What are the steps in PCR?

• PCR is closely patterned after the natural principle of DNA replication. It is a three-step process, referred to as a cycle, that is repeated a specified number of times.
• One PCR cycle consists of the following steps:
   1. Denaturation
   2. Annealing
   3. Extension
  
  
• This process takes place in a thermal cycler, an instrument that automatically controls and alternates the temperatures for programmed periods of time for the appropriate number of PCR cycles (usually between 30 and 40 cycles).

PCR Step 1: Denaturation by Heat

• Heat (usually >90°C) separates double-stranded DNA into two single strands, referred to as "Denaturation".

Since the hydrogen bonds linking the bases to one another are weak, they break at high temperatures, whereas the bonds between deoxyribose and phosphates, which are stronger covalent bonds, remain intact.

PCR Step 2: Annealing - Primer Binding to Target

• The goal is not to replicate the entire strand of DNA but to replicate a target sequence of approximately 100-600 base pairs that is unique to the organism.
• Primers mark the ends of the target sequence: AMPLICOR® Test primers are short, synthetic sequences of single-stranded DNA typically consisting of 20-30 bases, with a biotin-labeled 5' end to aid in detection. They are specific for the target region of the organism.
• Two primers are included in the PCR, one for each of the complementary single DNA strands that was produced during denaturation. The beginning of the DNA target sequence of interest is marked by the primers that anneal (bind) to the complementary sequence.
• Annealing Temperature: Annealing usually takes place between 40°C and 65°C, depending on the length and base sequence of the primers. This allows the primers to anneal to the target sequence with high specificity.

PCR Step 3: Extension

• Once the primers anneal to the complementary DNA sequences, the temperature is raised to approximately 72°C and the enzyme Taq DNA polymerase is used to replicate the DNA strands.
• Taq DNA polymerase is a recombinant thermostable DNA polymerase from the organism Thermus aquaticus and, unlike normal polymerase enzymes is active at high temperatures.
• Taq DNA polymerase, begins the synthesis process at the region marked by the primers. It synthesizes new double stranded DNA molecules, both identical to the original double stranded target DNA region, by facilitating the binding and joining of the complementary nucleotides that are free in solution (dNTPs)
• Extension always begins at the 3' end of the primer making a double strand out of each of the two single strands. Taq DNA polymerase synthesizes exclusively in the 5' to 3' direction. Therefore, free nucleotides in the solution are only added to the 3' end of the primers constructing the complementary strand of the targeted DNA sequence.

End of the First PCR Cycle

• At the end of the first PCR cycle, there are now two new DNA strands identical to the original target.
• Ending Point: The DNA polymerase does not recognise the end of the sequence. The newly formed strands have a beginning, which is precisely defined by the 5' end of the primer, but the 3' end is not precisely defined.
• As the number of cycles increases, a strand with more defined length frequently serves as the template for the newly synthesized sequence.
• The DNA strand synthesized from such a template then has a precisely defined length that is limited at either end by the 5' end of each of the two primers. These DNA strands are called an AMPLICON.
• After only a few cycles, DNA strands which correspond to the target sequence, are present in much larger numbers than the variable length sequences.
• In other words the sequence flanked or defined by the two primers is the section that is amplified.