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PCR - Polymerase Chain Reaction

PCR is a technique that amplifies a specific DNA sequence using DNA polymerase. It requires a DNA template, primers that flank the sequence of interest, DNA polymerase, dNTPs, and thermal cycling between high and low temperatures. During each cycle, the DNA denatures, primers anneal, and the polymerase extends the primers to exponentially amplify the target sequence. Proper primer design is important to ensure specific rather than non-specific binding. The annealing temperature is usually 5 degrees Celsius below the primer melting temperature. After 30 cycles of PCR, the target sequence is amplified over a billion-fold.

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27 views

PCR - Polymerase Chain Reaction

PCR is a technique that amplifies a specific DNA sequence using DNA polymerase. It requires a DNA template, primers that flank the sequence of interest, DNA polymerase, dNTPs, and thermal cycling between high and low temperatures. During each cycle, the DNA denatures, primers anneal, and the polymerase extends the primers to exponentially amplify the target sequence. Proper primer design is important to ensure specific rather than non-specific binding. The annealing temperature is usually 5 degrees Celsius below the primer melting temperature. After 30 cycles of PCR, the target sequence is amplified over a billion-fold.

Uploaded by

Pravin Vejan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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PCR Polymerase Chain Reaction

Powerful amplifies DNA sequences from a single copy


(theoretically) in vitro to millions of copies
DNA sequencing, southern blotting, fingerprinting, RT-PCR etc.

What do you need?


Template source of sequence to be amplified (Genomic DNA,
plasmid, any DNA for that matter)
Enzyme to catalyze the reaction. A DNA polymerase. Usually
Taq polymerase (thermostable, why?)
Primers remember you molecular biology
dNTPs, buffer, the right conditions

PCR Polymerase Chain Reaction

Taq polymerase from Thermus


aquaticus
PCR: 53, 94oC
Primers, template, dNTPs, Taq
polymerase, buffer
Multiple cycles, accumulation of
products
After 30 cycles 228 =
268435456
Products separated and visualized
on gels

Details
Designing primers:
Good primers important, poor primers can result in nonspecific binding

Lanes:
1 Good primer, specific binding, lots of product
2 Bad bad primer no product
3 Bad primer binding at wrong site
4 Bad primer non-specific binding

Example of primer design


Human a1-globin gene

How long should my primer be?


Depends on your sequence, Generally:
Too short not good non-specific binding
Too long not good 2o structure (hairpins, loops, etc.)
Just the right length (~ 15mer), GC content between 40-60%

What temperature to perform the reactions at?


General reaction as follows:
Denaturation at 94-98oC

What temperature to perform the reactions at?


General reaction as follows:
Denaturation at 94-98oC
Extension 72-74oC
(duration depends on length
of DNA)

What temperature to perform the reactions at?


General reaction as follows:
Denaturation at 94-98oC
Extension 72-74oC
(duration depends on length
of DNA)
Annealing temp?

Calculating annealing temperature:


You will need to calculate primer Tm (melting temperature) of
the primer defined as the temperature at which half of the
primer binding sites at the DNA template are occupied
Increases with length and depends on GC content
Tm = [(4[G+C]) + (2[A+T])] oC
The annealing temp is (Tm 5) oC

So for a typical reaction:


Segment

Cycles

Temp
(oC)

Deuration

94-98

45 s

25-30

94-98

45 s

Tm - 5

45 s

72

1-2 min /
kb

72

10 min

The reaction would contain:


dH2O
Buffer
dNTPs
DNA template (to be amplified)
Primers
DNA polymerase

Old school PCR machine

Current PCR machine

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