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

Abstract

The invention discloses a high-effective construction method for a T carrier based on a PCR method. The invention is that a pair of oligodeoxynucleotide sections which contain specific restriction enzyme sites are introduced into an initiation carrier (such as pUC18); the restriction enzyme site is characterized in that the corresponding enzyme (such as EcoRV) can generate blunt end, and the baseof fro and post tangency point are respectively T and A; the initiation carrier is linearized under the function of the enzyme which is then used as a PCR template; designed primers produce two single chain through single-primed PCR or asymmetric PCR amplification, the two single-chains are annealed so as to form the T carrier. The method for preparing the T carrier is simple and fast; the produced T carrier can effectively clone PCR products containing tail A, and the non-reconstitution background is nearly 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 for direct cloning of PCR products, and A-T cloning technology has proved to be extremely valuable for cloning of PCR products. The principle of this method is to use Taq DNA polymerase to add an additional template-independent dA base to the 3' end of the amplified product. This dA is just paired with the dT at the 3' overhang of the linear T vector, and the ligation is performed directly and efficiently. Cloning without restriction endonuclease digestion process. [0003] At present, there are two kinds of classical T vector construction methods. The first method is to first linearize the plasmid with an endonuclease that produces a blunt-end cut point such as SmaI or EcoRV, and then use a terminal transferase or a Taq enzyme ...

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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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