CDNA Synthesis

A cDNA is a polydeoxynucleotide duplex, the sequence of one strand which is complementary to RNA molecule. It is derived by the use of enzyme Reverse Transcriptase on the RNA as template.
There are many advantages of cDNA cloning because:
1. mRNA synthesis from cell is very easier and during cDNA synthesis from mRNA, ther would be no intervening sequence in it, so its expression like translation would be straightforward.
2. In plant cell or animal cell only about 1-10% of total DNA is functionally active because cDNA only represent the active genes which are smaller, hence simpler to construct and handle.
3. RNA viruses like influenza and reovirus which do not replicate via a DNA intermediate, only their cDNA can be cloned.
There are various step involved in cDNA cloning like mRNA enrichment, reverse transcription and selection process.
mRNA enrichment includes two phases as isolation of RNA and purification of mRNA. Isolation of RNA is used to obtain RNA from cell, firstly cell undergoes into cell lysis process. Cells contain ribonuclease and inactivation of this enzyme is very crucial for RNA Isolation. The ionic detergent, sodium dodecyl sulphate and sodium lauryl sarkosinate are frequently used to inhibit ribonuclease. After cell lysis, RNA is extracted with phenol or phenol chloroform mixture. The mixture pH should be bringing down to 4.7-5.2, saturate phenol and extracted RNA with acetate. This drives DNA to interphase of phenol and water and leaves RNA in the water phase. RNA is precipitated from aqueous phase by ethanol or isopropanol. By this process, we get 80-85% of rRNA, 10-15% tRNA and 1-5% mRNA. To obtain only mRNA, we should purify the mixture of RNA obtained from phenol chloroform method. In Prokaryotic cell, Sucrose density gradient centrifugation or gel electrophoresis can be done whereas for eukaryotic cell, poly adenylation can be done.
Reverse transcription is cDNA preparation which include production of mRNA-DNA molecule catalyzed by enzyme Reverse Transcriptase. dNTP’s are added to 3’end of exogenous primer. In eukaryotic cell, 3’end of the mRNA contain poly A tail so, primer used has almost been oligo dT.
Selection process includes various techniques as colony Hybridization, antibiotic sensitivity test, use of terminal transferase etc.