One-step multi-segment DNA molecule splicing method, kit and application thereof

Abstract

The invention discloses a one-step multi-segment DNA molecule splicing method, which comprises the following steps: mixing DNA fragments F1... Fn to be spliced, polynucleotide kinase and a reaction buffer solution to form a mixture, then carrying out gradient PCR (Polymerase Chain Reaction) on the mixture, and carrying out one-step reaction to obtain a completely spliced target DNA. According to the method, one-time splicing of multiple DNA molecules can be achieved, and the method can be used for solving the amplification problems, which cannot be solved by PCR, of primers due to reverse repetition, high and low GC and / or poly structures and the like. The invention also discloses a one-step type multi-segment DNA molecule splicing kit. According to the one-step multi-segment DNA molecule splicing method and the kit, the application effect of annealing assembly of the multi-segment DNA molecules is remarkably improved, and the method and the kit have application value for one-step integration of the multi-segment DNA molecules and splicing of difficult genes which cannot be completed through a PCR technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a one-step multi-segment DNA molecule splicing method, kit and application thereof. Background technique [0002] DNA splicing technology is one of the most important technical methods in current biological gene cloning research, and most modern molecular biology and cell biology are based on this technology. In the 1970s, researchers such as Cohen designed the first recombinant DNA molecule. The current mainstream DNA splicing method is still based on the original enzyme digestion ligation cloning method. This method has a complete operating system and involves a variety of biotechnological methods. , such as primer design, PCR, restriction endonuclease digestion, ligation transformation, etc. When there are too many actual experimental steps, the accuracy control of each step will test each practitioner, which increases the difficulty of operation. This traditional method cannot mee...

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Problems solved by technology

In the prior art, the strategy of using type II restriction endonucleases to first obtain suitable DNA fragments and then digest and connect clones is good, but due to the limited selection of restriction sites, when encountering gene cloning expression vectors, multiple fragments are spliced ​​and assembled into different DNA fragments. When designing the primers, although the restriction endonuclease site sequence has been designed at both ends of the functional DNA sequence to greatly reduce the complexity of the later stage of the splicing and assembly work, there are still the following problems: The throughput of seamless cloning or multi-segment DNA fragment assembly is low, and only two complementary sequences can be spliced ​​in one operation. If multiple fragments are to be spliced, multiple rounds of experiments are required, which is time-consuming, laborious, and inefficient

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