Highly efficient and rapid homogenized full-length cDNA library construction method

Abstract

The invention discloses a highly efficient and rapid homogenized full-length cDNA library construction method, which comprises the following steps: under the guide of PCR (Polymerase Chain Reaction) primers and oligonucleotide primrs, mDNA is reversely transcribed by a reverse transcriptase, so that first strand cDNA can be synthesized; when the purification of the first strand cDNA needs to be finished, the first strand cDNA is put onto ice to terminate reaction, the first strand cDNA is then put into a test tube, the test tube is then put onto a preheated PCR instrument, the first strand cDNA utilizes the PCR primers and anchor primers again, an improved Cap-trapper method is utilized to synthesize and amplify second strand cDNA in a PCR instrument and the second strand cDNA is stayed overnight under the condition of 14 DEG C to 20 DEG C, Lambda bacteriophage packaging reaction is respectively carried out on the second strand cDNA, and the unamplified second strand cDNA library is stored under the condition of 1 DEG C to 4 DEG C for 12 to 18 days. When constructing a bacterial cDNA library, the method can utilize characteristics to carry out reverse transcription to provide a theoretical basis, the recombination rate is high, and the requirement of current biological development is met.

Description

technical field [0001] The invention relates to the technical field of cDNA library construction, in particular to an efficient and rapid method for constructing a uniform full-length cDNA library. 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 ...

AI Technical Summary

Problems solved by technology

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 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.