RIBOZYME-ASSISTED METHOD FOR NUCLEIC ACID DETECTION

With the rapid increase in available DNA sequence information, nucleic acid-based diagnostics is a subject of intense interest. Improvements in the efficiency of sample preparation, nucleic acid amplification, and detection would permit greatly increased use of such methods for routine diagnostic purposes. The most widely used method for amplifying DNA prior to detection is the polymerase chain reaction (PCR), which is restricted to amplifying DNA and requires the use of costly thermal cyclers. Isothermal methods involve a sequence of enzymatic steps, with the attendant complexity, costs of the protein enzymes, and risk of contamination during multiple tube openings. Here we propose innovations in isothermal amplification based on the use of RNA as an amplifiable probe and the ability of RNA to catalyze its own cleavage and rejoining reactions. By taking advantage of RNA catalysis, we can eliminate requirements for all protein factors except an RNA polymerase and reduce the number of tube openings. Additional innovations in hybrid capture and wash procedures further increase the speed and ease of automation. The procedure lends itself to closed-tube, multiplex fluorescent detection during amplification, permitting rapid screening of many different targets while minimizing risks of exposure to pathogens or laboratory contamination by amplified targets.