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Method for improving specificity of multi-primer RCA (Rolling Circle Amplification)

A multi-primer, specific technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of high price, cumbersome steps, time-consuming and labor-intensive, etc., to fill non-specific amplification tails, improve specificity the effect of reducing non-specific amplification

Inactive Publication Date: 2018-02-13
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this document does not clearly indicate the amount of protein used. At the same time, this method needs to purify the active TthSSB protein first. The steps are cumbersome, time-consuming, expensive, and not conducive to transportation and storage. It is relatively difficult in practical applications.

Method used

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  • Method for improving specificity of multi-primer RCA (Rolling Circle Amplification)
  • Method for improving specificity of multi-primer RCA (Rolling Circle Amplification)
  • Method for improving specificity of multi-primer RCA (Rolling Circle Amplification)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: Improved multi-primer RCA system

[0019] A method for improving the specificity of multi-primer RCA, comprising the following steps:

[0020] (1) PEG8000 (polyethylene glycol 8000) solution with a concentration of 0.1 mg / ml is prepared for subsequent use;

[0021] (2) Denaturation: Using pUC19-hupB recombinant plasmid DNA as a template, in 5ul ddH 2 Add 1ng of pUC19-hupB recombinant plasmid DNA to O, mix well, heat at 95°C for 3min, then cool to room temperature or 4°C to obtain denatured sample solution; wherein, the gene sequence of recombinant plasmid pUC19-hupB is as shown in SEQ ID NO in the sequence table : 2 shown.

[0022] (3) Cultivation: Mix 2ul of 2.5mM dNTP, 2ul of 10×BSA, 1ul of phi29DNA polymerase buffer, and 0.2ul of the Phi29 DNA enzyme mixture in the TempliPhiTM DNA Sequencing Template Amplification Kit (the enzyme mixture includes Phi29 DNA polymerase Enzyme and primer hexamer), mix well, place on ice to obtain premixed enzyme solutio...

Embodiment 2

[0031] Example 2: Effect of PEG8000 on improving the specificity of multi-primer RCA (using the recombinant plasmid pUC19-hupB as a template)

[0032] A method for improving the specificity of multi-primer RCA, comprising the following steps:

[0033]1. Construction of recombinant plasmid pUC19-hupB: insert the hupB gene from Escherichia coli into the pUC19 plasmid, and obtain the recombinant plasmid pUC19-hupB through colony PCR, enzyme digestion identification and sequencing verification; among them, the hupB gene of Escherichia coli The sequence is shown as SEQ ID NO: 1 in the sequence listing.

[0034] 2. Denaturation: Using pUC19-hupB recombinant plasmid DNA as a template, in 5ul ddH 2 Add 1ng pUC19-hupB recombinant plasmid DNA to O, mix well, heat at 95°C for 3min, then cool to room temperature or 4°C to obtain denatured sample solution;

[0035] 3. Cultivation: Mix 2ul of 2.5mM dNTP, 2ul of 10×BSA, 1ul of phi29DNA polymerase buffer, and 0.2ul of the Phi29 DNA enzyme m...

Embodiment 3

[0042] Example 3: Identification of single enzyme digestion to improve the quality of multi-primer RCA products (using the recombinant plasmid pUC19-hupB as a template)

[0043] A method for improving the specificity of multi-primer RCA, comprising the following steps:

[0044] 1. Construction of the recombinant plasmid pUC19-hupB: Insert the hupB gene from Escherichia coli into the pUC19 plasmid, and obtain the recombinant plasmid pUC19-hupB through colony PCR, enzyme digestion identification and sequencing verification;

[0045] 2. Denaturation: Using pUC19-hupB recombinant plasmid DNA as a template, in 5ul ddH 2 Add 1ng pUC19-hupB recombinant plasmid DNA to O, mix well, heat at 95°C for 3min, then cool to room temperature or 4°C to obtain denatured sample solution;

[0046] 3. Cultivate: Mix 2ul of 2.5mM dNTP, 2ul of 10×BSA, 1ul of phi29DNA polymerase buffer, and 0.2ul of Phi29 DNA enzyme mixture in the TempliPhi™DNA sequencing template amplification kit (the enzyme mixtur...

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Abstract

The invention discloses a method for improving specificity of multi-primer RCA (Rolling Circle Amplification). The content of each component in a commercial multi-primer RCA kit TempliPhi (Amersham Biosciences) is improved, and the components are replaced with 2.5mMdNTP, 10*BSA, phi29DNA polymerase buffer which are easily purchasable in the market and low in price. Moreover, polyethylene glycol (PEG, 8000) with a proper concentration is added into the multi-primer RCA reaction process, so that the specificity of the multi-primer RCA can be obviously improved, and a tailing phenomenon producedby non-specific amplification which is always difficultly reduced in the multi-primer RCA reaction is filled. Therefore, the commercially available multi-primer RCA kit is obviously optimized.

Description

technical field [0001] The invention is based on the improvement of the commercial multi-primer RCA kit, and relates to a method for improving the specificity of the improved multi-primer RCA. Background technique [0002] Rolling circle amplification (multi-primer RCA) is an in vitro isothermal nucleic acid amplification method established in 1998. The method draws on the rolling circle replication method of DNA molecules of circular pathogenic microorganisms to realize high-speed circular DNA replication. As an efficient DNA amplification tool, RCA has many advantages. Such as low equipment requirements, simple operation, detection of circular DNA components in all infected bodies, and no need to know any gene sequence information. At present, RCA technology has been widely used in DNA sequencing, protein expression, biosensor, diagnosis, drug discovery and nanotechnology. [0003] Rolling circle amplification mainly utilizes DNA polymerases with strong stability and st...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2531/125C12Q2527/101C12Q2521/101
Inventor 张云威戚智青刁勇
Owner HUAQIAO UNIVERSITY
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