Figure 2.

Oligoduplexes. Synthetic oligonucleotides were annealed to give the duplexes shown. The species 24 s-Ax488 and 34 s-Ax488 carry the recognition sequence for FokI (underlined in bold), the downstream sites of DNA cleavage (arrows) and an Alexa Fluor 488 dye attached via a C₆ amino-linker to the 3′-end of the top strand (noted as Ax488): i.e. downstream of the recognition site. Two further duplexes, 24 s and 24 s-HEX (not shown), had the same sequence as 24 s-Ax488 but instead of the 3′-Ax488 label on the top strand have: in the case of 24 s, no label; in the case of 24 s-HEX, a hexachlorofluorescein moiety at the 5′-end of the bottom strand, again downstream of the site. A further derivative, 24 ns, has the same sequence as 24 s apart from alterations at all 5 bp of the specific sequence, to give a non-specific sequence for FokI. The duplex Ax488-28 s carries the recognition and cutting sites for FokI and an Ax488 dye (all noted as above), but the latter is attached, again via a C₆ amino-linker, to the 5′-end of the top strand: i.e. upstream of the site. The 13 s species has the recognition site but no sites for DNA cleavage.