Number Ten Ox
scottk at nuclease.berkeley.edu
Thu Jul 30 05:49:49 EST 1992
In article <36279 at sdcc12.ucsd.edu>, rounsley at jeeves.ucsd.edu
(Steve Rounsley) writes:
|> Quick question...
|> does anybody know what conditions to use for "Touchdown"
|> PCR. I came across this term, and I don't know what kind of
|> cycles to use.
|> thanks in advance
|> Steve Rounsley
The exact conditions will depend on what you're trying to do. The
reference for touchdown PCR is:
Don, et al. (1991) NAR 19(14): 4008
Basically, touchdown PCR involves decreasing the annealing temp
by 1 degree C every second cycle to a 'touchdown' annealing temp
which is then used for 10 or so cycles. It was originally intended
to bypass more complicated optimization processes for determining
optimal annealing temps. The idea is that any differences in Tm
between correct and incorrect annealing gives a 2-fold difference
in product amount per cycle (4-fold per degree C). You therefore
enrich for the correct product over any incorrect products.
Another use for this procedure is in determining DNA sequence for a
known peptide sequence. The strategy here is to use two sets of
degenerate primers that match potential coding sequences at the
two ends of a peptide of known sequence. In practice, this requires
that you know a stretch of peptide sequence of only 13 amino acids,
with left and right primers of 18 nt (6 a.a.) and a space in between
of one or more nt. Using these degenerate primers, you do a touchdown
PCR. You will get a huge number of products, but you can select the
desired product based on size since you know the exact interprimer
distance from the peptide sequence. The advantage of this technique is
that the touchdown PCR enriches for products containing correct matches
between primers and template. If you clone and sequence a dozen
PCR products, you can determine the correct coding sequence for
the peptide, design an oligo for hybridization, and ZOOOM...
cloned gene is practically in hand. The technique is obviously
especially useful for peptide sequences full of ser, lys, and arg
(six codons each).
I haven't seen this latter use of touchdown PCR published. I got
it from folks in the Tjian lab here at Berkeley, but I have no
idea whether they made it up or heard it from someone else.
____ Scott Keeney, DNA repair-queer ____
\ / scottk at mendel.berkeley.edu \ /
\/ Biochemistry and Molecular Biology \/
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