RIBOZYME-ASSISTED METHOD FOR NUCLEIC ACID DETECTION

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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. PROPOSED COMMERCIAL APPLICATION The increasing availability of DNA sequence information on microorganism genomes and genes of disease relevance, including oncogenes, makes nucleic acid-based methods extremely attractive for diagnosis of viral and microbial infections, detection of food contamination, and genetic screening. However, current method, while extremely sensitive, are too slow and expensive for routine use in clinical laboratories. According to NIST, the biotechnology industry projects the market for in vitro DNA diagnostics to grow from 500 million dollars in 1997 to 6 billion by 2005. This market will favor techniques that are inexpensive, simple, rapid, and easily automated, permitting completely automated diagnosis in a self- contained machine for routine use in clinical laboratories or as a method for testing large numbers of samples for multiple genetic targets that could be used in a center for high-throughput diagnostic testing. We propose several improvements in the state of the art toward that goal.