1.The PCR (polymerase chain reaction) protocol that is currently used in laborat
ID: 56070 • Letter: 1
Question
1.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?
Explanation / Answer
a. In PCR, denaturation, annealing and extension steps usually occurs at high temperatures that 94-96oC, 55-60oC and 72-74oC respectively. So that, in the PCR reaction mixture, we must have the thermostable enzyme for proper results, unlike most of the enzymes which are usually active at biological temperature.
b. If it is not heat stable, that will become denature and inactive. So that, the polymerization of new DNA strand will be halted or not initiated.
c. Primer length should be 18-24 nucleotides, the Tm (52-58oC) of the primers pair must be +/- 2oC because, in the PCR, annealing takes place simultaneously for both primers. The Tm of primers also depends on GC content, so that we must consider this for primer design, since, G pairs with C by 3 hydrogen bonds that require more energy to dissociate than A=T pairing. Next, self-complementarity also needed to check, otherwise the primer pairs will anneal each other and forms primer dimers that give non-specificity as well inefficient amplification of new DNA strand.
d. 163860 copies from all 5 dsDNA segments. 215x5
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