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DNA assembly and cloning method

A technology of inserting fragments and linear vectors, applied in the biological field, can solve the problems of no obvious advantages in time and economic cost, increased probability of non-specific hybridization, and increased cloning steps, so as to achieve high reaction efficiency and fidelity. , the effect of low mutation probability and short reaction time

Active Publication Date: 2015-02-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
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Problems solved by technology

Due to the limitation of DNA-specific sites, these two cloning methods still show great limitations for more complex or multi-fragment assembled DNA synthesis and cloning
[0004] Sequence-independent cloning methods are mainly based on the principle of homologous recombination, such as LIC, MAGIC, SLIC, Gibson, In-Fusion, PIPE, etc., but these cloning methods are completely independent of DNA sequences in the strict sense, and still need to be The overlapping region of the insert fragment and the target vector is missing or has some bases added to form a cohesive end that matches each other, which not only increases the cloning steps, but also uses an expensive combined enzyme system, so it is not necessary in terms of time and economic costs. showed no clear advantage
At the same time, the annealing step of these methods is usually carried out at room temperature, which increases the chance of non-specific hybridization, which in turn causes DNA misassembly
[0005] Each cloning method has its own advantages and disadvantages and the most applicable fields, but for the synthesis of complex gene fragment libraries, gene circuit diagrams, metabolic pathways, etc., more accurate, efficient, simple and economical DNA assembly and cloning methods are needed

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0080] Example 1: Cloning of a single gene.

[0081] The pAcGFP1 plasmid (3.3 kb, purchased from clontech) was used as the target vector, and the gentamycin resistance gene (531 bp) was used as the insert fragment for DNA recombination cloning.

[0082] 1. Acquisition of inserts and vectors with overlapping sequences: by respectively designing 2 pairs of corresponding primers (pAcGFP1-F and pAcGFP1-R, Gm-F and Gm-R) to initiate PCR to obtain inserts with overlapping sequence ends and linear carrier. See Table 2 for specific primer sequences.

[0083] The PCR reaction system in which the vector pAcGFP1 is linearized: 2ng pAcGFP1 plasmid as a template, 0.5μM primers (pAcGFP1-F and pAcGFP1-R), 0.8mM dNTP mixture (purchased from NEB, USA), 1×Phusion HF buffer (purchased from NEB Company of the United States), 0.5 μl of Phusion DNA polymerase (purchased from NEB Company of the United States), and replenish water to 50 μl. PCR reaction conditions: 98°C, 30s; (98°C, 10s; 58°C, 30s...

Embodiment 2

[0088] Example 2: Assembly and cloning of multi-gene fragments.

[0089] Take the assembly and cloning of 4 genes (3280bp cat2phaC, 2959bp pASK, 2047bp phaAB, 171bp terminator) in the synthetic pathway of a biodegradable plastic poly(3HB-co-4HB) as an example:

[0090] 1. According to the DNA sequences of the 4 genes, design and synthesize 4 pairs of primers respectively, and use the corresponding plasmids as templates to amplify the 4 genes. See Table 2 for specific primer sequences. Refer to Step 2 in Example 1 for the PCR reaction system and conditions. The PCR products were purified by electrophoresis and recovered.

[0091] 2. Four genes were added to the reaction system in equal molar amounts (3280bp gene 200ng) for assembly, and 4 sets of PCR reactions were performed in parallel, and the cycle numbers were 2, 5, 10 and 20, respectively. For the reaction conditions, refer to step 3 in Example 1. The product electrophoresis results were as image 3 shown. Depend on ...

Embodiment 3

[0093] Example 3: Assembly and cloning of a multi-gene fragment library.

[0094] Taking pAcGFP1N1 (4.7kb) as the target vector, and the gene fragment library (1.7kb) of the HIV envelope gene gp120 synthesized according to codon degeneracy as the insert fragment, DNA assembly and cloning were performed as an example. The specific operations are as follows:

[0095] 1. Chemically synthesize the HIV envelope gene gp120 gene fragment library containing merged bases, and use two methods to obtain recombinant plasmids containing the target gene:

[0096] Method a: Two-step assembly, that is, first assemble the small DNA fragments in the gene fragment library into a full-length gene, and then connect with the carrier;

[0097] According to the DNA sequences of the pAcGFP1N1 and gp120 gene fragment libraries, three pairs of primers were designed and synthesized: pAcGFP1N1-R and pAcGFP1N1-L (for the linearization of the pAcGFP1N1 circular vector), gp120-R and gp120-28L and gp120-L and...

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Abstract

The invention belongs to the field of biotechnology and discloses a simple DNA assembly and cloning method. According to the DNA assembly and cloning method, a polymerase chain reaction between an insert fragment and a linearized vector which are used as a primer and a template mutually is carried out to obtain double-stranded circular DNA, wherein two ends of the insert fragment respectively carry overlapping sequences overlapping with two ends of the linearized vector, and two ends of the linearized vector respectively carry overlapping sequences overlapping with two ends of the insert fragment. The DNA assembly and cloning method has a simple reaction system, is low-cost, is simple to operate, is easy for parallel operation, has obvious advantages for DNA assembly and cloning of a complex gene fragment library, a combined gene fragment library and polygene pathways, can widely be applied in DNA assembly and cloning of a single gene, polygenes and a gene fragment library, and is especially suitable for high-throughput gene synthesis and assembly.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for gene synthesis, in particular to a simple method for assembling and cloning DNA. Background technique [0002] DNA assembly and cloning are the most basic and commonly used techniques in molecular biology research. The rapid development of genomics and synthetic biology has put forward higher and higher requirements for DNA assembly and cloning technology. According to whether specific sites or sequences are required on the insert fragment and the vector, the current DNA assembly and cloning methods can be divided into two categories, namely, sequence-dependent and sequence-independent. [0003] Sequence-dependent cloning is based on restriction-ligation or site-specific recombination methods. Restriction digestion-ligation is a commonly used classical cloning method, that is, both the insert and the vector are cut with the corresponding restriction endonuc...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 王璐田敬东冯淼王丽娜
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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