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Module BB MMB3001
Principles of PCR
Uses DNA polymerase: Reads one sequence and makes a copy of it
Exponential amplification
Primer design
DNA is always written and read from 5' to 3' end
Forward and reverse primers (complement to the desired part of DNA), that
should have similar melting temperatures (difference <5 deg C)
GC clamp at the 3' end (since G-C bind more strongly, they are desired at the
end of the primer before DNA polymerase starts extension)
Avoid regions which have secondary structure, as this leads to the formation of
hairpin loops or primer-dimers (test primers beforehand)
Start PCR optimisation 3-5 deg C below the lowest melting temperature
Special rules
Add 5-6 "extra" bases if including a restriciton site (increases the efficiency of
enzyme binding)
Primers with 5' overhangs have much higher melting temperatures than just the
binding region
cDNA synthesis