Introduction of restriction sites via extended primers?

Kyle Legate legatek at
Wed Aug 4 12:16:32 EST 2004

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 from
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

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 an
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 certain
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.


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