In the last post, we discussed about the steps in recombinant DNA technology or gene cloning. In that post, we discussed genomic library as one of the source for getting our desired gene for cloning. In this post we will be discussing the second source of getting the gene of interest that is cDNA library.
Definition cDNA library: cDNA library represents the collection of clones containing cDNA of an individual organism under study or It represents the collection of mRNA complement of a single type of cell.
What is the advantage of cDNA library over genomic library?
Majority of the cases, we need to express a eukaryotic gene in a prokaryotic system like bacteria. The default choice of gene expression is E.coli. Eukaryotic gene is made of both introns and exons. For a gene to synthesize protein, prokaryotic system should have splicing mechanism or pre-mRNA processing mechanism where introns are removed and exons are joined together. As prokaryote like E.coli lack this mechanism, it is very difficult to express a eukaryotic gene in prokaryotic host like E.coli. This problem is solved by using cDNA for gene cloning. cDNA id synthesized form processed mRNA after splicing, therefore introns are absent. Thus cDNA can be used directly for gene expression in E.coli.
Construction of cDNA library
The procedure is same for all other organism
Step 1: Isolation of mRNA from the cells and synthesis of cDNA from this eukaryotic functional mRNA
cDNA synthesis from processed or mature mRNA is achieved using the enzyme reverse transcriptase; an RNA dependent DNA polymerase.
Step2: Cloning of synthesized cDNAs in suitable vector like plasmid or λ phage
The cDAN synthesized are joined with suitable adaptors using T4 ligase and 5’ P group is removed using alkaline phosphatase. The most common vectors for cDNA cloning include plasmid and λ phages
Step 4: Introduction to a suitable host like bacterium.
Introduce this rDNA molecule with cDNA fragments into a host, most often E.coli bacterium. Plasmid will multiply inside forming numerous copies. We need to identify the host cells with these fragments from the rest of cells without rDNA molecule.
Step 5: Screening and Maintenance of set of clones containing cDNA
Screening of colonies with rDNA is similar to genomic library. The most common screening procedure includes DNA hybridization or immunological assays. The clones with cDNA (rDNA) are selected. Maintenance of such clones or colonies containing cDNAs is the cDNA library