LGC eBook - 2017 - 6

Accelerating Science Using Integrated PCR Tools * Advances in PCR Technology and Its Applications in Healthcare

they call "SuperSelective" primers to amplify
these extremely rare DNA fragments, and they
have successfully used them to detect as few as
ten mutant fragments among 1,000,000 wild-type
sequences-even when they only differed by
a single nucleotide polymorphism (SNP).
SuperSelective primers have a unique three-part
design that enables this selectivity: a 20-24
nucleotide-long "anchor" sequence that binds
both wild-type and mutant fragments on the
5' end, a short seven-nucleotide-long "foot"
sequence that perfectly complements the
mutant sequence without binding the wild-type
on the 3' end, and a "bridge" sequence designed
not to bind any regions in the target DNA
between the two ends.
To function effectively as a diagnostic tool,
however, the assay needs to test for several
different mutations simultaneously, and according to
Dr. Kramer, "clinically-relevant [cancer] mutations are
often located within the same gene or even within
the same or adjacent codons of the same gene," making it extremely difficult to design multiplexed assays.
6

| GENengnews.com

For the past two years, Dr. Kramer's lab has
combated this difficulty to generate a protocol
that can simultaneously detect multiple
clinically relevant cancer mutations using their
SuperSelective primers. The protocol they
developed hearkens back to the lab's earlier
invention of molecular beacon probes in 1992.
These hairpin-shaped oligonucleotide probes
contain a fluorophore on one end and a
quenching molecule on the other, which are held
together by short, complementary sequences
when un-hybridized. An identifying "tag"
sequence on the 5' end of the SuperSelective
primers hybridizes with the molecular beacons
to "turn on" the fluorophore by increasing the
distance between it and the quencher following
each round of successful amplification. Finetuning the different bridge designs helped to
further resolve independent threshold cycles
for different mutation sequences of interest.
Now that they have worked out the principles
behind the design of their multiplexed assay,
Dr. Kramer is meeting with different diagnostic
companies to test the technology: "companies

know how to take an assay and, if it works, get
it approved and make it available to hospitals
and clinics everywhere."
But getting discoveries from bench to bedside
often requires innovation in bioprocessing as
well. "If you look back fifteen years ago and
evaluate the kinds of technology platforms that
were being used for DNA testing-a lot of it was
simply cost-prohibitive," remarked Ross Higgins,
Director of Laboratory Operations at OneOme,
a pharmacogenomics startup company in
Minneapolis. "With platforms like the
IntelliQube® [For research use only. Not for
use in diagnostic procedures.] and other
highly-automated systems, the reaction
volumes have been driven down to a level
that makes testing more affordable on a
per sample basis," he added.
The IntelliQube® recently released by
LGC Douglas Scientific*, is one example of the
automated, high-throughput PCR instruments
that are helping drive down the cost of real-time
PCR. "It's not necessarily doing something
http://www.GENengnews.com
LGC eBook - 2017

Table of Contents for the Digital Edition of LGC eBook - 2017
