Learn.
Polymerase chain reaction (PCR) is a major tool in molecular biology which allows for rapid replication of DNA. It allows for fast, accurate duplications of DNA from low quality, however, it requires knowledge of target region and it’s sequence and it can replicate other genomes if they are contaminants.
The technique was founded by Kary Mullis in the 1980s, reportedly envisioning the idea while on a camping trip.
Denaturation – typically 1 minute
In order to start the reaction, the DNA must first be denatured – this is done around 94-96 °C – this unzips the DNA meaning G-C rich DNA will “stick” together for longer then the A-T rich DNA. At this temperature a bacterium polymerase from hot springs is required. The thermus aquaticus bacteria polymerase (Taq) has a half life of 1.6 hours at 95 °C. This means it can withstand this high temperature and still remain active afterword’s if it occurs for a short period of time.
This starting step means we can use DNA of low quality.
- Requires template strand of DNA
Annealing – typically 1 minute
The primers are typically longer then or equal to 18 nucleotides, melting temperature of the DNA strand and primer must be close and be close to the region of interest we wish to applify.
During the annealing phase, the primer binds to the DNA, for this reason the temperature is brought down to 60 °C.
- Requires primers
Elongation – typically 1 minute
The elongation is what allows us to double the DNA each subsequent round. The dNTP’s are added in a 5′ to 3′ order by the Taq Polymerase. The optimum temperature for Taq polymerase is 72 °C, allowing it to add approximately 1000 bases a min to a DNA strand. Therefore, the reaction is carried out at 72 °C.
- The substrates and the Taq polymerase is required for this step
This sequence is often repeated 30 to 40 times depending on how much amplification of the DNA is required.
It is carried out with:
- Buffer which optimizes pH, [Mg2+] – polymerase optimization, [KCl] – ionic strength optimization
Memorize.
Master.
Question 1.
Explain the specific temperature choice for each stage in the PCR reaction.
Question 2.
Outline the reactants required for a PCR reaction and what their functions are.