T-carrier capable of directionally cloning promoter and studying its activity as well as constructing method thereof

Abstract

The invention discloses a T carrier that can directly and directionally clone a promoter and research the activity of the promoter and a construction method thereof. The construction method comprises the steps: firstly: an anterior T carrier with a Xcml box (or an AhdI box) is prepared, and the anterior T carrier comprises a DNA spacer sequence that has different resistance genes or other selective markers with the initiating carrier and two Xcml enzyme cutting site sequences that are closely connected with two sides of the DNA spacer sequence respectively; secondly, the T carrier is obtained by enzyme cutting the anterior T carrier in the first step with the Xcml enzyme or isoschizomer of the Xcml enzyme; the Xcml box in the first step is obtained by amplifying PCR, and the recognition site of the upstream primer Xcml enzyme still contains the first half part of the recognition site of the rare restriction enzyme Pmel which is used for the directional cloning analysis of the promoter PCR products. The method in the invention is also applicable to the preparation of the T carrier of a common cloning type and an expressive T carrier, and plays an important role in the genetic engineering field.

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 T vector capable of directly cloning a promoter in a high-throughput direction and studying the function of the promoter. 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 to use Taq DNA polymerase 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. without the need for restriction endonuclease digestion. [0003] In the field of promoter research, PCR amplification products of promoters often need to be cloned into vectors with reporter genes such as enhanced green fluorescent protein (EGFP...

AI Technical Summary

Problems solved by technology

However, this method has two disadvantages: first, the efficiency of subcloning is restricted by many factors such as enzyme digestion effect, target gene recovery quality, yield and ligation efficiency; second, in the process of promoter research, usually the longer the fragment , the more restriction endonuclease sites it contains, it will inevitably be limited by the selection of restriction endonuclease sites during the cloning process

However, a new problem posed by the introduction of spacer DNA is that, since circular supercoiled plasmids surge faster than linear plasmids of equivalent molecular weight in agarose gel electrophoresis, completely undigested circular supercoiled It is often mixed with the T carrier (completely cut) whose molecular weight is smaller than it, so the background of non-recombinant transformants is higher. If the pre-T carrier lacks a screening marker that is different from the T carrier, it will face more cumbersome screening work

[0010] 2. Directional cloning cannot be realized, and the screening workload is heavy

If the T vector is only used for cloning, amplification and preservation of the target gene, there is no hindrance; but if it is used for gene expression or promoter analysis, etc., more tedious screening work is required to screen out the clones in the correct direction , time-consuming and labor-intensive, especially not suitable for high-throughput screening of promoters

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