reply: SSCP: Restriction Enzymes vs. Shorter Fragments
Gregory S. Buzard 'Buz'
buzardg at MAIL.NCIFCRF.GOV
Tue Aug 6 18:01:55 EST 1996
"Conventional wisdom be damned" someone once said.
Sometimes you get lucky and long PCR products reveal single
nucleotide changes in a particular SSCP gel system. The
published record is 800+ bp, and we have recently had a
shift of a 1200 bp mutant! Try the long fragments.
Your discription of disappointing results with restriction
digests is less than clear. If it is fuzzy, you may be
seeing the affects of the restriction enzyme or the buffer
on the structure of the molecules as they enter the
SSCP gel, or you may have a mild case of exonuclease
activity that is undetected with dsDNA, but is apparant
with the SSCP analysis. And then there is the old question,
do you have positive controls for your system to tell you
if you in fact have a problem at all?
The original posting:
I am just starting to use SSCP as a technique (labelling
with 35S dATP) to screen for variation in a population
genetic structure study. At the moment all my primers are
designed to amplify gene fragments (mtDNA) in the 400-700bp
range. All the literature that I have read on the subject
suggests that SSCP is run optimally with 250bp fragments,
and still provides reasonable resolution up to about 350bp.
It also suggests that restriction enzymes are usually a
waste of time when trying SSCP. My main question here is
can I use restriction enzymes to cut the fragments I am
getting from my current primers to try and save time?
have done some preliminary experiments and the results on
the SSCP gel were a little difficult to interpret. Has
anyone tried this with any success, or will I have to
design some new primers to get shorter fragments? The other
possibility was to try and run several short fragments in
the same lane on the gel to try and save time (I have a few
thousand to run!). Has anyone done this with success?
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