Method for efficient preparation of linear DNA based on simple large-scale PCR

Abstract

The invention provides a method for the rapid preparation of linear DNA based on large-scale PCR. The large-scale PCR method with the volume 50-1200 times higher routine PCR is established through utilizing an optimized PCR program based on high-characteristic NDA polymerase RKOD obtained in a Chinese patent (having a patent number of ZL201010229160.5) in a low cost manner, and the content of a PCR product obtained in the invention can reach 300mug / mL. In the invention, dNTPs, a primer and a PCR buffer in a PCR system are added according to a routine PCR concentration, the plasmid template concentration is 1mug / mL, the DNA polymerase amount is 10U / mL, and the large-scale PCR method comprises the following steps: 1, placing a reaction system in 98DEG C water bath for 0.5min; 2, carrying out 72DEG C water bath for 1min; and 3, repeating step 1 and step 2 40 times, and recovering the PCR product. The method provided by the invention can realize of the low-cost, high-rate and large-scale production of the linear DNA, and lays a foundation for the further low-cost, efficient and rapid preparation of linear DNA vaccines for coping with new infectious diseases.

Description

technical field [0001] The invention relates to a low-cost and high-speed method for preparing linear DNA by large-scale PCR (polymerase chain reaction, polymerase chain reaction), which belongs to the field of industrial microorganism applications. Background technique [0002] PCR (polymerase chain reaction, polymerase chain reaction) is a technique for amplifying linear DNA in vitro first invented by Kary B. Mullis (Nobel Laureate for procedure to replicate DNA, science, 1985), and it is the most important basic principle of modern molecular biology. One of the research methods. [0003] PCR simulates the natural replication process of DNA. It utilizes the DNA polymerase-catalyzed reaction mediated by artificially synthesized primers. The primers start to synthesize a DNA strand that is complementary to the template DNA. After a cycle of denaturation, annealing, and extension, the number of DNA strands doubles, and the template is subsequently cycled, and the newly synt...

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Problems solved by technology

However, they still mainly use the traditional PCR method to produce linear DNA. For example, a large-scale PCR platform developed by Vandalia can achieve a daily output of 12L, but due to high production costs and slow production rates, it is still impossible to realize industrial production. need

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