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Every chart of palindromic restriction enzymes I've seen lists their restriction sites from 5' to 3', something like this:

EcoR1 cuts GAATTC between the G and A:
5' NNNGAATTCNNN 3' --> 5' NNNG____ AATTCNNN 3'
3' NNNCTTAAGNNN 5' --> 3' NNNCTTAA____ GNNN 5'

Will restriction enzymes match and cut a site if it's running antiparallel? For example, will EcoR1 cut this sequence?

5' NNNCTTAAGNNN 3'
3' NNNGAATTCNNN 5'

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Whatever sequence (plus direction) is target, so enzyme will cut. In your example "correct" and "antiparallel" sequences are two different sites.

Actually, your example of 5'-CTTAAG-3' is cut by AflII enzyme.

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    $\begingroup$ Additionally, the definition of "palindrome" means that it does not matter which direction the read is in, as either direction is identical. $\endgroup$ – March Ho May 4 '15 at 20:54
  • $\begingroup$ Another way to think about it is to look at the 3-D structure of GAATTC, I believe there is also a 3-D structure of the Eco RI enzyme. The sequence CTTAAG cannot be superimposed on top of GAATTC--well, when you superimpose them they don't align significantly. Likewise ds GAATTC fits perfectly into the enzyme active site while ds CTTAAG will fit, but poorly. $\endgroup$ – mdperry May 4 '15 at 22:36
  • $\begingroup$ @mdperry that is what behind (in some part) specificity :) $\endgroup$ – aaaaa says reinstate Monica May 4 '15 at 22:42

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