DzyNA-PCR: Use of DNAzymes to Detect and Quantify Nucleic Acid Sequences in a Real-Time Fluorescent Format Alison V. Todd, * Caroline J. Fuery, Helen L. Impey, Tanya L. Applegate, and Margaret A. Haughton Background: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic se- quences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. Methods: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are pro- duced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluores- cence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. Results: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r  0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA- PCR to analyze DNA that was isolated from human serum. Conclusion: DzyNA-PCR is a simple, rapid, and sensi- tive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens. © 2000 American Association for Clinical Chemistry In vitro evolution has been used to discover nucleic acids capable of catalyzing a broad range of reactions. The 10-23 DNAzyme is capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds under simulated physiological conditions (1). This DNAzyme has a cata- lytic domain of 15 deoxynucleotides flanked by two substrate-recognition domains (arms). The DNAzyme in- teracts with the substrate through Watson-Crick pairing and cleaves between an unpaired purine and a paired pyrimidine. These molecules have potential as therapeutic agents through suppression of gene function by inactiva- tion of target cellular RNA (1–3 ). DNAzymes can also be exploited as molecular tools in genetic diagnostic assays. This study describes the use of 10-23 DNAzymes to facilitate the detection of the products of in vitro amplifi- cation by PCR (4, 5). The strategy, which is known as DzyNA-PCR, is illustrated in Fig. 1. PCR is performed using a DzyNA primer that contains a target-specific sequence and the complementary (antisense) sequence of a 10-23 DNAzyme. During PCR, amplicons are generated that contain both target sequences and active (sense) copies of DNAzymes. A DNA/RNA chimeric reporter substrate, containing fluorescence resonance energy transfer fluorophores incorporated on either side of a DNAzyme cleavage site, is included in the PCR mixture. Cleavage of this reporter substrate produces an increase in fluorescence that is indicative of successful amplifica- tion of the target gene or transcript. DzyNA-PCR is a generic and flexible strategy that provides an alternative to other homogeneous amplification and detection sys- tems, including the TaqMan TM (6), Molecular Beacon (7), Sunrise TM primer (8), and HybProbe assays (9). Johnson & Johnson Research Pty Limited, Australian Technology Park, 1 Central Ave, Eveleigh NSW 1430, Australia. *Author for correspondence. Fax 61-2-8396-5811; e-mail atodd@medau. jnj.com. Received January 11, 2000; accepted February 28, 2000. Clinical Chemistry 46:5 625– 630 (2000) Molecular Diagnostics and Genetics 625