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July 28, 2010

Shaking Paves Way To Clean DNA

Nucleic Acid Chemistry: Method purifies by catching desired product in a gel

Carmen Drahl

CATCH AND RELEASE The linker (red) is attached to the DNA via phosphoramidite chemistry, standard in nucleotide coupling.
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Using a polymer commonly used for gel electrophoresis, chemists have developed a simple way to purify synthetic DNA. The technique might be applicable to oligonucleotides in development as antisense drugs for cancer and other diseases.

Typically done by chromatography, DNA purification is a bottleneck in antisense drug development. The method Shiyue Fang and graduate student Suntara Fueangfung of Michigan Technological University have devised relies on shaking and extraction instead. They attach a cleavable linker to their desired DNA during the last step of an automated synthesis. This ensures that incomplete DNAs, which are capped during the synthesis, don't get a linker, Fang explains. Through the linker, the team incorporates their product into a polyacrylamide gel (Org. Lett., DOI: 10.1021/ol101316g). Gentle shaking washes impurities away, and then cleaving the linker yields purified DNA.

"The beauty of Fang and Fueangfung's catching-by-polymerization method is that it allows the capture of full-length oligonucleotides from the crude synthesis product without using reverse-phase chromatography or solid-phase extraction cartridges, thus saving time and materials," says nucleic acid expert William H. Pearson of Pearson Chemistry Solutions. But it still doesn't overcome every limitation of established technology, he says. For instance, it still cannot remove sequences where one nucleotide is deleted as a result of incomplete capping, because those will still have a linker.

When the method is scaled up, it could be hard for reagents to access the linker cleavage site, and impurities might become trapped in the gel, says Willi Bannwarth, an expert in oligonucleotide purification at the University of Freiburg, in Germany. Still, "the method is very innovative and differs significantly from all purification strategies out there," he says.

The gel swells in the solvents used to wash away impurities and to cleave the linker, which might help in scaling up the technology, Fang notes. He says his team plans to apply the technique to purifying other biopolymers such as peptides and oligosaccharides.

Chemical & Engineering News
ISSN 0009-2347
Copyright © 2011 American Chemical Society
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