Co-immobilization method for DNA polymerase and reverse transcriptase, and application of co-immobilized DNA polymerase and reverse transcriptase

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

Active Publication Date: 2018-07-31
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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  • Co-immobilization method for DNA polymerase and reverse transcriptase, and application of co-immobilized DNA polymerase and reverse transcriptase
  • Co-immobilization method for DNA polymerase and reverse transcriptase, and application of co-immobilized DNA polymerase and reverse transcriptase
  • Co-immobilization method for DNA polymerase and reverse transcriptase, and application of co-immobilized DNA polymerase and reverse transcriptase

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Embodiment 1

[0050] The method for co-immobilization of Taq DNA polymerase and MMLV reverse transcriptase in this embodiment includes 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 at 30℃ and fix for 2 hours , Vacuum dry, get immobilized Taq DNA polymerase;

[0052] (2) Add 5mL of Tris-HCl buffer containing 0.2mol / L sodium chloride to the immobilized Taq DNA polymerase, the pH of the reaction is 9.0, then add 0.0030g of reverse transcriptase, and mix at 30℃ Fix for 3 hours, vacuum dry, and then wash with the Tris-HCl buffer containing 0.2mol / L sodium chloride until free DNA polymerase and reverse transcriptase can not be detected in the washing solution;

[0053] (3) Freeze-drying at a temperature of -20°C and a pressure of 40 Pa to obtain co-immobilized Taq DNA polymerase and reverse transcriptase.

Embodiment 2

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

[0056] (1) Add 0.20g of sodium alginate to 6mL of phosphate buffer containing 0.1mol / L sodium chloride at a reaction temperature of 7.0, and then add 0.0016g of Taq DNA polymerase and mix at 33°C After fixation for 1.5h, 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, and fix for 2.5 hours after mixing at 33°C , Vacuum dry, and then wash with Tris-HCl buffer containing 0.3mol / L sodium chloride until free DNA polymerase and reverse transcriptase can not be detected in the washing solution;

[0058] (3) Freeze drying at a temperature of -15°C and a pressure of 50 Pa to obtain co-immobilized Taq DNA polymerase and reverse transcriptase.

Embodiment 3

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

[0061] (1) Add 0.30g of sodium alginate to 4mL of phosphate buffer containing 0.3mol / L sodium chloride at a reaction temperature of 6.0, and then add 0.0024g of Taq DNA polymerase and mix at 27°C After fixation 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, and fix for 3.5 hours after mixing at 27°C , Vacuum dry, and then wash with Tris-HCl buffer containing 0.1 mol / L sodium chloride until free DNA polymerase and reverse transcriptase can not be detected in the washing solution;

[0063] (3) Freeze drying at a temperature of -25°C and a pressure of 30 Pa to obtain co-immobilized Taq DNA polymerase and reverse transcriptase.

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Abstract

The invention specifically relates to a co-immobilization method for DNA polymerase and reverse transcriptase, belonging to the field of enzyme engineering. The method comprises the following steps: adding sodium alginate into a first buffer, then adding DNA polymerase, carrying out mixing and immobilizing, and then carrying out vacuum drying so as to obtain immobilized DNA polymerase; adding a second buffer and reverse transcriptase into the immobilized DNA polymerase, carrying out mixing and immobilizing, then carrying out vacuum drying and then carrying out washing with the second buffer; and then carrying out lyophilization so as to obtain the co-immobilized DNA polymerase and reverse transcriptase. According to the invention, DNA polymerase and reverse transcriptase are co-immobilizedon a same carrier, so one-step conversion of a RT-PCR reaction is realized, and the catalytic properties of DNA polymerase and reverse transcriptase are integrated to eliminate certain interference factors, and thus, the overall catalytic efficiency of DNA polymerase and reverse transcriptase to RT-PCR reactions is improved.

Description

Technical field [0001] The invention belongs to the field of enzyme engineering, and specifically relates 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 (PCR) of cDNA. Two enzymes commonly used in RT-PCR reactions are DNA polymerase and reverse transcriptase. At present, the storage 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 a certain liquid reagent and stored at -20°C, which can only achieve a short time Storage and easy introduction of other impurities, it is difficult to maintain good activity of polymerase. [0003] Multi-enzyme immobilization technology refers to a technology in which multiple enzymes are simultaneously immobilized in ...

Claims

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Application Information

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Patent Type & Authority Applications(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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