Method for constructing cDNA library

Abstract

The invention discloses a method for building a cDNA library which is characterized in that the method is simple, quick and does not need endonuclease. The method essentially comprises the following steps: (1) a first cDNA chain is synthesized with general RNA (or mRNA) as the template through reverse transcription with the pair of primers. (2) a second cDNA chain is synthesized with the pair of primers through PCR catalyzed by Tap DAN synthetase. (3) the PCR product obtained in step (2) is directly connected with a T-carrier through DNA ligase, and then colon bacillus competence is converted to obtain the cDNA library. The method has very low cost in building the cDNA library, needs a very small number of RNA templates, and is applicable to biological sample RNA of any source.

Description

technical field [0001] The invention discloses a method for constructing a cDNA library. Using a T-vector to rapidly construct a cDNA library is a method for separating and identifying functional genes in genetic engineering technology, which belongs to the category of molecular biology. Background technique [0002] Construction of cDNA library is an effective means to identify functional genes of unknown biological genomes. Since Rougeon et al. successfully constructed cDNA library for the first time in 1975, various methods of library construction have emerged, and the technical means have become more and more mature. The main The technical means include nothing more than: using polyd(T) oligosaccharide nucleotides and poly(A) complementary pairing of mRNA as primers, and using mRNA as a template to synthesize the first cDNA strand catalyzed by RNA reverse transcriptase. The second cDNA strand is then synthesized by DNA polymerase. In order to clone the synthesized cDNA ...

AI Technical Summary

Problems solved by technology

These methods all involve complex procedures such as linker primers and enzyme digestion, the operation is too cumbersome, and the success rate is not very high, let alone the library construction of trace precious samples.

Although recently there are also phage insertion / excision site-specific recombination systems (ie, GATEWAY TM system) to connect cDNA to the vector, but the efficiency of this recombination needs to be improved, and it is used for recombination Enzymes are very expensive and difficult to promote

In short, there is no simple, economical, fast and efficient method that does not require a large number of RNA samples and can construct cDNA libraries with high throughput and high success rate.

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