Cycling probe technology utilizes a cyclic, isothermal process that begins with the hybridization of the chimeric probe with the target DNA. Once hybridized, the probe becomes a suitable substrate for RNase H. RNase H, an endonuclease, cleaves the RNA portion of the probe, resulting in two chimeric fragments. The melting temperature (T) of the newly cleaved fragments is lower than the melting temperature of original probe. Because the CPT reaction is isothermally kept just above the melting point of the original probe, the cleaved fragments dissociate from the target DNA. Once dissociated, the target DNA is free to hybridize with a new probe, beginning the cycle again.
After the fragments have been cleaved and dissociated, they become detectable. A common strategy for detecting the fragments involves fluorescence. With this method, a fluorescent marker is attached to the 5’ end of the probe and a quencher is attached to the 3’ end of the probe. When RNase H cleaves the probe, the quencher and fluorescent marker separate, increasing the intensity of the fluorescent marker. Cleaved fragments can alternatively be detected via amplification (e.g., PCR) or further modification to allow for other chemical means of detection.
When working with small concentrations of target DNA, the CPT protocol can be modified to increase specificity and efficiency. Increasing allotted time has been shown to improve probe cleavage efficiency. Both increasing RNase H concentrations and use of a probe that isn't prone to inter-probe and intra-probe interactions has been shown to increase specificity.