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A method for de novo synthesis of DNA shuffled libraries using chips to synthesize oligonucleotide libraries

An oligonucleotide library and library technology, applied in DNA preparation, recombinant DNA technology, chemical library, etc., to achieve the effect of reducing synthesis cost, increasing recombination rate, and increasing diversity

Active Publication Date: 2020-10-30
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the de novo design and synthesis of gene mutation libraries using chip-synthesized oligonucleotide libraries has not been reported yet.

Method used

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  • A method for de novo synthesis of DNA shuffled libraries using chips to synthesize oligonucleotide libraries
  • A method for de novo synthesis of DNA shuffled libraries using chips to synthesize oligonucleotide libraries
  • A method for de novo synthesis of DNA shuffled libraries using chips to synthesize oligonucleotide libraries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Design, synthesis and amplification of RFP oligo pool

[0038] 1. Design of RFP oligo pool

[0039] First, the 21 RFP parent sequences selected were codon-optimized according to the codon usage frequency of Escherichia coli, and the restriction endonuclease sites contained in the sequences were removed to form the optimized REP gene full-length sequence (SEQ ID No. .5-25). A pair of primer sequences are then added to the two segments of these sequences to facilitate subsequent amplification of the full-length sequence. These sequences are then compared, and the aligned sequences at the ends of their homologous regions are as follows figure 1 Interleaved cuts into multiple oligos were performed as shown. A pair of universal primers containing a type IIS restriction endonuclease Mly I site was added to each of the two segments of these oligos, which can be removed by enzymatic digestion before subsequent assembly reactions. These oligonucleotides can be ass...

Embodiment 2

[0056] Example 2: Analysis of Correction Efficiency of RFP oligo pool Synthesis Errors

[0057] In this implementation case, a round of error correction processing is performed on the RFP oligo pool described in Embodiment 1 by using MICC. The error-corrected sample is used as a template to re-amplify the ED-oligo pool by PCR, and then the oligo pool is assembled into a full-length recombinant library sequence by the method of LCR-PCA. Finally, these assembled full-length genes were cloned into the pMD18-T vector (Bao Bioengineering (Dalian) Co., Ltd.). Sequencing was performed, and the DNA analysis software Bioedit was used to compare the sequencing results with the full length of the parental genes.

[0058] The specific implementation steps are:

[0059] 1.MICC processing re-hybridized RFP oligo pool:

[0060] 1) Sample loading. Take 60 μl denatured and annealed RFP oligo pool, add 180 μl binding buffer and mix, the final concentration of DNA is 12.5ng / μl. Then load th...

Embodiment 3

[0110] Example 3, Plate induced expression of RFP recombinant library, positive clonal ratio analysis and positive clone sequencing analysis

[0111] 1. Construction of RFP recombinant library expression library

[0112] The full-length gene sequence of the recombinant library RFP-lib-NX prepared in Example 2 and the pET-21c vector were double digested with Nhe I and Xho I respectively, and the RFP-lib-NX sequence was inserted into the pET-21c vector by ligation , the plasmid pTaqMutS was obtained. The specific operation is as follows:

[0113] The amplified product RFP-lib-NX gene sequence and the pET-21c vector were digested with Nhe I and Xho I respectively, and ligated to construct the plasmid library "pet-RFP lib".

[0114] Enzyme digestion system of RFP-lib-NX gene sequence:

[0115]

[0116] Enzyme digestion system of pET-21c vector:

[0117]

[0118] Ligation system of RFP-lib-NX gene sequence and pET-21c vector:

[0119]

[0120]

[0121] The obtained...

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Abstract

The invention discloses a method for de novo synthesis of a library by using a chip synthesized oligonucleotide library for DNA shuffling. The method mainly comprises the following steps: cutting a parent sequence into oligonucleotide sequences by combining comparison software; carrying out high-throughput error correction on an oligonucleotide library by using a mismatch binding protein MutS immobilized cellulose column; and carrying out full-length recombinant library assembly by using the cut oligonucleotides. Compared with the existing method, the method disclosed by the invention has theadvantages of high recombination rate, reduced difficulty of assembling into a full-length gene sequence, reduced synthesis cost, improved diversity of the sequence, improved accuracy of the oligo pool, improved fidelity of the synthesized gene library and improved probability of recombination.

Description

technical field [0001] The invention relates to a method for constructing a DNA recombination library, in particular to a method for de novo synthesis of a DNA shuffling library by using a chip to synthesize an oligonucleotide library. Background technique [0002] DNA shuffling (DNA shuffling) technology is an important in vitro molecular directed evolution method, which can be used to generate a large number of recombinant mutants that combine effective mutations from multiple individuals. The DNA shuffling method randomly fragments some functionally related gene fragments into a merger fragment library, and the DNA in these fragment libraries acts as primers and templates through the mutual homologous regions through recombination reactions. During the assembly process, Fragments derived from different parental sequences were assembled into a full-length mutant gene library. The advantage of DNA shuffling is that when recombining a gene family, homologous parental genes ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C40B50/06
Inventor 宛雯洪泂
Owner XUZHOU NORMAL UNIVERSITY
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