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Theory
• Purpose: To amplify a specific region
of DNA to a high copy number (1 copy
of a sequence is not enough for us to
study)
• Use a DNA polymerase from a
bacterium to replicate the region for us
• Use heat to denature the “template”
DNA to permit replication (whereas the
cell uses enzymes)
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Primers
• During normal DNA replication in the
cell, DNA polymerases need primers
(WHY?)
• We make short oligonucleotide primers
(18 - 30 bases) to prime DNA replication
in vitro
• By using a two specific primers, we can
delimit the region to be amplified by the
polymerase
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DNA P
dNTPs
THEN ELONGATE:
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• Primers
– Mismatch: change sequence
– Degenerate: relax constraints
– Multifunctional: add sequences
• Polymerases
– Thermostability
– Proofreading
– “Hot Start”
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Denaturation
• 94ºC is standard
• For 30 - 60 seconds
• The high heat stops enzymatic reactions
and melts DNA
• A longer (120 seconds +) initial
Denaturation step is common
• Taq does lose some activity over many
cycles
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Annealing
• A random process of Brownian motion!
• Hydrogen bonds are constantly being
formed and broken
• “Correct” annealing depends on:
– Concentration of primers (want huge
excess)
– Availability of annealing sites
– Presence of competing, non-ideal
annealing sites
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TM of a “perfect” primer =
4(#Gs + #Cs) + 2(#As +#Ts)
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Extension
• 72ºC for 30 - 90+ seconds
• Why don’t the primers just fall off at this
higher temperature before extension
begins?? (trick question)
• Taq can synthesize thousands of base
pairs per minute under ideal conditions,
so optimal extension time depends on
the length of your target sequence…
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Figure 7-27
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Figure 7-29a
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