Biotechnology and Bioinformatics (1+1)

2.6.1.Construct ion and screening of a complete expression of cDNA library

A complete cDNA library theoretically contains all cDNA clones corresponding to all mRNAs expressed in a cell or tissue type.

An expression cDNA library refers to one in which all cDNAs are cloned in the sense orientation so that all the cDNAs in the library can be induced to express their mRNAs and proteins.

As a result, this cDNA library can be screened with specific antibodies against the expressed protein of interest or with a specific nucleic acid probe. In theory, this type of cDNA library preserves as much of the original cDNAs as possible, which can allow one to ‘fish” out any possible cDNA clones by screening the cDNA library as long as specific probe or specific antibodies are available.

Jiang et al. (1989) showed that antifreeze protein (AFP) existed in the serum of winter flounder, Psuedopleuronectes americanus and they prepared and purified the AFP mRNA. A gene probe was synthesized according to the AFP gene sequence of Pseudopleuronectes americanus, and subsequently hybridized with the mRNA from P. americanus.

A single-stranded cDNA was enzymatically synthesized with reverse transcriptase. Double-stranded cDNA synthesize was carried out with polymerase and SI nuclease treatment. The cDNA was then placed in an E. coli JM83 cell using pUC 19 as the vector, thus enabling the cloning of P. americanus cDNA.

The construction of gene libraries for common carp and grass carp were first reported by Zhu et al. (1990). GH of common carp and grass carp were screened and hybridized with the GH gene probe of Salmo salar and gene cloning was also carried out in fishes.