New and effective method for building complex carrier

Abstract

The invention provides a simple, general and effective method for building complex carrier. A randomly designed linker is added on a target fragment 5' in PCR primer design, the target fragment is cloned by PCR, then the PCR cloned target fragment is processed by 3'-5' excision enzyme activity of T4DNA polymerase, a plurality of end-to-end sticky ends are generated, multi-fragment orientation connection, conversion and recon identification are carried out. The method for building complex carrier has high efficiency of rearrangement, connection and conversion and is applicable to building various complex carriers.

Description

technical field [0001] The present invention relates to a method for constructing vectors used in genetic engineering or molecular biology. In particular, a highly efficient method for constructing complex vectors. Genomic DNA and PCR amplification products suitable for cloning. Background technique [0002] Vector construction technology is one of the commonly used technologies in molecular biology and genetic engineering research. Traditional vector construction methods not only need to design multiple restriction sites when constructing complex vectors with multiple fragments spliced, but There are DNA fragments with restriction sites, and site-directed mutagenesis is required to remove these cut points on the DNA fragments. It is necessary to construct multiple intermediate vectors and perform multiple connections and transformations. Not only is the design complicated, the operation is cumbersome, time-consuming, laborious and expensive. Also the success rate is low. ...

AI Technical Summary

Problems solved by technology

[0002] Vector construction technology is one of the commonly used technologies in molecular biology and genetic engineering research. Traditional vector construction methods not only need to design multiple restriction sites when constructing complex vectors with multiple fragments spliced, but There are DNA fragments with restriction sites, and site-directed mutagenesis is required to remove these cut points on the DNA fragments. It is necessary to construct multiple intermediate vectors and perform multiple connections and transformations. Not only is the design complicated, the operation is cumbersome, time-consuming, laborious and expensive. also low success rate

The splicing of some gene fragments that require high connection accuracy is even more difficult

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