Gibson assembly carrier, preparation method therefor and applications thereof

A carrier and application technology, applied in the field of DNA synthesis, can solve the problems of cumbersome cloning process and instability, and achieve the effect of simple design and efficient assembly results

Inactive Publication Date: 2014-02-19
SHENZHEN HUADA GENE INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of cloning the spliced ​​DNA fragments into the vector, the homology region between the DNA fragment and the vector must be redesigned every time. Due to the difference between the DNA fragment and the vector, the properties of the homology region are very different, making The cloning process becomes cumbersome and highly unstable

Method used

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  • Gibson assembly carrier, preparation method therefor and applications thereof
  • Gibson assembly carrier, preparation method therefor and applications thereof
  • Gibson assembly carrier, preparation method therefor and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 - Preparation of vectors pSBGAA and pSBGAK

[0057] The specific implementation is as follows:

[0058] 1) Use the vectors pSB1A3 and pSB1K3 as templates for PCR amplification:

[0059] The reaction system includes: Ex Taq (5U / μL) 0.25μL, 10×PCR buffer 2.5μL, primers pSB-F, pSB-R 1μL each, dNTP (each 2.5mM) 2μL, DNA template 1μL, ddH 2 O17.25 μL.

[0060] Reaction program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 5 min. The PCR product was recovered with a Qiagen PCR purification kit, and the method was referred to its instruction manual.

[0061] 2) BamHI digestion:

[0062] Reaction procedure: BamHI (15U / μL) 1 μL, 10×K buffer 2 μL, recovered PCR product DNA 1 μg, add ddH2O to make up 20 μL. Enzyme digestion at 30°C for 3h. The digested product was recovered using Qiagen PCR purification kit, and the method was referred to its instruct...

Embodiment 2

[0073] Example 2 - Assembly of long yeast DNA fragments using the vector pSBGAA

[0074] 1) Sequence design:

[0075]Select the DNA fragment of yeast chromosome 2, numbered yeast_chr02_3_22.A2, with a length of about 6.8kb, add the overlapping region R and overlapping region L of the vector to both ends of the DNA fragment, and then divide the DNA fragment into three fragments of about 2kb, named respectively They are yeast_chr02_3_22.A2.F1, yeast_chr02_3_22.A2.F2, yeast_chr02_3_22.A2.F3, with a 40bp homologous region in the middle. Add an XhoI restriction site at the end of the fragment and send it to Gene Synthesis Company for synthesis.

[0076] 2) Assembly of DNA fragments and vector preparation:

[0077] The synthesized DNA fragment and the constructed vector were enzymatically cut into linear DNA fragments respectively.

[0078] Vector digestion: BamHI (15U / μL) 1μL, 10×K buffer 2μL, pSBGAA 1μg, add ddH 2 Make up 20 μL with O; digest at 30°C for 3 hours.

[0079] Dig...

Embodiment 3

[0092] Example 3 - Assembly of long yeast DNA fragments using vector pSBGAK

[0093] 1) Sequence design:

[0094] Select the DNA fragment of yeast chromosome 2, numbered yeast_chr02_3_22.A5, with a length of about 11kb, and add the overlapping region R and overlapping region L of the vector to both ends of the DNA fragment, and then divide the DNA fragment into five fragments of about 2kb, named respectively Yeast_chr02_3_22.A5.F1, yeast_chr02_3_22.A5.F2, yeast_chr02_3_22.A5.F3, yeast_chr02_3_22.A5.F4, yeast_chr02_3_22.A5.F5 have a homologous region of 40 bp in the middle. Add an XhoI restriction site at the end of the fragment and send it to Gene Synthesis Company for synthesis.

[0095] 2) Assembly of DNA fragments and vector preparation:

[0096] The synthesized DNA fragment and the constructed vector were enzymatically cut into linear DNA fragments respectively.

[0097] Vector digestion: BamHI (15U / μL) 1μL, 10×K buffer 2μL, pSBGAK 1μg, add ddH 2 Make up 20 μL with O; ...

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Abstract

The invention relates to the DNA synthesis field, and concretely relates to large fragment assembly of DNA and especially a carrier used for DNA fragment assembly. The invention provides a Gibson assembly carrier, a preparation method therefor and applications thereof.

Description

technical field [0001] The invention relates to the field of DNA synthesis, more specifically to splicing of large DNA fragments, in particular to a carrier for DNA fragment assembly. Background technique [0002] With the development of DNA synthesis technology, it has reached the level of simple, fast and efficient synthesis of DNA fragments at this stage. play an increasingly important role. Due to the limitations of oligonucleotide synthesis length, precision and cost, it is necessary to gradually assemble short fragments of oligonucleotides or double-stranded DNA when performing gene synthesis or even whole genome synthesis, thus developing high-throughput, low-error High-efficiency and fast DNA fragment assembly methods have become another important problem to be solved in synthetic biology. Among the many assembly methods that have been developed, Daniel D Gibson et al. developed a technique for splicing large fragments of DNA, called the Gibson assembly strategy, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/11
Inventor 陈泰康康沈玥王俊汪建杨焕明
Owner SHENZHEN HUADA GENE INST
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