Polynucleotide kinase detection method, intracellular monitoring system and application
A polynucleotide kinase and detection method technology, applied in the field of biochemical analysis and diagnosis, can solve the problems of harmful environment and human body, poor repeatability and high background signal, and achieve the effect of improving sensitivity, strong specificity and low background signal
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Embodiment 1
[0038] In order to prove the cleaving effect of DNAzyme on MB, we divided the experiment into two groups, one group added MB with a final concentration of 200 nM to 100 μL system, and the other group added MB with a final concentration of 200 nM and 100 nM DNAzyme to 100 μL system. All groups were reacted at 37°C for 3.5h, and the fluorescence intensity was measured (Ex / Em=450 / 521, scanning range 495-580nm, slit width 10nm).
[0039] Experimental results such as figure 1As shown in A. It can be seen that in the absence of DNAzyme, there is no obvious fluorescent signal (1A, MB). However, after DNAzyme was introduced into the reaction buffer, an obvious fluorescent response appeared (1A, DNAzyme+MB). The above results show that DNAzyme can cleave MB, and the fluorescence signal increases significantly.
Embodiment 2
[0041] In order to demonstrate the function of RNase H to release DNAzyme from template ligation, we divided the experiments into the following 6 groups:
[0042] Group 1: Add DNAzyme with a final concentration of 100 nM to 100 μL of the system, and 100 nM template DNA3 for complementary pairing at 37°C for 45 minutes, followed by reaction at 37°C for 60 minutes. Add MB with a final concentration of 200nM to continue the reaction at 37°C for 45min, and measure the fluorescence intensity (Ex / Em=450 / 521, scanning range 495-580nm, slit width 10nm).
[0043] The second group: add DNAzyme with a final concentration of 100nM to 100μL system, 100nM template RNA complementary pairing at 37°C for 45min, react at 37°C for 60min, add MB with a final concentration of 200nM and continue to react at 37°C for 45min, measure the fluorescence intensity (Ex / Em =450 / 521, scanning range 495-580nm, slit width 10nm).
[0044] The third group: add DNAzyme with a final concentration of 100nM to 100μ...
Embodiment 3
[0050] In order to prove the ligase repair function of T4 DNA ligase, we divided the experiments into 4 groups, as follows:
[0051] The first group: add p-DNA1 with a final concentration of 100nM, DNA2 with a final concentration of 100nM, and template RNA with a final concentration of 100nM in 100μL system for complementary pairing at 37°C for 45min, then add RNase H with a final concentration of 0.03U / μL at 37°C for 60min, and finally add MB with a final concentration of 200nM was reacted at 37°C for 45min, and the fluorescence intensity was measured (Ex / Em=450 / 521, scanning range 495-580nm, slit width 10nm).
[0052] The second group: add p-DNA1 with a final concentration of 100nM, DNA2 with a final concentration of 100nM, and template RNA-DNA with a final concentration of 100nM in 100μL system for complementary pairing at 37°C for 45min, then add RNase H with a final concentration of 0.03U / μL and react at 37°C for 60min. Finally, MB with a final concentration of 200nM was ...
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