Touchdown PCR reactions
p-kaub at csu.murdoch.edu.au
Wed Aug 24 23:21:17 EST 1994
>Frederick Garbrecht (FRED at bmt.mcw.edu) wrote:
>: I am curious as to how people go about optimizing PCR reaction
>: conditions when trying out new primer pairs. One specific question I
>: have is, how reliable is the 'calculated' annealing temperature.
>: That is, say the calculated temperature is 60oC, and you don't get
>: specific bands in preliminary experiments, do you assume that some
>: other condition is not optimal (eg Mg++ concentration), or do you
>: begin to lower the annealing temperature in subsequent experiments.
>: Are there any hints as to how to begin to optimize, based on what you
>: see in initial experiments ( for example, no bands, or ladders, etc)?
>: My problem is that my thermal cycler is real sloooooow, so to run a
>: bunch of different temperature trials takes days, so I would like to
>: eliminate as much of that as possible up front. I know there is
>: probably no easy way out, but I'd be very interested to hear how
>: you plan your strategies for optimization to get the right conditions
>: fast. Thanks alot.
szcooley at chip.ucdavis.edu () writes:
>You might try "touchdown PCR". In this method it is not neccessary to
>optimize. You, in essence, run a ramp of the annealling temperature. The
>first cycles start at a high temp and subsequent cycles at lower and
>lower temps. The system works well for new primers where the problem may
>be something else in the reaction as well. Also you can "optimize" at the
>same time by making duplicate reactions and slipping a new tube into the
>machine after every couple of cycles. Depending on which ones work and
>which ones don't the optimal temp will be evident
I must put in my vote for "Touchdown PCR" (Roux, K.H., Using Mismatched
Primer-Template Pairs in Touchdown PCR, Biotechniques, 16(5):812)
particularly when going gene fishing in between species. It saves on
having to use degenerate primers, but allows you to use existing primers
from one species. Basically by dropping your temperature every few cycles
from an initially high annealing temp. to 40C you get the best of both
worlds i.e. initial high specificity followed by low enough annealing
temp. to incorporate mismatches that may exist in a homologous sequence.
If you can get bands at 40C with a primer pair, then touchdown PCR
could help you to get one product that is highly homologous, but not
identical to the sequence your chasing form another species/individual
Pete Kaub <p-kaub at possum.murdoch.edu.au>
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