Biology Reference
In-Depth Information
8.4.15 Reverse-Transcription PCR
Messenger RNA can be reverse transcribed and the resultant cDNA then can
be amplified using
Taq
DNA polymerase.
R
everse
T
Transcriptase-PCR (RT-PCR)
allows detection of gene expression in small numbers of specific cells or tis-
sues. Reactions have been carried out with RNA isolated from as few as
10-1000 cells.
The process involves 1) isolation of mRNA, 2) reverse transcription of mRNA
into cDNA, and 3) amplification of cDNA by DNA polymerase. Primers for the
amplification should be 18-22nt long, and should occur in separate exons to
inhibit amplification of any contaminating genomic DNA in the RNA prepara-
tion. Multiple reverse transcriptases can be used in RT-PCR to increase sensitivity
and product yield (
Nevett and Louwrier 2000
).
RT-PCR has been used to monitor for the presence of rabbit hemorrhagic dis-
ease virus in fly species in Australia (
Asgari et al. 1998
). This calcivirus causes a
lethal disease in European rabbits, but little was known about how it spreads
in the field. RT-PCR provided a sensitive and reliable method for detecting the
virus in flies and fly spots, which allows it to be used to study the epizootiology
and vector biology of the virus.
8.4.16 TaqMan PCR
TaqMan PCR is a type of real-time PCR. TaqMan PCR uses a nucleic-acid probe
complementary to an internal segment of the target DNA. The probe is labeled
with two fluorescent moieties. The emission spectrum of one overlaps the exci-
tation spectrum of the other, resulting in “quenching” of the first fluorophore
by the second. The probe is present during the PCR and if product is made, the
probe is degraded via the 5
′
-nuclease activity of
Taq
polymerase that is specific
for DNA hybridized to template (
=
TaqMan activity). The degradation of the
probe allows the two fluorophores to separate, which reduces quenching and
increases intensity of the emitted light. Because this assay involves fluorescence
measurements that can be performed without opening the PCR tube, the risk of
contamination is greatly reduced. Furthermore, no electrophoresis is required,
so labor costs are reduced (
Kalinina et al. 1997, Sambrook and Russell 2001,
Baker 2011
).
8.4.17 Digital PCR
Digital PCR (dPCR) involves diluting a sample of DNA and partitioning it into
hundreds or even millions of separate reaction chambers so that each cham-
ber contains one or no copies of the target sequence (
Lo et al. 2007
). After
