Question

A typical PCR program is listed below. Define what is happening to promote DNA in steps 2‐4 below amplification (i.e., what happens to DNA and enzyme in those steps). PCR program – C487 Amplification 1. 95° C for 1 minute 30 seconds 2. 95° C for 30 seconds (melting) 3. 52° C for 30 seconds (annealing) 4. 72° C for 60 seconds (DNA elongation) 5. Repeat steps 2‐4, 29 times (30 cycles total) 6. 72° C for 5 minutes 7. Hold at 4° C

Answer 1

In step 2: Denaturation of the double-strand occurs.

In step 3: Annealing of the primer to the single strands.

In step 4: Extension or elongation takes place in this step.

Explanation:

In the PCR program an enzyme Taq Polymerase is used because it can withstand high temperature without altering its functions.

PCR is required for the amplification of DNA into multiple copies for experimental purpose. The artificial environment is created to form new DNA molecules from the sample in question.

The first step in replication is the opening of the double helix which is done by temperature treatment in PCR. The temperature would be 90 degrees for some 30 sec to two minutes.

The next step of primer annealing would be done at 52 degrees, this is the primer melting temperature.

The elongation of the DNA strand to be synthesized will take place at 72 degrees as Taq Polymerase can withstand that temperature.

Nearly one million copies of DNA will be made after 30 cycles of PCR.

PCR products can be stored at 4 degrees for some two months.