The appropriate oligonucleotide primer (oligo- T for eukaryotic mRNA) is annealed with the mRNA; this primer will base-pair to the 3'-end of mRNA. Reverse transcriptase extends the 3'-end of the primer using mRNA molecule as a template. This produces a RNA.DNA hybrid molecule, the DNA strand being the cDNA.

The RNA strand is digested either by RNase H or alkaline hydrolysis; this frees the single-stranded cDNA. Curiously, the 3'-end of this cDNA serves as its own primer and provides the free 3'-OH required for the synthesis of its complementary strand; therefore, a primer is not required for this step.

The complementary strand of cDNA single strand is synthesized by either the reverse transcriptase itself or by E. coli DNA polymerase; this generates a hairpin loop in the cDNA. The hairpin loop is cleaved by a single strand specific nuclease to yield a regular DNA duplex.

2. When the protein produced by a gene is known, it is purified and used to produce antibodies specific to it. These antibodies are used to precipitate the polysomes (mRNAs associated with ribosomes. and newly synthesized polypeptide chains), and the mRNA is isolated from them.
3. Some genes are expressed only in specific tissues, e.g., seed storage proteins in developing seeds, chicken ovalbumin gene in oviduct, etc. Therefore, mRNA preparations from such tissues are exceptionally rich in the concerned mRNA or may even contain only this mRNA.
Use of cDNA is absolutely essential when the expression of an eukaryotic gene is required in a prokaryote, e.g., a bacterium. This is because eukaryotic genes have introns, which must be removed from their transcripts to yield mature mRNA, and bacteria do not possess the enzymes necessary for removal of introns.
For example, cDNAs for interferon, blood clotting factor VIIIC (both human) and several other mRNAs have been expressed in bacteria.