High throughput directional T carrier cloning process

Abstract

The invention provides a method for cloning high-flux directed T vector. The method is as follows: an appropriate IIS type restriction enzyme site and lactose operator gene sequence (LacO) are selected; through reconstructing integrate lactose operator gene sequence (LacO) between vector mutagenesis cleavage joint transformation and cloning fragment, directed cloning and non-identification cloning are realized; moreover, fusion and non-fusion T vector construction is realized. The method has simple primer design, high cloning efficiency, less time consumption and low cost; moreover, the obtained recon does not need subsequent identification and is compatible with the prior cloning method.

Description

technical field [0001] The present invention relates to biological gene cloning and expression technology, especially a visible high-throughput directional T-vector cloning method, which is especially suitable for large-scale, accurate and fast cloning of PCR products, and expressing corresponding ORFs on the same vector. Background technique [0002] With the completion of the whole genome sequence of more and more species, the function of genes and the interaction between genes have increasingly become the focus of molecular biology research. Researchers use various bioinformatics tools to analyze the whole genome sequence, obtain valuable gene sequence information, design primers from known sequences to amplify DNA and clone and express, and then conduct related structure and function research. The process of life science research has been accelerated, but how to accurately clone these gene fragments of various lengths on a large scale has become a bottleneck restricting ...

AI Technical Summary

Problems solved by technology

Researchers use various bioinformatics tools to analyze the whole genome sequence, obtain valuable gene sequence information, design primers from known sequences to amplify DNA and clone and express, and then conduct related structure and function research. The process of life science research has been accelerated, but how to accurately clone these gene fragments of various lengths on a large scale has become a bottleneck restricting the work of many researchers

[0003] The traditional method of cloning PCR products utilizes the 5′ end of PCR primers to introduce restriction enzyme sites for cloning, which reflects its own shortcomings when dealing with high-throughput cloning. There are four shortcomings: the first point is insufficient, the synthesis of primers high cost

Due to the introduction of enzyme cutting sites and protective bases at the 5' end of the primer, the cost of primer synthesis will increase by 1 / 3

The second point is insufficient. For each ORF (open reading frame) or gene fragment, it is necessary to consider that the restriction enzyme site introduced at the 5' end of the two PCR primers may also exist in the ORF or gene fragment at the same time. When these gene fragments will be cut off, the gene will be incomplete or unable to be cloned, and flux cannot be achieved

The third point is that the fragments amplified by PCR need to be recovered-digested-recovered before the enzymatic ligation reaction, which will consume a lot of manpower and material resources

The fourth point is that the efficiency of cloning is not high, and the cloned transformants need to be digested or identified by PCR, which is not conducive to high-throughput operations

(Gateway Entry clone requires an additional 61nt additional base sequence, Topo Cloning requires 2×(9~16)nt, and longer base sequence means higher synthesis cost and error probability, which are not conducive to the development of high-throughput technology and need to be overcome urgently

[0006] Second, in terms of cloning efficiency, In-Fusion TM PCR Cloning, Gateway cloning, Topo Cloning requires enzymatic reactions in vitro, and these reactions are reversible. If the cloning fragment is large, the cloning efficiency will be greatly reduced.

[0007] Third, the downstream identification of these cloning methods takes up a lot of time, manpower and material resources. The transformants obtained by common cloning methods need to undergo traditional and cumbersome downstream identification. These identifications include: identification of the size of the extracted plasmids, PCR identification, Enzyme digestion identification and sequencing identification (traditional T-A cloning cannot achieve directional cloning, and at this time it is necessary to perform directional enzyme digestion and PCR re-identification)

As far as the current high-throughput cloning is concerned, the identification process generally requires more than 48 hours, which is difficult to adapt to the growing pace of gene functional omics research

[0008] Fourth, these cloning methods are not suitable for gene non-fusion expression research

Since the upstream or downstream of the gene introduces more additional essential bases through primers, these cloning methods cannot achieve seamless cloning in the true sense. essential bases to generate extra amino acids that are not needed by a researcher who is striving for excellence in experimental results

[0009] Fifth, the time period for cloning is long. It generally takes more than 72 hours from the time when the researchers obtain the gene to actually find the certain recombinant containing the insert fragment, which is difficult to meet the needs of the booming gene function and protein-protein interaction research.

[0010] Sixth, the cost of the experiment is still relatively high. First, whether it is the In-Fusion reaction enzyme mixture, topoisomerase, or lambda integrase, integrated host factor protein and excision enzyme, they are all relatively expensive; Take up a large proportion of research costs

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