Question

Which of the following lives not essential for polymerase chain reaction(PCR).
(a) Restriction enzyme
(b) Denaturation enzyme
(c) Primers
(d) DNA polymerase

Answer 1

PCR is an effective procedure for generating large quantities of a specific sequence of DNA in vitro. PCR generally requires a target DNA strand, two oligonucleotide primers, an excess of four deoxyribonucleotides, and a heat-stable DNA polymerase.PCR is now a common technique used in medical and biological research labs for a variety of applications.

Complete step by step answer:
PCR generates microgram quantities of a DNA segment up to billion copies from a single copy within a few hours. Requirements of PCR; a target sequence in a DNA sample that lies between a pair of polymers and can be from 100 to 35000bp long, two synthetic oligonucleotide primers complementary to target DNA sequences present at 3’ ends of two strands of the desired segment. The primers flank the target DNA sequence. After annealing to the source DNA, the 3’ hydroxyl ends of the primers are oriented toward each other. An excess of four deoxyribonucleotides, namely dATP, dGTP, dCTP, dTTP, A heat- stable DNA polymerase. e.g., Taq polymerase, isolated from the bacterium Thermus aquaticus, growing in hot springs, pfu isolated from Pyrococcus furiosus or vent polymerase from Thermococcus literalism, various buffers and cofactors like magnesium ions, required by DNA polymerase. Each cycle of PCR consists of 3 steps; Denaturation, annealing, and extension. PCR does not require the difficult and costly storage of restriction enzymes, ligase, and vector DNA.

So, the correct answer is ‘Restriction enzyme’.

Additional Information:
- At the start of PCR, the DNA from which, a segment to be amplified, an excess of the two primer molecules, the four deoxyribonucleotide triphosphate and Taq- DNA polymerase are added to the tube.
- Denaturation is the first step in which the target DNA melts at 95°C for 15 to 30 seconds to separate the strands.
- In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA.
- The selectivity of PCR results from the use of primers that are complementary to the DNA specific thermal cycling conditions.
- In the third step, which is the primer extension; temperature is raised above 75°C. At this step, DNA synthesis begins at 3’- hydroxyl end of each primer, with the help of nucleotide triphosphate and thermostable DNA polymerase.

Note: PCR technique was developed by Kary Mullis (American scientist) in the year 1985 and he was awarded the Nobel prize in chemistry in the year 1993.PCR is more efficient as it needs only much less amount of desired DNA, a single copy is enough. It requires monogram quantities of DNA while gene cloning requires microgram amounts. PCR requires far less work, time, and skill. PCR is fully automated and it has more applications than gene cloning. PCR techniques are used in studies of molecular evolution, production RADP, the study of polymorphism, etc.