Deg. primer PCR w/genomic DNA: Problems
dspinella at chugaibio.com
Tue May 20 09:14:45 EST 1997
Ken Soderstrom writes:
> Dear PCR Wizards:
> I have degenerate primers that will reliably amplify a 715 bp fragment
> using cDNA but unreliably amplify the same sized fragment from genomic DNA.
> Actually these primers only worked the first two times I tried PCR w/
> genomic DNA. Initial success was obtained using a standard 100ul reaction
> w/ 100ng DNA, 100uM dNTP, 1.5mM MgCl2, 1uM each primer, and 2U Promega Taq.
> Positive CTL (1ng cloned fragment) has been consistently beautiful.
> Failed solutions include:
> 1) Isolating additional (fresh) genomic DNA.
> 2) Dissolving DNA in H2O instead of TE.
> 3) Using multiple temperatures (94, 100C) and durations (1', 5', 10') for
> initial denaturation.
> 4) Digesting with Not1.
> 5) Using ranges of [Mg++] (1.25, 2.5, 3.75 mM), [DNA] (.1, 1, 10, 100, 1000
> ng), [primer] (1uM, 3uM), all w/ & w/o 5% DMSO and 10% glycerol
> (BTW, + CTLs only looked weak w/ 10% glycerol - does this stuff really
> enhance some amplifications?).
> I'm hoping for suggestions of other potential solutions (besides giving-up
> which I'm almost beginning to look forward to).
> Thanks in advance.
> Ken Soderstrom
I doubt I qualify as a PCR wizard, but I have a sneaking suspicion that
the reason your genomic DNA amplified the first few times you tried it
is that you accidentally contaminated the DNA prep with PCR product
derived from cDNA, or the cloned control fragment you have been using
(something that is very easy to do unless you are extremely careful to
prevent it). It may well be that your priming sequences are interspersed
by one or more long introns in genomic DNA, and you cannot amplify up
the same fragment as in cDNA. One way to check this possibility is to
look for unique restriction sites close to the priming sites in the
cDNA, and then use those restriction enzymes to do a Southern blot on
genomic DNA, using the cDNA-derived PCR product as a probe. Do you get
the same fragment size as predicted by the cDNA sequence?
I could be wrong of course, but it sounds to me like you've tried a lot
of things. If the real PCR wizards can't give you a tip that changes
your luck, you might consider this possibility. Just a thought. Good
luck. -- D.G. Spinella
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