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Method for quantitatively determining activity of T4 polynucleotide kinase

A technology of polynucleotide and kinase activity, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problems of quality limit, cumbersome operation process, and easy radioactive pollution, etc., and achieve easy operation and operation Simple steps and quick response

Inactive Publication Date: 2015-01-21
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has high sensitivity, it is limited by the quality of markers, and the operation process is cumbersome, which is prone to radioactive contamination.

Method used

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  • Method for quantitatively determining activity of T4 polynucleotide kinase
  • Method for quantitatively determining activity of T4 polynucleotide kinase
  • Method for quantitatively determining activity of T4 polynucleotide kinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Establishment of a method for measuring T4 polynucleotide kinase activity by chemiluminescence

[0023] Phosphorylation reaction:

[0024] T4 polynucleotide kinase is NEB M0201;

[0025] Oligonucleotide substrate: 5'-acggattcatatttcatcct-3'

[0026]

[0027] temperature time 37℃ 30 minutes 4℃ Keep

[0028] Luciferase reaction:

[0029]

[0030] *T4 polynucleotide kinase reaction product corresponding to tube number was added to tubes 1-12.

[0031] Chemiluminescence detection

[0032] Detection was performed with a GloMax® 96 Microplate Luminometer (promega Cat.# E6501). The test results are as follows:

[0033] Enzyme amount of T4 polynucleotide kinase (mU) Chemiluminescence value 1024 241. 512 1050. 256 1086. 128 1578. 64 3236. 32 14675. 16 49947. 8 91598. 4 116186. 2 117052. 1 117184. 0 117963

[0034] This result indicated that T4 PNK could...

Embodiment 2

[0036] Example 2. Drawing of T4 polynucleotide kinase activity-fluorescence intensity curve and EC50 calculation

[0037] Take the log value of the T4 polynucleotide kinase activity unit (mU) of 2#-12# as the abscissa, and the fluorescence intensity as the ordinate, and use GraphPad Prism5 to make a nonlinear regression curve. We found that these data points can be well fitted with an inverted sigmoid curve, such as image 3 shown.

[0038] By repeating the experiment several times and calculating the half-maximal effect concentration (EC50) of the curve, we found that the EC50 value remained stable across multiple experiments:

[0039] experiment EC50 (mU) 1 13.76 2 13.54 3 14.37 4 13.58 5 14.07

[0040] Mean (mU) 13.86 Standard deviation SD (U) 0.35 Coefficient of Variation CV (%) 2.54

[0041] Therefore, this EC50 value can be used as the definition of T4 polynucleotide kinase activity in this method. T...

Embodiment 3

[0042] Example 3. Fluorometric assay of different batches of T4 polynucleotide kinase activity, and unit calibration

[0043] We selected two commercial T4 polynucleotide kinases and expressed and purified T4 polynucleotide kinases by ourselves. Self-made T4 polynucleotide kinase was cloned from T4 phage polynucleotide kinase expression gene, cloned into pET28a expression vector and transformed into Escherichia coli BL21 for expression. The expression product was purified to a purity of about 95% by conventional chromatography. All T4 polynucleotide kinases are assayed for activity using the radioisotope method. International unit (IU) is defined as: within 30 minutes at 37°C, the amount of enzyme required to transfer 1 nmol of γ-phosphate group on ATP to the 5'-OH end of DNA is defined as 1 activity unit. According to the method of Example 1, the activity-fluorescence intensity standard curve was drawn and EC50 was calculated. curve like Figure 4 and the results are disp...

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Abstract

The invention discloses a method for quantitatively determining activity of T4 polynucleotide kinase. The method for quantitatively determining the activity of T4 polynucleotide kinase comprises the following steps: in a reaction system used for phosphorylation, adding ATP by taking oligonucleotide as a substrate for phosphorylating an end 5' of oligonucleotide by utilizing T4 polynucleotide kinase, so as to generate phosphorylated oligonucleotide; then adding a luciferase reporter gene system into the reaction system, wherein luciferase in the luciferase reporter gene system utilizes residual ATP in the reaction system to oxidize luciferin to form oxygenated luciferase and produce chemiluminiscence at the same time; and detecting numerical value of luminescence, and deriving activity degree of T4 polynucleotide kinase by virtue of detection results, wherein the activity degree of T4 polynucleotide kinase is in negative correlation with luminous quantity. Compared with the prior art, the method for quantitatively determining the activity of T4 polynucleotide kinase has the advantages that no radioactive contamination is produced, operation is easy, and quantitative detection analysis can be carried out on the activity of T4 polynucleotide kinase.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a quantitative T4 polynucleotide kinase activity assay method. Background technique [0002] T4 Polynucleotide Kinase (T4 Polynucleotide Kinase), referred to as T4 PNK, is a protein encoded by the pseT gene of T4 phage, which was originally isolated from E. coli cells infected by T4 phage, so it is also called T4 polynuclear nucleotide kinase. The enzyme can catalyze the transfer of γ-phosphate of ATP to the 5' hydroxyl of single-stranded or double-stranded DNA, RNA, oligonucleotide or mononucleotide with 3' phosphate group. The enzyme also has 3' phosphatase activity, which hydrolyzes 3'-phosphate groups from the 3' phosphate termini of oligonucleotides, deoxy 3'-monophosphate nucleosides and deoxy 3'-diphosphate nucleosides. [0003] As an important tool enzyme, T4 polynucleotide kinase is widely used in the field of genetic engineering technology and molecul...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/66C12Q1/48
CPCC12Q1/66C12Q1/485
Inventor 徐晓昱刘来花王静
Owner VAZYME BIOTECH NANJING
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