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Method and kit for detecting DNA polymerase activity

A technology of polymerase and enzyme activity, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as difficult to achieve high-throughput detection, radioactive contamination, and complicated operation.

Active Publication Date: 2017-08-11
FAPON BIOTECH INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is prone to radioactive contamination, cumbersome operation, and difficult to achieve high-throughput detection

Method used

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  • Method and kit for detecting DNA polymerase activity
  • Method and kit for detecting DNA polymerase activity
  • Method and kit for detecting DNA polymerase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Standard curve established from T4 DNA polymerase standard

[0092] 1.1. Provide a template to be filled in including the first template strand and the second template strand, the base sequence of the first template strand is shown in SEQ ID No.1, and the base sequence of the second template strand is shown in SEQ ID No.2 shown. Take 10 μL of T4 DNA polymerase standard (3U / μL) and add it to 90 μL of 1×TE buffer, and perform six serial 10-fold serial dilutions to form six standard product gradients. Configure the fill-in reaction system as shown in Table 1 respectively. The fill-in reaction conditions are: 37°C, 1h. After fill-in, a fill-in template is obtained.

[0093] Table 1: Filling Reaction System

[0094] Reagent volume 10×T4 DNA polymerase buffer 5μL dNTP (10mmol / L) 0.2 μL Template to be filled (0.025μmol / L) 10μL Serial dilution of T4 DNA polymerase 5μL wxya 2 o

Supplement to a total volume of 50 μL

[009...

Embodiment 2

[0108] Detection of the enzymatic activity of unknown T4 DNA polymerase

[0109] Run the SDS-PAGE gel of the sample to be tested, and initially calibrate the enzyme activity of T4 DNA polymerase in the sample to be tested according to the protein concentration. Take 10 μL of the sample to be tested for 100-fold dilution, and make 8 parallel dilutions. Prepare the leveling system according to Table 1 in Example 1, configure the PCR reaction system according to the PCR reaction system in Table 2, and perform PCR amplification according to the PCR amplification conditions in Table 3, and obtain the CT values ​​corresponding to the PCR amplification of 8 groups of samples , and the results are shown in Table 5.

[0110] Table 5: CT values ​​of T4 DNA polymerase samples

[0111] Sample number 1# 2# 3# 4# 5# 6# 7# 8# CT value 16.67 16.68 16.61 16.65 16.67 16.62 16.58 16.6

[0112] Bring the CT value corresponding to the sample into Figure 4 In...

Embodiment 3

[0118] Detection of enzymatic activity of T4 DNA polymerase from different sources

[0119] In order to further verify the feasibility of the detection method, two commercial T4 DNA polymerases were selected for detection. The commercial T4 DNA polymerase 1 was NEB-T4 DNA Polymerase, and the commercial T4 DNA polymerase 2 was Enzymatics-T4 DNA Polymerase. In addition, the enzymatic activity of self-expressed and purified T4 DNA polymerase was detected. 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 expressed product was purified to a purity of about 98% by conventional chromatography.

[0120] The two commercially available enzymes and self-expressed and purified T4 DNA polymerase were first determined by the standard radioisotope incorporation method. The International Unit (IU) was defined as the amount required to incorporate 1 nmol of isotope-lab...

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Abstract

The invention relates to a method and kit for detecting DNA polymerase activity; the method comprises: mixing DNA polymerase under detection and a sufficient template to be filled in, and using, in the presence of dNTP, the DNA polymerase under detection to induce a first DNA fragment in the template to be filled in so as to form a filling-in fragment complementary with a base of a projecting fragment, thereby acquiring a filled-in template; digesting to remove the base U of the filled-in template through sufficient UDG (uracil DNA glycosylase) to obtain a template suitable for PCR (polymerase chain reaction) amplification, designing a forward primer for the projecting fragment and a reverse primer for 5' end of the first DNA fragment, and inducing first template chain amplification, wherein a template which is not filled in and has the base U removed via a digestive enzyme is unable to pair with the forward primer, and PCR amplification cannot be performed; CT (cycle threshold) value of DNA polymerase under detection can be brought to a standard curve established via a DNA polymerase standard, and activity of the DNA polymerase under detection can be acquired just by calculating.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting DNA polymerase activity and a detection kit. Background technique [0002] DNA polymerase (DNA polymerase) is an important enzyme in the replication of DNA in cells, which can induce the replication and elongation of DNA chains under certain conditions. The relatively important DNA polymerases in organisms include, for example, T4 DNA polymerase, Klenow enzyme (also known as Klenow fragment, Klenow Fragment) and DNA polymerase I (DNA-pol I). Wherein T4 DNA polymerase is encoded by T4 phage polymerase I gene, has 5'-3' polymerase activity and 3'-5' exonuclease activity. T4 DNA polymerase is an important tool enzyme in genetic engineering technology, and plays an important role in the process of high-throughput sequencing (next generation sequencing) library construction. [0003] The traditional method for detecting the activity of DNA polymerase generally ado...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/48
CPCC12Q1/48C12Q1/6851C12Q2521/101C12Q2521/531C12Q2545/114
Inventor 蔡统聪邓艳华杨浩黄田田孙康成王益琼
Owner FAPON BIOTECH INC
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