New restriction sites can be generated by ligation of DNA fragments with compatible ends. These ends may be generated by:

1. Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
2. Cleavage with two restriction endonucleases that produce blunt ends.
3. Cleavage with two restriction endonucleases that produce compatible overhangs.

Compatible ends, generated by each of the above methods, can be ligated to produce DNA sequences that often contain useful restriction endonuclease sites. Generation of these sites is detailed in the following tables.

The table below lists restriction endonucleases that generate 5´ overhangs which, if filled-in and ligated, result in new restriction sites. The combinations listed were identified by computer analysis, and although we have tried to ensure their accuracy, they have not necessarily been confirmed by experimentation.

Restriction enzymes which have degenerate recognition specificities (i.e. recognize more than one sequence) have been excluded from this list. Where isoschizomers exist, only one member of each set is listed. Only commercially available enzymes have been listed.

Table Notes:

• Enzymes in red have recognition sequences of 6 or 8 bases. Sequence length is indicated by superscript (e.g., AscI⁸ = 8-base cutter).
• Enzymes in parenthesis indicate that the new sequence is still a substrate for the original enzyme.
• A superscript ² indicates that two identical sites have been generated within the filled-in/ligated sequence. For example, fill-in/ligation of AflII generates the sequence CTTAATTAAG which contains two MseI sites (TTAA).

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