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A one-step, seamless, non-homologous, multi-segment gene splicing transformation method and its kit

A gene splicing and genetic modification technology, applied in the field of molecular biology, can solve the problems of DNA tail sequence modification, failure to realize standardization and reuse of DNA fragments, low efficiency, etc., and achieve the effect of high-efficiency transformation reaction

Active Publication Date: 2019-10-29
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the standardization and reuse of DNA fragments are still not realized, and the sequence modification at the end of DNA still exists
In addition, the multi-fragment DNA connection is still not reliable enough, the efficiency of assembly of more than 4 fragments is low, and the number of transformants is very rare

Method used

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  • A one-step, seamless, non-homologous, multi-segment gene splicing transformation method and its kit
  • A one-step, seamless, non-homologous, multi-segment gene splicing transformation method and its kit
  • A one-step, seamless, non-homologous, multi-segment gene splicing transformation method and its kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Circular DNA Molecular Splicing

[0079] Table 5 shows the DNA sequences used and the corresponding fragment sizes. During the primer design process, the Tm values ​​of all the half-bridge primers and the Tm values ​​of the amplification primers were 60 degrees. The designed bridging primers were all in the range of 30-60bp in length. The sequences of the bridging primers and amplification primers used are shown in Table 6.

[0080] Table 5 Fragments used in circular DNA molecule splicing

[0081]

[0082] Table 6 The bridging primers and amplification primers used in the splicing of circular DNA molecules

[0083]

[0084]

[0085] The specific components and concentrations used in the bridging method, the thermocycling program are listed in Table 7, and the buffer components are listed in Table 8. The bridging method completes the one-time splicing of 8 target fragments in one step, and at the same time obtains a high concentration of ligation pr...

Embodiment 2

[0094] Example 2 Linear DNA Molecular Splicing

[0095] Table 9 shows the DNA sequences used and the corresponding fragment sizes.

[0096] Table 9 Fragments used in linear DNA molecular splicing

[0097]

[0098] During the primer design process, the Tm values ​​of all the half-bridge primers and the Tm values ​​of the amplification primers were 60 degrees. The designed bridging primers were all in the range of 30-60bp in length. Both the bridging primer sequence and the amplification primer sequence used in the bridging method are listed in Table 10.

[0099] Table 10 Bridging primers and amplification primers used in linear DNA molecule splicing

[0100]

[0101]For conventional splicing of two fragments to form fusion fragments, fusion PCR is used in most cases. However, the fusion PCR method has low repeatability and poor stability. And for different DNA fragments, it is necessary to explore the parameters to obtain a better fusion reaction. However, if the br...

Embodiment 3

[0106] Example 3 One-step point mutation of plasmid DNA molecule

[0107] The plasmid DNA used is pZS-ssMBP-LKGFP (SEQ ID NO: 2), and the size of the plasmid is 5967bp. The resulting high-concentration mutated circular DNA ligation product can be directly used for transformation to obtain a large number of transformants.

[0108] The bridging primer sequences and amplification primer sequences used in the bridging method are listed in Table 11.

[0109] Table 11

[0110]

[0111] In principle, point mutations using the bridging method are equivalent to the last two steps in the full bridging method. The introduced amplification primers with mutations completely amplify the entire plasmid, and the linear amplification products complete the circularization reaction in the thermal cycle under the guidance of the bridging primers to obtain high-concentration target products.

[0112] For conventional point mutations, in most cases, fusion PCR reaction needs to be carried out...

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Abstract

The invention discloses a one-step seamless, non-homologous and multi-fragment gene splicing method. The steps of DNA fragment phosphorylation, DNA fragment bridging and exponential type amplification of a ligation product are finished in one pipe through thermal cycling similar to PCR. The method has the following advantages: (1) ligation reaction is finished by one step, so that high cost caused by using a phosphorylation primer is avoided; (2) the concentration of DNA, the concentration of a bridging primer and components and a proportion of a buffer solution are optimized, and the ligation reaction with high reliability and good repeatability is reached; (3) DNA fragments are seamlessly ligated with one another without introduction of excess nucleotide; (4) non-DNA sequence dependency is realized; (5) the multi-fragment gene splicing ability is efficient; (6) the ligation product is amplified while ligation, so that the concentration of a product is greatly increased, and efficient conversion and subsequent experiment needs are guaranteed; and (7) the cost is low compared with that of the conventional restriction enzyme ligation method.

Description

technical field [0001] The invention relates to the technical field of molecular biology, more specifically, to a one-step, seamless, non-homologous, multi-segment gene splicing and transformation method and a kit thereof. Background technique [0002] DNA splicing technology is one of the most important technical methods in current biological research, and most of molecular biology and cell biology are based on this technology. However, since Cohen and other researchers recombined the first DNA molecule 40 years ago, the mainstream DNA manipulation method is still based on the original enzyme-cut ligation method. There are 6 bp nucleotide scars left in the generated DNA products; at the same time, this traditional method cannot meet the complex and diverse application requirements of today. For example, multi-fragment ligation in metabolic engineering, standardization and reuse of DNA fragments in synthetic biology, and efficient and reliable splicing of DNA fragments of d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1027C12Q2521/101C12Q2521/501
Inventor 陆勇军阿迪亚
Owner SUN YAT SEN UNIV
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