Hoxful Monsters

One of the principle objectives of performing a PCR reaction to have DNA amplified with no errors.The amplified product will be of no use if its gets copied with altered bases.

DNA replication ( which forms the crux also in in vitro PCR reaction) in vivo is carried out with high fidelity ,thanks to proof reading ability of the DNA polymerase.During the replication of mammalian genomes ,only one base in about (3 × 10 power 9) is copied incorrectly.Proof reading property of DNA polymerase enables it to ensure a check on wrong base from getting integrated.

Unlike RNA polymerases, DNA polymerases absolutely require the 3′-hydroxyl end of a base-paired primer strand as a substrate for chain extension. Additionally, DNA molecules with a mismatched nucleotide at the 3′ end of the primer strand are not effective templates for DNA synthesis.Most of the DNA polymerases contain an integral 3′to 5′ exonuclase activity. whenever an in correct base gets inserted during synthesis of DNA ,the elongation of chain stops and instead the 3′ → 5′ exonuclease activity removes one nucleotide at a time from the 3′ hydroxyl terminus until a correctly base-paired terminus is obtained, enabling DNA synthesis to proceed again.

In this way DNA polymerases are self correcting and thus ensures great accuracy while replication of genomes.

The most widely used enzyme for PCR’s is Taq DNA polymerase derived from T. aquaticus. This DNA polymerase, however, has no associated 3′ → 5′ exonuclease to confer a proofreading function, and the error rate due to base misincorporation during DNA replication is rather high.

In order to overcome this problem and to ensure error free amplification researchers looked for various alternatives.The problem of infidelity of Pcr was reduced considerably with the use of heat stable DNA polymerase with 3′ → 5′ exonuclease activity ( eg :Pyrococcus furiosus DNA polymerase) .There are so many efficient DNA polymerases are available now a days in market and most of them do considerable great job.Even I used quite a few in last two years,but pretty much influenced by the Phusion taq from FINZYMES. The error rate with this enzyme is very less, zero one can say.Sometimes people in lab also amplify a cDNA around 5KB without any mutations ,which is remarkable to say the least.I cloned around 30-40 constructs for making transgenics and thanks to phusion it made my job rather simple.Unfortunately I discovered this enzyme slightly late (after finishing cloning for 20 constructs or so) but better late then never.