Experimental and theoretical analysis of the invasive signal amplificationreaction

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstr eam and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running t he reaction at an elevated temperature, the downstream oligonucleotide cycl es on and off the target leading to multiple cleavage events per target mol ecule without temperature cycling. We have examined the performance of the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and t he DNA polymerase I homologues from Thermus aquaticus and Thermus thermophi lus in the invasive signal amplification reaction. We find that the reactio n has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavag e. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates th at arise during the course of a reaction were measured and compared with th e rate of the specific reaction. We find that the different 5' nuclease enz ymes display a much greater variability in cleavage rates on the nonspecifi c substrates than on the specific substrate. The experimental data are comp ared with a theoretical model of the invasive signal amplification reaction .