Frequency controllable GC specificity DNA mutagenesis method

Abstract

The invention belongs to the genetic engineering technology field, and specifically relates to a frequency controllable GC specific DNA mutation method. Sodium hydroxide firstly denatures a gene to be mutated to a single chain, hydroquinone and sodium bisulfite are further used for processing, thereby allowing C in the DNA chain to become U by deamidization; the modified DNA is taken as a template, U is matched with A by PCR amplification, and A is continually matched with T in the follow-up amplification, thereby allowing GC in the original DNA chain to be converted to the AT direction and obtaining mutant DNA molecules; and the mutant DNA molecules are loaded to an expression vector, thereby constituting a DNA molecular mutant library. As the processing time of the sodium bisulfite is prolonged, the final mutation frequency of DNA is gradually increased, and the DNA with the mutation frequency of about 5 percent can be finally obtained, thereby overcoming the problem of excessively low mutation frequency in a plurality of conventional random mutation methods. The frequency controllable GC specific DNA mutation method is designed against GC base groups in the DNA molecules, the total mutation rate of GC is more than 90 percent, and the frequency controllable GC specific DNA mutation method is particularly applicable to the mutation of genes which are rich in GC. The invention can be used for the in vitro improvement of DNA molecules.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for artificially mutagenizing genes, which is related to molecular evolution in vitro. Background technique [0002] In vitro molecular evolution technology simulates the process of natural evolution in the laboratory, artificially introduces mutations and recombinations, constructs a diverse mutation library, and cooperates with appropriate selection pressure to select mutant enzymes with required properties in a short period of time. Through this technology, people can endow natural enzymes with different characteristics, making them more suitable for industrial and agricultural production requirements, such as high activity, high temperature and high pressure resistance, extreme pH, etc. Mutagenizing the target gene and constructing a mutant library is a key step in this technology. The current method of mutagenesis is mainly error-prone PCR (error-pron...

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Problems solved by technology

The current method of mutagenesis is mainly error-prone PCR (error-prone PCR), which is based on the low fidelity of Taq DNA polymerase or using Mn 2+ Replace Mg 2+ The characteristics that reduce the fidelity of the enzyme, or the use of base analogs to cause mismatches and incorporation of wrong bases during the PCR process, the mutation frequency of these methods is limited to a low range, and the mutation frequency is not easy to control, mutation The spectrum mainly tends to transform from AT to GC, which greatly limits the diversity and randomness of mutagenesis of enzyme molecules. Especially to obtain highly active enzymes with large structural changes or enzymes with new functions, multiple rounds of mutagenesis are often required. Change

For genes with high GC content (such as from higher animals and plants, molds, streptomyces), the above method is not applicable

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