By identifying a 30-year-old mistaken assumption,
Johns
Hopkins Kimmel Cancer Center scientists have found that
substituting a simple bleach solution for more complex
tools makes a DNA separation technique called
electrophoresis five times faster and also less costly.
Reported in the February issue of
BioTechniques, the scientists say that using the
compound sodium boric acid in DNA electrophoresis may speed
genetic discoveries. The scientists searched old literature
and dozens of compounds to find one that could replace
antiquated solutions used to conduct the electric current
necessary to separate negatively charged DNA molecules, the
building blocks of genetic code.
In electrophoresis, DNA is fed through porous,
jellylike slabs of sugar (also known as a DNA gel) to
reveal the outlines of the code, with small DNA molecules
crawling up the gel faster than less mobile larger
particles as the current passes through.
Like the two poles on a battery, the difference in the
positive and negative charges represents a voltage. "DNA
just needs to know it's in a voltage, and it will move,"
said Scott Kern, professor of oncology and
pathology at Johns
Hopkins. "So the most important feature of a solution in
electrophoresis is its ability to carry a voltage."
Solutions historically used for DNA electrophoresis
are called Tris-acetic acid-disodium EDTA, called TAE, and
Tris-boric acid-disodium EDTA , or TBE. For 30 years,
scientists have mistakenly assumed these solutions were
good conductors and "buffers." Buffers serve to reduce
acidity or pH and were used for protein electrophoresis in
the 1950s. They are still used today and work well in
protein electrophoresis because pH balance is important for
separating proteins, which in their natural state carry an
unpredictable charge.
However, Kern and postdoctoral fellow Jonathan Brody
conducted experiments demonstrating that TBE and TAE
provide only some buffering — which they say isn't
important
anyway for DNA separation — and too much conductivity. "In
fact, TBE and TAE essentially short-circuit DNA gels by
creating too much current and heat," Kern said. Gels using
TBE and TAE at high voltage are known to melt and distort
DNA particle movement.
"Our work opened up the door to try different
solutions," Brody said. "But there was no theory to predict
which compound would work better."
After more than two months of trial and error
experimentation, Brody settled on sodium boric acid. "It
has great resolution at high voltages. I can now run a gel
in 15 minutes using sodium borate as opposed to an hour and
a half with TBE or TAE," Brody said. "It's like switching
from a dial-up modem to a high-speed Internet connection.
You never want to go back."
Cost per gel of sodium boric acid is $0.07 as compared
to TAE at $0.27 and TBE at $0.67. The scientists estimate
industry savings of $37 million annually based on the total
market for DNA gels in the United States.
"In science, as in any other profession, to some
extent you accept what you're taught, and everybody was
taught to use the same tools for extracting the components
of DNA," Kern said. "Scientists 30 years ago made no claim
that these were the best tools, but when other scientists
asked them what they used, they told them, and then, like a
herd, everyone followed and accepted it."
Kern and Brody have filed for a provisional patent on
the sodium boric acid solution. They are entitled to
royalty payments from and own equity in FasterBetterMedia,
LLC, the company that is commercializing this product.
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