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

A technology for identifying modules and connecting methods, applied in the field of genetic engineering, can solve the problems of difficulty in DNA preservation, material cost, time cost, high sequencing cost, cumbersome operation, etc.

Active Publication Date: 2014-01-08
上海煦顼技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
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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

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Connection between TALENs recognition modules and construction of recombinant vector

[0064]1. 4 identification modules (modular)

[0065] (1) Synthesis of recognition modules NI, NG, HD, and NN that recognize four single bases A, T, C, and G respectively. The sequences are shown in Table 1, as shown in SEQ ID No.1-4, respectively.

[0066] Table 1 Sequences of four single-base recognition modules

[0067]

[0068] 2. Make 100 plasmid libraries by adding restriction sites and linkers to the recognition module

[0069] (1) PCR amplification to add enzyme-cut recognition sequences and connection adapters

[0070] F1 / R1; F2 / R2; F3 / R3; F4 / R4; F5 / R5; F6 / R6; F7 / R7; F8 / R8; F9 / R9; F10 / R10; F11 / R11; F12 / R12; F13 / R13; F14 / R14; F15 / R15; F16 / R16; F17 / R17; F18 / R18 were used as primers, and the T vector containing 4 single-base recognition modules was used as a template for PCR, a total of 4×18=72 ( The first part connects the unit, taking n=18 as an example);

[...

Embodiment 2

[0131] TALENs (SEQ ID NO: 47 and 48) that recognize the following DNA sequences were ligated:

[0132] Fragment 1: CGCGCGCGCGCGCGCGCGT;

[0133] Fragment two: CCCACTCCCCATCCAGT.

[0134] (1) Select the desired recognition module from the PCR library

[0135] For Fragment 1 (CGCGCGCGCGCGCGCGCGT), 18 linking units need to be connected (the carrier already contains the 19th T recognition module); select figure 1 Single-base recognition modules C1, G2, C3, G4, C5, G6, C7, G8, C9, G10, C11, G12, C13, G14, C15, G16, C17, G18; the connection vector is pEF1a-NLS -TALE backbone-Fok1(R)-pA;

[0136] For Fragment 2 (CCCACTCCCCATCCAGT), 16 linking units need to be connected (the recognition module of the 17th position T was already contained on the carrier); select figure 1 Single-base recognition modules C1, C2, C3, A4, C5, T6, C7, C8, C9, C10, A11, T12, C13, C14, A20, G18 in the single-base recognition module; the first cohesive end of A20 is connected with the first section 14 The...

Embodiment 3

[0151] The steps and methods of Example 1 were used to construct the recognition module and recombinant vector.

[0152] 1. The synthesis of four single-base recognition modules is the same as in Example 1.

[0153] 2. Make 100 plasmid libraries by adding restriction sites and adapters to the recognition module.

[0154] (1) PCR amplification to add enzyme-cut recognition sequences and connection adapters

[0155] Taking n=18 as an example, the construction method for the first part of the connection unit is the same as that in Embodiment 1.

[0156] In the second part of connecting units, the primer pairs used for connecting units 19-25 are: F8 / R15, F9 / R15, F10 / R15, F11 / R15, F12 / R15, F13 / R15, F14 / R15. That is, m=7, q=14, p=7~13.

[0157] Restriction sites, cohesive end sequences and primer sequences are the same as in Example 1. All the other are with embodiment 1.

[0158] (2) Gel recovery and purification of 100 PCR products, and connection into the PMD18-T system

[...

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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 ...

Claims

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Application Information

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IPC IPC(8): C12N15/66C40B40/06C40B50/06
Inventor 吴昭孙文页
Owner 上海煦顼技术有限公司
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