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3'-5' excision enzyme activity measurement method

An activity assay, exonuclease technology, applied in the field of biochemistry, can solve the problems of difficult screening, long cycle, difficult to achieve high-throughput, automation, etc., and achieve the effect of simple and fast operation steps

Active 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

This method is prone to radioactive contamination, and has many steps and a long cycle, making it difficult to achieve high-throughput and automation, thus bringing difficulties to screening

Method used

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  • 3'-5' excision enzyme activity measurement method
  • 3'-5' excision enzyme activity measurement method
  • 3'-5' excision enzyme activity measurement method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Preparation of M13 template / primer complexes:

[0028] M13 ssDNA: M13mp18 ssDNA (NEB);

[0029] Primer: M13 primer 5'-aagccatccgcaaaaatgacctct-3';

[0030] M13 template / primer complex: M13mp18 single-stranded DNA was mixed with M13 primer at a molar ratio of 1:1, heated at 70°C for 5 minutes in annealing buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl), slowly Cool down to room temperature to anneal the template primers.

[0031] 2. Preparation of M13 template / mismatched primer complex:

[0032] M13 ssDNA: M13mp18 ssDNA (NEB);

[0033] Primer: M13 mismatch primer 5'-aagccatccgcaaaaatgacctct a -3'; (The underline indicates that the base does not match the template)

[0034] M13 template / mismatched primer complex: M13mp18 single-stranded DNA and M13 mismatched primer were mixed at a molar ratio of 1:1, heated at 70°C for 5 Minutes later, slowly cool down to room temperature to anneal the template primers.

[0035] 3. 3'-5' exonuclease reaction:

[0036]

[0037] ...

Embodiment 2

[0052] Example 2. Drawing of 3'-5' exonuclease activity-fluorescence intensity curve and EC50 calculation

[0053] Take the log value of the Pfu 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 fit a S-shaped curve very well, such as figure 2 shown.

[0054] 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:

[0055]

[0056] Mean (mU) 17.75 Standard deviation SD (U) 0.47 Coefficient of Variation CV (%) 2.65

[0057] Therefore, this EC50 value can be used as the definition of Pfu 3'-5' exonuclease activity in this method. That is, in the reaction system of Example 1, the amount of enzyme that makes the fluorescence intensity reach half of the maximum value is define...

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Abstract

The invention discloses a non-radioactive 3'-5' excision enzyme activity measurement method. The method comprises the following steps: firstly, synthesizing a primer with 3' mismatching according to a single chain template; mixing and annealing the primer and the single chain template; adding 3'-5' excision enzyme activity to reaction liquid and carrying out 3' base circumscribed reaction; adding a plenty of polymerase extension primers free of 3'-5' excision enzyme activity until a dual-chain DNA is obtained; detecting the relative quantity of the dual-chain DNA in a reaction system; and deriving the excision enzyme activity degree according to the detection result. Compared with an existing technique, the method is free of radioactive pollution, and simple and rapid in operation steps; high-flux automatic activity measurement can be achieved; and the method can be used for screening the closed activity of 3'-5' excision enzymes.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring 3'-5' exonuclease activity. Background technique [0002] Pfu DNA polymerase (Pfu for short) is a highly thermostable DNA polymerase derived from the thermophilic bacterium Pyrococcus furiosus. Pfu has two activities: 5'-3' polymerase activity and 3'-5' exonuclease activity (or proofreading activity). The 3'-5' exonuclease activity of Pfu is very strong, which can degrade the base-mismatched nucleotides in the DNA synthesis chain, greatly increase the correct rate of base pairing, and ensure the high fidelity of DNA synthesis. Therefore, the enzyme is currently the highest fidelity thermostable DNA polymerase and is suitable for use in PCR applications that require high precision, including cloning, gene expression, and mutation analysis. [0003] Although the high 3'-5' exonuclease activity of Pfu can bring high fidelity, it can also cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/44
CPCC12Q1/44
Inventor 徐晓昱王静
Owner VAZYME BIOTECH NANJING
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