challenges while multiplexing.
PCR further evolved to Digital
PCR or dPCR which builds on the
amplification possible in
traditional PCR and uses the
qPCR fluorescent-probe–based
detection to provide a highly
sensitive, precise quantification of
DNA. The reagents involved in the
assay remain the same but the
instrumentation changed such
that the total number of
individual target molecules can be
counted directly in a digital
format, thereby doing away the
need of a reference standard. The
sensitivity of qPCR is improved to
such an extent that rare alleles,
rare events which were missed out
can now be captured by dPCR.
A look at the background and
methodology
Pioneering work on digital PCR
was done by Sykes et al in 1992
and by Vogelstein and Kinzler in
1999. The principle involves
dilution of DNA sample to obtain
a single copy of the target which is
amplified by an end-point PCR.
The distribution of the target
DNA follows Poisson distribution
providing an absolute measure of
nucleic acid concentration. These
products were detected using
fluorescent probes. Error can
happen only if two targets are
collocated in the same well and
this is calculated by Poisson
distribution. If a particular
reaction well contains the target it
will become brightly fluorescent
while those wells without the
target will have only background
fluorescence thus an absolute
measure of the target in the
sample is possible.
The techniques further developed
and could overcome many of the
drawbacks of qPCR. The term
“digital PCR” was coined to
The term “digital PCR”
was coined to describe
these technologies.
Fluidgm Corporation first
commercialized dPCR in
2006 while the first
instrument for dPCR was
launched in 2013. It has
several advantages over
qPCR in terms of
absolute quantification,
enhanced sensitivity,
greater precision and
multiplexing capabilities.
describe these technologies.
Fluidgm Corporation first
commercialized dPCR in 2006
while the first instrument for
dPCR was launched in 2013.
Multiplexing is facilitated by
dPCR since there are only 0 or 1
target molecules per well and
specific probes can be employed
simultaneously. The problems of
competition and cross reactivity
are eliminated.
Droplet dPCR (ddPCR) partitions
DNA using water-oil emulsion
droplets in which amplification
takes place. It is a combination of
microfluidics and proprietary
surfactant chemistries. The
droplets are analysed using Flow
cytometry. Emulsion dPCR is a
PCR variation that some NGS
technologies use to replicate DNA
sequences. It is conducted on a
bead surface within tiny water
bubbles floating on an oil solution.
Adoption of standards for qPCR
and dPCR experiments
A major issue with experiments
using qPCR and dPCR has been a
lack in standardized protocols and
data analysis. In most
publications the experimental
details are not clearly outlined
and therefore the quality of
experiments can get questioned.
As a result, the Minimum
Information for Publication of
Quantitative Real-Time PCR
Experiments (MIQE) guidelines
were established in 2009
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