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A kind of dna ligase activity assay method

A DNA ligase and activity determination technology, applied in the field of biochemistry, can solve the problems of low experimental cost, complex probe design, unsatisfactory, etc., and achieve the effect of simple operation steps, easy primer design, and fast operation steps

Active Publication Date: 2016-09-07
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The experimental cost of this method is low, but its biggest defect is that the analysis of the electrophoretic pattern is quite subjective.
Even if image scanning and grayscale analysis are used to determine the ratio of ligation products, it is still a semi-quantitative method, so the activity of DNA ligase cannot be accurately measured, so the stability between batches cannot be well maintained
[0005] Although the molecular beacon method and AP labeling method developed based on fluorescence technology are simple and fast, the probe design is complex, the cost is high, and false positive signals are prone to be generated during the detection process, which cannot meet the different needs of people's research.

Method used

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  • A kind of dna ligase activity assay method
  • A kind of dna ligase activity assay method
  • A kind of dna ligase activity assay method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Feasibility verification of T4 DNA ligase activity by "transformation method"

[0024] Preparation of M13 template / normal primer-3' terminal phosphorylated primer complex:

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

[0026] Normal M13 primer: 5'-aagccatccgcaaaaatgacctct-3';

[0027] 3'-terminal phosphorylated M13 primer: 5'-tatcaaaaggagcaattaaagg-3' (3'-terminal hydroxyl group is phosphorylated)

[0028] M13 template / normal primer-3'-terminal phosphorylated primer complex: M13mp18 single-stranded DNA was mixed with normal M13 primer, 3'-terminal phosphorylated M13 primer in a molar ratio of 1:1:1, in annealing buffer (10 mM Tris -HCl pH 8.0, 50 mM NaCl), after heating at 70 °C for 5 min, slowly cool to room temperature to anneal the template primers.

[0029] Preparation of M13 template / 3' terminal phosphorylated primer complexes:

[0030] M13 single-stranded DNA: M13mp18 single-stranded DNA (NEB);

[0031] 3'-terminal phosphory...

Embodiment 2

[0050] Example 2. Drawing of T4 DNA ligase activity-fluorescence intensity curve and EC50 calculation

[0051] Take the log value of the T4 DNA ligase 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 to an inverted sigmoid curve, such as figure 2 shown.

[0052] By repeating the experiment multiple times and calculating the half-maximal effect concentration (EC50) of the curve, we found that the EC50 value remained stable across experiments:

[0053] experiment

[0054] Average (mU)

Embodiment 3

[0055] Example 3. Drawing of DNA ligase activity-fluorescence intensity standard curve from different sources and definition of "transformation method" activity

[0056] We also selected a different species of DNA ligase, namely E. coli DNA ligase (enzymaticsL6090L), and according to the methods of Example 1 and Example 2, the activity-fluorescence intensity standard curve was drawn and EC50 was calculated. All DNA ligase activities were determined using standard semi-quantitative assays. The unit commonly used to describe ligase activity is cohesive end unit (CEU): 1 cohesive end activity unit refers to 50% of the ligase digested by HindIII in 20μL of 1×T4 DNA ligation reaction buffer at 16°C for 30min. The amount of enzyme required for ligation of the λ DNA fragment [5' end at a concentration of 0.12 μM (300 μg / mL)]. The result is as image 3 shown.

[0057] species

source

EC50 (mU)

T4 phage

NEB E. coli recombinant expression

12.84±0.17*

...

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Abstract

The invention discloses a method for determining activity of DNA ligase. The method comprises the following steps: firstly, synthesizing two adjoined primers according to a single-chain DNA template, wherein for the direction from 5' to 3' of the primers, the 3' end of the primer in the 5' direction is adjacent to 5' of the primer in 3' direction, and the primer in the 5' direction is a normal primer, and the primer in the 3' direction is a 3' terminal phosphorylated primer; then annealing the two primers and the single-chain DNA template, performing ligation reaction in the presence of DNA ligase by taking the formed single-chain DNA template / normal primer-3' terminal phosphorylated primer compound as a substrate; and by taking the product of ligation reaction as a substrate, performing polymerization reaction under the action of polymerase with chain replacement activity, then detecting the relative amount of a dual-chain DNA or single-chain DNA, and deriving the activity degree of the DNA ligase on the basis of the relative amount. The method has no radioactive pollution, is simple and fast to operate, and can perform quantitative detection analysis on the activity of the DNA ligase.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring DNA ligase activity. Background technique [0002] DNA ligase was discovered in 1967, it can close the gap on the DNA chain, and the energy provided by the hydrolysis of ATP or NAD catalyzes the formation of phosphodiester bonds at the 5'-phosphate end and 3'-hydroxyl end of the DNA chain, so that the same One complementary strand binds in pairs and joins two oligonucleotide strands in close proximity (without a gap between them). Escherichia coli DNA ligase, derived from Escherichia coli, is the first DNA ligase discovered and can be used to connect sticky ends; T4 DNA ligase, derived from T4 phage, can connect sticky ends and blunt ends, but the ligation efficiency is low; The thermostable DNA ligase is isolated and purified from the strain of Actinomyces thermophilus. It is a nuclease that can catalyze the ligation of two oligonucleotid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/48C12Q1/25
CPCC12Q1/25C12Q1/48
Inventor 徐晓昱王静
Owner VAZYME BIOTECH NANJING
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