Method for detecting abundance of mutant gene as well as compositions and application of mutant gene
A gene and point mutation technology, applied in the field of gene mutation abundance detection, can solve the problems of inability to judge the abundance of tumor-sensitive mutations and the reduction of drug efficacy
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Embodiment 1
[0070] Example 1, plasmid extraction and linearization enzyme digestion
[0071] Heat-shock transformed the required plasmid into E. coli DH5α, picked the monoclonal strain into 100ml LB medium (yeast extract 5g, protein jelly 10g, NaCl 10g, water 1000mL, pH7.4~7.6) the next day, and put it in 37 Cultivate overnight in an incubator at 180rpm / min. The cultured bacterial solution was centrifuged at 10,000 rpm / min to collect the bacterial cells, and the plasmid was extracted with HiSpeed Plasmid Midi Kit (Qiagen), and the steps were performed according to the kit instructions. For the determination of the concentration of the extracted plasmid, use a microplate reader Spectrophotometer (Thermo Fisher) to measure the concentration at a wavelength of 260 nm, and take a certain amount to run agarose (0.7%) electrophoresis.
[0072] The extracted plasmid was single-digested with the restriction enzyme Xba I (the restriction site of Xba I is not included in the gene of interest) to...
Embodiment 2
[0077] Example 2. Quantitative Calibrators for Linearized and Circular Plasmids
[0078] Serial dilution and storage of linearized and circular plasmid standards: the dilution storage solution is TE-glycerol buffer (Tris-HCl0.1M, EDTA1mM, glycerol 20%, pH8.0).
[0079] Linearized plasmid standard gradient is: 10 5 , 10 4 , 10 3 , 10 2 , 10 copies / ul.
[0080] Circular plasmid standard gradient: 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 copies / ul.
[0081] The reaction system is shown in Table 4.
[0082] Table 4
[0083] components
Dosage (25ul reaction system)
2×Realtime PCR Master Mix
12.5ul
F-primer
300nM
R-primer
300nM
MGB fluorescent probe
100nM
plasmid
2ul
wxya 2 o
Replenish water to a volume of 25ul
[0084] After the PCR system is prepared, run the PCR on the machine, using the quantitative PCR instrument ABI-7300, and the PCR setting conditions are shown in Table 5....
Embodiment 3
[0089] Example 3, Quantitative Results of Linearized Plasmid and Circular Plasmid Standards on Cell Line Genome Mutant Genes
[0090] 3.1 Cell line genome extraction
[0091] KB, H1975, H1650, HCC827 and other cell lines use RPMI-1640+10%FBS medium, at 37°C, 5%CO 2 Cultured in an incubator, the cultured cells were extracted with the High Pure PCR Template Preparation Kit (Roche), and the operation was carried out according to the kit instructions. The concentration of the extracted gDNA was measured with a Spectrophotometer (Thermo Fisher), and the concentration of the corresponding mutant gene was adjusted to 2500 copy / ul. The mutations corresponding to the cell lines are shown in Table 6.
[0092] Table 6
[0093] EGFR gene mutation type
[0094] 3.2 Fluorescent PCR quantification
[0095] The known concentration of mutant gDNA of the cell line was determined by real-time fluorescent quantitative PCR of linearized and circular plasmid standard products, and th...
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