POLYMERASE CHAIN REACTION (PCR): 

The invention of polymerase chain reaction technique has revolutionized the field of research, using this technique we can make million copies from a single molecule of DNA, within few hours. This technique was discovered by Kary Mullis in 1983 for which he was awarded a Nobel prize in 1993.using this technique makes sequence analysis, cloning, mutagenesis, diagnostics, identification of infections as well as criminals and establishing family relationships very easy.

PRINCIPLE:

This PCR mixture undergoes replication cycles which involve the following three steps:

 PCR machine under process.                                            The monitor showing the details of polymerase chain reaction.

Enzyme Used

Taq polymerase is widely used DNA polymerase for PCR and is stable at high temperature. However, there are other heat stable polymerase like Thermus thermophillus(Tth) DNA polymerase, which can also perform reverse transcription in presence of Mn⁺² ions(Myers T. W. and Gelfand H.D., 1991). Thermococcus litoralis DNA polymerase also known as Vent polymerase is very good alternative for Taq polymerase, it also performs 3’-5’ proof reading exonucleases activity and facilitate the highly accurate DNA synthesis in vitro.(Mattila P. et.al., 1991)

Concentration of polymerase used may range between 0.5-2.5units/100µl.Depending upon the experiment it is optimized in between 0.5 to 5 units /100µl. Over all, it should be high enough to bind the template at correct position but low enough to avoid non specific annealing.

Primers

Primers used are very important in successful amplification of the target sequence. The two primers should be complimentary to the 3’ end of two strands and should not have self complimentary region which can result in hairpin formation with in primer region. Secondly, the two primers should not have region complimentary to each other which will lead to primer dimer formation.

Length of primer is also very important to successfully hybridize with correct position. For instance if the primer sequence is of 8 bp; 4⁸=65,536bp, this would give about 46,000 possible sites in human genome and with increase in length of the primer the possibility of primer to bind precisely at wanted position increases. However, if the length of the primer is too long then its possibility to hybridize completely with template sequence decreases. So the length of primer should be long enough to hybridize at correct position and short enough to get it completely hybridize with the 3’ end of the DNA to be amplified.

Annealing Temperature

Temperature which will support the proper annealing is very important. If the temperature is too low it supports the non specific hybridization of the primers and if it is too high the annealing do not occur.

Annealing temperature is set by calculating the melting temperature(T_m) and is set 1-2^oC below it. Formula for melting temperature is:

T_m=4[(G+C) + 2(A+T)]

Where G, C, A and T are number of G,C,A and T present in the primer sequence.

And both the primer designed should be designed talking in mind that the annealing temperature should be same for both the primers.

In total, the successful PCR product is obtained by selecting appropriate length of primer, annealing temperature, extension temperature for the appropriate chosen polymerase, ionic strength of metal required and length of target sequence.

Advantages of PCR

It is very sensitive technique and even a single template is enough to produce sufficient amount of copies. The PCR requires 45-90min to make copies unlike gene cloning which require many hours to grow the cloning vector. PCR can also be used to produce cDNA copies of the mRNA by using the Tth polymerase in presence of Mn⁺² ions in less time. It can be modifies according to the target sequence and has been used in various ways like Inverse PCR, Anchored PCR, RT PCR, Site directed mutagenesis using PCR, Overlap Extension PCR, Asymmetric PCR, Thermal Cycle sequencing PCR, Nested PCR, Touchdown PCR, Hot start PCR  and ARMS PCR. So PCR is very powerful tool and has very vast use in field of Life Sciences.

Limitation of PCR 

PCR involves hybridization of primer with known sequence which flanks the target sequence or sequence to be amplified. So cannot be use if sequence of flanking region is not known.

With the increase in length of amplicon(sequence to be amplified) the efficiency of the PCR decreases. So very long sequences i.e. sequences upto several mega bases can only be done by breaking the sequence into pieces which is not always suitable and gene cloning fits to be better option for very long sequences

All DNA polymerase including Taq polymerase commit error and require proof reading which is done by them self but Taq polymerase lacks the ability if proof reading and as a result with every cycle the error get copied and multiplied which reaches to 1 error per 300bp after 30 cycles. For this reason most of the labs set the cycle between 20-30. This could be overcome by using Vent polymerase which is heat stable and posses 3’-5’ proof reading exonuclease activity.

Exponential amplification by PCR change to linear after 20-25 cycles because of the limited reagents, exhaustion of the enzyme and high concentration of product which tend to re-anneal with each other.

References:

·              Burke J.F., 1996, PCR Essential Technique. Chichester: John Wiley & Sons Ltd.

·       Mattila P. et.al., 1991, Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading                 activity. Nucleic Acids Research, Vol. 19, No. 18 4967-4973.

·       Erlich H.A. , 1989, Polymerase Chain Reaction, Journal of Clinical Immunology, Vol. 9, No. 6.

·       Innis M.A., 1990; PCR Protocols A guide to Methods and Applications. London: Academic Press Limited.

·       Myers T.W. and Gelfand D.H., 1991, Reverse Transcription and DNA Amplification by a Thermus                             thermophilus DNA Polymerase,Accelerated Publications Biochemistry, Vol. 30, No. 31.

·       Pherson M.J., Ouirke P. and Taylor G.R., 1991; PCR A Practical Approach. Oxford: Oxford Press University.

·       Sambrook J. and Russel W.D., 2001, Molecular Cloning Laboratory Manual, 3^rd Edition. New York: Cold spring Harbour laboratory Press.