Fluorescence detection method for DNA and kit thereof
A fluorescence detection, DNA probe technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve the problem of reducing the distinguishability of complementation and mutation, prolonging the reaction and detection time, and increasing the operation steps to improve mutation detection, improve sensitivity, and improve selectivity
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Embodiment 1
[0041] As shown in Figure 1, according to the requirements of the product specification, MMPs were first washed with 0.5×SSC buffer three times before use, and then the biotin-labeled stem-loop structure capture probe DNA 1 was added to the MMPs containing TTL buffer, Mix gently for about 10 minutes. The surface density of capture probes is about 4-6×10 11 chain / cm 2 . Then the complex of MMPs and capture probe (MMPs-capture probe, denoted as MMPs-cp 1) was washed twice with TTA buffer, suspended in the hybridization solution, and refrigerated at 4°C for use.
[0042] Then the mixture of competitive capture probe DNA 2, targeting DNA 4 (or mutant targeting DNA 5) and linear signal probe DNA 3 of stem-loop structure was added to the suspension of MMPs-cp 1 prepared in advance, wherein The concentration of targeting DNA or mutant targeting DNA 5 is 1 nM, and the molar concentration ratio of competitive capture probe DNA2:capture probe DNA 1:complementary targeting DNA4 or mut...
Embodiment 2
[0045] The reaction was carried out with reference to the conditions in Example 1, except that the temperature of the hybridization reaction was 29°C. The result was a signal ratio of 2.25 for complementary targeting to mutant DNA.
Embodiment 3
[0047] The reaction was carried out with reference to the conditions in Example 1, except that the temperature of the hybridization reaction was 39°C. The result was a signal ratio of 2.31 for complementary targeting to mutant DNA.
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