Biomedical Engineering Reference
In-Depth Information
from the untreated control and treated samples to determine effects of a
particular experimental treatment on the regulation of target genes (Bustin,
2002; Livak and Schmittgen, 2001). In this section, important basics of quan-
titative real-time PCR, such as fl uorescence chemistry, primer optimization,
reference gene choice, and data analysis, are discussed.
Fluorescence-based PCR product detection methods
Several fl uorescence-based detection methods are available for qPCR
product measurement. Two detection methods used in real-time PCR sys-
tems are (1) the Förster resonance energy transfer (FRET)-based approach
that relies on fl uorescence generated by distance-dependent energy transfer
between reporter and quencher fl uorophores during PCR, and (2) the DNA
binding dye approach that utilizes the property of specifi c fl uorescence dyes
designed to bind only to double-stranded DNA to produce fl uorescent sig-
nals (Fig. 4.1) (Keer, 2008; Machay and Landt, 2007).
Among the existing approaches, the 5'-nuclease method, also called the
TaqMan
TM
method, and the SYBR
®
Green I method are the most commonly
used FRET and DNA binding dye detection approaches, respectively. The
5'-nuclease method attaches an oligonucleotide probe with a fl uorescent
(a) The 5
-nuclease method
FRET
Fluorphore
Quencher
Probe
Primer
Target DNA
Ta q
a.
Primer
Target DNA
Ta q
b.
Fluorescence
signal
c.
Primer
Target DNA
Ta q
(b) The SYBR
®
Green I method
SYBR Green 1
Primer
Target DNA
Ta q
a.
Primer
Target DNA
b.
4.1
Quantitative real-time PCR detection mechanisms.
