DzyNA-PCR: Use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format

Background: DzyNA-PCR is a general strategy for the detection and quantific ation of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification couple d 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 produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate include d in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter subst rate 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 DzyN A-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resol ve as little as twofold differences in the amount of starting template. Dzy NA-PCR allowed the detection of 10 or fewer copies of the target. The clini cal utility of the assay was demonstrated using DzyNA-PCR to analyze DNA th at was isolated from human serum. Conclusion: DzyNA-PCR is a simple, rapid, and sensitive technique for homog eneous amplification and quantification of nucleic acids in clinical specim ens. (C) 2000 American Association for Clinical Chemistry.