Gene Synthesis by Using mRNA
The mRNAs are the transcripts of genes that have to be translated into polypeptides or proteins. It is rather difficult to identify a particular gene on a chromosome. However, it is easier to pick up the mRNAs and synthesize a gene. The total population of mRNA in a cell remains high. The majority of mRNA contains a long polyadenylated tract i.e.
about 100 (A) residues at 3' terminus (Kates, 1970). Therefore, the mRNA can be separated from the rest of RNA population of the cell.
Synthesis of cDNA by using mRNA
The mRNAs are passed through an oligo-dT cellulose affinity column (Fig. 2.13). The poly (A) binds to (T). The oligo-dT segment contains 10-20 nucleotides which hybridizes the poly (A) tract of mRNA. The oligo-dT provides primer at poly (A) region with a free 3'-OH end. The reverse transcriptase uses the free end and synthesizes a single stranded cDNA in the presence of dCTP, dGTP, dATP and dTTP, and results in mRNA-cDNA hybrid. At the end the enzyme forms a loop by using the last few bases as the template. This results in synthesis of a short 'hairpin' loop at 3'end of the cDNA (Leis and Hurwitz , 1972). The mRNA is degraded from mRNA-cDNA hybrid by using alkali. This phenomenon is known as hydrolysis. Consequently mRNA is separated.
In the next step the single stranded DNA acts as template for the synthesis of double stranded DNA in the presence of polymerase I and all the four deoxynucleotides. The hairpin acts as primer for chain elongation. Finally, a double stranded cDNA is synthesized. SI nuclease is used to cleave hairpin loop and results in double stranded cDNA (Fig. 2.13).
In 1970, S. Mizutami, H.M. Temin and D. Baltimore discovered the RNA dependent DNA polymerase i.e.
reverse transcriptase in retroviruses. This enzyme yields single stranded DNA on RNA template. For the discovery of reverse transcriptase R. Dulbecco, Temin and Baltimore were awarded Nobel prize in 1975.
Land et al
(1981) have given an improved method for cDNA synthesis. The single stranded cDNA complementary to mRNA is tailed with oligo-dC tail. This process is facilitated by using the enzyme terminal transferase and adding dCTP nucleotide. The tailing is followed by oligo-dG priming of the second strand synthesis. This checks the formation of hairpin loop. Therefore, SI nuclease is not required.