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. 2007 Sep 7;35(18):e117. doi: 10.1093/nar/gkm654

Figure 1.

Figure 1.

Nicking endonuclease signal amplification. (A) Double-stranded target DNA containing an Nt.Alw I site is first denatured at 95°C and then annealed to a fluorescently labeled oligonucleotide probe at 58°C. Theoretically, the fluorescent label can be anywhere in the probe as long as it does not interfere with Nt.AlwI cleavage; we have used it successfully at either the 5′ or 3′ positions. Nt.Alw I cleaves the probe but not the target DNA and the two probe pieces spontaneously dissociate from the target. New full-length probe anneals to the target and the reaction is repeated. (B) Analysis of NESA using capillary electrophoresis. The top trace shows only full-length probe, indicating no cleavage activity occurred while the bottom trace shows an example of positive cleavage activity; the two peaks correspond to cut probe and full-length probe.