Polymerase Chain Reaction
DNA replication in vitro – allows the amplification of DNA up to a billionfold in a
test tube
Primers are made of DNA (rather than RNA – in vivo)
Discovered by Kary Mullis (1990)
Now can be done using machines (“thermocyclers”) commercially available
Process
1. The template DNA is denatured by heating (95 oC)
2. Mixture cooled to 72oC. Two artificial DNA oligonucleotide primers flanking the
target DNA are present in excess, ensuring that most template strands anneal
to a primer (don’t form primer dimers) as the mixture cools to 2-5 oC below the
Tm of primers
Tm important: too low, primers won’t be specific, higher annealing temperature
will ensure that the primer fully base pairs with correct DNA segment
(hopefully) – reduces chances of non-specific hybridisation
3. DNA polymerase then extends primers using original DNA as the template
(Extension time depends on target DNA length: typically 1kb/min) at 72 oC
4. After appropriate extension time, mixture is reheated again to denature the
strands, then cooled to allow primers to hybridise with complementary regions
of newly synthesised DNA (whole process is repeated)
One primer extension becomes templates or next cycle. Consequently, each cycle
doubles the amount of original target DNA
Usually 20-30 (35) cycles, yielding a 106-109 fold increase
25 rounds = 33.5 million copies, 35 rounds = 34 billion copies
Though PCR is never this efficient – 35 cycles gives enough DNA for further
genetic analysis via sequencing or cloned in to various vectors
a) In first cycle, product extends beyond sequence defined by primers – terminate
when polymerase falls off – product is of indeterminate length
b) Cycle 2: Genomic DNA as template but also using cycle 1 products to begin PCR –
products of cycle are the exact length required
c) Over time, the concentration of products of correct length increases
DNA replication in vitro – allows the amplification of DNA up to a billionfold in a
test tube
Primers are made of DNA (rather than RNA – in vivo)
Discovered by Kary Mullis (1990)
Now can be done using machines (“thermocyclers”) commercially available
Process
1. The template DNA is denatured by heating (95 oC)
2. Mixture cooled to 72oC. Two artificial DNA oligonucleotide primers flanking the
target DNA are present in excess, ensuring that most template strands anneal
to a primer (don’t form primer dimers) as the mixture cools to 2-5 oC below the
Tm of primers
Tm important: too low, primers won’t be specific, higher annealing temperature
will ensure that the primer fully base pairs with correct DNA segment
(hopefully) – reduces chances of non-specific hybridisation
3. DNA polymerase then extends primers using original DNA as the template
(Extension time depends on target DNA length: typically 1kb/min) at 72 oC
4. After appropriate extension time, mixture is reheated again to denature the
strands, then cooled to allow primers to hybridise with complementary regions
of newly synthesised DNA (whole process is repeated)
One primer extension becomes templates or next cycle. Consequently, each cycle
doubles the amount of original target DNA
Usually 20-30 (35) cycles, yielding a 106-109 fold increase
25 rounds = 33.5 million copies, 35 rounds = 34 billion copies
Though PCR is never this efficient – 35 cycles gives enough DNA for further
genetic analysis via sequencing or cloned in to various vectors
a) In first cycle, product extends beyond sequence defined by primers – terminate
when polymerase falls off – product is of indeterminate length
b) Cycle 2: Genomic DNA as template but also using cycle 1 products to begin PCR –
products of cycle are the exact length required
c) Over time, the concentration of products of correct length increases
