Many current nucleic acid amplification systems for detecting pathogenic agents may not yield consistent results because optimal amplification conditions vary with each target sequence. Cascade rolling circle amplification (CRCA) is a novel isothermal amplification system that uses generic primers to amplify circularized probes instead of the target. It is rapid, highly sensitive, and easily multiplexed, consistently yielding over a billion-fold amplification within one hour without using a thermocycler. If one of the primers is an energy transfer-labeled primer, homogeneous fluorescence detection of the CRCA product is possible within a closed system. The speed and ease of the assay make this technology suitable for field work or use in developing countries, as well as for simpler automation in diagnostic applications. The feasibility of this system has been demonstrated in preliminary experiments. Phase I objectives include optimization of ligation and amplification conditions to achieve maximum signal strength and sensitivity, and optimization of energy transfer-labeled primer structures that yield the highest fluorescence signal to background ratio without compromising amplification efficiency. The speed and sensitivity of the proposed method will be compared with traditional techniques using a pathogenic target Phase II studies will focus on clinical applications of this system.
This amplification and detection system can form the basis of any DNA diagnostic procedure where the presence or absence of a specific nucleic and target sequence is determined, such as an infectious disease agent, inherited germline mutation, or somatic mutation in a cancer screening test The assay is highly sensitive, fast and simple, and we estimate that it will be less costly than current methods. Because it can be performed in a closed system, false-positive results from carry-over contamination will be minimized. The system is readily amenable to simple automation.
| Thomas, D C; Nardone, G A; Randall, S K (1999) Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction. Arch Pathol Lab Med 123:1170-6 |
