PCR Technique
1.
Polymerase Chain Reaction (PCR) is a nucleic
acid amplification technique used to amplify the DNA or RNA in vitro
enzymatically.
2.
It is a temperature-dependent enzymatic process
where either a specific targeted region of DNA or the whole DNA is replicated
to quickly make millions of copies of the target DNA .
1.
It is temperature dependent cyclic process, and
by the end of the 20th to 25th cycle, over a million copies of the target
nucleic acid segment will be produced.
2.
It denatures the double-stranded DNA (dsDNA)
into single-stranded DNA (ssDNA), selects a segment of DNA to amplify using a
specific primer, and then follows the method of replication using DNA
polymerase enzyme to amplify that segment.
3.
Developed by Kary Mullis in 1993.
Components
of PCR
1.
Nucleic acid template (DNA)
2.
DNA Polymerase (Taq Polymerase
extracted from Thermus aquaticus)
3.
Primer (short sequences of
oligonucleotides)
4.
dNTp
5.
PCR buffer
6.
PCR machine
Steps of
PCR
Note : These following steps are performed inside thermocycler machine.
1. Denaturation
Ø
It is the 1st step of the
amplification reaction where the double-stranded DNA is thermally denatured
into two single-stranded DNA templates.
Ø
Temperature is raised to about 94°C (90 to 95°C)
for about 30 to 90 seconds.
Ø
At this temperature, the thermal energy
overcomes the weak hydrogen bonds holding the two DNA strands together,
allowing them to separate.
1. 2. Annealing
Ø
Denaturation is followed by the annealing step,
where the primer anneals (attach) the ssDNA templates at their complementary
sites.
Ø
The forward primer anneals at the
complementary site of the antisense strand, and the reverse primer anneals
at the complementary site of the sense strand of the template DNA.
Ø
For annealing to occur, the temperature is
reduced to 55°C-70°C (the annealing temperature differs based on the GC content
of the primer).
Ø
About 30 to 60 seconds are enough for annealing
in most of the PCR processes.
1. 3. Elongation
Ø It
is the final step in the amplification reaction where the temperature is raised
to 72°C so that the Taq DNA polymerase enzyme begins synthesizing new DNA
strands in the 5’ to 3’ direction.
Ø
The DNA polymerase enzyme adds nucleotides from
the reaction mixture to the 3’ OH- end of the annealed primer forming a new
complementary strand.
Ø Generally, elongation takes place at the rate of 1 kbp per 0.5 to 1 minute.
Ø At the end of elongation, two new dsDNA will be formed from a single dsDNA template at the beginning of the reaction.
