Kit for forecasting treatment effect of epidermal growth factor receptor inhibitor
An epidermal growth factor and kit technology, applied in the field of medicine, can solve problems such as unclear clinical significance, and achieve the effects of reducing medical costs, simple operation, and overcoming blindness.
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Embodiment 1
[0070] Example 1 Construction of Epidermal Growth Factor Receptor Gene Wild / Mutant Genotype Positive Control Plasmid
[0071] Materials and methods
[0072] Instruments: centrifuge (Eppendorf 5415D), PCR instrument (ABI 2700), gel imaging system (Huadian DH2000)
[0073] Materials: pET28 vector plasmid, E. coli strain DH5α
[0074] Steps:
[0075] 1. Tissue genomic DNA extraction:
[0076] The paraffin-embedded non-small cell lung cancer tumor tissue was selected, and after dewaxing, the genomic DNA in the tumor tissue was extracted using a DNA extraction kit (QIAmp DNA kit).
[0077] 2. Amplification of EGFR exon 18 gene fragment
[0078] To amplify the epidermal growth factor receptor target gene fragment, use the following primers to amplify the EGFR exon 18 gene fragment.
[0079] SEQUENCE 5: CTCGAATTCTCCAATGAGCTGGCAAGTG,
[0080] SEQUENCE 6: TCCAAGCTTTCCCAACACTCAGTGAAACAAA
[0081] At the 5' end of the primers, respectively contain restriction sites for EcoR1 and H...
Embodiment 2
[0097] Example 2 Determination of the genotype of non-small cell lung cancer samples using the kit
[0098] 1. Sample preparation: Genomic DNA was extracted from 46 tumor tissues (fresh tissues or paraffin blocks) of non-small cell lung cancer patients taking gefitinib.
[0099] 2. Configuration of PCR amplification mixture
[0100] Configure the PCR amplification mixture according to Table 1.
[0101] 46 PCR reaction mixtures were prepared respectively. In addition, positive control sample mixtures of two genotypes and a negative sample mixture were prepared.
[0102] Table 1 PCR amplification mixture configuration table
[0103] components
Volume ul
Taq DNA polymerase 5U / ul
0.5
10×PCR buffer
2.5
Mg2+25mM
3
dNTP 1.25mM
4
Primer 1SEQUENCE3
0.4
Primer 2SEQUENCE4
0.4
Probe 1SEQUENCE1
0.25
Probe 2SEQUENCE2
0.25
DNA template
11.25
h 2 o
...
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