Question

Replication is a process that occurs naturally in a cell, where PCR is done by scientists in a laboratory. Compare replication and PCR by discussing the components and processes that are similar and the components and processes that are different. Finally, tell me why PCR is important in RFLP analysis.

Answer 1

In the process of DNA replication, this occurs in 5’ to 3’ direction, use nucleotides and the Polymerases for addition of nucleotides in both (In vivo in the cell and in vitro in PCR).

DNA replication which is naturally occurs in the cell is carried out at body temperature (37˚C in humans) and it involves more complex machinery than that of PCR. The natural DNA replication in the cell use many enzymes like helicase which unwinds the double stranded DNA, single-strand-binding proteins (SSB proteins) which stabilize the unwounded form of DNA strands, Topoisomerases which are involves in the removal of over and under winding of the DNA strands and the DNA polymerases whereas the PCR involves many reactions of temperature cycles (70-90˚C) for the denaturation and annealing of DNA strands.

In the eukaryotes cell the polymerases are responsible for the addition of nucleotides are different than that of the polymerases used in PCR. The polymerases which are used in PCR are thermostable polymerases. The PCR polymerases are isolated from many living sources like from archaea and from bacteria like Taq polymerase (thermostable polymerases). In the body the DNA of every cell (whole genome) is routinely replicated by the cell’s DNA polymerases whereas in PCR reaction, the polymerases which are only efficient for the smaller fragment of DNA and it less half-life.

The DNA polymerase of the cell has high speed and fidelity, proofreading activity and repair activity which are desirable features and are required for more accuracy of the DNA replication whereas PCR used simpler polymerases which have no proofreading activity.

	In vivo replication in living cell	In vitro replication in PCR
Primers	RNA	DNA
Denaturation	By Helicase	By Heat
Polymerases	DNA polymerase	Taq polymerase and other thermophillic DNA polymerase like ampliTaqGold polymerase, PlatinumTaqPolymerase, Pfu DNA polymerase, etc
common in both situations	Nucleotides which are needed for elongation, DNA serves as template for new strand	Nucleotides which are needed for elongation, DNA serves as template for new strand

In short PCR produce large amount of DNA from less amount of nucleic acid. In the cells, the in vivo DNA replication involves a well defined but more complex biological compound like enzymes and other factors whereas in PCR the in vitro DNA synthesis is simpler in comparison with in vivo replication by using a smaller set of enzymes and reaction at relatively high temperatures.

For the analysis of the RFLPs there are sufficient amount DNA is needed which takes much more time and also labor intensive. As we knows that the PCR can amplify very less amounts of DNA and takes less time in comparison with DNA isolation which required for RFLP analysis. Therefore, by using PCR in RFLP analysis we can analyze more samples in shorter time. So that PCR is important in RFLP analysis.