DNA labeling alternatives
crasmussen at anat.med.ualberta.ca
Fri May 26 09:24:05 EST 1995
In article <3q3bb1$a3s at sunserver.lrz-muenchen.de>,
salger at wap4.zi.biologie.uni-muenchen.de (Klaus Salger) wrote:
> I think the PCR primers would give longer labeled fragments because
> every single one would start at the very end of the template. With random
> priming you get different sizes. I think by far most of the resulting
> fragments are much shorter than the template. Having a probe of 600bp would
> be better for hybridization than an average of say 300bp.
> There seems to be a trend away from hexamer primers to longer ones which
> prime less frequently and more efficiently at 37deg C.
> This points to the same direction. Using longer, specific primers might be
> the optimal choice (if one has them anyway). And even if this approach isn't
> better than random priming, I can't see any reason why it should be worse.
> Risc nothing and have the chance to win something. I think that's worth a try.
> BTW, The additional advantage of beeing able to synthesize a strand specific
> probe isn't a desireable feature for southerns but might be interesting for
> northerns and other techniques.
True, but using an end-labelled molecule results in lower specific
activity. Depending on the application it may not be sensitive enough
(e.g. genomic Southern or Northern of a rare mRNA). For Southerns of
phage or isolated plasmids it will work fine and does improve the
specificity due to the longer probe length.
More information about the Methods