An efficient technique on the road to personal genome sequencing
by Shelby Martin
Imagine getting your genome sequenced in as little time as getting your film developed. You'd walk into a clinic, get a quick cheek swab, and quickly learn your risk for all sorts of genetic diseases. This could soon be possible through breakthroughs in DNA sequencing, a method that determines the exact order of the base pairs in a segment of DNA.
Today, the technology for DNA sequencing is exploding. ÒThe doubling time for the technology is something like 12 months,Ó says Stanford Ph.D. student in Applied Physics Will Greenleaf. ÒItÕs the MooreÕs Law of DNA sequencing.Ó Greenleaf is working in the lab of Dr. Steven Block, Professor of Applied Physics and of Biological Sciences, to develop a revolutionary way to sequence DNA. Their publication in the August 11th issue of Science describes a method that uses ten trillion fewer molecules than traditional sequencing techniques. This technique may drastically reduce sequencing time and cost, making it practically feasible for individuals to have their own genomes sequenced.
Conventional DNA Sequencing
For the last 30 years, almost all DNA sequencing has been done by the chain termination method, originally developed by Fredrick Sanger. This method involves hybridizing a short DNA primer to the template DNA to be sequenced, and subsequent elongation of that primer via DNA polymerase, an enzyme that adds nucleotides complementary to the template strand. In addition to the normal set of nucleotides - adenine (A), cytosine (C), guanine (G) and thymine (T) - in the reaction mixture, relatively small numbers of dideoxy nucleotides are also present. The dideoxy nucleotides are essentially crippled 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 ÒtaggedÓ 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 GreenleafÕs 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 pushes the polymeraseÕs 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 Ògiant painÓ. 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 DNAÑmore than any of the other methods to date. The future of having your genome "sequenced while you wait" may be here soon.