Introduction of restriction sites via extended primers?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Aug 4 13:11:23 EST 2004


Oligos are cheap, so I usually add 6 extra bases of "GC-clamp", that 
is GCGCGC.  Presumably G and C are preferred because of the higher 
melting temperature they'd impart to the ends. This strategy has 
generally worked well for me. Of course, if you were introducing 
GCGCGC as your restriction site, something else might be better of 
the bases on the end.

One thing to keep in mind if you add any extra bases on the other 
side of the restriction site (if for example you are adding a 
translational terminator or other desired sequence), make sure you 
don't introduce a methylation site.  It won't matter in the PCR 
product, but once the fragment is cloned, it'll be trouble!

Mike

>
>
>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?
>
>Peter


-- 

Michael L. Sullivan, Ph.D
Research Plant Molecular 
Geneticist                                                            
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5397 Phone
(608) 264-5147 Fax
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