The ability to synthesize DNA from an RNA template, via reverse transcription, enables researchers to study RNA with the same molecular approaches used for DNA investigations. cDNA generated by reverse transcription can be amplified using polymerase chain reaction (PCR). The combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample. In the first step of the PCR process, the cDNA is denatured by heating to 95°C, which disrupts the hydrogen bonds between complementary strands, yielding single-stranded molecules. The temperature is then lowered in order to allow primers complementary to the sequence(s) of interest to anneal. The DNA polymerase included in the reaction will then begin DNA synthesis. At this point, the temperature is raised to the optimal activity temperature of the DNA polymerase (usually 72°C) to synthesize a new strand complementary to the template. The process of denaturing, annealing, and extension can be repeated multiple times, with a two-fold increase in the amount of DNA molecules with each cycle. Because PCR can selectively amplify a template, it is an important method for detecting specific nucleic acid molecules in a particular cell or small populations of cells. PCR Products can be used in many downstream applications, such as cloning into plasmid vectors and sequencing using next generation sequencing platforms.
- Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D.C.: ASM
Press, 2007. 129-132.