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Method for improving rolling circle amplification efficiency with integration host factor

A technology of integrating host factors and rolling circle amplification, which is applied in the field of improving the efficiency of rolling circle amplification by integrating host factors, can solve the problems of slow response speed and low sensitivity of RCA technology, and achieve the effect of improving efficiency and enhancing sensitivity

Inactive Publication Date: 2016-03-30
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a method for improving the efficiency of rolling circle amplification by integrating host factors, which largely solves the problems of slow response speed and low sensitivity of RCA technology

Method used

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  • Method for improving rolling circle amplification efficiency with integration host factor
  • Method for improving rolling circle amplification efficiency with integration host factor
  • Method for improving rolling circle amplification efficiency with integration host factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Preparation of IHF-β

[0021] ① Insert the IHF-β gene derived from Escherichia coli into the pET-22b expression vector, and through ligation transformation and verification by colony PCR, enzyme digestion and sequencing, the recombinant expression vector pET-22b-IHF-β can be obtained;

[0022] ②Transform the constructed recombinant vector pET-22b-IHF-β into Escherichia coli BL21, take the positive strain containing the recombinant plasmid and add it to LB liquid medium for overnight cultivation, and then inoculate the bacterial liquid into 50ml of LB culture medium at a ratio of 1%. , placed in a shaker at 37°C until the OD value is 0.6-0.8; add IPTG, put the bacterial solution in a shaker at 37°C to induce expression for 3 hours, and the shaker speed is 160rpm;

[0023] ③Centrifuge the bacterial liquid after induced expression to obtain the bacterial cells, lyse and release the protein. SDS-PAGE electrophoresis to detect protein expression, electrophoresis results ...

Embodiment 2

[0028] Example 2: Activity detection of IHF-β

[0029] Configure a 10 μl reaction system according to Table 1 below:

[0030] Table 1

[0031]

[0032] In the reaction solution, different concentrations of the IHF-β protein obtained in Example 1 were added and allowed to stand at room temperature for 10 minutes. Afterwards, 5 μl of the reaction solution was taken for 1% agarose gel electrophoresis (105V, 25min), then stained in 0.5μg / mL ethidium bromide staining solution for 15min, and observed with a UV transilluminator. For specific experimental results, see figure 2 .

[0033] figure 2 Each lane indicates: M is 1kbDNALadderMarker; 1, 2, 3, 4, 5, 6, 7, 8 indicate that 0μl, 0.5μl, 1μl, 2μl, 4μl, 6μl, 8μl and 9μl of IHF-β protein were added to the reaction solution .

[0034] Depend on figure 2 It can be seen that as the amount of IHF-β protein added increases, the DNA bands gradually move up. It shows that the purified protein IHF-β is active and can combine with...

Embodiment 3

[0035] Example 3: Effect of IHF-β on the efficiency of rolling circle amplification (using the recombinant plasmid pUC19-hupB as a template)

[0036] 1. Construction of recombinant plasmid pUC19-hupB: Insert the hupB gene from Escherichia coli into the pUC19 plasmid, transform by ligation and use colony PCR, enzyme digestion identification and sequencing verification to obtain the recombinant plasmid pUC19-hupB;

[0037] 2. Denaturation: Using the recombinant plasmid pUC19-hupB as a template, add 1 ng of pUC19-hupB plasmid DNA to 5 μl of sample buffer, mix well, heat at 95°C for 3 minutes, and then cool to room temperature or 4°C to obtain a denatured sample solution;

[0038]3. Incubation: Add 0.2 μl Phi29 DNA polymerase to 5 μl reaction buffer, mix well, and place on ice to obtain a premixed enzyme solution; add 5 μl premixed enzyme solution to the denatured sample solution, mix well to obtain an RCA reaction solution; add IHF-β protein 0ng, 2.04ng, 5.1ng, 15.3ng, 25.5ng and...

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Abstract

The invention provides a method for improving rolling circle amplification efficiency with an integration host factor. The method is characterized in that RCA reaction liquid is prepared, IHF-beta protein is added into the RCA reaction liquid, and the concentration of the IHF-beta protein in the RCA reaction liquid is 0.204-4.08 nanogram / microliter. The rolling circle amplification efficiency can be substantially improved, the sensitivity of rolling circle amplification is enhanced, and the blank of the method and application fields of improving RCA amplification efficiency with heat-resistant DNA double-strand binding protein is filled up.

Description

【Technical field】 [0001] The invention relates to a method for improving rolling circle amplification efficiency by utilizing integrated host factors. 【Background technique】 [0002] Rolling circle amplification (Rolling circle amplification, RCA) is an in vitro isothermal nucleic acid amplification method established in 1998. This method draws on the rolling circle replication method of DNA molecules of circular pathogenic microorganisms in nature, and can realize high-speed circular DNA replication. It has the characteristics of high sensitivity, strong specificity, high throughput, easy operation and mild reaction conditions. Single nucleotide polymorphism analysis, DNA chips, protein chips, nucleic acid sequencing, etc. have been widely used. [0003] The mechanism of rolling circle amplification mainly lies in the use of polymerases with strong processivity and strand displacement activity, such as Phi29 DNA polymerase, BstDNA polymerase, etc. In the rolling circle am...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 戚智青侯丹齐晓雪唐黎
Owner HUAQIAO UNIVERSITY
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