ARMS fluorescent quantitative PCR technology, that is, samples are subjected to enzyme digestion, PCR enrichment and ARMS fluorescent quantitative PCR identification in one reaction system. . The specific process is:

[0088] 1. Sample preparation: Genomic DNA was extracted using Tiangen Tissue Sample Genomic DNA Extraction Kit.

[0089] 2. Detection method: first, the samples and the negative control were incubated in 25 μl reaction system at 37°C for 30 minutes, and the endonuclease Msel (NEB, England) or Mscl (NEB, England) was used to treat EGFR wild-type exon 19 respectively. Or 21 for enzyme digestion to reduce the interference of wild-type DNA on mutated bases. Then carry out the PCR reaction and run the first cycle to enrich the ...

Embodiment 3

[0097] Example 3 Clinical tissue sample detection

[0098] 1. Materials and methods

[0099] A total of 73 lung cancer tumor tissue samples were collected from Union Medical College Hospital and China Institute for the Identification of Pharmaceutical and Biological Products, and the whole genome DNA was extracted using the Tiangen Tissue Sample Genomic DNA Extraction Kit.

[0100] 2 μl each was taken for mutation enrichment ARMS fluorescent quantitative PCR detection, and sterile deionized water was used as a negative control. See Example 2 for specific implementation methods.

[0101] 2. Experimental results

[0102] In 73 tissue samples, 12 E746_A750del deletion mutations and 11 L858R mutations were detected. The detection method is determined to be the same as in Example 2.

[0103]

[0104]

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Abstract

The invention discloses a method for detecting exon 19 deletion mutation and exon 21 point mutation of an epidermal growth factor receptor gene. In method, enzyme digestion enrichment PCR (Polymerase Chain Reaction) reaction and ARMS (amplification refractory mutation system) fluorescence quantitative PCR reaction are carried out in one reaction system to respectively detect E746_A750del deletionmutation and L858R point mutation; and the enzyme digestion mutation enrichment PCR and ARMS fluorescent quantitative PCR are integrated together, the temperature and time are effectively controlled,and one reaction process is carried out to complete the enzyme digestion mutation enrichment and ARMS fluorescent quantitative PCR reaction.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to enzyme digestion enrichment PCR technology and ARMS fluorescent quantitative PCR technology. Background technique [0002] Lung cancer is a common disease that seriously endangers people's life and health. At present, the incidence and mortality of lung cancer are still rising at home and abroad. The therapeutic effect of lung cancer has not been significantly improved for many years, and the total cure rate is only about 10%. The reason is that its biological characteristics are very complicated, its malignancy is high, and it is multidrug resistant. On the other hand, the key point is that more than 80% of lung cancers have been diagnosed at an advanced stage. The traditional treatment for advanced non-small lung cancer is chemotherapy-based comprehensive treatment. Although new chemotherapy drugs have been discovered in recent years, their efficacy has not been sign...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 陈唯军赵金银刘利成吴伟力姜君王鹏志
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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