Question

The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95°C.a. Why would such a heat-stable polymerase be beneficial in PCR?b. What would happen if it weren’t heat stable?c. How might you choose a region of DNA for a PCR primer so as to increase the temperature necessary for primer annealing (to minimize nonspecific PCR products)?d. A PCR reaction begins with 5 double stranded segment of DNA. Estimate the number of double-stranded copies of DNA that are present after the completion of 15 amplification cycles?

Answer 1

Answer and Explanation:

a. Why would such a heat-stable polymerase be beneficial in PCR?

- Because in PCR, DNA is heated up 95 °C to denature DNA (see first figure)

b. What would happen if it weren’t heat stable?

- If it weren't heat stable we had to add it in every PCR cycle and please note that PCR can take 20 to 35 cycles. Imagine being researcher that you need to open 20 small tubes every 5 minutes and add polymerase enzyme into these tubes for 20 to 35 times. It is very labor intensive and Taq polymerase relives the researchers from this work.

c. How might you choose a region of DNA for a PCR primer so as to increase the temperature necessary for primer annealing (to minimize nonspecific PCR products)?

- You need to calculate melting temperature (Tm) of your primers and use the calculated values to prevent non specific bindings. Primers usually binds non-specifically if the low annealing  temperature is used (lower than 5 °C of your Tm value)

d. A PCR reaction begins with 5 double stranded segment of DNA. Estimate the number of double-stranded copies of DNA that are present after the completion of 15 amplification cycles?

In every amplification cycle copies of DNA are doubled. So the answer is 5 x 2^15.