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An endonuclease VIII activity measuring method

A technology for measuring endonuclease and activity, which is applied in the field of biochemistry, can solve the problems of inaccurate measurement of nicking endonuclease activity, inability to maintain stability between batches, lack of quantitative guidance, etc., to achieve primer design Easy, easy to operate, and the effect of simple operation steps

Active Publication Date: 2015-01-28
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, the analysis of electrophoretic patterns is quite subjective, lacks quantitative guidance, and cannot accurately measure the activity of nicking endonucleases, so that the stability between batches cannot be well maintained.

Method used

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  • An endonuclease VIII activity measuring method
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  • An endonuclease VIII activity measuring method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Determination of Endonuclease Activity of Endonuclease VIII by Fluorescence Method

[0024] Preparation of M13 template / end blocking primer complex:

[0025] M13 single-stranded DNA: M13mp18 single-stranded DNA (NEB);

[0026] Primer M13 end blocking primer: 5’-aagccatccgcaaaaa u gacctct-3' (3' includes but not limited to phosphorylation, amination, spacer-C7, biotinylation and other modifications);

[0027] M13 template / end-blocking primer complex: Mix M13mp18 single-stranded DNA with M13 end-blocking primer at a molar ratio of 1:1, and heat at 70°C for 5 in annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl) After minutes, slowly lower to room temperature to anneal the template primer.

[0028] Preparation of M13 template / normal primer complex:

[0029] M13 single-stranded DNA: M13mp18 single-stranded DNA (NEB);

[0030] Primer M13 Normal primer: 5’-aagccatccgcaaaaa-3’

[0031] M13 template / normal primer complex: Mix M13mp18 single-stranded DNA with M13 normal primer ...

Embodiment 2

[0056] Example 2. EndoⅧ activity-fluorescence intensity curve drawing and EC50 calculation

[0057] Take 2#-12#'s EndoⅧ activity unit (mU) log as the abscissa and fluorescence intensity as the ordinate, and use GraphPad Prism5 to make a nonlinear regression curve. We found that these data points can fit an S-shaped curve well, such as figure 2 Shown.

[0058] experiment EC50 (mU) 16.550 26.724 37.139 46.729 56.903

[0059] Mean (mU)6.81 Standard deviation SD (U)0.22 Coefficient of variation CV (%) 3.27

[0060] Therefore, this EC50 value can be used as the definition of endonuclease activity in this method. That is, in the reaction system of Example 1, the amount of enzyme that makes the fluorescence intensity half of the maximum value is defined as 1 fluorescence unit (FU); 1 FU = 6.81 mU (NEB unit).

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Abstract

An endonuclease VIII activity measuring method is disclosed. The method includes: a step of synthesizing a primer according to a single-strand DNA template, wherein the primer comprises two parts which comprise a normal primer part at the 5' end and an end-capped primer part at the 3' end, and the two parts are connected by dUTP; a step of annealing the synthesized primer and the single-strand DNA template to form a composite; a step of generating an apyrimidinic site on the dUTP position of the primer under the functions of uracil-DNA glycosylase (UDG) and endonuclease VIII (Endo VIII); a step of synthesizing double-strand DNA by utilization of a polymerase with strand displacement activity; a step of measuring the relative amount of the double-strand DNA and single-strand DNA in a reaction system; and a step of deriving the degree of the endonuclease VIII activity according to the measurement result, wherein the degree of the endonuclease VIII activity is positively correlated with the amount of the double-strand DNA in the reaction system. Compared with the prior art, the method is free of radioactive contamination, simple and convenient in operation and capable of performing quantitative detection analysis on the Endo VIII activity.

Description

Technical field [0001] The invention belongs to the field of biochemical technology, and specifically relates to an endonuclease VIII activity determination method. Background technique [0002] E. coli EndoⅧ (Endonuclease VIII, Endo VIII) is encoded by the E. coli nei gene. EndoVIII has both N-glycosylase activity and AP-lyase activity. N-glycosylase activity can release damaged pyrimidine bases on double-stranded DNA, creating an apyrimidinic (AP) site. AP-lyase activity cleaves the phosphodiester bond at the 3'and 5'ends of the AP site, respectively, to produce 3'phosphate and 5'phosphate ends. The damaged bases that can be recognized and removed by Endo Ⅷ include: urea, 5,6-dihydroxythymidine, thymine glycol, 5-hydroxy-5-methyrolone, uracil glycol, 6- Hydroxy-5,6-dihydrothymine and methyl hydroxymalonyl urea. [0003] Endo VIII can be applied to single-cell gel electrophoresis or comet experiment (Collins, A. et al. (1993). Carcinogenesis. 14, 1733-1735.). Comet experiment...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/34
CPCC12Q1/34C12Q1/44
Inventor 曹林徐晓昱王静
Owner VAZYME BIOTECH NANJING
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