DNA polymerase immobilization method and application thereof

A technology of polymerase and chitosan, applied in the field of enzyme engineering, can solve the problems of low activity of TaqDNA polymerase and poor stability of TaqDNA polymerase, and achieve the effect of wide application range and good thermal stability

Active Publication Date: 2018-06-08
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] For this reason, the first technical problem to be solved by the present invention is the problem that the activity of the immobilized TaqDNA polymerase prepared by the prior art is low, and the second technical problem to be solved by the present invention is the

Method used

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Experimental program
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Effect test

Embodiment 1

[0053] The immobilization method of Taq DNA polymerase of the present embodiment comprises the following steps:

[0054] Add 5.0 g of modified chitosan to 200 mL of water, heat to dissolve, sterilize with moist heat for 40 minutes, and cool to 40°C to obtain a modified chitosan aqueous solution;

[0055] 10g of Taq DNA polymerase was added to the modified chitosan aqueous solution, and an adsorption reaction was carried out. The reaction temperature was 37° C., the pH value of the reaction was 7.0, and the reaction time was 120 minutes to obtain an adsorption reaction liquid of Taq DNA polymerase;

[0056] Add 50 mL of dialdehyde dextran aqueous solution with a volume fraction of 0.05% to the adsorption reaction solution of Taq DNA polymerase to carry out cross-linking reaction. The reaction temperature is 32 ° C and the reaction time is 90 min. Potassium solution, let stand, then wash with 4-hydroxyethylpiperazineethanesulfonic acid buffer solution containing 0.5mol / L sodium ...

Embodiment 2

[0060] The immobilization method of the above-mentioned Taq DNA polymerase of the present invention comprises the following steps:

[0061] Add 4.0 g of modified chitosan to 240 mL of water, heat to dissolve, sterilize with moist heat for 35 minutes, and cool to 45°C to obtain a modified chitosan aqueous solution;

[0062] 8g of Taq DNA polymerase was added to the modified chitosan aqueous solution to carry out adsorption reaction, the reaction temperature was 40°C, the pH value of the reaction was 6.5, and the reaction time was 140min to obtain the adsorption reaction liquid of Taq DNA polymerase;

[0063] Add 40 mL of dialdehyde dextran aqueous solution with a volume fraction of 0.06% to the adsorption reaction solution of Taq DNA polymerase to carry out cross-linking reaction. The reaction temperature is 30 °C and the reaction time is 100 min. Potassium solution, let it stand, and then wash with buffer to obtain the cross-linking reaction solution of Taq DNA polymerase;

...

Embodiment 3

[0067] The immobilization method of the above-mentioned Taq DNA polymerase of the present invention comprises the following steps:

[0068] Add 6.0 g of modified chitosan to 160 mL of water, heat to dissolve, sterilize with moist heat for 55 minutes, and cool to 35°C to obtain a modified chitosan aqueous solution;

[0069] 12g of Taq DNA polymerase was added to the modified chitosan aqueous solution for adsorption reaction, the reaction temperature was 35°C, the pH value of the reaction was 7.5, and the reaction time was 100min to obtain the adsorption reaction liquid of Taq DNA polymerase;

[0070] Add 60 mL of dialdehyde dextran aqueous solution with a volume fraction of 0.04% to the adsorption reaction solution of Taq DNA polymerase to carry out cross-linking reaction. The reaction temperature is 35 °C and the reaction time is 80 min. Potassium solution, let it stand, and then wash with buffer to obtain the cross-linking reaction solution of Taq DNA polymerase;

[0071] Mi...

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Abstract

The invention belongs to the field of enzyme engineering and particularly relates to a DNA polymerase immobilization method and an application thereof. The immobilization method comprises steps as follows: a modified chitosan water solution is prepared firstly, an adsorption reaction solution of DNA polymerase is prepared, a crosslinking reaction solution of the DNA polymerase is prepared, the crosslinking reaction solution and wood pulp sponge are mixed, a carrageenin water solution is added, the mixture is centrifuged and washed and finally freeze-dried, and the immobilized DNA polymerase isobtained, and the prepared immobilized DNA polymerase has higher activity and better thermal stability; the prepared immobilized DNA polymerase has the highest activity and the best thermal stabilityby selecting a specific protein flocculant, specific adsorption reaction conditions, specific crosslinking reaction conditions, a specific crosslinking agent, a specific buffer solution, specific carriers, a specific PCR accelerator and specific freeze-drying conditions.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, and in particular relates to a DNA polymerase immobilization method and application thereof. Background technique [0002] Polymerase chain reaction (PCR) refers to: the DNA will be denatured into a single strand at a high temperature of 95°C in vitro, and the single strand will combine with the primer according to the principle of complementary base pairing at a low temperature of about 60°C. When the optimum reaction temperature of DNA polymerase is around 72°C, DNA polymerase will synthesize complementary chain properties along the direction from phosphoric acid to five-carbon sugar (5'-3'), and realize the molecular biology technology of amplifying and amplifying specific DNA fragments . [0003] Taq DNA polymerase is the heat-resistant DNA polymerase with the highest amplification efficiency. It can amplify DNA fragments below 6kb very well, and the amplification base error rate is 10 -5 ...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N11/02C12N11/10C12P19/34
CPCC12N9/1252C12N11/02C12N11/10C12P19/34C12Y207/07007
Inventor 车团结徐进章赵芳
Owner SUZHOU BAIYUAN GENT CO LTD
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