Myeloid Leukemia

Real-Time RT-PCR Strategies 33
2.1.1.2. LIGHT-UP PROBE
Some reporters are intercalating asymmetric cyanine dyes that exhibit a large
increase in fluorescence intensity upon binding to DNA: oxazole yellow (YO),
thiazole orange, and 4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-
1,3-thiazole)-2-methylidene)]-1-methyl-pyridinium iodide (BEBO) (8,9). The
light-up probe is a peptide nucleic acid (PNA) that consists of a sequencerecognizing
element linked to a single fluorescent dye that serves as a group
reporter. The PNA is an uncharged nucleic acid analog to which an asymmetric
cyanine dye (thiazole orange) is tethered (10). The light-up probe is flexible,
allowing the dye to interact with the target nucleic acid upon hybridization.
PNA are uncharged analogs to minimize internal complex formation. Upon
probe hybridization, PNA binds specifically to the target nucleic acid, bringing
the dye to it, which results in a large enhancement in dye fluorescence (10,11).
2.1.1.3. AMPLIFLUOR HAIRPIN PRIMERS
Amplifluor hairpin primers (Amplifluor Technology) are designed so that a
fluorescent signal is generated only when the primer is unfolded during its
incorporation into an amplification product (12). The fluorescence signal produced
directly correlates with the accumulation of PCR product at each cycle.
Each Amplifluor primer consists of a 3� 18-base oligonucleotide tail (Z
sequence) and a 5� intracomplementary sequence labeled with paired energytransfer
molecules (the fluophore and the quencher DABSYL). The Z sequence
acts as a universal PCR primer and is specifically designed to reduce PCR
background due to heterodimer formation. The Z sequence is added to the 5�
end of one of the target-specific primers. At the annealing temperature, the Z
sequence anneals to the complementary sequence contained in an amplicon
generated in the initial cycles of the reaction. The hairpin is unfolded, allowing
an increase in fluorescence emission (Fig. 3).
Light upon extension (LUX) primers (Invitrogen) are a variation on hairpin
primers. They are oligonucleotides of 20–30 bases labeled with a single
fluorophore (FAM or JOE) close to the 5� end in a hairpin structure (13). This
fluorogenic primer has a short sequence tail of 4–6 nucleotides on the 3� end
that is complementary to the 5� end of the primer (Fig. 4). This configuration
intrinsically renders fluorescence quenching capability, so that a separate
quenching moiety is not needed. When the primer is incorporated into the
double-stranded PCR product, the PCR LUX primer is dequenched, resulting
in a significant increase in fluorescence signal. The fluorogenic primer may be
the forward or the reverse primer. The characteristics of the primers, such as
length and Tm, are included in the primer design by proprietary software, called
LUX Designer (Invitrogen, http://www.invitrogen.com/lux). These design
rules enable the software to output numerous primer pairs that are located
throughout the target (input) sequence.