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Non-homology, multi-long-fragment, high-efficiency and zero-background assembly method

A technology of homology and long fragments, applied in the field of bioengineering, can solve the problems of no homology of gene fragments, inability to link 6 fragments into a circle, and many steps

Pending Publication Date: 2021-06-08
通用生物(安徽)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, due to the lack of homology between gene fragments, it is usually impossible to connect six fragments in sequence at one time to form a ring. Conventional gene synthesis methods are completed in stages and in multiple steps, requiring more steps for the round manager

Method used

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  • Non-homology, multi-long-fragment, high-efficiency and zero-background assembly method
  • Non-homology, multi-long-fragment, high-efficiency and zero-background assembly method
  • Non-homology, multi-long-fragment, high-efficiency and zero-background assembly method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] see Figure 1-3 As shown, no homology, multiple long fragments, efficient zero-background group method, including the following steps:

[0029] The first step is to add a B fragment Yesatori at the only place of the restriction endonuclease NotI site of the entire circularization plasmid, and at the same time ensure that there are exactly two NotI restriction sites on the left and right sides of the B fragment, so that the subsequent NotI single-cut self-ligation Remove the B fragment;

[0030] The second step, design two Primer 1 / 2 primers between fragments A and B without homology, design two Primer 3 / 4 primers between fragments B and C without homology, and use the primers as two fragments The bridges connected between them, and the design of other fragments are analogized in turn;

[0031] The third step is to combine all the fragments with a total of 7 ABCDEFGs. These fragments can be obtained by PCR amplification or plasmid digestion, and a total of 7 pairs of p...

Embodiment 2

[0042] No homology, multiple long fragments, high-efficiency zero-background group method, including the following steps:

[0043] The first step is to add a B fragment Yesatori at the only place of the restriction endonuclease NotI site of the entire circularization plasmid, and at the same time ensure that there are exactly two NotI restriction sites on the left and right sides of the B fragment, so that the subsequent NotI single-cut self-ligation Remove the B fragment;

[0044] The second step, design two Primer 1 / 2 primers between fragments A and B without homology, design two Primer 3 / 4 primers between fragments B and C without homology, and use the primers as two fragments The bridges connected between them, and the design of other fragments are analogized in turn;

[0045] The third step is to combine all the fragments with a total of 7 ABCDEFGs. These fragments can be obtained by PCR amplification or plasmid digestion, and a total of 7 pairs of primer1-14 with a pair...

Embodiment 3

[0056] No homology, multiple long fragments, high-efficiency zero-background group method, including the following steps:

[0057] The first step is to add a B fragment Yesatori at the only place of the restriction endonuclease NotI site of the entire circularization plasmid, and at the same time ensure that there are exactly two NotI restriction sites on the left and right sides of the B fragment, so that the subsequent NotI single-cut self-ligation Remove the B fragment;

[0058] The second step, design two Primer 1 / 2 primers between fragments A and B without homology, design two Primer 3 / 4 primers between fragments B and C without homology, and use the primers as two fragments The bridges connected between them, and the design of other fragments are analogized in turn;

[0059] The third step is to combine all the fragments with a total of 7 ABCDEFGs. These fragments can be obtained by PCR amplification or plasmid digestion, and a total of 7 pairs of primer1-14 with a pair...

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Abstract

The invention discloses a non-homology, multi-long-fragment, high-efficiency and zero-background assembly method. The method comprises the following steps: firstly, adding a B fragment Yeast ori at the unique position of the Not I site of restriction enzyme of whole cyclization plasmid, and simultaneously ensuring that the left and right of the B fragment have just two Not I enzyme digestion sites; designing two Primer 1 / 2 primers between an A fragment and a B fragment which are not homologous for example, designing two Primer 3 / 4 primers between the B fragment and a C fragment which are not homologous, and using the primers as bridges for connecting the two corresponding segments; transferring all 7 fragments ABCDEFG and 7 pairs of primer1-14 at the joints of the corresponding fragments into competent yeast cells for shaking culture for two days; extracting yeast plasmid cultured for two days; and finally, performing amplification and detection. The invention provides the non-homology, multi-long-fragment, high-efficiency and zero-background assembly method, the zero-background cloning is completed through yeast assembly and Escherichia coli screening, no empty vector is produced, and the method saves a large amount of time compared with a traditional cloning mode.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an assembly method with no homology, multiple long fragments, and high efficiency and zero background. Background technique [0002] At present, due to the lack of homology between gene fragments, it is usually impossible to connect six fragments in sequence at one time to form a ring. Conventional gene synthesis methods are completed in stages and in multiple steps, requiring more steps for the round manager . Contents of the invention [0003] The purpose of the present invention is to provide an assembly method with no homology, multiple long fragments, high efficiency and zero background. [0004] The technical problem to be solved in the present invention is: [0005] In the prior art, due to the lack of homology between the gene fragments, it is usually impossible to connect six fragments in order to form a ring at one time. The conventional gene synth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/70C12N15/66
CPCC12N15/81C12N15/70C12N15/66C12N2800/80
Inventor 喻明军崔康乐王维坤骆晓雯纪世春
Owner 通用生物(安徽)股份有限公司
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