Module 2: DNA and genes
A quick primer on how to set up a PCR profile
The PCR reaction, despite being brilliantly simple, has also been the bane of many a graduate student’s existence (current author included)! The reaction that we are using here is a known reaction that should be fairly forgiving in terms of variation in the preparation.The following is a short explanation of the “typical” PCR profile. A “typical” PCR profile consists of three steps: denaturing, annealing and extension. The initial step denatures the target DNA by heating it to 94°C or higher for 15 seconds to 2 minutes. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase. In the next step of a cycle, the temperature is reduced to approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the denatured target DNA and serve as primers for the DNA polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70–74°C. The extension step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C for denaturation.