19 Polymerase Chain Reaction.


a. Principle :


Polymerase chain reaction (PCR), first described by Saiki R.K. et.al. 1985(1) is relatively simple technique by which a DNA or c-DNA template is amplified many thousands or million-fold quickly & reliably. By amplifying just a small portion of a nucleic acid target, it is possible to isolate that portion from the rest of the nucleic acid sample.  The PCR process is exquisitely sensitive. While most biochemical analysis, including nucleic acid detection with radioisotopes requires the input of significant amounts of biological material, the PCR process requires very little.  This feature makes the techniques extremely useful, not only in basic research, but also in commercial uses including genetic identity testing, forensics, industrial quality control, and in vitro diagnostics.


As originally developed, the PCR process amplifies short (approximately 100-500 bp) segments of a longer DNA molecule. A typical amplification reaction includes the sample of target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium & optional additives.  The components of the reaction are mixed and the reaction mixture is placed in a thermal cycler, which is an automated instrument that takes the reaction through a series of different temperatures for varying amounts of time.  This series of temperature and time adjustments is referred to as one cycle of amplification.  


Each PCR cycle theoretically doubles the amount of targeted template sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor of more than a million in a matter of hours.



Each cycle of PCR amplification consists of a number of steps, which produce two oligonucleotide-primed single stranded DNA templates, set up the polymerization reaction and synthesize a copy of each strand of the template being targeted.  These steps should be optimized for each template and primer pair combination.  The initial step in a cycle denatures the target DNA by heating it to 95°C or higher for 15 seconds to 2 minutes.  In the denaturtion process, the two interwined strands of DNA separate from one another producing the necessary single-stranded DNA template for the thermostable polymerase.


The next step of cycle reduces the temperature to approximately 40°-60° C. At this temperature, the oligonucleotide primers can form stable associations (anneal) with the separated target DNA strands and serve as primers for DNA synthesis by a thermostable DNA polymerase. This step lasts approximately 30-60 seconds.  


Finally the synthesis of new DNA begins when the reaction temperature is raised to the optimum for the thermostable DNA polymerase. For most thermostable DNA polymerases this temperature is approximately 74° C.  Extension of the primer by the thermostable polymerase lasts approximately 1-2 minutes. This step completes one cycle, and the next cycle begins with a return to 95° C for denaturation.  After 20-40 cycles, the amplified nucleic acid may then be analyzed for size, quantity, sequence, etc. or used in further experimental procedures, e.g. cloning.  


B. Materials :


a)Template DNA(B.malayi mf) - 100 ng/µl.


b)Taq Polymerase (1-2 units) (Appendix III)


c)Two oligonucleotide Primers (Appendix I) *


   e.g. forward and reverse Primer (200 ng/µl)


d) Deoxynucleotide triphosphates (2.5 mm) mix solution.


e)10 X Taq Polymerase assay buffer with magnesium Chloride (1 x for final reaction volume).


f)Autoclaved Double Distilled Water/Milli Q water.


* Primers designed to amplify the gene for ES Ag (Excretory-Secretory) from B.malayi genomic DNA.




C. Method :


For 50 µl Reaction, the components are added as mentioned below.


1)Add 38 µl of Milli Q or autoclaved double distilled water into 0.2/0.5 ml microfuge tube.


2)Add 5 µl of 10 x Taq Polymerase assay buffer with Magnesium chloride.


3)Add 3 µl of 2.5 mM dNTP mix solution.


4)Add 1 µl of template DNA isolated from B. malayi microfilariae (200 ng/µl)


5)Add 1 µl each of forward & reverse primers (250 ng/µl)


6)Add 1-2 units of Taq DNA Polymerase (1µl)


7)Mix the solution gently.


8)Layer the reaction mix with 50 µl of mineral oil to avoid any evaporation.


9)Carry out the amplification using following reaction conditions for at least 35 cycles. Initial denaturation temperature at 94°C for 1 minute


1)Denaturation at 94° C for 30 Sec.


2)Annealing at 55° C for 30 Sec.


3)Extension at 72° C for 1 minute.


Steps 1 to 3 are repeated to 35 times.


4)Final extension at 72° C for 2 minutes.  


After the reaction is over, take out the reaction mixture and run 10 µl of it on 1% Agarose gel