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Measuring method of activity of T4 DNA polymerase

A technology for activity determination and polymerase, which is applied in the field of T4 DNA polymerase activity determination, can solve the problems of difficult determination of polymerase activity and inapplicability of T4 DNA polymerase activity determination, etc., to achieve automatic activity determination and overcome the interference of polymerase activity determination , simple and fast operation steps

Inactive 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

[0006] However, the inventors have found that this method is not suitable for the determination of the activity of T4 DNA polymerase
Because T4 DNA polymerase has a strong 3'-5' exonuclease activity, in this assay system, the 3'-5' exonuclease activity will offset the polymerase activity to a large extent, resulting in polymerase activity Difficult to measure

Method used

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  • Measuring method of activity of T4 DNA polymerase
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  • Measuring method of activity of T4 DNA polymerase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Fluorometric assay of polymerase activity of T4 DNA polymerase

[0028] Preparation of M13 template / primer complexes:

[0029] M13 ssDNA: M13mp18 ssDNA (NEB);

[0030] Primer: M13 primer 5'-aagccatccgcaaaaatgacctct-3';

[0031] 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.

[0032] 2. Polymerase reaction

[0033]

[0034] 3. Picogreen fluorescence detection:

[0035] Add 0.5 μl of picogreen dye (invitrogen P11495) to the reaction product; detect with a fluorescent microplate reader, the excitation wavelength is 480 nm, and the emission wavelength is 520 nm. The test results are as follows:

[0036] Escherichia coli DNA polymerase I enzyme amount (mU) Fluorescence value 800.00 2288. 400.00 2199. 200.00 ...

Embodiment 2

[0040] Example 2. Establishment of a method for measuring exonuclease activity in T4 DNA polymerase by fluorescence method

[0041] 1. Preparation of double-stranded DNA substrate

[0042] Primer F: 5'-aataacgtcggcaactttgg-3';

[0043] Primer R: 5'-gttacgccaccagtcatcct-3';

[0044] PCR template: λ DNA

[0045] PCR reaction:

[0046] components volume DDW to 50μl 10x Taq buffer 5 dNTPs (10 mM) 1μl Primer F (10 μM) 2μl Primer R (10 μM) 2μl lambda DNA 10ng Taq enzyme 1 U

[0047]

[0048] The PCR product was extracted with phenol-chloroform and purified by ethanol precipitation. The concentration of the purified PCR product was determined by A260.

[0049] 3. Exonuclease reaction:

[0050] components volume DDW to 50μl 10x T4 buffer 5 PCR product 100ng T4 DNA polymerase (NEB M0203) 0-1024 mU

[0051] temperature time 37℃ 30 minutes 4℃ hold ...

Embodiment 3

[0060] Example 3. Drawing of T4 DNA polymerase activity-fluorescence intensity standard curves from different sources and definition of "fluorescence method" activity

[0061] We selected two commercial T4 DNA polymerases and expressed and purified T4 DNA polymerases by ourselves. The self-made T4 DNA polymerase was cloned from T4 phage DNA polymerase I 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 DNA polymerase activities were measured by standard radioisotope incorporation. International unit (IU) is defined as: 1 unit (U) is defined as the amount of enzyme required to incorporate 1 nmol of isotope-labeled dATP into the acid-insoluble precipitate within 1 hour. According to the method of Example 1, the activity-fluorescence intensity standard curve was drawn and EC50 was calculated. The result is as follows:

[0062]...

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Abstract

A measuring method of activity of a T4 DNA polymerase is disclosed. The method includes: under the circumstance of no dNTP, adding the T4 DNA polymerase into a reaction system adopting a double-strand linear DNA template as a substrate, detecting the relative amount of double-strand DNA after a reaction is finished so as to measure the excision enzyme activity of the T4 DNA polymerase, and allowing the excision enzyme activity of the T4 DNA polymerase to be related to the polymerase activity so as to obtain the polymerase activity degree of the T4 DNA polymerase. The polymerase activity is measured by conveniently measuring the excision enzyme activity and by utilization of relevance between the excision enzyme activity and the polymerase activity of the T4 DNA polymerase. Compared with methods in the prior art, the method is free of radioactive contamination and simple and rapid in operation, and the method overcomes interference of the excision enzyme activity on the measurement of the polymerase activity, so that a result of the method is more accurate.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring T4 DNA polymerase activity. Background technique [0002] T4 DNA polymerase is encoded by the T4 phage polymerase I gene and has 5'-3' polymerase activity and 3'-5' exonuclease activity. T4 DNA polymerase is an important tool enzyme in genetic engineering technology, which can be used to smooth the ends of double-stranded DNA. In the process of high-throughput sequencing (next generation sequencing) library construction, the fragmented DNA ends first need to be smoothed with T4 DNA polymerase, and then phosphoricated by T4 polynucleotide kinase before they can be connected with double-stranded DNA to connect. [0003] The standard T4 DNA polymerase assay method uses the radioisotope method. T4 DNA polymerase can catalyze the 32 Incorporation of P-labeled dNTPs into calf thymus DNA. After TCA precipitation and Whatman filter paper filtr...

Claims

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Application Information

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