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By Shauna Farr-Jones

 

The Stanford Genome Technology Center, having already created instruments to increase the throughput and lower the cost of DNA sequencing and analysis, is now tackling post-genomic problems. “We are moving more to functional genomics tools from developing sequencing tools,” says biologist Paul Hardenbol, a postdoc at the center.

Previously, to speed genome sequencing efforts, the center developed a DNA shearing device, which is now sold by GeneMachines as HydroShear. It produces 2,000-base-long oligonucleotides — the appropriate length for plasmid sequencing libraries — and solves two major limitations of slicing nucleic acid into pieces, which were highly varied fragment lengths and fragments that weren’t random enough.

For example, digestion with restriction enzymes is sequence dependent. The non-random distribution of the resulting libraries isn’t useful for shotgun cloning. And other mechanical stress techniques, such as sonication, result in a huge range of fragment sizes, and therefore require another size-separation step before researchers can use them for cloning.

The HydroShear has been a commercial success. In fact, GeneMachines, which was founded by former Stanford center researchers to commercialize its innovations, calls the HydroShear its best-selling product.

The length of the oligos it produces, however, limits its use to sequencing prep. So, building on their experience, Hardenbol and his colleagues have now built a prototype of a second-generation DNA shearer. It consistently generates oligos of only about 200 bases.

Like the HydroShear, it chops DNA into random fragments within a narrow size distribution — 90 percent of the DNA fragments are between 100 and 300 bases. But reducing the length by an order of magnitude opens the technology for a whole new suite of applications.

The DNA shearer forces filtered genomic DNA through a narrow orifice at supersonic speed. To create the tiny hole, the Stanford researchers slice open a 15-to-50-micron diameter fused-glass capillary tube. “The sharp edges are important to increase acceleration,” says Hardenbol.

They then glue the tube into a metal fitting and attach it to a high-pressure pump, which splits the DNA into the desired size. “We have only taken it up to 80,000 PSI, but the pump can go as high as 100,000,” Hardenbol says.

But at the high pressures necessary to create such small fragments, intense friction heats and degrades the nucleic acids. Hardenbol, along with DNA shearer codevelopers physicist Tom Willis and engineer Roger Donaldson, solved the problem by adding glycerol to the genomic DNA. Because heat decreases glycerol’s viscosity, friction becomes less an issue. “You get the benefit from the glycerol in increased shearing, but you don’t pay the price later because the viscosity drops as the temperature goes up,” Hardenbol explains.

The fragments include native ends — that is 3’ and 5’ hydroxyl groups — that can be used for PCR or cloning and produce plasmid libraries of whole genomic DNA. They also are small enough for mass spec analysis, which the Stanford center is coupling with an HPLC separation step.

Because the sheared DNA consists of random, whole-genome fragments, it could also be used as a target sample for microarray probes to compare genomes of organisms or strains. Hardenbol says he plans to hybridize the random fragments to Affymetrix chips. “You could do the same cleavage with RNA,” he says.

He is also working on other applications for the whole-genome oligos, but for now is keeping the ideas to himself. “I want to be the first person to do those experiments,” he says.