Method for determining activity of terminal deoxynucleotidyl transferase and application of method
A deoxynuclease and detection method technology, which is applied in the field of determination of terminal deoxynuclease activity, can solve the problems of time-consuming and labor-consuming, and achieve the effects of enhanced detection sensitivity, high sensitivity and good selectivity
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Embodiment 1
[0068] Embodiment 1 Principle and feasibility verification of the inventive method
[0069] 1. The detection schematic diagram of TdT (Terminal deoxynucleotidyl transferase, terminal deoxynucleotidyl transferase) of the present invention is as follows figure 1 shown. TdT can perform catalytic addition reaction at the 3'-OH end of ssDNA in a template-free manner, and the specific implementation process is as follows:
[0070] (1) A 14nt primer ssDNA1 (5'ACC CCC CAC CCC CA 3') was designed and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0071] (2) Add primer ssDNA-1 containing 2.5μM, 1×TdT buffer (20mM Tris-HAc, 50mM KAc, 10mMMg(Ac) 2 , pH 7.9), 250 μM CoCl 2 , 0.75mM dTTPs, and different concentrations of TdT (the amount added is: 0U, 0.5U, 1U, 2U, 5U; the concentration of TdT in the system is 0U / mL, 25U / mL, 50U / mL, 100U / mL, 250U / mL) samples were incubated at 37°C for 80min, then incubated at 72°C for 10min (to inactivate TdT), and five samples were obtaine...
Embodiment 2
[0085] The optimization of embodiment 2 experimental system
[0086] 1. Optimization of TdT incubation time
[0087] The extended incubation time of TdT has a decisive effect on the yield of poly-T sequence formation and its subsequent detection of the corresponding value of the fluorescent signal. Therefore, we conducted a systematic investigation on the optimal incubation time of TdT. Specific steps are as follows:
[0088] According to the method in Example 1.3 (feasibility verification) (wherein the final concentration of TdT in step (2) is 250U / mL, HgCl in step (3) 2 The final concentration is 2.5μM), the difference is: the incubation condition of TdT in step (2) is to first incubate at 37°C for 0, 10, 20, 30, 40, 50, 60, 70, 80min, and then at 72°C Incubate for 10 min.
[0089] The result is as Figure 5 As shown in a: by Figure 5 a It can be seen that with the increase of time (0, 10, 20, 30, 40, 50, 60, 70, 80 min), the fluorescence detection signal increases, b...
Embodiment 3
[0094] Example 3 Detection of TdT activity
[0095] As a biomarker of leukemic disease and a nucleic acid modification tool, detection of TdT activity has clinical and molecular biological implications. Therefore, we tested the activity of TDT. The specific implementation process is as follows:
[0096] (1) Contain 2.5μM primer probe ssDNA, different absolute amounts of TdT (0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10U) 1×TdT buffer, 250μM CoCl 2 , 0.75mM dTTPs samples were mixed and incubated at 37°C for 70min, and then incubated at 72°C for 10min. Among them, the total amount of sample is 20 μL, that is, the concentration of TdT in the sample is 0.5U / mL, 1U / mL, 2.5U / mL, 5U / mL, 10U / mL, 25U / mL, 50U / mL, 100U / mL, 250U / mL, 500U / mL.
[0097] (2) 2.5μM HgCl 2 (final concentration) and the resulting sample obtained in step (1) were incubated at room temperature for 20 min.
[0098] (3) Finally, after adding 0.245 μM SYBR green I dye to the reaction system obtained in step (2...
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