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Solid-phase synthesis method of transcription activator-like effector

A transcriptional activation and effector technology, applied in peptide preparation methods, microbial-based methods, chemical instruments and methods, etc., can solve problems that are not suitable for large-scale applications, time-consuming, and labor-intensive

Inactive Publication Date: 2012-07-18
BEIJING VIEWSOLIDBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the existing synthetic preparation method, a large number of enzyme digestion, linking and product purification operations are required, which not only takes a long time (~3 weeks), but also requires a lot of labor, and is not suitable for large-scale applications

Method used

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  • Solid-phase synthesis method of transcription activator-like effector
  • Solid-phase synthesis method of transcription activator-like effector
  • Solid-phase synthesis method of transcription activator-like effector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. TALE 5-connected sub-unit assembled from 1 TALE unit (1-connected sub-unit)

[0070] The DNA linker sequence, the 5 TALE linker unit sequences, the DNA sequence to be combined with the 5-linker and the final assembled TALE 5-linker unit sequence are shown in Attached Table 1.

[0071] First, before DNA immobilization, mix the 5-terminal modified biotin DNA linker with the first linking unit digested by BbsI, and connect in solution; secondly, after treating the linking unit immobilized on the magnetic beads with BbvI enzyme digestion , wash the magnetic beads 3 times; moreover, before adding the second TALE linking unit, digest it with BbsI, purify it, and connect it to the magnetic beads under the action of T4 DNA ligase; repeat the above digestion and purification , connection and other steps 3 times, you can get 5 consecutive pieces.

Embodiment 2

[0072] Example 2 TALE 10-connected sub-unit assembled from 1 TALE unit (1-connected sub-unit)

[0073] The DNA linker sequence, the 5 TALE linker unit sequences, the DNA sequence to be combined with the 5-linker and the final assembled TALE 5-linker unit sequence are shown in Attached Table 2.

[0074] First, before DNA immobilization, mix the 5-end biotin-modified DNA linker with the first linking unit digested by BbsI, and connect in solution; secondly, treat the linking unit immobilized on the magnetic beads with BbvI enzyme digestion , wash the magnetic beads 3 times; moreover, before adding the second TALE linking unit, digest it with BbsI, purify it, and connect it to the magnetic beads under the action of T4 DNA ligase; repeat the above digestion and purification , connection and other steps 8 times, you can get 10 consecutive pieces.

Embodiment 3

[0075] Embodiment 3 is assembled into TALE 16 linker by 4 TALE quadruple linker units

[0076] The DNA sequence to be combined with the DNA linker, the 16-linker, and the sequence of each TALE linking unit are shown in Attached Table 3.

[0077] On the basis of TALE quadruplets, 4-step assembly can be carried out on the magnetic beads, and finally assembled into 16 TALE linkers. In the first step, the biotinylated ds-DNA linker (Biotinylated ds-DNA linker) has a SpeI restriction site, so it will produce sticky ends after digestion. After purification, it will be incubated with magnetic beads and will be fixed to the surface of the magnetic beads. The second step is to purify and amplify the TALE4 linker. In the third step, the TALE 4-linkage was digested with SpeI. In the fourth step, add the enzyme-digested first 4-linker to the magnetic bead solution with the DNA linker, connect for 15 minutes to 1 hour, and wash 3 times. In the fifth step, use NheI to digest the magnetic...

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Abstract

The invention relates to a solid-phase synthesis method of transcription activator-like effector (TALE). The method comprises the following steps of: immobilizing a segment of nucleic acid adaptation sequence (DNA (Deoxyribonucleic Acid) linker) to a solid-phase interface, and shearing with a restriction endonuclease to generate 3'-cohesive end; contacting a product with TALE linkage unit which is treated by the restriction endonuclease and is provided with 5'-cohesive end, connecting under the action of a DNA ligase, and eluting unconnected matrix; treating the product obtained in the previous step with restriction endonuclease to shear and generate 3'-cohesive end on DNA again, contacting a product with TALE linkage unit which is treated by the restriction endonuclease and is provided with 5'-cohesive end, connecting under the action of a DNA ligase, and eluting unconnected matrix; repeatedly operating the steps for at least one time; shearing the connected DNA molecule from the solid-phase interface with restriction endonuclease, and separating and purifying to remove the unconnected matrix; and expressing the DNA molecule into protein molecule, namely fusion protein (such as TALEN (TALE nuclease) or TALEA (TALE activator)) containing target TALE.

Description

technical field [0001] The present invention relates to a solid phase synthesis method of transcription activator like effectors (Transcription Activator Like Effectors, TALE), in particular to a large-scale, Low-cost, high-efficiency synthetic method. Background technique [0002] In the post-genome era, we urgently need new bioengineering technologies such as efficient gene manipulation and synthesis. For example, we often need to suppress or silence the expression of a gene. Traditional gene knockout (Gene Knockout) technology relies on homologous recombination that occurs naturally in cells, and its efficiency is very low, usually 10 -6 level; RNAi technology is simple and easy to implement, but it is difficult to obtain a 100% inhibitory effect. In addition to suppressing or silencing specific genes, we often need to modify a few bases or even a certain sequence of specific genes. Also only relying on homologous recombination technology, it is difficult to obtain th...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/04C12N9/22C12P19/34
CPCC12N15/66C12N15/10C12P19/34C12N9/22C12N15/63C07K14/195C12R1/64
Inventor 庄峰锋
Owner BEIJING VIEWSOLIDBIOTECH
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