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PCR-Based Assays For a New Assay Technology, Try Playing with Tadpoles

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In the grand scheme of molecular biology, it’s hard to overstate the impact of PCR in allowing researchers to detect and manipulate genetic material. That’s why it comes as good news that researchers at the Molecular Sciences Institute have come up with a technique for harnessing PCR’s power to create sensitive and quantifiable assays for proteins, peptides, small organic molecules, and most other compounds of biological interest.

Led by Roger Brent, the team at MSI has developed a technique, described in the January issue of Nature Methods, for wedding protein or peptide capture molecules to a nucleic acid tail that functions (with the help of PCR) as the reporter. The trick to doing this is expressing the capture agent as a fusion protein linked to an intein, which is a segment of the protein that is able to excise itself from the polypeptide chain. Ian Burbulis, working in Brent’s lab, designed the intein excision to occur in such a way as to allow a strand of DNA to attach to the protein capture agent. The DNA strand, when properly configured with a T7 RNA polymerase transcription binding and start site and a PCR amplification region, can function as a tag that can be amplified with PCR. The molecule as a whole — the resulting protein-DNA chimera — is known as a “tadpole.”

Piggybacking on PCR makes the tadpole construct 109 more sensitive than an ELISA assay, and an additional advantage to this scheme lies in the precision with which the DNA tag is coupled to the capture agent. Writing in a companion commentary in the same journal, immunologist Garry Nolan of Stanford University says that in contrast to other, more established techniques for linking a DNA tag or fluorophore to a capture agent, Brent and his group provide evidence in their paper that their technique consistently links only one DNA tag per capture agent. The result, Brent says, is an assay that can more sensitively quantify the concentration of a target compound.

Stanford biochemist Ron Davis, however, has reservations about how readily the tadpole strategy can be adopted for high-throughput experiments. “Assays like this are going to be critical for dealing with protein concentration problems,” Davis says. “It has good characteristics, but how well will it work in a large, multiplexed format?” He and a collaborator, Simon Fredriksson at the Rudbeck Laboratory in Sweden, are developing a similar PCR-based assay technology that Davis says “at the moment has better sensitivity” than the tadpole approach.

But Brent argues that the significant amount of characterization and supplementary material his group has provided should make the tadpole approach relatively easy to adopt, and furthermore, “it’s no half a million dollar gizmo,” Brent says.

Burbulis, I.; Yamaguchi, K.; Gordon, A.; et. al. "Using protein-DNA chimeras to detect and count small numbers of molecules" Nature Methods 2005, 2, 31 – 37.

Fredriksson, S.; Gullberg, M.; Jarvius, J.; et. al. "Protein detection using proximity-dependent DNA ligation assays" Nature Biotechnology, 2002, 20, 473 – 477.

— John S. MacNeil

 

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