Single-module DNA (deoxyribonucleic acid) library and connecting method for TALENs (transcription activator-like effector nucleases) identification modules

Abstract

The invention discloses a DNA (deoxyribonucleic acid) library, a construction method for TALENs (transcription activator-like effector nucleases) plasmids and a connecting cell library for TALEs (transcription activator-like effectors). The DNA library comprises all TALE connecting cells in the TALE connecting cell library. The construction method comprises the following steps of determining identification modules according to a target sequence of a target gene, and selecting a DNA fragment corresponding to each identification module from the DNA library; recombining and connecting the connecting cells in each DNA fragment into a TALENs vector by adopting type-two restriction enzymes and DNA ligases in the same system; and performing digestive treatment by using linear DNA digestion enzymes to obtain the TALENs plasmids. Compared with the prior art, the DNA library, the construction method and the connecting cell library have the advantages that the DNA library is used for constructing the TALENs plasmids, and 12 to 19 identification modules can be connected at one time, so that high speed, high efficiency, simplicity in operation and low cost are ensured, and materials are easy to store.

Description

technical field [0001] The present 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, a method for connecting TALENs recognition modules, 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 ...

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. In many practical applications, TALENs with more than 14 recognition modules and 14 fragment lengths are required. The limitation not only affects the application of this method, but also is not conducive to the improvement of the specificity of TALENs, 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 the single-stranded DNA. tail easily degrades

[0006] In the past, some researchers connected TALENs 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

Images