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Primer combination for detecting mutation of single cell EGFR gene and application of primer combination

A combination of primers and single-cell technology, applied in the fields of biotechnology and medicine, can solve problems such as the inability to detect EGFR gene mutations and limit the detection of geographical heterogeneity, and achieve good specificity and high sensitivity

Active Publication Date: 2017-12-15
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is largely due to the fact that previous studies have relied primarily on tissue biopsies, which greatly limited the detection of geographic heterogeneity
Moreover, none of the existing technologies can realize the detection of EGFR gene mutation at the single-cell level

Method used

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  • Primer combination for detecting mutation of single cell EGFR gene and application of primer combination
  • Primer combination for detecting mutation of single cell EGFR gene and application of primer combination
  • Primer combination for detecting mutation of single cell EGFR gene and application of primer combination

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Experimental program
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Effect test

Embodiment 1

[0030] 1. Primer design:

[0031] Primers targeting exons 18, 19, 20, and 21 were designed according to the EGFR genome sequence NG_007726.3 in the American Gene Database (Genbank), and a general sequence was added before the upstream and downstream primer sequences. The specific primer sequences are shown in Table 1.

[0032] Table 1

[0033]

[0034]

[0035] 2. PCR: artificial sequence

[0036] Genomic DNA of A549 lung cancer cells was extracted, and 200ng was taken as a template for PCR amplification. The PCR reaction conditions were: 95°C for 3min, 95°C for 30s, 57°C for 45s, 72°C for 45s, after 25 cycles, 72°C for 5min.

[0037] PCR products were subsequently identified by 2.5% agarose gel electrophoresis. The result is as figure 1 As shown, the target bands were observed within the target length range, and there were no miscellaneous bands, which proved that the primers designed by the present invention had good specificity.

[0038] 3. Quadruple PCR amplific...

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Abstract

The invention relates to the field of biotechnology and medicine and specifically discloses a primer combination for detecting mutation of single cell EGFR gene and application of the primer combination. According to the invention, by adopting a high-specificity amplification primer and a multiple-amplification system, all common mutations on exons 18, 19, 20 and 21 of the EGFR gene are obtained at a time, and new type of mutation can be discovered in time. By adopting the primer combination disclosed by the invention to detect the mutation of single cell EGFR gene, the specificity and sensitivity are high, DNA extraction is not needed, two copies in as few as single cell can be amplified, and more systematic research of tumor heterogeneity is facilitated.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a primer combination for detecting single-cell EGFR gene mutation and its application. Background technique [0002] Activating mutations in the epidermal growth factor receptor (EGFR) sensitize most non-small cell lung cancer (NSCLC) patients harboring such mutations to EGFR tyrosine kinase inhibitors (TKIs). First-generation inhibitors such as erlotinib and gefitinib target receptors by reversibly binding to the tyrosine kinase domain, while second-generation inhibitors such as afatinib bind covalently to the target to exert an inhibitory effect. However, patients will always develop tolerance after 9-16 months of treatment, and 60% of them are due to the secondary mutation (T790M) of codon No. 790. The third-generation covalent mutation-selective EGFR TKisCO-1686 and AZD9291 simultaneously target activating and T790M mutations and can be used in T790M-positive NSCL...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143
Inventor 岳春燕李仁马艳朱光宇胡志远
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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