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

Active Publication Date: 2019-03-29
JINAN UNIVERSITY
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

These methods require multiple processes using radioactively or fluorescently labeled DNA or nucleotides, which is relatively time-consuming and labor-intensive

Method used

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  • Method for determining activity of terminal deoxynucleotidyl transferase and application of method
  • Method for determining activity of terminal deoxynucleotidyl transferase and application of method
  • Method for determining activity of terminal deoxynucleotidyl transferase and application of method

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a method for determining activity of terminal deoxynucleotidyl transferase (TdT) and application of the method. The method comprises the steps of: (1) designing a single-stranded DNA as a DNA probe; (2) incubating TdT with different final concentrations with DNA probes, CoCl2, dTTPs and a buffer solution separately to obtain a single-stranded probe DNA solution rich in a poly-T sequence; then adding a mercury salt to incubate to obtain a T-HgII-T structure-mediated double-stranded DNA solution; adding a nucleic acid dye to obtain a mixed solution; and finally detectinga fluorescence intensity signal of the mixed solution, and drawing a detection curve according to the fluorescence intensity signal and the final concentration of TdT; and (5) replacing the TdT with asample to be tested, repeating the above steps, and then comparing the detection result with the detection curve to calculate the content of TdT in the sample to be tested. The method has high sensitivity and good selectivity, and provides a new means for medical detection and biological research.

Description

technical field [0001] The invention belongs to the technical field of biological detection, in particular to a method for measuring terminal deoxynuclease activity and its application. Background technique [0002] Terminal deoxynucleotidyl transferase (TdT) randomly adds deoxyribonucleoside triphosphate (dNTP) to the 3'-OH end of ssDNA in a template-free manner. It is a tool enzyme widely used in many fields for target DNA, RNA , metal ions, apoptotic cells, modified enzymes and other biological samples. Also, abnormal expression of TdT is closely related to leukemia and cancer, being overexpressed in about 90% of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cases. Many clinical studies have shown that abnormal expression of TdT plays an important role in cancer development and may reduce cancer response to anticancer chemotherapy. In addition, TdT activity in blast cells also serves as an important biomarker. Therefore, it is of great significanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
CPCC12Q1/48
Inventor 邢曦雯王静茹赖淑妍吴纯刘奋勇
Owner JINAN UNIVERSITY
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