Enzymaticbased Signal Amplification

Another category of signal amplification assays utilizes enzymatic reactions that can detect conformation changes in a target. The most frequently used method in this category utilizes the Cleavase enzyme, which can detect single-basepair changes in the Invader assay (Third Wave Technologies, Madison, WI) (43,44) using linear probes, Whereas another method (RAM) combines RCA and strand displacement amplification to amplify a circular DNA probe that serves as the reporter molecule (45).

3.2.1. Cleavage Reactions Flap enzymatic reactions utilize enzymes that can detect changes in the secondary structure of hybridized molecules (Fig. 8). In Fig. 8, the invader oligomer overlaps a single base of interest in the target.

Unligated Circular Probe Ligated Cjrcular probe

■ Rolling Circle

^^^ Strand displacement amplification

Fig. 9. Enzymatic signal amplification ramification amplification.

■ Rolling Circle

^^^ Strand displacement amplification

Fig. 9. Enzymatic signal amplification ramification amplification.

A specific probe that is complementary base-pairs with the target's base of interest. The combination of these three molecules forms a structure that is recognized by the cleavase enzyme with then cleaves the primary probe. These cleaved probes then base-pair to the detection molecule, which has a flourochrome and flourochrome-quencher bordering the single base of interest. Again, the structure of the three strands of molecules forms a substrate for the cleavase enzyme. The enzyme cleaves the secondary molecule and releases the flourochrome, which then is detected. None of these reactions takes place if the primary molecule does not bind to its complementary base. Different flourochromes can be used to detect different bases.

3.2.2. Ramification Amplification Ramification amplification is also known as hyperbranched rolling circle or cascade rolling circle amplification (20,24). As its synonyms suggest, this method is similar to the rolling circle amplification with the exception that a ligase closes a circular probe, which then serves as the template for subsequent amplification using reverse and forward primers. As a result, a linear target can be detected using this methodology (Fig. 9).

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