Myeloid Leukemia

36 Picard, Silvy, and Gabert
Fig. 5. Double-stranded DNA detection with DzyNA. Reverse transcription of the
transcript from total RNA and amplification of cDNA occur sequentially in a single
tube. cDNA amplification is achieved by using both the standard and a DzyNA primer
containing both target sequence and 5� tag sequences that are complementary to 10–
23 deoxyribozymes (DNAzymes). During polymerase chain reaction (PCR),
amplicons are synthesized that contain the target sequences linked to catalytic (sense)
DNAzymes, which cleave reporter substrates included in the PCR mixture. The accumulation
of amplicon is monitored during PCR by the change in fluorescence produced
by separation of the reporter (circle) and the quencher (pentagon) moieties
incorporated into opposite sides of DNAzyme cleavage sites within the generic substrates
(dotted line) (14).
duces an increase in fluorescence that is indicative of successful amplification
of the target gene or transcript (Fig. 5).
The accumulation of amplicons during PCR is monitored by an increase in
reporter fluorescence produced by separation of fluoro/quencher dye molecules
incorporated into opposite sides of the 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. This technology has been used at diagnosis and for molecular
monitoring of acute promyelocytic leukemia to quantify PML/RARA
fusion transcripts (14).