A kind of method for preparing hot start taq enzyme

Abstract

A method for preparing hot start Taq polymerase comprises mixing the Taq polymerase with treatment solution to obtain the hot start Taq polymerase, wherein the treatment solution is heparin solution, heparin sodium solution or heparin potassium solution.

Description

technical field

[0001] The invention relates to the technical field of preparation methods of Taq enzymes, in particular to a method for preparing hot-start Taq enzymes. Background technique

[0002] Polymerase chain reaction, Polymerase chain reaction, referred to as PCR, is a molecular biology technique used to amplify specific DNA fragments. This method can be carried out in vitro without relying on organisms such as Escherichia coli or yeast. The technology of PCR is widely used in medical and biological laboratories, such as for determining whether a certain genetic disease will appear in a sample, diagnosis of infectious diseases, gene replication, and paternity testing.

[0003] PCR is used to amplify a small known segment of DNA, which may be a single gene, or just a part of a gene. Unlike living organisms, PCR can only replicate very short DNA fragments, usually no more than 10kbp. The currently applied PCR reaction requires several basic components: DNA template ...

Examples

Embodiment 1

[0025] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0026] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0027] Its specific operation method is:

[0028] Heparin, heparin sodium or heparin potassium is dissolved in deionized water until the final concentration of the solution is above 0.0001U / ml, and Taq enzyme is added into the solution. The reaction product is purified by dialysis, column or ultrafiltration to obtain the hot start Taq enzyme.

Embodiment 2

[0030] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0031] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0032] Its specific operation method is:

[0033] Add heparin, heparin sodium or heparin potassium to the PCR reaction solution, so that the final concentration of heparin, heparin sodium or heparin potassium is above 0.0001U / ml, after the Taq enzyme in the PCR reaction solution is mixed with heparin, heparin sodium or heparin potassium solution , that is, hot-start Taq enzyme.

Embodiment 3

[0035] Apply the hot-start Taq enzyme of the present invention to PCR, take Foregene PCR Easy as an example, use template 2.5ul (2.5ng), 2×PCR Easy Mix25ul, 10uM Primer11ul, 10uM Primer21ul, add ddH 2 From 0 to 50ul, react according to the following reaction conditions: heat denaturation at 94°C for 3min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 1min, a total of 25-40 cycles, and finally hold at 72°C for 5min. At the same time, untreated DNA polymerase (5U / ul) was used as a parallel control. After completing the PCR reaction, take 5ul of the reaction product and test it by agar gel electrophoresis. The results are shown in figure 1 ,From figure 1 It can be seen that the amplification intensity of the hot-start high-temperature-resistant DNA polymerase is significantly increased. In the figure, 1, 2, 3: ordinary Taq enzyme amplification, 1a, 2a, 3a: hot start Taq enzyme amplification, M: 100bp-1KB Marker, 1-1a: ZmIVR (225bp), 2-2a: Bn...

technical field

[0001] The invention relates to the technical field of preparation methods of Taq enzymes, in particular to a method for preparing hot-start Taq enzymes. Background technique

[0002] Polymerase chain reaction, Polymerase chain reaction, referred to as PCR, is a molecular biology technique used to amplify specific DNA fragments. This method can be carried out in vitro without relying on organisms such as Escherichia coli or yeast. The technology of PCR is widely used in medical and biological laboratories, such as for determining whether a certain genetic disease will appear in a sample, diagnosis of infectious diseases, gene replication, and paternity testing.

[0003] PCR is used to amplify a small known segment of DNA, which may be a single gene, or just a part of a gene. Unlike living organisms, PCR can only replicate very short DNA fragments, usually no more than 10kbp. The currently applied PCR reaction requires several basic components: DNA template ...

Examples

Embodiment 1

[0025] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0026] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0027] Its specific operation method is:

[0028] Heparin, heparin sodium or heparin potassium is dissolved in deionized water until the final concentration of the solution is above 0.0001U / ml, and Taq enzyme is added into the solution. The reaction product is purified by dialysis, column or ultrafiltration to obtain the hot start Taq enzyme.

Embodiment 2

[0030] The invention relates to a method for preparing a hot-start Taq enzyme. The Taq enzyme is mixed with a treatment solution to obtain the hot-start Taq enzyme.

[0031] The treatment solution is heparin solution, heparin sodium solution or heparin potassium solution, and the concentration of heparin, heparin sodium or heparin potassium in the mixture of Taq enzyme and treatment solution is above 0.0001 U / ml.

[0032] Its specific operation method is:

[0033] Add heparin, heparin sodium or heparin potassium to the PCR reaction solution, so that the final concentration of heparin, heparin sodium or heparin potassium is above 0.0001U / ml, after the Taq enzyme in the PCR reaction solution is mixed with heparin, heparin sodium or heparin potassium solution , that is, hot-start Taq enzyme.

Embodiment 3

[0035] Apply the hot-start Taq enzyme of the present invention to PCR, take Foregene PCR Easy as an example, use template 2.5ul (2.5ng), 2×PCR Easy Mix25ul, 10uM Primer11ul, 10uM Primer21ul, add ddH 2 From 0 to 50ul, react according to the following reaction conditions: heat denaturation at 94°C for 3min, denaturation at 94°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 1min, a total of 25-40 cycles, and finally hold at 72°C for 5min. At the same time, untreated DNA polymerase (5U / ul) was used as a parallel control. After completing the PCR reaction, take 5ul of the reaction product and test it by agar gel electrophoresis. The results are shown in figure 1 ,From figure 1 It can be seen that the amplification intensity of the hot-start high-temperature-resistant DNA polymerase is significantly increased. In the figure, 1, 2, 3: ordinary Taq enzyme amplification, 1a, 2a, 3a: hot start Taq enzyme amplification, M: 100bp-1KB Marker, 1-1a: ZmIVR (225bp), 2-2a: Bn...

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