Non-false positive T vector and preparation

Abstract

The invention relates to a non-false-positive T vector for cloning PCR fragments, and preparation thereof. The T vector is a linearization plasmid vector, of which two 3' ends both have a prominent dT, wherein sites of the T vector in which the PCR fragments of a dA prominent at the 3' ends, namely two tails ends of a linearization T vector, are positioned between an initiation codon of positive-clone selective marker gene and an upstream ribosome bind site. When the T vector designed and constructed according to the invention is used for cloning the PCR fragments, false-positive cloning cannot be produced because of the frame shift of the positive-clone selective marker gene, which is caused by the self connection of the T vector. The T vector overcomes the technical-mechanism disadvantage that the prior T vector produces false-positive clones during application because of the self connection of the vector. Therefore, from a technical mechanism (namely excluding the interference of other experimental factors), the T vector designed by the invention is void of the rate of false-positive cloning when the T vector is applied to T-A cloning.

Description

technical field [0001] The content of the invention belongs to the field of biotechnology, and specifically relates to a T carrier and its preparation. Background technique [0002] 1. Plasmid vector [0003] Molecular cloning technology is the basis of genetic engineering technology. Molecular cloning technology is used to insert a target gene (DNA fragment) into a DNA vector for long-term preservation and mass production of the target gene for subsequent research. The most commonly used molecular cloning vector is the plasmid vector: a closed circular double-stranded DNA molecule capable of autonomous replication and passage in bacterial cells. A plasmid vector for molecular cloning must contain at least the following three functional units: genetic elements necessary for the autonomous replication of the plasmid, 1-2 antibiotic resistance genes, and restriction sites for inserting target DNA fragments. The modern positive screening plasmid vector also includes a screen...

AI Technical Summary

Problems solved by technology

[0010] 3. Defects in the technical mechanism of existing T carriers

However, when the T vector is prepared by this method, a small fragment between the two enzyme cutting sites will be produced after digestion of the pre-T vector. If this small fragment is not completely removed in the recovered T vector (large fragment), In T-A cloning, the small fragments will be rejoined into the large fragments of the vector (because they have complementary sticky ends), and can be rejoined in both forward and reverse directions, and the positive clones in the vectors rejoined in the reverse direction can be used to screen the genes. Changes in the ORF of the gene may disrupt its function and form false positive clones

It is very difficult to remove this small fragment 100% in actual production, so false positive clones will also be produced when using this T vector

[0018] Self-ligation of linearized T-carrier molecules with 1 overhanging dT at both 3' ends is more difficult because their two cohesive ends are non-complementary, but self-ligation of such T-carrier molecules does in practice occur and lead to the generation of false positive clones

Of course, the efficiency of this kind of self-connection is not high, which is also consistent with the actual situation: the false positive clone rate in the use of various existing T vectors on the market is usually about 20%.

Although the false positive rate of 20% seems not high, what the user needs must be a true positive clone containing the target fragment! Therefore, when people use the traditional T vector, they have to screen the white colonies again (such as colony PCR screening) to obtain real positive clones after the blue-white screening, which obviously increases the experimental operation steps and increases the experimental cost.

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