Method for preparing circular DNA in vitro

A circular and purposeful technology, applied in the field of in vitro preparation of circular DNA, can solve the problems of residual pollution process, complexity, high cost, etc., and achieve the effect of strong operability, simple process, and reduced biosafety risks

Pending Publication Date: 2022-03-25
SHENZHEN HUADA GENE INST
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0008] Aiming at the problems of residual pollution, complicated process and high cost in the method for preparing circular DNA in vitro, the present invention provides a method for preparing circular DNA in vitro

Method used

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  • Method for preparing circular DNA in vitro
  • Method for preparing circular DNA in vitro
  • Method for preparing circular DNA in vitro

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Embodiment 1

[0032] 1. Select the green fluorescent protein (GFP) gene and its promoter (CMV) as the target linear DNA fragment.

[0033] 2. According to the CMV-GFP sequence information, design the Gibson assembly junction fragment according to "target DNA upstream 25bp + BsaI recognition site sequence + target DNA downstream 25bp". They are: cacat, ctgag, tacac, cgcct, acccg, acaca, cagaa, accgt, accca, tgccg, tttta, ccaga, caccc, gtggg, aggtg, ccaaa, attgg, ctgag, gacgg, gaata. The designed Gibson assembly connection fragments are as follows:

[0034]

[0035]

[0036]

[0037] 3. Synthesize the junction fragments by Gibson assembly, dilute to 10 μmol / L with sterile water, mix 10 μL each of the forward F and reverse R fragments, and anneal on the PCR instrument according to the following procedure to form double-stranded fragments: 95 ° C, 10 min; 85°C, 1min; 75°C, 1min; 65°C, 1min; 55°C, 1min; 45°C, 1min; 35°C, 1min; 25°C, 1min. After annealing, the 20 groups of fragments we...

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Abstract

The invention discloses a method for preparing circular DNA (Deoxyribose Nucleic Acid) in vitro. The method comprises the following steps: designing a Gibson assembly connection fragment according to a target linear DNA fragment, introducing a recognition site sequence of restriction enzyme Bsa I, mixing the target linear DNA fragment and the Gibson assembly connection fragment, carrying out Gibson assembly, carrying out rolling circle amplification and purification by taking a Gibson assembly product as a template to obtain an RCA product, carrying out Bsa I enzyme digestion on the purified RCA product, and carrying out purification to obtain the RCA. Carrying out agarose gel electrophoresis after enzyme digestion, carrying out gel cutting to recover an enzyme digestion band consistent with the target linear DNA fragment in size, and finally cyclizing and purifying an enzyme digestion product by using T4 DNA ligase to obtain a cyclized product of the target linear DNA fragment. The method can replace traditional plasmids, only an incision enzyme recognition sequence with the length of 11 basic groups is additionally introduced except necessary elements, the molecular weight of circular DNA is reduced to the minimum, meanwhile, the biological safety risk is reduced, and in-vitro cyclization of linear DNA fragments is achieved.

Description

technical field [0001] The invention belongs to the technical field of DNA synthesis and relates to a method for preparing circular DNA in vitro. Background technique [0002] As a molecular biology tool, plasmids are widely used in gene expression, RNA transcription, gene regulation and other fields. In the process of plasmid application, plasmid construction, Escherichia coli competent cell transformation experiment, yeast cell transformation experiment, animal cell transfection experiment, etc. are involved. Efficiency is especially critical in eukaryotic cell transfection experiments. The efficiency of plasmid entry into eukaryotic cells directly determines whether the experimental effect meets the requirements. The transfection efficiency of eukaryotic cells is related to many factors, among which the size of the plasmid is one of the key factors, and smaller plasmids are easier to transfect into cells under the same conditions. Therefore, reducing the size of the pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12P19/34
CPCC12N15/10C12P19/34
Inventor 高杨刘超高峰顾颖沈玥
Owner SHENZHEN HUADA GENE INST
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