Myeloid Leukemia

Real-Time RT-PCR Strategies 31
cific detection) or an oligonucleotide labeled with a fluorophore and a quencher
moiety (specific detection). In 2004, the most frequently used chemistry for
detection and quantification of fusion gene transcripts in AML utilizes dual
labeled probes (TaqMan probes). Only a few articles have described other
chemistries for these applications (3). However, this situation may change with
the advance of new chemistries. Indeed, all chemistries allow quantification,
and some of them provide other advantages such as multiplexing or greater
specificity (double-stranded DNA detection vs specific fragment detection).
The aim of this chapter is to give an overview of the various possibilities either
with double-stranded DNA detection or with specific fragment detection.
2.1.1. Double-Stranded DNA Detection
For systems employing double-stranded DNA detection, the detection is not
specific for the amplified sequences. Rather, the specificity relies on the primers
chosen and the PCR conditions. At least four different chemistries can be
described.
2.1.1.1. SYBRGREEN I
SybrGreen I is an intercalating dye that binds only to the minor groove of
double-stranded DNA (dsDNA) in a sequence-independent manner (Fig. 2).
SybrGreen I fluorescence increases over 100-fold upon binding. This dye is
excited at 497 nm and emits at 520 nm. Increasing fluorescence is observed
during the polymerization step and decreases during DNA denaturation. Accordingly,
in RQ-PCR, fluorescence measurements are performed at the end of
the elongation step (4,5).
This method is inexpensive and easy to set up. However, only one target can
be amplified, therefore precluding multiplexing. Furthermore, longer
amplicons create stronger amplification signals, occasionally causing chargecoupled
device (CCD) camera saturation. The advantage and disadvantage of
SybrGreen I is that it binds to any dsDNA. Therefore design and optimization
of probes are not required. However, all amplifications, specific or not, are
detected equally well. Primer dimers are the most frequent cause of nonspecific
amplification and limit the sensitivity of assays. To reduce the formation
of primer dimers, a Roche technical note (LC 1/1999) provides different optimization
strategies. The use of two step reactions (reverse transcription and
PCR) and DNAase treatment of RNA prior to cDNA synthesis decreases the
formation of primer dimers (6).
Generation of a melting curve at the end of the reaction is indispensable for
identifying amplification products (7). dsDNA amplification is melted into
ssDNA by a stepwise increase in temperature from 40°C to 95°C while continuously
monitoring the fluorescence. PCR products of different length and
sequence will be melted at different temperatures and will be observed as dis-