Biomedical Engineering Reference
In-Depth Information
reporter molecule at the 5'-end and a quencher molecular at the 3'-end, to
a specifi c sequence of target DNA to detect PCR products. When the probe
is not complementarily bound to the target DNA sequence and remains
intact, very little fl uorescence is emitted from the 5'-reporter due to effi cient
intramolecular quenching by the 3'-quencher molecule. However,
Ta q
poly-
merases cleave the probe from the 5'- to 3'-end with their 5'-exonuclease
activity during primer extension that separates the reporter molecule
from the quencher to emit measurable fl uorescence. In this way, fl uores-
cence is detected only when the target DNA region is amplifi ed so detec-
tion of false signals due to non-specifi c binding of primers is avoided. The
TaqMan
TM
-based approach requires optimization of primers and probes to
effectively detect signals, which can prove challenging for less experienced
researchers. Many commercial sources offer a solution by providing opti-
mized primer and probe sets for detecting various genes.
Another quantitative real-time PCR approach employs DNA binding
fl uorescent dye, such as SYBR
®
Green I, to detect double-stranded DNA
products produced during PCR cycles. The fl uorescence emission from
DNA-bound SYBR
®
Green I drastically increases (up to 1000 times) com-
pared to that from the unbound SYBR
®
Green I, which allows SYBR
®
Green I to detect dynamic amplifi cation of PCR products. Though con-
venient and simple, the SYBR
®
Green I-based PCR detection approach
detects all double-stranded DNA in a solution, including primer dimers
and non-specifi c PCR products. Therefore, false signals for double-stranded
DNA may be detected. Compared to the TaqMan
TM
method, the SYBR
®
Green I method is less technically demanding and can only accurately mea-
sure PCR products if highly specifi c primers are used (Newby
et al.
, 2003;
Schmittgen
et al.
, 2000).
Quantitative real-time PCR primer optimization
Accurate real-time PCR quantifi cation is highly dependent on the quality of
primers. False signals from non-specifi c PCR products and/or primer dim-
ers are attributed by an improper primer sequence design, which increases
diffi culties in accurately interpreting data. In general, there are three steps
for primer optimization: (1) primer search, (2) validation of PCR effi ciency
with designed primers, and (3) verifi cation of PCR products.
The primer search is the fi rst step, focusing on screening a target mRNA
sequence and selecting primers to detect and amplify the gene of interest.
Functional real-time PCR primers should meet common criteria for primer
design (Table 4.3) (Applied Biosystems, 2010c; Raymaekers
et al.
, 2009;
SABiosciences, 2008). Modern bioinformatics make the selection process
easier by allowing researchers to search for the mRNA region of interest
