There are two ways to synthesize specific deoxyribonucleic acid (DNA) fragments to insert for recombinant DNA. They can be synthesized in vitro prior to cloning. Those two ways are as follows:
• The DNA can be amplified from genomic sequence by using polymerase chain reaction (PCR).
• By copying the m RNA sequences into complementary DNA (cDNA) using enzyme reverse transcriptase.
In the in vitro method, the reaction takes place inside the test tube. Polymerase chain reactions occur inside the test tube. A particular gene or important section of DNA is isolated and amplified by DNA polymerase. This DNA polymerase is obtained from the heat-tolerant bacteria. PCR finds the target DNA by the complementary binding of certain short primers to the ends of that sequence. Then the replication process was directed by the primers. The replication process leads to the production of huge amount of the aimed DNA as an isolated DNA fragments.
Another technique to produce specific DNA fragments is by copying the mRNA sequence into cDNA. The cDNA is a DNA form of mRNA molecule. The cDNA is used more than mRNA for the reason that RNAs are naturally less stable than the DNA. The other reason is the methods for regularly developing and purifying individual RNA molecules are not available. From the mRNA molecule cDNA can be produced with the use of reverse transcriptase, isolated from retroviruses.
Just before creating cDNA, the purified mRNA is added to the test tube containing the enzyme reverse transcriptase, the four dNTPs, and a short primer of polymerized dTTP residue called an oiligo-dT primer. The primer molecule anneals to the poly (A) tail of the mRNA being copied. By utilizing the mRNA molecule as a template, reverse transcriptase catalyzes the production of a single-stranded DNA molecule beginning from the oligo-dT primer. Then, the cDNA is copied into a double-stranded cDNA by DNA polymerase.
Thus, the genomic DNA from PCR or double stranded cDNA can be introduced into recombinant DNA for further development.