PCR (POLYMERASE CHAIN REACTION)
Content
Introduction .............................................................................................................................................2
Materias for PCR ......................................................................................................................................2
Method for PCR ................................................................................................................................... 3-4
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PCR (POLYMERASE CHAIN REACTION)
Introduction
PCR, or polymerase chain reaction, is a biochemical technique that can generate millions of copies of
a template strand of DNA. The technique relies on the same enzymes that cells use to replicate DNA,
however it is performed in a simple test tube using controlled cycles of heating and cooling. PCR has
revolutionized the field of biotechnology, making it quick and inexpensive to replicate, or amplify,
specific segments of DNA.The process of PCR has made it possible to perform DNA sequencing and
identify the order of nucleotides in individual genes. PCR techniques are applied in many areas of
biotechnology including protein engineering, cloning, forensics (DNA fingerprinting) and for analysis
of environmental samples.
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PCR (POLYMERASE CHAIN REACTION)
Materials for PCR
Template DNA (genomic, plasmid, cosmid, bacterial/yeast colony, etc.)
Forward primer (F)
Reverse primer (R)
Buffer (usually 10X, usually sold with Taq polymerase)
MgCl2 (50mM is convenient)
Taq DNA polymerase
dNTP mix
Sterile ddH20
Thermal cycler machine
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PCR (POLYMERASE CHAIN REACTION)
Method for PCR
1. Vortex all solutions for good mixing.
2. Adding nuclease free water to a pcr tube.
3. Adding Taq polymerase enzyme buffer.
4. Adding dNTP mix.
a. dNTP(Deoxyribonucleotide triphosphate) Mix is a premixed solution containing
sodium salts of
i. dATP,
ii. dCTP,
iii. dGTP
iv. and dTTP
5. Adding template DNA.
6. Adding
a. Forward primer (F)
b. and Reverse primer (R).
i. A primer is a strand of nucleic acid that serves as a starting point for
DNA synthesis.
7. Adding MgCl2.
a. It helps in the binding of primer (and the subsequent target DNA) to the
template DNA.
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PCR (POLYMERASE CHAIN REACTION)
8. Adding Taq DNA polymerase enzyme.
a. Taq polymerase, an enzyme originally isolated from the bacterium Thermus
aquaticus.
b. This DNA polymerase enzymatically assembles a new DNA strand from DNA
building-blocks, the nucleotides, by using single-stranded DNA as a template
and DNA oligonucleotides.
9. Place the prepared mixing tube to thermal cycler machine for starting cycling.
10. Setting PCR program:
a. Initialization step: This step consists of heating the reaction to a temperature of 94–
96°C which is held for 1–9 minutes.
b. Denaturation step: This step is the first regular cycling event and consists of heating
the reaction to 94–98 °C for 20–30 seconds. It causesDNA melting of the DNA
template by disrupting the hydrogen bonds between complementary bases, yielding
single-stranded DNA molecules.
c. Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds
allowing annealing of the primers to the single-stranded DNA template.
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PCR (POLYMERASE CHAIN REACTION)
d. Extension/elongation step: The temperature at this step depends on the DNA
polymerase used; Taq polymerase has its optimum activity temperature at 75–80 °C,
and commonly a temperature of 72 °C is used with this enzyme.
i.
At this step the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template strand by adding dNTPs that are
complementary to the template in 5' to 3' direction, condensing the 5'phosphate group of the dNTPs with the 3'-hydroxyl group at the end
of the nascent (extending) DNA strand.
e. Final hold: This step at 4–15 °C for an indefinite time may be employed for shortterm storage of the reaction.
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