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A kind of co-immobilization method and application of dna polymerase and reverse transcriptase

A technology of reverse transcriptase and polymerase, which is applied in the field of enzyme engineering, can solve the problems of no co-immobilization method of DNA polymerase and reverse transcriptase, and achieve the effects of improving catalytic efficiency, stability and high enzyme activity

Active Publication Date: 2021-04-27
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] For this reason, the technical problem to be solved by the present invention is that the prior art does not have the problem of the co-immobilization method of DNA polymerase and reverse transcriptase

Method used

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  • A kind of co-immobilization method and application of dna polymerase and reverse transcriptase
  • A kind of co-immobilization method and application of dna polymerase and reverse transcriptase
  • A kind of co-immobilization method and application of dna polymerase and reverse transcriptase

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Embodiment 1

[0050] The co-immobilization method of Taq DNA polymerase and MMLV reverse transcriptase in this embodiment comprises the following steps:

[0051] (1) Add 0.25g of sodium alginate to 5mL of phosphate buffer containing 0.2mol / L sodium chloride, the pH of the reaction is 6.5, then add 0.0020g of Taq DNA polymerase, mix and fix at 30°C for 2 hours , vacuum-dried to obtain immobilized Taq DNA polymerase;

[0052] (2) Add 5 mL of Tris-HCl buffer solution containing 0.2 mol / L sodium chloride to the immobilized Taq DNA polymerase, the pH value of the reaction is 9.0, then add 0.0030 g of reverse transcriptase, after mixing at 30 ° C Fix for 3 hours, vacuum-dry, and then wash with the Tris-HCl buffer solution containing 0.2mol / L sodium chloride, until no free DNA polymerase and reverse transcriptase are detected in the washing solution;

[0053] (3) Freeze-dry under the condition of temperature of -20°C and pressure of 40Pa to obtain co-immobilized Taq DNA polymerase and reverse tra...

Embodiment 2

[0055] The co-immobilization method of Taq DNA polymerase and MMLV reverse transcriptase in this embodiment comprises the following steps:

[0056] (1) Add 0.20g of sodium alginate to 6mL of phosphate buffer solution containing 0.1mol / L sodium chloride, the reaction temperature is , and the pH value of the reaction is 7.0, then add 0.0016g of Taq DNA polymerase, and mix at 33°C Post-fix for 1.5h, and vacuum-dry to obtain immobilized Taq DNA polymerase;

[0057] (2) Add 6 mL of Tris-HCl buffer containing 0.1 mol / L sodium chloride and 0.0036 g of reverse transcriptase to the immobilized Taq DNA polymerase, the pH of the reaction is 8.5, mix at 33°C and fix for 2.5 h , vacuum-dried, and then washed with a Tris-HCl buffer solution containing 0.3mol / L sodium chloride, until no free DNA polymerase and reverse transcriptase were detected in the washing solution;

[0058] (3) Freeze-dry under the condition of temperature of -15°C and pressure of 50Pa to obtain co-immobilized Taq DNA ...

Embodiment 3

[0060] The co-immobilization method of Taq DNA polymerase and MMLV reverse transcriptase in this embodiment comprises the following steps:

[0061] (1) Add 0.30g of sodium alginate to 4mL of phosphate buffer solution containing 0.3mol / L sodium chloride, the reaction temperature is , the reaction pH value is 6.0, then add 0.0024g of Taq DNA polymerase, mix at 27°C After fixing for 2.5 hours, vacuum-dry to obtain immobilized Taq DNA polymerase;

[0062] (2) Add 4 mL of Tris-HCl buffer containing 0.3 mol / L sodium chloride and 0.0024 g of reverse transcriptase to the immobilized Taq DNA polymerase, the pH of the reaction is 9.5, mix at 27°C and fix for 3.5 h , vacuum-dried, and then washed with a Tris-HCl buffer solution containing 0.1mol / L sodium chloride, until no free DNA polymerase and reverse transcriptase were detected in the washing solution;

[0063] (3) Freeze-dry under the condition of temperature of -25°C and pressure of 30Pa to obtain co-immobilized Taq DNA polymerase...

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Abstract

The invention belongs to the field of enzyme engineering, in particular to a co-immobilization method of DNA polymerase and reverse transcriptase. The method comprises the following steps: adding sodium alginate to the first buffer solution, then adding DNA polymerase, mixing, fixing, and vacuum-drying to obtain immobilized DNA polymerase; adding the second DNA polymerase to the immobilized DNA polymerase The second buffer solution and the reverse transcriptase are mixed, fixed, vacuum-dried, and then washed with the second buffer solution; freeze-dried to obtain co-immobilized DNA polymerase and reverse transcriptase. By co-immobilizing DNA polymerase and reverse transcriptase on the same carrier, the present invention can not only realize the one-step conversion of RT-PCR reaction, but also can combine the catalytic properties of the two, and can eliminate some interference factors, thereby improving The overall catalytic efficiency of the two on the RT-PCR reaction.

Description

technical field [0001] The invention belongs to the field of enzyme engineering, in particular to a co-immobilization method and application of DNA polymerase and reverse transcriptase. Background technique [0002] Reverse transcription polymerase chain reaction (RT-PCR) is a technology that combines reverse transcription (RT) of RNA and polymerase chain amplification reaction (PCR) of cDNA. Two enzymes commonly used in RT-PCR reactions are DNA polymerase and reverse transcriptase. At present, the preservation of DNA polymerase and reverse transcriptase is mainly concentrated in the form of RT-PCR reaction mixture, dried and stored at room temperature, or added with some liquid reagents and frozen at -20°C, which can only be achieved for a short period of time. Preservation and easy introduction of other impurities, it is difficult to maintain good activity of polymerase. [0003] Multi-enzyme immobilization technology refers to a technology that simultaneously immobilize...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/18C12N11/10C12Q1/686
CPCC12N9/1252C12N9/1276C12N11/10C12N11/18C12Q1/686C12Y207/07007C12Y207/07049C12Q2521/101C12Q2521/107C12Q2563/107C12Q2565/625
Inventor 车团结沈颂东赵芳
Owner SUZHOU BAIYUAN GENT CO LTD
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