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Recall the different steps within one cycle of PCR (names, temps, purpose of stage) recall that many cycles of these 3 steps occur when the reactions are in the thermocycler. why are many cycles performed?

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PCR, or Polymerase Chain Reaction, consists of three main steps in each cycle, which are:

  1. Denaturation:

    • Temperature: Typically around 94-98°C
    • Purpose: This step involves heating the reaction mixture to separate the double-stranded DNA template into single strands. The high temperature breaks the hydrogen bonds between the complementary bases, resulting in two single-stranded DNA molecules.

  2. Annealing:

    • Temperature: Usually around 50-65°C, depending on the melting temperature (Tm) of the primers used
    • Purpose: During this step, the temperature is lowered to allow the primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates. Primers are short sequences that initiate the synthesis of new DNA strands.

  3. Extension (or Elongation):

    • Temperature: Typically around 72°C, which is the optimal temperature for the DNA polymerase enzyme (commonly Taq polymerase)
    • Purpose: In this step, the DNA polymerase synthesizes a new DNA strand by adding nucleotides to the 3' end of the annealed primers, extending the new DNA strand complementary to the template strand.

Why Many Cycles Are Performed

Many cycles of these three steps (usually between 25 to 35 cycles) are performed in PCR for several reasons:

  • Amplification: Each cycle doubles the amount of DNA, allowing for exponential amplification of the target DNA sequence. A single target sequence can be amplified to millions of copies after just a few cycles.

  • Sensitivity: Multiple cycles allow for the detection of even very small amounts of target DNA. This is especially important in applications like forensic biology or medical diagnostics where the initial amount of target DNA may be limited.

  • Specificity: Repeated cycles enhance the specificity of the amplification process. As the target sequence is amplified over multiple cycles, non-specific products are less likely to accumulate in significant amounts, leading to a cleaner and more specific product.

Overall, the repeated cycling in PCR allows researchers and scientists to obtain a sufficient quantity of the desired DNA fragments for further analysis, manipulation, or application in various fields such as research, diagnostics, and forensics.