Introduction of restriction sites via extended primers?

Peter Frank peter_frankde at
Wed Aug 4 09:04:02 EST 2004


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

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?


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