Biomedical Engineering Reference
In-Depth Information
Table 4.5
Fluorophores and quenchers for quantitative multiplex PCR
Fluorophore
Excitation
(nm)
Emission
(nm)
Quencher
Absorption
maximum
(nm)
FAM
495
515
Iowa Black FQ
532
TET
523
540
Black Hole
quencher-1
534
HEX
535
555
Black Hole
quencher-2
580
Cy3
550
570
Iowa Black RQ
645
TEMRA
555
575
Texas red
585
605
Cy5
650
670
4.2.3 Multiplexing technology
Multiplex PCR is a modern quantitative PCR technique by which multiple
target genes of interest can be detected simultaneously in the same reac-
tion (Markoulatos
et al.
, 2002). Another advantage of using this technique
is the saving of cDNA samples and reagents while shortening the reaction
time compared to conventional real-time PCR. Measurements using this
technology can be performed in regular real-time PCR machines equipped
with multiple optical fi lters. For example, multiplex real-time PCR based
on the TaqMan
TM
approach can detect multiple target genes labeled with
different fl uorophores (see section 'Fluorescence-based PCR product
detection methods' for the 5'-nuclease method). The choice of fl uoro-
phores depends on the availability of optical fi lters and fl uorescent dyes
(Table 4.5). The general rule is that the emissions spectra of fl uorophores
used in the same PCR reaction should not interfere with each other. The
major technical requirement in multiplex PCR is to set a PCR operating
condition that amplifi es all target genes with similar effi ciency (Applied
Biosystems, 2010a). This is especially challenging when cDNA amounts of
target genes are signifi cantly different. Higher concentrations of cDNA
can consume synthesis chemicals, such as dNTPs, more effectively during
PCR cycles than the lower concentrations of cDNA, which could possibly
result in an extremely high
C
T
value for the lower concentration cDNA
gene. To overcome this challenge, various primer concentrations should be
tested to determine an optimal concentration for the multiplex PCR target
cDNAs. Several companies have developed multiplex quantitative PCR
reaction mixes that have been optimized for effi cient binding of primers
and probes.
