Multi-module DNA (deoxyribonucleic acid) library and method for constructing transcription activator like effector nuclease plasmid

Abstract

The invention discloses a DNA (deoxyribonucleic acid) library and a method for constructing a transcription activator like effector nuclease plasmid. A transcription activator like effector connection unit library comprises 16 groups*n double-basic group identification modules and 4 groups*m single-basic group identification modules, wherein two tail ends of each double-basic group identification module or single-basic group identification module are both formed into a first cohesive tail end and a second cohesive tail end by being subjected to enzyme digestion through a second class restriction enzyme; n expresses the quantity of each group of double-basic group identification modules, and m expresses the quantity of each group of single-basic group identification modules. The DNA library comprises plasmids, wherein the plasmids are the same as the connection units of the Tale connection unit library in quantity, each plasmid comprises a connection unit with basic group sequences mutually different, the connecting positions of the two tail ends of the connection unit and an original carrier are provided with BsaI enzyme digestion sites. The DNA library disclosed by the invention is used for constructing the transcription activator like effector nuclease plasmid, can realize that 19-26 identification modules are connected for one step by identifying the enzyme digestion of the modules and connecting in same reaction, and has the advantages of high speed, high efficiency, easiness for operation, easiness for material preservation and low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a TALE junction unit library, a DNA library comprising all TALE junction units in the TALE junction unit library, and a method for constructing a transcription activator-like effector nuclease plasmid. Background technique [0002] It has always been the dream of many scientists to modify the genome according to human wishes. Specifically delete or add the sequences we need on the endogenous genome. On the one hand, various animal models can be constructed for basic biological research and disease mechanism research. On the other hand, animal reactors can be produced to produce what we need cheaply. Biological components that are difficult to obtain from other sources. People have not found a simple and efficient method for genome-targeted modification of the genome. Traditional gene targeting technology relies on the random exchange of homologous chromosomes naturally occurr...

AI Technical Summary

Problems solved by technology

But they all have their own defects, because the chemical synthesis of highly repetitive sequences of DNA to be synthesized is very difficult and the cost is very high; the two-step method requires two steps of connection, so the material cost, time cost, and sequencing cost are all high; the now public The only method that can be connected in one step can only connect up to 14 recognition modules, and the length of common recognition modules in nature is 12-23. The limitation not only affects the application of this method, but also is not conducive to the improvement of TALEN specificity, and because of the need for enzyme digestion, purification, and re-ligation, the operation is cumbersome, the process is complicated, and the DNA after enzyme digestion and purification is difficult to store, especially single-stranded DNA. tail easily degrades

[0006] In the past, some researchers connected TALEN with a single module, such as connecting 18 recognition modules. It is very difficult to connect 18 fragments in one reaction, and no one in the world has been able to achieve it; Can connect 14 identification modules

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