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