A method for improving the mutation efficiency of kunkel reaction

A technology of reaction and efficiency, applied in the direction of biochemical equipment and methods, microorganisms, nucleic acid carriers, etc., can solve the problem of low mutation efficiency of Kunkel reaction, and achieve the effect of improving mutation efficiency and efficiency

Active Publication Date: 2021-07-20
ABLINK BIOTECH CO LTD
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Problems solved by technology

[0006] In order to solve the technical problem of low Kunkel reaction mutation efficiency, the present invention discloses a method for improving the Kunkel reaction mutation efficiency. The method significantly improves the Kunkel reaction at a single site and The efficiency of multi-site mutations, thereby increasing the diversity and capacity of phage libraries

Method used

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  • A method for improving the mutation efficiency of kunkel reaction
  • A method for improving the mutation efficiency of kunkel reaction
  • A method for improving the mutation efficiency of kunkel reaction

Examples

Experimental program
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Effect test

Embodiment 1

[0039] 1. Synthesis of dU-ssDNA

[0040] Phagemid pShort (ABLINK Biotech, Chengdu, China) was transformed from pBluescript (by inserting the secretion signal peptide, restriction site and pIII gene into the pbluescript plasmid (genescript) in order to construct PShort phagemid) for scFvs fused to M13 pill protein are displayed. Escherichia coli dut - ung -CJ236 can be used to synthesize dU-ssDNA due to the lack of dUTPase and uracil-N-glycosidase. dut + / ung + Strains are used to synthesize new strands and digest uracil-containing templates.

[0041] The phagemid was electroporated into CJ236 cells. On the second day, five clones were picked and inoculated in 3 ml of 2YT medium containing carbenicillin (50 μg / ml) and chloramphenicol (15 μg / ml), and 10 10 pfu / ml M13K07 helper phage (New England Biological Laboratory) was cultivated at 37°C and 200rpm for 2h. Then kanamycin (25 μg / ml) was added and cultured for 6 hours. Finally, all the cultures were transferred into 50m...

Embodiment 2

[0047] The difference between this example and Example 1 is that the prepared dU-ssDNA is WT D2E7 scFv; the primers are one or more of WTL3-Oligo, WT H1-Oligo, WT H2-Oligo and WT H3-Oligo, using Single-stage annealing procedure: 90°C, 3min; 50°C, 5min; 20°C, 5min. The nucleotide sequences of the primers are shown in Table 2, and the mutation efficiency of the Kunkel reaction is shown in Table 3. image 3 shown.

Embodiment 3

[0049] The difference between this example and Example 1 is that the prepared dU-ssDNA is LGC D2E7 scFv; the primers are one or more of LGCL3-Oligo, LGC H1-Oligo, LGC H2-Oligo and LGC H3-Oligo, using Single-stage annealing procedure: 90°C, 3min; 50°C, 5min; 20°C, 5min. The nucleotide sequences of the primers are shown in Table 2, and the mutation efficiency of the Kunkel reaction is shown in Table 3.

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Abstract

The invention discloses a method for improving the mutation efficiency of the Kunkel reaction. By optimizing the codons of the DNA template and its mutation primer, the GC content in the complementary region of the DNA template and the mutation primer is reduced, and the upstream and downstream of the complementary region of the mutation primer are reduced. The GC content difference, combined with a multi-stage annealing procedure, can effectively improve the Kunkel reaction mutation efficiency. Experiments have proved that the above method can significantly improve the efficiency of Kunkel reaction in single-site and multi-site mutations, thereby effectively increasing the diversity and storage capacity of phage display libraries.

Description

technical field [0001] The invention relates to the technical field of gene site-directed mutation, in particular to a method for improving the efficiency of Kunkel reaction mutation. Background technique [0002] Site-directed mutagenesis is an important technology widely used in genetic engineering and protein engineering. Among them is a site-directed mutagenesis method invented by TA Kunkel, which introduces mutations by annealing primers containing coding mutations to specific sites on uracil-containing single-stranded DNA (dU-ssDNA) templates. The Kunkel mutation method is a common method for constructing peptide, protein and antibody phage display libraries. In addition, this approach enables simultaneous mutagenesis of multiple sites in all complementarity determining regions (CDRs) of an antibody in a single reaction. [0003] During protein or antibody library construction, degenerate primers can introduce billions of mutations in specific regions, such as mutati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/102C12N2800/22
Inventor 刘江海刘彬龙爽何奕佳
Owner ABLINK BIOTECH CO LTD
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