6.1 Polymerase Chain Reaction (PCR)
Watch the PCR video below to get a better understanding of the process.
To actually start a PCR experiment, several things need to take place:
- DNA has to be isolated and verified from the target organisms.
- Primers have to be designed, ordered, resuspended, and diluted to the proper concentrations.
- The experiment has to be designed, and the workspace has been prepared.
- A program has to be created on the thermocycler.
- The QiAxcel has to be prepared for a gel read.
6.1.1 Primers
Primers are the cornerstone of any PCR. Many vendors sell primers that already work with a particular gene. However, many scientists have to design their own primers, because the gene of interest is not available from a commercial distributor. After the primers have been obtained, they need to be resuspended in DNAse free molecular grade water. This is called the “stock solution”. A “working solution(s)” should also be made to preserve the stock solution.
6.1.2 Restriction Fragment Length Polymorphism (RFLP)
RFLP is a genotyping technique that exploits and simultaneously identifies genetic variations in homologous DNA sequences. Restriction enzymes are used to break DNA into 2 or more fragments that are then analyzed with gel electrophoresis. Different alleles can be detected when the associate polymorphisms cause the restriction fragments to be differing sizes.
6.1.3 Thermocycler
A thermocycler is a machine that amplifys DNA using PCR. The thermocycler systematically raises and lowers the temperature of the holding block using a Peltier device. The end user must program each step into the thermocycler, based on the methods of the PCR reaction. Generally, a thermocycler program will take the PCR reaction through 4 different steps:
- Inital Denaturation
- Amplification (30-40 cycles)
- Final Extension
- Cooling
The amplification cycles in step two can be further broken down:
- Denaturation
- Annealing
- Extension
While the temperatures and timing of each step may change with each PCR reaction, most of the time these basic steps will be worked into the thermocycler program.