Method for high-effective construction of T-carrier based on polymerase chain reaction

Abstract

The invention discloses a method for efficiently constructing a T vector based on the PCR method. The main method is to introduce a pair of oligonucleotide fragments containing a specific restriction enzyme site into the starting vector (such as pUC18). The restriction enzyme site is characterized by The corresponding enzyme (such as EcoRV) can produce blunt ends, and the bases before and after the cutting point are T and A, respectively. Then use the enzyme to linearize the starting vector as a PCR template, design primers to generate a single strand through single primer PCR amplification or asymmetric PCR amplification, and then anneal the two single strands to form a T vector. The preparation of the T vector by the method is simple and fast, and the prepared T vector can efficiently clone the PCR product with an "A" tail, and its non-recombination background is almost zero.

Description

technical field [0001] The invention relates to a method for constructing a vector in the field of genetic engineering, in particular to a method for constructing a high-efficiency T vector. Background technique [0002] T vector is a linear vector used for direct cloning of PCR products, and A-T cloning technology has been proved to be extremely valuable for the cloning of PCR products. The principle of this method is that Taq DNA polymerase can be used to add a non-template-dependent dA base to the 3' end of the amplified product. This dA has just paired with the dT at the 3' overhang of the linear T vector for direct high-efficiency connection and Cloning process without restriction endonuclease digestion. [0003] Currently, there are two classic methods for constructing T vectors. The first method is to linearize the plasmid with an endonuclease such as SmaI or EcoRV that produces a blunt end cut point, and then use terminal transferase or Taq enzyme with terminal tra...

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Problems solved by technology

[0005] 1. High non-recombinant background and low cloning efficiency

[0006] The preparation of T vectors by terminal transferase or Taq enzyme is sometimes due to the low efficiency of adding T to the enzyme, and the ends of some vector molecules are not modified at all, resulting in the existence of blunt-ended linear plasmids with no T added at both ends. It is difficult to avoid the loop of the vector itself during the ligation process. Sometimes there is also the problem that the sequences at both ends of the linear plasmid are partially deleted during the T-tailing process

The method of preparing T vectors with specific restriction endonucleases such as XcmI, AhdI and other enzymes also often produces incomplete digestion due to relatively low digestion efficiency, and the problem of excessive non-recombination background in the cloning process

Therefore, the key problem in the construction of T vector is the high non-recombination background caused by the low efficiency of the enzyme, which leads to low cloning efficiency.

[0007] 2. There are limitations of endonucleases

But the premise of adopting this method is that the starting vector itself does not have recognition sites such as XcmI, AhdI (or their isozymes), otherwise mutagenesis must first be carried out to make this site disappear, and the more such sites, The mutagenesis process is more cumbersome, time-consuming and labor-intensive

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