engineering

+ Photo Credit: U.S. Department of Energy Human Genome Program

technology http://www.ornl.gov/hgmis.

Sequencing Made Simple

by Shelby Martin

An efficient technique on the road to

personal genome sequencing

I
magine getting your genome sequenced in as little than traditional sequencing techniques. This technique
time as getting your film developed. You’d walk may drastically reduce sequencing time and cost, making it
into a clinic, get a quick cheek swab, and quickly practically feasible for individuals to have their own genomes
learn your risk for sequenced.
all sorts of genetic diseases. Photo Credit: Will Greenleaf
This could soon be possible Conventional DNA
through breakthroughs in DNA Sequencing
sequencing, a method that For the last 30 years, almost
determines the exact order of all DNA sequencing has been
the base pairs in a segment of done by the chain termination
DNA. method, originally developed by
Today, the technology for Fredrick Sanger. This method
DNA sequencing is exploding. involves hybridizing a short
“The doubling time for the DNA primer to the template
technology is something like 12 DNA to be sequenced, and
months,” says Stanford Ph.D. subsequent elongation of that
student in Applied Physics Will primer via DNA polymerase, an
Greenleaf. “It’s the Moore’s RNA polymerase (green) is bound to a bead (blue) which is enzyme that adds nucleotides
Law of DNA sequencing.” held in place by a laser (pink). The DNA (dark blue) is bound to complementary to the template
Greenleaf is working in the lab another bead (blue), which is also held by a laser (pink). The RNA strand. In addition to the
polymerase makes RNA (red), from the DNA template. As the RNA
of Dr. Steven Block, Professor polymerase moves down the DNA template, the distance between
normal set of nucleotides -
of Applied Physics and of the beads increases. adenine (A), cytosine (C),
Biological Sciences, to develop guanine (G) and thymine
a revolutionary way to sequence (T) - in the reaction mixture,
DNA. Their publication in the August 11th issue of Science relatively small numbers of dideoxy nucleotides are also
describes a method that uses ten trillion fewer molecules present. The dideoxy nucleotides are essentially crippled

Their publication in the August 11th issue of Science

describes a method that uses ten trillion fewer molecules
than traditional sequencing techniques.
48 stanford scientific

This technique may drastically reduce sequencing
time and cost, making it practically feasible for individuals
to have their own genomes sequenced.
nucleotides; when they are added to the growing DNA
strand, they prevent it from further elongation.
There are dideoxy versions of all four nucleotides, and
each are labeled to emit a different color under ultraviolet
light. The result is a series of DNA chains of different lengths,
each ending with a “tagged” dideoxy nucleotide. For instance,
if the fifth nucleotide in a DNA sequence is cytosine, the 5-
nucleotide long fragment will fluoresce the color of dideoxy
cytosine. A special computer reads the colors of all the
fragments and displays the full sequence.
No Primers Needed
Block and Greenleaf’s method represents an interesting
paradigm shift: instead of using DNA polymerase for
sequencing purposes, they employ RNA polymerase. In
a cell, RNA polymerase reads a DNA strand and makes
a complementary RNA strand. However, unlike DNA
polymerase, RNA polymerase doesn’t need any primers. In
the Block and Greenleaf experiment, RNA polymerase reads
a length of DNA that is tethered at one end to a stationary
polystyrene bead. The RNA polymerase is tethered to a
second polystyrene bead. As the RNA polymerase reads the
DNA, it moves down along the DNA strand. This motion
Photo Credit: Will Greenleaf
Stanford Ph.D. student in Applied Physics Will Greenleaf
layout design: Melanie Kanter
engineering
+
technology
pushes the polymerase’s bead farther away from the first
stationary bead. A laser tracks this movement with angstrom-
level precision.
The experiment is run in four different media. Each
medium has all four nucleotides, but one is in short supply.
When the RNA polymerase needs to add that limited
nucleotide, it takes longer to find it and stalls at that point.
The location where it stalled is measured, and the results
from all four buffers are combined to determine the entire
DNA sequence.
Current Challenges
Although the technique is ingenious, Greenleaf admits
that it is a “giant pain”. The setup is difficult. Furthermore,
whenever RNA polymerase incorrectly incorporates a
nucleotide, it pauses to correct itself. These random pauses
occur around once per thousand nucleotides, and they
require the researchers to compare the results of several
experiments to maintain accuracy. Finally, RNA polymerase
cannot process more than approximately two thousand
nucleotides before it falls off the DNA template strand.
The solution lies in parallelizing the technique by running
thousands of reactions at the same time. Eventually, says
Greenleaf, “Such parallelization is not out of the question,
but would be a major technical challenge.”
Rapid Progress
The Block and Greenleaf method is one of several “next
generation” DNA sequencing techniques. Another strategy
measures electrical conductance changes as DNA is sucked
through tiny membranes via nanopores. Another, called
pyrosequencing, uses enzyme-catalyzed light flashes to track
the addition of different nucleotides. While these techniques
have had limited success, the Block and Greenleaf method
has shown the most promise. In their published study, they
sequenced 30 nucleotides of DNA—more than any of the
other methods to date. The future of having your genome
“sequenced while you wait” may be here soon. S
SHELBY MARTIN is a junior majoring in Biological Sciences. She
enjoys studying LGBT fruit flies in the Baker lab at Stanford.
To Learn More
Visit the Block lab website: http://www.stanford.edu/
group/blocklab
Read the full-text version of the Greenleaf and Block’s
Science paper “Single molecule, motion-based DNA
sequencing using RNA polymerase”.
volume v 49