Nanobiotechnology Sorts DNA
Source: Cornell University
February 15, 2001
Cornell researchers replace test tube with tiny silicon
devices to rapidly measure, count and sort biological molecules
SAN FRANCISCO -- Up to now, most biologists have studied
the molecules of life in test tubes, watching how large numbers of
them behave.
But now researchers at Cornell University in Ithaca,
N.Y., are using nanotechnology to build microscopic silicon devices
with features comparable in size to DNA, proteins and other biological
molecules -- to count molecules, analyze them, separate them, perhaps
even work with them one at a time. The work is called
"nanofluidics."
"This will expand the methods for analyzing very small
amounts of biochemicals, and create new abilities unanticipated by the
test-tube methods," says Harold Craighead, Cornell professor of
applied and engineering physics and director of the Cornell
Nanobiotechnology Center (NBTC).
Craighead will describe some of his laboratory's work in
a talk, "Separation and Analysis of DNA in Nanofluidic Systems," at
the annual meeting of the American Association for the Advancement of
Science (AAAS) at the Hilton San Francisco today (Feb. 15, 2 p.m.).
The talk is part of a two-day seminar on nanotechnology.
Craighead's work began with a quest for an "artificial
gel" that would replace the organic gels used to separate fragments of
DNA for analysis. Traditionally this has been done by a process
called gel electrophoresis. Enzymes are used to chop DNA strands into
many short pieces of varying
length. The sample is placed at one end of a column of organic gel
and an electric field is applied to force the DNA to move through the
gel. As they slowly snake their way through the tiny pores of the
material, DNA fragments of different lengths move at different speeds
and eventually collect in a series of bands as a ladder-like structure
that can be photographed using fluorescent or radioactive tags. The
resulting image, Craighead explains, is just a list of the lengths of
the fragments, from
which scientists can read out genetic information. So he looked for
other ways to sort DNA fragments by length that would allow scientists
to work rapidly with small amounts of material. Craighead and his
colleagues manufactured a variety of silicon-based nanostructures with
pores comparable to the size of a large DNA molecule.
They have explored three approaches to DNA separation:
* Devices with openings that are less than the "radius
of gyration" of the DNA fragments -- When suspended in water, a DNA
chain quickly coils into a roughly spherical blob. When pressed
against a barrier with openings smaller than the radius of this form
it must uncoil to pass through. Paradoxically, larger molecules do
this more quickly, because they press a broader area against the
obstacle, offering more places where a bit of the chain can be drawn
in to start the uncoiling. When an electric field is used to drive a
mix of DNA fragments along a channel with several such barriers,
fragments of different lengths will move at different speeds, arriving
at the far end in a series of bands not unlike those seen in gel
electrophoresis.
* Sorting by physical length -- A DNA sample is placed
just outside a "forest" of tiny pillars arranged in a square grid, and
an electric field applied to force the molecules to move into the
grid. (Imagine pulling a coiled watch spring into a long, straight
alley.) If the electric field is
turned on just briefly and turned off before the molecule gets all the
way in, the uncoiled portion will snap back out, just as the watch
spring will pull back into its coil. But once the entire molecule is
inside the grid, there is nothing to pull it back out. By varying the
length of the electric field pulse, the researchers can control the
length of the DNA strands that
are collected in the grid. In addition to providing a way to measure
strand length, Craighead says, this tool could be used to separate DNA
for other work. If two molecules of different length are present at
the start, the shorter molecules could be moved into the grid,
leaving a pure sample of the longer strands outside.
* Lateral diffusion by length -- When moving through a
grid of tiny pillars, DNA chains are constantly buffeted by moving
water molecules that can knock them off-track, a process called
"Brownian motion." If the pillars are flat vanes, all angled in the
same direction, movement of all
the chains will be skewed to one particular side. Shorter, lighter
molecules will be pushed farther, so molecules can be sorted or
measured based on the distance they are moved across the track when
they emerge from the grid. Craighead calls these devices "Brownian
ratchets." These techniques all work with molecules en masse, but
Craighead's group is also studying ways to work with single molecules,
or at least to work with molecules one at a time. They have built
microscopic tunnels just large enough for DNA molecules to run through
in single file. Nanofabricated light pipes are placed on either side
of the tunnel. Although very large, DNA molecules are still too small
to be seen directly by visible light, but they can be tagged with
other molecules that fluoresce when exposed to an
ultraviolet laser, and the fluorescence can be detected, with larger
molecules giving off longer pulses of light. In addition to counting
the number of molecules of a given size in a sample, these devices
could incorporate switches that could shunt molecules of different
sizes into different channels, Craighead says.
While most of the work up to now has been with DNA,
Craighead says, these methods could also be applied to the study of
other large organic molecules, including proteins, carbohydrates and
lipids.
by David Brand - deb27@cornell.edu
Bill Steele - ws21@cornell.edu
607-255-7164
http://www.news.cornell.edu/releases/Feb01/AAAS.nanofluidics.ws.html