It represents only transcribed genes i.e., DNA copies of the RNA sequence (mRNA) and clone them.
Libraries of these types are highly feasible as they represent not only an expressed sequence of the gene, but also the sequences produced after post-transcriptional modification such as removal of introns etc.
Besides, cDNA molecules to be cloned are only a few kilobase long and conveniently inserted in plasmid cloning vectors.
However, viral lambda phage vector is preferred due to subsequent screening process:
(i) cDNA preparation by RNase H method:
This approach involves the synthesis of complementary DNA strands by reverse transcription to make an RNA: DNA duplex.
The cDNA synthesis requires separation of polyadenylated RNA (mRNA) from other RNA.
This can be accomplished by fractionation of RNA on oligo-dT cellulose in which short deoxy T residues have been covalently attached via hydroxyl groups of the cellulose.
mRNA is separated from the rest of the RNA.
This is accomplished by passing a solution containing RNA through a column of oligo-dT cellulose.
The poly A tail of the RNA forms hydrogen bond with oligo-dT, and poly A + RNA (mRNA) retains in the column.
After washing all other brand RNA from the column, the poly A + RNA is eluted with a low-salt buffer.
The purified mRNA is mixed with a reaction mixture containing reverse transcription and four deoxyribonucleotides.
The first step in cDNA synthesis is the annealing of chemically-synthesized oligo-dT primer to the 3′ poly A tail of the RNA.
Addition of 10-15 residue long primers initiates synthesis of the first DNA strand with reverse transcriptase and dNTPs.
As a result RNA: DNA duplex is formed.
In the next step, RNA strand is replaced by DNA strand.
This process can be initiated by adding a low concentration of RNase H together with DNA polymerase and dNTPs.
Treatments of RNA: DNA duplex with RNase H result in the nicking of RNA and produced free of 3′- hydroxyl groups.
This acts as primers at 3′ end to initiate DNA synthesis.
As DNA chains are synthesised, any molecule that is base-paired to the DNA template further down are degraded and leaves DNA duplex strand.
(ii) cDNA synthesis by self-priming approach:
In the second approach, once DNA: RNA duplex is secured, it is then subjected to treatment with dilute sodium hydroxide for alkaline hydrolysis of RNA leaving a single DNA strand.
Since oligo-dT cannot facilitate second strand synthesis, self-priming takes place by chance occurrence of complementary sequence between region near 3′-end and region to 5′-end which consequently attains hair pin loop.
The base paired region acts as a primer for the synthesis of the second DNA strand.
Presence of loop is removed by treatment with SI nuclease, which degrades single-strand regions (loops) and subsequently results in the formation of double-stranded blunt-ended DNA molecules.
The generation of blunt ended cDNA molecules is then attached with linkers before inserting them into the vector.
