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Mechanical engineer Reg Beer (right) and electronics engineer Gary Johnson test a new polymerase chain reaction (PCR) instrument developed at Lawrence Livermore National Laboratory that can process biological samples in less than three minutes. Photos: Jacqueline McBride/LLNL
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Picture
this: You've brought your sick child to the doctor's office. After checking her
pulse and blood pressure, he takes a nasal or throat swab and inserts it into a
mysterious black box. Before the doctor finishes his examination, the black box
beeps, indicating that the pathogen that's making your child sick has been
identified.
Sound
far-fetched? Actually, this scenario is closer to becoming a reality. Thanks to
work by Reginald Beer and his team of scientists and engineers at Lawrence
Livermore National Laboratory (LLNL), sub-three-minute amplification of nucleic
acids (DNA and RNA) via polymerase chain reaction (PCR) is now possible.
With
three school-age children of his own, Beer knows all too well the frustration
of waiting 24 to 48 hours for lab results to learn whether his sick youngster
can be treated with antibiotics. "I was sitting in the pediatrician's
office with my daughter several years ago, and it struck me that if we could
make PCR fast and easy enough for use in doctors' offices, it would have a huge
impact."
PCR
is an indispensible technique in medical and biological research laboratories
around the world. It allows researchers and clinicians to produce millions of
copies from a single piece of DNA or RNA for use in genome sequencing, gene
analysis, inheritable disease diagnosis, paternity testing, forensic
identification, and the detection of infectious diseases.
The
standard approach to PCR typically takes about an hour, which is a vast
improvement over pre-PCR techniques that required days. However, PCR for
point-of-care, emergency-response, or widespread monitoring applications needs
to be faster still—on the order of a few minutes.
Beer
set out to find out just how fast PCR could go. His goal was to develop a
device where the limiting factor would be enzyme kinetics or thermodynamics,
not mechanical considerations. He also wanted to use "volumes that are
easy to interact with," not microfluidic-scale, knowing that in a
diagnostic setting multiple analyses are often needed and the ability to load
samples by hand is vital.
As
described in a paper published in Analyst, Beer and his colleagues created such a
device and demonstrated PCR times of less than three minutes. Their work was
funded by the Defense Advanced Research Projects Agency and internal LLNL money.
The
device achieves its extremely fast thermal cycling through the use of a porous
material and a thin-film resistive heater, making possible heating and cooling
rates of 45 C/sec, for a thermal cycle speed of less than 2.5 sec. "This
device is unique in that it cools as fast as it heats," notes Beer.
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A researcher loads a sample into Lawrence Livermore National Laboratory's new fast polymerase chain reaction (PCR) instrument.
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With
the device in hand, Beer's next challenge was to see if any commercially
available polymerase enzymes could work rapidly enough. Out of a group of 10
polymerases advertised by their manufacturers to be compatible with fast
thermal cycling, two enzymes worked in the LLNL device right out of the box.
"We
were really encouraged by the fact that two off-the-shelf enzymes worked at
these speeds," says Beer, "and there are a lot of parameters that can
be adjusted to potentially go even faster."
The
researchers demonstrated their PCR device by amplifying genomic DNA from an Enterobacter bacterium and a portion of
SARS DNA. The first tested the device's ability to rapidly amplify a large DNA
segment, and the second showed the device's utility in handling a public health
threat virus. The device achieved 30-cycle (billion-fold) PCR amplification of
the target DNA in as little as two minutes and 18 sec.
"When
I set out to do this, people said it would never work," Beer recountes.
"They gave mechanical reasons or enzyme kinetic reasons. But I answered,
'nobody really knows.'"
Now
that Beer and his team have demonstrated sub-three-minute PCR, they are working
to develop a real-time-detection device. They envision a PCR instrument that
can complete a test, from sample to results, in five to 10 min.
The
market for such a device would be huge. In addition to the traditional public
health and medical research applications, an easy-to-use real-time PCR device
would be enormously useful in the livestock, poultry, agricultural, and
processed food industries for ensuring food safety.
"We
have a lot of ideas for solving the scientific and technical challenges unique
to these applications," says Beer. "But this next step would benefit
from finding the right commercial partner. We're very good at inventing
technologies, but we need commercial partners to bring our innovations to market."
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