Myeloid Leukemia

130 Mokany et al.
total RNA extracted from bone marrow or peripheral blood specimens from
patients with APL.
The general principle of QZyme analysis is similar to those of other quantitative
PCR systems; however, the mechanism is quite different. The protocol
exploits the catalytic activity of DNAzymes (deoxyribozymes), which are DNA
enzymes that have a conserved catalytic core and variable hybridizing arms.
The arms bind complementary nucleic acid substrates and cleave them at specific
phosphodiester bonds. The strategy of QZyme, as specifically applied to
single-tube, duplex RT-PCR of PML-RARα and BCR transcripts, is illustrated
in Fig. 1. In the first step, 3� primers specific to RARα exon 3 and BCR exon 15
are extended by reverse transcriptase to make cDNA copies of the two transcripts.
This cDNA is then amplified by PCR using the same two 3� primers
and two 5� QZyme primers included in the mix. The 5� QZyme primer for
PML-RARα has a 3� terminus, complementary to either PML exon 3 (for Stype
transcripts) or exon 6 (for V-type or L-type transcripts), and a 5� tag of
the inactive antisense of DNAzyme B. Similarly, the 5� QZyme primer for
BCR has a 3� terminus complementary to BCR exon 14, and a 5� tag of the
inactive antisense of a second DNAzyme D. During PCR, amplicons are produced
which contain either PML-RARα or BCR sequence joined to catalytically
active DNAzymes. Two DNAzyme substrates are also present in the
reaction mix: (1) the B-FAM substrate, cleaved by B DNAzymes on the termini
of PML-RARα amplicons, and (2) the D-CAL Orange substrate, cleaved
by D DNAzymes on the termini of BCR amplicons. Cleavage of the substrates
results in separation of the fluorophore and quencher dye pairs on each substrate.
Real-time monitoring of the resulting increase in fluorescence from
FAM and CAL Orange allows quantification of PML-RARα and BCR transcripts,
respectively.
2. Materials
2.1. Calibrators and Controls
1. Plasmid L-type PML-RARα (8194 bp) contains full-length L-type PML-RARα
cDNA (2888 bp), containing PML exons 1–6 and RARα exons 3–9 cloned into
the pTL2 expression vector between the restriction enzymes BglII and EcoRI.
This L-type plasmid was derived with permission from the plasmid MyP-RARα
(16) kindly provided by Prof. P. Chambon, Université Louis Pasteur, Paris,
France.
2. Plasmid S-type PML-RARα (7725 bp) contains full-length S-type PML-RARα
cDNA (2420 bp), containing PML exons 1–3 and RARα exons 3–9 derived from
patient material and cloned into the pTL2 expression vector between the restriction
enzymes BglII and EcoRI. This S-type plasmid was derived from the L-type
PML-RARα described in step 1.