"‘Digital PCR’ has
become one of the latest buzz words," says Mike
Lucero, EVP of sales & marketing for Fluidigm
Corporation. Indeed, the term digital PCR has found its
way into the molecular biology literature — a simple
PubMed search returns hundreds of items.
"While people have found ways to do digital PCR,
they’re all fairly complicated," Lucero contends.
In what are known as limiting dilution experiments,
samples are appropriately diluted and pipetted into
1,000s of microtiter wells. Only those wells that are
positive for the target give a signal. In practice,
digital PCR in microwell plates is complex and prone to
errors. Also, "The reagents are prohibitively
expensive for a 1,000-well experiment at microvolumes,"
says Lucero. "Further, it’s done at a dilution
where each one of the reaction vessels will either have
0 or 1 target molecule."
Fluidigm’s digital array device simplifies the
process, making digital PCR easier to perform and more
reliable, while using nanovolumes limits cost
constraints. "The digital array is a type of
integrated fluidic circuit," Lucero explains.
"Running the array requires one step." The
user loads a mixture of the diluted sample and an
analyte-specific reagent for the target of interest. A
network of channels and valves partition it into about
1,000 distinct reaction vessels. The array can then be
amplified by PCR, making it possible to detect target
present in any of the partitions.
Slicing and dicing the mixture effectively enriches
the target molecule. After PCR amplification on the
array, fluorescent signals for any positives are clearly
visible in the chambers. "The ability to assay each
of the 1,000 distinct partitions, from an appropriately
dilute sample allows you to accurately count how many
target molecules are in a sample," says Lucero. In
theory, the digital array could be applied to any
fluorogenic PCR assay currently done in closed tubes or
microwell plates.
Detection of Tumor-Derived DNA as Surrogate for Tumor
Frank McCormick, director of the Comprehensive Cancer
Center at University of California, San Francisco, uses
the Fluidigm digital array to detect mutant DNA in the
serum of patients with cancer. Such an application of
the technology could enable clinicians to detect disease
early, or monitor patient response to therapy.
Typically, tumor cells shed a tiny amount of mutant
DNA among an enormous background of normal DNA released
from healthy cells, McCormick explained. Being able to
detect one mutant within an excessive amount of normal
material is technically very difficult, but Fluidigm’s
digital array facilitates the process.
"Partitioning a sample into thousands of smaller
aliquots separates out the proverbial needle in the
haystack, making it easier to detect," says
McCormick, acknowledging that while it’s too early to
say whether the digital array will be used clinically
for early detection of disease, it has been shown to
work for that purpose.
A second application is for guiding treatment.
Gleevec serves as a prominent paradigm: Mutations in the
bcr-abl oncogene render patients refractory to the drug.
Detecting mutant clones early could be critical in
selecting which drugs patients get next. "I believe
that [this] application of the technology is more likely
than the first to go straight into clinical
practice," McCormick notes. Oncology may be the
most prominent indication, but the same principle
applies to other areas. For example, as with Gleevec,
patients can develop resistance and become refractory to
their HIV treatments.
McCormick touts the advantages of the Fluidigm
digital array over other liquid-handling methods for
sample separation. "The sample size is tiny,
material is accurately distributed, plus the PCR
reaction can be measured in real time," McCormick
notes. Each step in the process could be done in a
clunky way using different technology, but "this
streamlines distribution, amplification, and sensitive
detection, on one system."
"Digital arrays may be a novel approach to
catching disease early," McCormick concludes.
"And detecting DNA mutations may be the most
specific way to do that for cancer."
