Method for Touchdown PCR

Ron Kagan rkagan at ewald.mbi.ucla.edu
Mon Sep 19 18:41:18 EST 1994


In article <35bn1k$cif at mserv1.dl.ac.uk> LOGAND,
logand at msdos.montpellier.inra.fr writes:
>I wish to determine the specificity of a PCR reaction with regard to the 
>anealing temperature and "think" that Touchdown PCR is an efficient
means to 
>this end. However, I do not entirely understand the method (having only
seen it 
>mentioned in brief on the net) and would thus be very grateful to anyone
who 
>could enlighten me via the net or by faxing the method of Roux, K.H. In 
>Biotechniques 16(5) : 812. We do not get Biotechniques here. 


Basically, you start program your thermocycler to start off with a high
annealing temp. say, 62C or 60C, and drop the annealing temp. by 1 degree
every 2 cycles, until you get to 52C or 50C annealing, and then continue
the PCR at that annealing temp. for another 10-15 cycles.  

The idea is that primers that perfectly match the template will anneal at
a higher temp. than those with mismatches to the template, so you get
preferential amplification of the perfectly matched templates over the
ones with mismatches.  For every 1 degree difference between the
annealing temp. of the perfectly matched template and artifacts, you get
a 4-fold amplification (2 PCR cycles) of the perfectly matched template
over the artifacts.

Ron Kagan
rkagan at ewald.mbi.ucla.edu



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