Method for constructing RNA (Ribonucleic Acid) interference vector by directly annealing multi-primers

Abstract

The invention provides a method for constructing a RNA (Ribonucleic Acid) interference vector by directly annealing multi-primers. The method mainly comprises the following three steps of: modifying the vector; annealing an oligonucleotide sequence; and cloning annealing products to RNAi. The method does not need to synthesize an oligonucleotide primer sequence with a length larger than 50nt and does not need to carry out operations including PCR (Polymerase Chain Reaction), recovery of small molecular DNA by enzyme digestion, ligase treatment and the like, but generates recombinant plasmids by using junction of respective protruding cohesive ends of the linear vector and the annealing products in Escherichia coli. The method is simple, convenient and fast and has the advantages of economy, visibility, convenient screening and high accuracy.

Description

technical field [0001] The present invention relates to biological gene cloning and expression technology, in particular to a method for constructing RNA interference (RNA interference, RNAi) vector quickly, efficiently, at low cost and with high throughput. It is especially suitable for large-scale construction of animal and plant RNAi expression vectors for functional research on animal and plant genes. Background technique [0002] RNA interference (RNA interference, RNAi) is a ubiquitous, sequence-specific, post-transcriptional gene silencing process that exists in organisms. At present, the rapid realization of gene silencing in vivo by using RNAi technology has become a powerful experimental tool in the study of gene function in animals and plants. [0003] Due to the characteristics of animals and plants and the differences in their mechanisms of RNAi, the molecules of RNAi used in the study of gene functions in animals and plants are also different. At present, the...

AI Technical Summary

Problems solved by technology

Method (II) (III) is not only time-consuming and cumbersome, but also has a high probability of linking or self-ligating the vector to non-target fragments, which complicates the subsequent screening and identification process

[0007] 2. Not suitable for high-throughput operations

[0008] The fragments to be cloned in the above three methods all need to be purified in advance, and all rely on the ligation reaction between the carrier and the foreign fragments. Although this is feasible for the construction of a single or a small number of RNA interference vectors, it is difficult for large-scale construction of RNA interference vectors. but very difficult and inefficient

The oligonucleotide single strand used in method (I) and (II) is usually 60-100nt, and the oligonucleotide single strand of synthesizing this length not only improves synthetic cost greatly, and base synthesis error rate increases greatly, and then Greatly increased the workload of recombinant plasmid screening, time-consuming and laborious

The exogenous fragments in methods (II) and (III) all need to go through complicated steps such as double enzyme digestion and recovery. Because the exogenous fragments are generally small (within 200bp), the recovery and purification efficiency is low. These two methods not only The cost of the experiment is expensive, and the follow-up screening is difficult and the workload is heavy

Images