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Hot-start Taq DNA (deoxyribonucleic acid) polymerase and preparation method thereof

A polymerase and hot-start technology, applied in the field of DNA polymerase and its preparation, can solve the problems of high price, uncommercialization, infeasibility, etc., and achieve the effect of strong specificity

Active Publication Date: 2016-03-02
刘未斌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The manual hot start operation is cumbersome and is not feasible in practical applications. The wax protective coating method is very unstable and cannot be commercialized. The antibody used in the antibody method is obtained through animal immunization, and the preparation process is complicated and expensive.

Method used

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  • Hot-start Taq DNA (deoxyribonucleic acid) polymerase and preparation method thereof

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Embodiment 1

[0046] Preparation of hot start TaqDNA polymerase of the present invention

[0047] 1. Preparation of aldylated polysaccharides: Dextran 40 (molecular weight 40kDa) 2.5g was dissolved in NaIO 4 (0.12M) in 100ml of acetate buffer (pH6), react at 4°C in the dark for 24 hours, dialyze 3 times with distilled water, concentrate, and dry to obtain powdery formylated dextran; figure 1 It is the molecular formula of aldylated polysaccharides.

[0048] 2. Combination of TaqDNA polymerase with formylated polysaccharides:

[0049] Add 8 mg of powdery formylated dextran to 3 ml of an aqueous solution of 50 mg / LTaq DNA polymerase (molecular weight 75 kDa) produced by Promga to form an oxidation mixture. Based on the total weight of the reaction mixture, 1% bovine serum albumin BSA with a mass fraction of 10wt% was added, and the reaction was blocked at 4° C. in the dark for 24 hours;

[0050] Put the reaction mixture containing TaqDNA polymerase after the reaction on a 1.6×55cm, Sephade...

Embodiment 2

[0052] Preparation of hot start TaqDNA polymerase of the present invention

[0053] 1. Preparation of aldylated polysaccharides: Dextran 70 (molecular weight 70kDa) 4.375g was dissolved in NaIO 4 (0.12M) in 100ml of acetate buffer (pH4), react at 4°C in the dark for 24 hours, dialyze 4 times with distilled water, concentrate, and dry to obtain powdery formylated dextran;

[0054] 2. Combination of TaqDNA polymerase with formylated polysaccharides:

[0055] Add 14 mg of powdered aldylated dextran to 3 ml of 50 mg / L high-fidelity TaqDNA polymerase (molecular weight 90 kDa) produced by Takara Company in an aqueous solution to form an oxidation mixture, the enzyme activity unit is greater than 150 U / mg, and react in the dark at 4°C 24h, then based on the total weight of the reaction mixture, add 1% of BSA with a mass fraction of 10wt%, and block the reaction at 8°C for 30h in the dark;

[0056] The reaction mixture containing TaqDNA polymerase after the reaction was completed wa...

Embodiment 3

[0058] Preparation of hot start TaqDNA polymerase of the present invention

[0059] 1, prepare aldylated polysaccharides: Chitosan 50 (molecular weight 50kDa) 3.125g is dissolved in NaIO 4 (0.12M) in 100ml of acetate buffer (pH4-6), react at 4°C in the dark for 24 hours, dialyze 3 times with distilled water, concentrate, and dry to obtain powdery formylated chitosan;

[0060] 2. Combination of TaqDNA polymerase with formylated polysaccharides:

[0061] Add 10 mg of powdery formylated chitosan to 3 ml of an aqueous solution of 50 mg / L fast TaqDNA polymerase (molecular weight 94 kDa) produced by Promega to form an oxidation mixture. , then based on the total weight of the reaction mixture, add 1% of BSA with a mass fraction of 10wt%, and block the reaction at 6° C. in the dark for 40 h;

[0062] The reaction mixture containing TaqDNA polymerase after the reaction was completed was dialyzed with a molecular weight cut-off of 70KD and dialyzed for 48h at 4°C with a storage buffe...

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Abstract

The invention provides hot-start Taq DNA (deoxyribonucleic acid) polymerase and a preparation method thereof. The hot-start Taq DNA polymerase is obtained by combining Taq DNA polymerase with aldehyde polysaccharide, the combination is achieved by reaction of an amino group of the Taq DNA polymerase with an aldehyde group of the aldehyde polysaccharide, and an amide bond can keep unbroken for more than five minutes at the temperature of 95 DEG C, so that polymerization activity of the Taq DNA polymerase is released; meanwhile, the polysaccharide capable of protecting the Taq DNA polymerase inhibits 3'-5' polymerization activity of the Taq DNA polymerase at the low temperature and is beneficial to improvement in PCR (polymerase chain reaction) efficiency as a protective agent of the Taq DNA polymerase. The hot-start Taq DNA polymerase prepared by the method can be widely applied to the fields of molecular biology, legal medical expert DNA detection and the like.

Description

technical field [0001] The invention relates to a DNA polymerase and a preparation method thereof, in particular to a hot-start Taq DNA polymerase and a preparation method thereof. Background technique [0002] PCR reaction (Polymerase Chain Reaction) is an in vitro nucleic acid amplification technology developed in the mid-1980s. Its purpose is to rapidly amplify DNA fragments in a short period of time, and it has outstanding advantages such as specificity, sensitivity, high yield, rapidity, simplicity, good repeatability, and ease of automation, making it rapidly recognized in the fields of molecular biology and medicine. Wide range of practical applications. It is regarded by many scientists as the most important technological breakthrough in the field of molecular biology in recent decades. [0003] PCR technology has set off a revolution in life sciences. It allows people to amplify DNA by 10% through a DNA polymerization reaction in a test tube in a few hours. 9 tim...

Claims

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Application Information

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IPC IPC(8): C12N9/12
CPCC12N9/1252C12Y207/07007
Inventor 刘未斌
Owner 刘未斌
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