Fast and efficient construction method of normalized full-length cDNA library

Abstract

The invention relates to a fast and efficient construction method of a normalized full-length cDNA library. The method consists of: using specific 5-OH oligonucleotide to close non-full-length mRNA, then employing tobacco acid pyrophosphatase to remove a cap structure from the mRNA5' end to make a phosphate group at the cap structure of the mRNA5' end exposed, adopting an efficient RNA connection system to connect a section of optimized oligomeric RNA to the mRNA 5' end to serve as a primer binding site for triggering synthesis of second strand cDNA, finally conducting reverse transcription, two-strand synthesis as well as graded purification, and cloning the double-stranded DNA into a vector. The method can concisely and efficiently screen full-length mRNA, and realizes full-length mRNA capture of eukaryotes. The mRNA5' tail end contains a biotin label, which can be used for full-length mRNA separation. The used oligonucleotide primer contains optimized modification method and a nucleotide sequence, thus being able to realize full-length mRNA capture of eukaryotes, reverse transcription and DNA synthesis.

Description

technical field [0001] The invention relates to a fast and efficient method for constructing a uniform full-length cDNA library, which belongs to the technical field of modern molecular biology. Background technique [0002] The full-length cDNA library can not only greatly improve the process of gene sequencing and bioinformatics analysis, but also facilitates later protein expression and functional analysis. For organisms that cannot carry out whole genome sequencing, it is an important way to conduct functional genome research. Even after the complete genome sequencing of model organisms, such as Arabidopsis thaliana, rice, and Caenorhabditis elegans, molecular biologists still constructed full-length cDNA libraries of the corresponding organisms, and performed large-scale full-length cDNA sequencing to in-depth Learn about the function of genes. Such as: Arabidopsis (A.thaliana), mouse (M.musculus), fruit fly (D.melanoga ster), rice (O.sati2va), etc., have produced a l...

AI Technical Summary

Problems solved by technology

[0017] Existing methods for constructing full-length cDNA libraries have their own characteristics and have certain defects. Some of them involve PCR amplification, which can easily change the representativeness of clones in the library and affect the cloning of difficult-to-amplify genes; Plasmids are carriers, which are not conducive to the cloning of large fragments of genes; some experiments have long procedures and cumbersome steps; some have high costs and low efficiency, etc.

Moreover, none of the four existing construction methods includes the key technical step of homogenization, but requires a separate homogenization process, which greatly increases the difficulty of operation, time cost and workload, and has relatively large technical defects.

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