Rf: RE; touchdown PCR
gqva12 at udcf.gla.ac.uk
Tue Sep 20 06:00:51 EST 1994
Here's how we do it -- I should say that I got this method from Ian Campbell
at the University of SOuthampton (UK) Department of Obstetrics and Gynaecology
The object of the method is to gradually decrease the anealing temperature
for the initial cycles until it comes just below (or to, or above) the
theoretical melting temperature of your oligo. Then you can go ahead with your
30 cycles at 60 degrees, or whatever.
We usually start the touchdown part of the sequence at our normal extension
temperature (72) and drop it by one degree per cycle until we reach the
desired anealing temp. I have seen a range of steps from 2 degrees to 0.2 used
but this seems extreme to me.
The theory (I think) is that at any given temperature, some fraction of the
oligo will bind to its complimentary sequence; the higher the temperature
the more energy will be in the system therfore the more likely it is that any
binding will be very specific. As the temperature is gradually lowered, you
reach a point where enough of the oligo is in place for long enough to allow
a synthesis to take place. Hence, the first few rounds of synthesis are based
on very specific annealing. As the reaction proceeds, these specific products
come to dominate the target sequences and so become preferred binding sites
for the oligo as the annealing temp. is lowered and the potential for
miss-priming goes up.
So, touchdown is a way of improving the specificity of your PCR. We've used it
most often where we have degenerate primers, but it is also useful for getting
an insert out of a vector or any situation where your first strategy provided
a high-class smear.
You'll have to devise your own protocol to suit your primers, but as a first
go, try a 2 degree drop from 72 and shrten your regular number of cycles by 5.
You might also post a specific request to Tracy Aquilla, since she regularly
provided good, useable answers to this board.
Good luck, Grant Gallagher, University of Glasgow Department of Surgery
More information about the Methods