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:

  1. DNA has to be isolated and verified from the target organisms.
  2. Primers have to be designed, ordered, resuspended, and diluted to the proper concentrations.
  3. The experiment has to be designed, and the workspace has been prepared.
  4. A program has to be created on the thermocycler.
  5. 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:

  1. Inital Denaturation
  2. Amplification (30-40 cycles)
  3. Final Extension
  4. Cooling

The amplification cycles in step two can be further broken down:

  1. Denaturation
  2. Annealing
  3. 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.