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Protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as preparation method and application of protein-type PCR accelerant

A technology of deoxyuridine triphosphate and uridine triphosphate, which is applied in the field of genetic engineering and can solve the problems of inhibiting DNA polymerase activity, transversion-type base mutation, and reducing the effective length of DNA amplification and amplification yield and other problems, to achieve the effect of improving PCR amplification yield, good versatility, and increasing the effective length of DNA amplification

Inactive Publication Date: 2015-07-22
YUANJIAN BIOTECH SHANGHAI CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to thermal cycle-based PCR, high temperature will hydrolyze deamination of deoxycytidine triphosphate (dCTP), one of the PCR reaction substrates, to generate deoxyuridine triphosphate (dUTP), which will deoxycytidine triphosphate (dUTP) Incorporated into the synthesized DNA molecule by DNA polymerase, the uracil base incorporated into the DNA severely inhibits the activity of DNA polymerase, ultimately reducing the effective length of DNA amplification and amplification yield
On the other hand, the deoxyuridine base inserted into the DNA will also pair with guanine, resulting in a transversion-type base mutation
However, traditional PCR modifiers are powerless to deal with these problems

Method used

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  • Protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as preparation method and application of protein-type PCR accelerant
  • Protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as preparation method and application of protein-type PCR accelerant
  • Protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as preparation method and application of protein-type PCR accelerant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of embodiment 1 pyrococcus deoxyuridine triphosphate hydrolase

[0035] The first step is to design and synthesize the Pyrococcus deoxyuridine triphosphate hydrolase gene and insert it into the pET28 expression vector to construct the recombinant expression plasmid of the deoxyuridine triphosphate hydrolase. The N-terminus of the recombinant pyrococcus deoxyuridine triphosphate hydrolase has six consecutive histidine affinity purification tags derived from the pET28 vector, and is used for purification by immobilized nickel ion affinity chromatography.

[0036] The gene sequence of Pyrococcus deoxyuridine triphosphate hydrolase is shown in SEQ ID NO:1.

[0037] In the second step, the recombinant expression of pyrococcus deoxyuridine triphosphate hydrolase. The pyrococcus deoxyuridine triphosphate hydrolase recombinant expression plasmid is transformed into Escherichia coli expression host BL21 (DE3), and the pyrococcal deoxyuridine triphosphate hydrola...

Embodiment 2

[0042] Example 2 Application of Pyrococcus deoxyuridine triphosphate hydrolase as PCR accelerator

[0043] Purified Pyrococcus deoxyuridine triphosphate hydrolase was used as a PCR promoter to improve the PCR reaction catalyzed by Pfu DNA polymerase. The target gene for amplification is Escherichia coli nuclease IV gene, PCR reaction buffer: 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 0.1mg / mL BSA, 0.1% (v / v) Triton X-100, 2mM MgSO 4 ; Other components are 0.2mM dNTP, 0.3μM primer, 20ng Escherichia coli genomic DNA, 2.5 units of Pfu DNA polymerase. The agarose gel electrophoresis pictures of PCR amplification results are shown in image 3 , the results showed that Pyrococcus deoxyuridine triphosphate hydrolase increased the yield of PCR amplification by 2-3 times.

Embodiment 3

[0044] Example 3 The application of deoxyuridine triphosphate hydrolase from the Methanogenic Archaea Jannae as a PCR accelerator

[0045] According to the process of Example 1, the deoxyuridine triphosphate hydrolase of Jannai methanogenic archaea was prepared, the amino acid sequence of the protein (nitrogen terminal → carbon terminal) is shown in SEQ ID NO: 5, the gene encoding the enzyme protein The sequence is shown in SEQ ID NO:2. Purified deoxyuridine triphosphate hydrolase from the methanogenic archaea Jannai was used as a PCR promoter to improve the PCR reaction catalyzed by KOD DNA polymerase. The target gene for amplification is Escherichia coli endonuclease III gene, PCR reaction buffer: 20mM Tris-HCl (pH 8.8), 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 0.1mg / mL BSA, 0.1% (v / v) Triton X-100, 2mM MgSO 4 ; Other components are 0.2mM dNTP, 0.3μM primer, 10ng Escherichia coli genomic DNA, 2.5 units of KOD DNA polymerase. The agarose gel electrophoresis pictures of PCR amplif...

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a protein-type PCR (polymerase chain reaction) accelerant based on deoxyuridine triphosphate hydrolase as well as a preparation method and an application of the protein-type PCR accelerant. Deoxyuridine triphosphate hydrolase is subjected to enzymatic activity and thermal stability detection, so that specifically hydrolyzed dUTP (deoxyuridine triphosphate) and unhydrolyzed dCTP (deoxycytidine triphosphate), dTTP (deoxy-thymidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate) are screened out; at the same time, deoxyuridine triphosphate hydrolase with enzymatic activity half-life period not smaller than 30 minutes at 95 DEG C serves as the protein-type PCR accelerant. The protein-type PCR accelerant has the benefits that the PCR accelerant is the protein-type PCR accelerant, is good in universality, high in activity and good in thermal stability, can be used as accelerants of various commercialized high-fidelity DNA (deoxyribonucleic acid) polymerases, and can increase the PCR amplification yield by 2-6 times and improve the effective amplification length of DNA fragments.

Description

technical field [0001] The invention relates to a new PCR accelerator preparation and application technology, in particular to a protein-type PCR accelerator preparation and application technology based on deoxyuridine triphosphate hydrolase, which belongs to the technical field of genetic engineering. Background technique [0002] The emergence of PCR technology has made it possible to quickly prepare specific DNA molecules in vitro. It is currently widely used in various nucleic acid amplification and detection fields, and has greatly promoted the development of nucleic acid diagnostic technology, modern genetic engineering, and recombinant protein engineering. Since the amplification yield of PCR and the effective length of DNA amplification are affected by many factors, a variety of techniques to improve the performance of PCR have been developed. These techniques mainly include optimizing the components of the PCR reaction system, such as adding dimethyl sulfoxide (DMSO...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/14C12N15/70C12N15/10
CPCC12N9/14C12N15/10C12P19/34C12Y306/01023
Inventor 刘喜朋杜飞
Owner YUANJIAN BIOTECH SHANGHAI CO LTD
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