[ajithnandanam]

Creating cDNA Library involves the following Steps:

Isolation / Extraction of mRNA

Construction of cDNA / complementary DNA

For the synthesis of cDNA from mRNA poly A tail is used as priming site, a short tag of oligo dT with a free 3'OH group will bind and which will be extended by reverse transcriptase to create cDNA. Next job is to remove the mRNA which is achieved by treating it with RNase enzyme resulting in the single stranded cDNA. The single stranded cDNA need to be converted to double stranded cDNA which is achieved with the help of DNA polymerase. The free 3'OH group for polymerase extension is provided by the single stranded cDNA itself by forming a hairpin loop like structure, which can later be cleaved using nuclease.

Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.