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Method for replicating or amplifying circular DNA

A circular and linear technology, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem that template circular DNA cannot be multiplied

Pending Publication Date: 2019-11-12
ORICIRO GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, when trying to use the E. coli small chromosome replication system to generate circular amplification products, there is a problem that the template circular DNA cannot even be multiplied

Method used

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  • Method for replicating or amplifying circular DNA
  • Method for replicating or amplifying circular DNA
  • Method for replicating or amplifying circular DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0267] Example 1: Replication of circular DNA accompanied by DNA multimer inhibition using terminator sequence ter and Tus protein

[0268]

[0269] The circular DNA used as a template is prepared as follows. Insert the oriC fragment into the M13mp18 plasmid vector to make an 8.0kb circular DNA. Insert a DNA fragment ((5’-ACTT) containing two opposing ter sequences (underlined) into the region on the opposite side of oriC in the 8.0kb circular DNA TAGTTACAACATAC TTATT-N 176 -AATAA GTATGTTGTAACTA AAGT-3' (SEQ ID NO: 26)), made into 8.0kb circular DNA with ter inserted ( image 3 (A)). The 8.0 kb circular DNA inserted with ter was used as the template DNA, and the 8.0 kb circular DNA was used as the control DNA without ter sequence.

[0270] Tus is produced and prepared from the E. coli expression strain of Tus through a process including affinity column chromatography and gel filtration column chromatography.

[0271] A reaction solution having the composition shown in Table 1 an...

Embodiment 2

[0291] Example 2: Circular DNA with DNA multimer inhibition using site-specific recombination sequences dif and XerCD copy

[0292]

[0293] The dif sequence (SEQ ID NO: 22) is contained in the region on the opposite side of oriC of the circular DNA, and a 12 kb circular DNA with dif inserted as a template ( Figure 4 (A)). Specifically, using Escherichia coli expressing the recombinant proteome of lambda phage, a cassette containing oriC and kanamycin resistance genes and the 4.2 kb region and downstream of the dif upstream side of the chromosome of Escherichia coli were prepared by intracellular recombination reaction. A circular DNA with a target length of 6.0 kb on the side.

[0294] As a control DNA without dif sequence, the 8.0 kb circular DNA described in Example 1 was used.

[0295] XerCD is purified and prepared from the E. coli co-expression strain of XerC and XerD through a process including ammonium sulfate precipitation and affinity column chromatography.

[0296] The ...

Embodiment 3

[0301] Example 3: The influence of the position of ter sequence or dif sequence in circular DNA In Examples 1 and 2, the template DNA was prepared in such a way that the ter sequence or the dif sequence was located in the region on the opposite side of oriC in the circular DNA. In Example 3, a replication / amplification reaction was performed on circular DNA in which the ter sequence or the dif sequence was arranged near or adjacent to oriC.

[0302]

[0303] In order to construct a 15kb circular DNA, the E. coli genome was used as a template to amplify and prepare a 15kb DNA fragment without oriC.

[0304] As the circular DNA in which the ter sequence was arranged in the vicinity of oriC, a 15kb-ori-ter circular DNA was prepared as follows. The ori-ter cassette was connected to the 15 kb DNA fragment described above and circularized to produce ( Figure 5 ). The sequence of the ori-ter box (0.38 kb) is as follows, with ter sequences (underlined) outward at both ends of the oriC b...

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Abstract

The present invention provides a method that enables replication or amplification of circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for inhibiting generation of a DNA multimer which is a by-product, during replication or amplification of circular DNA having a replication initiation sequence (origin of chromosome (oriC)) by using the following enzymes: (1) first enzymes which catalyze replication of the circular DNA; (2) second enzymes which catalyze an Okazaki fragment linking reaction, to synthesize two sister circular DNA molecules forming a catenane; and (3) third enzymes which catalyze a separation reaction of the two sister circular DNA molecules. Further provided is a method that comprises introduction of oriC into circular DNA by using a transposon.

Description

Technical field [0001] The present invention relates to a method for replication or amplification of circular DNA. In more detail, it relates to a method that can efficiently replicate or amplify circular DNA in a cell-free system. The present invention also relates to nucleic acids that can be utilized as a functional cassette for preparing circular DNA. Background technique [0002] The DNA cloning technology, which has become the basis for the development of biotechnology, is a method in which circular DNA prepared by cutting and pasting DNA fragments is amplified as a plasmid in cells such as E. coli. When using cell-based DNA cloning technology to amplify circular DNA, cumbersome steps such as cell culture and extraction and purification of amplified products are required. In addition, in order to perform DNA cloning using cells, it is necessary to produce genetically recombined organisms, and therefore there are restrictions on the experimental environment. [0003] As a m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/11
CPCC12N15/10C12Q1/6844C12Q2521/513C12Q2521/519C12Q2531/125C12Q1/686C12Q2521/507C12Q2522/101C12Q1/6806C12P19/34
Inventor 末次正幸奈良圣亚
Owner ORICIRO GENOMICS INC
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