PCR

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 Polymerase Chain Reaction (PCR)


Polymerase Chain Reaction (PCR) is an in vitro biochemical reaction used in molecular biology laboratories to produce multiple copies of a specific gene sequence. A particular fragment of gene or a gene fragment is amplified by using primers complimentary to sequence needs to be amplified. The technique was first invented by a scientist Kary Mullis in 1983. The procedure functions similar to the DNA replication a natural phenomenon making multiple copies. However, in PCR, we make multiple copies of gene by using a DNA polymerase isolated from a microbe surviving hot springs "Thermus Aquaticus", as the enzyme can withstand the high temperature needs to undergo the process of polymerization. One of the most important ingredients of the chemical reaction are primers, i.e., forward and reverse primers. Forward primers adds nucleotides in 5' to 3' direction whereas reverse primers add nucleotide from 3' to 5' direction. Primers must be complimentary to the sequence we want to amplify. Once primers are bound to that sequence, DNA polymerase starts adding nucleotides to the existing strand. The most critical step during PCR is annealing step where primer bind with the specific sequence that we want to amplify. So, suppose we don't get anything on gel after conducting experiment, we need to lower the annealing temperature. Other important consideration is that GC content of the primer must be around 50%. We keep GC content lower as it can give non-specific products that we were not looking for. So, annealing temperature must be optimized to get best results. 

Following Steps are involved to under to undergo the process

Denaturation               95 ℃ for 1min

Annealing                    58-65℃for 30 Seconds

Extension                     72 to 75℃ for 30Sec

Ingredients

DNA template                       

Primers                                  

dNTPs                          

DNA Polymerase                   


PCR is used to diagnose several viral infections and genetic orders. PCR can be also be applied in forensic science to trace the criminal or suspect. 

There are different types of PCRs

Nested PCR

qPCR 



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