The FEN-type Cleavase enzymes that are used in Invader DNA assays are not able to recognize RNA targets; instead, pol-type Cleavase enzymes that recognize both DNA and RNA targets are used. The altered substrate specificity of the pol-type Cleavase enzymes requires an adaptation to the reaction used to cleave the FRET oligo. RNA assays occur in two sequential steps: the primary reaction and the secondary reaction. During the primary reaction, the Invader oligo and primary probe form an invasive structure on the RNA target. An additional oligo, the stacker oligo, is designed to coaxially stack with the 3' end of the primary probe. The stacker oligo allows the primary probe to cycle on and off the target at a higher temperature and increases the sensitivity of the assay. In the secondary reaction, the cleavage product of the primary reaction (the cleaved 5'-flap plus one base of the TSR) forms a one-base overlap structure with a secondary reaction template (SRT) and a FRET oligo. The enzymatic cleavage of the FRET oligo separates a fluorophore from a quencher molecule, as described for DNA assays, generating fluorescent signal. In contrast to the DNA format, the released 5'-flap remains bound to the SRT while the FRET oligos cycle on and off the SRT. Consequently, multiple FRET oligos are cleaved per 5'-flap, resulting in a linear accumulation of fluorescence signal. An arrestor oligo, which hybridizes to the TSR plus a portion of the 5'-flap of uncleaved primary probes, is also included in the secondary reaction. The arrestor oligo allows for greater signal generation by sequestering uncleaved primary probes and preventing competitive inhibition of 5'-flap hybridization with the SRT during the secondary reaction (Fig. 2).[3,4,6]
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The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.