Introduction of restriction sites via extended primers?

salli nospam at
Fri Aug 13 09:10:28 EST 2004

"Kyle Legate" <legatek at> skrev i en meddelelse
news:2ncjvfFvfrspU1 at
> Peter Frank wrote:
> > Hello,
> >
> > I want to introduce new restriction sites to the ends of a DNA
> > sequence via extended primers, i.e. the primers will have a matching
> > region (around 20 bases) and then the non-complementary restriction
> > site (around 6-8 bases).
> > I found a source saying "Extra bases are usually added to the 5' end,
> > as restriction enzymes do not cleave DNA efficiently at the end of a
> > fragment.".
> >
> > How many extra bases are usually added and does it matter which ones?
> > Are there any other criteria that should be considered when designing
> > primers for introducing new restriction sites by PCR?
> >
> The NEB catalogue has a table in the appendix entitled "Cleavage close to
> DNA ends", which applies to oligos of various lengths cut by a good
> selection of enzymes. Facing that page is a table for linear fragments cut
> first with one enzyme to create an end, then a second enzyme close to the
> end to give an indication of how many extra bases are required and while
> this would more closely approximate a PCR product, I've also used info
> the oligo table with great success. Use an older version of the catalogue,
> because the latest version seems to be missing much of the data from the
> tables.
> Most enzymes cut 90% or greater with an addition of 4 bases, but you can
> sometimes use fewer than that. By default I always add AATT to the end of
> oligo that introduces a RE site because 1. It fulfills the requirement for
> 99% of the enzymes out there (I've never had a failure, but I favour
> RE sites so the sample size is not large) and 2. It doesn't contribute as
> much to the melting/annealing temp as using G's and C's.

Hi Kyle.
I don't understand the last bit, where you say that AATT contributes less to
annealing that G and C? This is of cause correct, however this overhang is
NOT going to anneal to template - so do you include it when calculating
annealing temp?
Just wondered

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