New gene mutation recombination method and application thereof

A mutant, target gene technology, applied in combinatorial chemistry, chemical library, library creation, etc., can solve the problems of not widely used and few literature reports.

Inactive Publication Date: 2009-09-16
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to DNA shuffling, methods such as random-priming in vitro recombination (RPR) and staggered extension process developed in recent

Method used

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  • New gene mutation recombination method and application thereof
  • New gene mutation recombination method and application thereof
  • New gene mutation recombination method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Amplification of target gene

[0014] This method intends to select the kanamycin resistance gene (kan) as the target gene for error-prone PCR. The kan gene expression unit from the plasmid vector pCMABIA1302 is amplified by PCR and then inserted into the pUC18 vector to obtain a gene containing kan. and ampicillin (amp) resistant recombinant plasmid p18Kan, and then using the p18Kan vector as a DNA template, the kan gene fragment was amplified with a pair of artificially synthesized primers (see below), and the conditions were optimized. As shown in the table below:

[0015]

[0016] PCR primers are: upstream primer; 5′-CCTGTGATCA CCGCGG TTTC-3', the underline is the restriction enzyme SacII cleavage site. Downstream primer; 5′-CTGTTTCTTCCCC GATATC CTCC-3', the underline is the restriction enzyme EcoRV cleavage site. The pair of primers amplified the expected PCR product with a size of 0.89 bp, which included the 5' end coding region and non-coding ...

Embodiment 2

[0018] Example 2: Error-prone PCR of a gene of interest

[0019] According to Example 1, error-prone PCR was performed with the kanamycin resistance gene (kan) as the target gene. This method is different from the conventional error-prone PCR method, but nucleotide analogs (dITP) are randomly incorporated into the product sequence through PCR technology, thereby forming random point mutations. In order to control the frequency and number of dITP incorporated into the target sequence during the PCR reaction, so that small fragments of the target gene of appropriate size can be obtained in the subsequent enzyme digestion step, it is first necessary to adjust the ratio of dNTP and dITP in the PCR reaction system. optimization. The specific steps required are: in a 100 μl PCR system, adjust the ratio of dNTP:dITP in the system to 8:0, 6:2, 5:3, 4:4, 3:5, and 2:6 as shown in the table below.

[0020] dNTP (2.5mM) 8μl 8μl 8μl 4μl 3μl 2μl dITP (2.5mM) 0μl...

Embodiment 3

[0022] Example 3: Random Fragmentation of Mutant Genes

[0023] Use endo V to digest the obtained mutant gene. The enzyme digestion reaction system is 20 μl, including 10 μl of PCR product, 2 μl of endo V buffer, 1 U of endonuclease endo V (MBI company), and 60 μl of enzyme digestion at 65 ° C. Minutes, the digestion reaction was terminated by heating at 95°C for 15 minutes, and the product was detected by 1.5% agarose gel electrophoresis. The results are shown in the attached figure 2 Shown in B: As the ratio of dNTP:dITP decreases, the fragments produced after endoV digestion become smaller, which also indicates that the site of incorporated dITP increases. When the ratio of dNTP:dITP is 2:6, the digested DNA fragments are mainly concentrated below 150bp; when the ratio of dNTP:dITP is 3:5, the digested fragments are mainly distributed at 200-50bp. This range is generally considered suitable for DNA assembly.

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Abstract

The invention belongs to the field of gene engineering and relates to a gene mutation recombination method and application thereof in construction of protein (proteinase) gene mutation library. In a polymerase chain reaction (PCR) system for amplifying a target sequence, nucleotide analogue 2-deoxyinosine-5-triphosphoric acid (dITP) is randomly doped in a target gene order by a PCR reaction so as to generate multi-point random mutation, the endonuclease V (endo V) cutting the dITP is distinguished by specificity so as to segment the amplified target gene order, and then the obtained the mutation target gene segments are extended and recombined by the PCT without a primer, and finally the mutation gene library of the recombined mutation gene is obtained. The method of the invention overcomes the shortage that the traditional DNA shuffling method is not easily controlled and wastes time and labour in the process of randomly cutting the target sequence by endonuclease (DNase I). The method can be widely applied to the construction of random mutation library of all protein (proteinase) coding genes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for in vitro molecular evolution of genes and the application of the method in the construction of a random mutation library of protein (enzyme) coding genes. Background technique [0002] Gene mutation technology is a common strategy in molecular biology research and is widely used in the modification and research of various gene functions. Gene mutation technology can not only obtain information on the relationship between specific sequences in genes and gene functions, but also modify the corresponding proteins by mutating genes encoding proteins (especially various enzymes). This strategy can be used to study Protein function or for the acquisition of mutant proteins (enzymes) with novel catalytic properties. In the field of protein engineering, it is often necessary to modify a certain natural enzyme to obtain a mutant enzyme with improved enzymatic properties in all asp...

Claims

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Application Information

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IPC IPC(8): C12P19/34C40B50/06
Inventor 冯红万民远杨庆军李念张义正
Owner SICHUAN UNIV
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