Lab Notes

Posted August 2008

Looking for a Reaction

A new DNA amplification method is better, faster, and cheaper than traditional tests.

When the polymerase chain reaction (PCR) process was developed in the early 1980s, it revolutionized DNA cloning and analysis by allowing investigators to take a few strands of DNA and replicate them until there were so many that the odds of detecting them significantly increased. Now researchers at Lincoln Laboratory say they've brought PCR to a new level by developing reagents that make the process faster, cheaper, and more sensitive. This innovation opens up new possibilities for detecting pathogens for military uses, food safety, and clinical diagnostics.

Chemist Christina Rudzinski and applied biologist Amanda Stephens of the Biodefense Systems Group, along with molecular biologist Laura Bortolin, have developed an artificial molecule that enhances PCR and that requires less sample preparation and provides heightened sequence specificity. The molecules designed by the trio are a modification of traditional PCR primers, which initiate the DNA amplification reaction. The new primers rely on a synthetic component, a peptide nucleic acid (PNA). PNA is made up of the same four nucleic acids as DNA.

Instead of the sugar phosphate molecules that make up the backbone to which the nucleic acids attach, PNA has a backbone based on peptide bonds. Because DNA backbones have a negative electrical charge, opposing strands tend to repel each other although the tendency of complementary nucleic acids to bind overcomes that repulsion. PNA backbones, however, lack charge, so they bind much more strongly to DNA. Because of this strong binding, PNA has been used for more than a decade to block certain DNA reactions in assays. But it has never been used for DNA replication in real-time PCR before. As it turns out, the lack of charge gives the molecule new properties that improve the workings of PCR, which up to now has been the gold standard for gene amplification.

The researchers’ primer is a strand of nucleic acids specific to the DNA they're trying to replicate. Say they want to detect anthrax. Using a computer program Stephens developed to design the primers, they pick a portion of a DNA sequence that's unique to anthrax and have an outside company synthesize a matching primer. The primer starts with one to seven units of PNA, followed by a string of DNA; the Lincoln Laboratory team calls it a chimeric primer, named for the mythical Chimera, which had the head of a lion and the body of a goat. The PNA, which is more strongly attracted to the target DNA, snaps onto the beginning of the target sequence, and the DNA follows, just as in natural replication. Rudzinski says the presence of PNAs essentially jumpstarts the reaction.


Diagram indicating the use of PNA as a starter for binding DNA to a target string of DNA

DNA and PNA are both composed of the same nucleotides (top). But while the DNA backbone is made up of sugar phosphate molecules, the PNA has a polyamide backbone similar to that found in proteins. A chimeric primer (below) starts with a few units of PNA (red) followed by DNA (blue), which together bind to a target string of DNA (black).

On rare occasions, all-DNA primers sometimes bind to the wrong spot on the target molecule. For a number of complex reasons, the PNA-DNA primers generate fewer binding errors. Such specificity is important if you're trying to tell, say, whether a sample contains anthrax or Bacillus thuringiensis kurstaki, a common insecticide that is often mistaken for anthrax.

Because the PNA at the head of the primer isn't charged, it's much less sensitive to salt levels in the sample, whereas traditional PCR won't work unless the salt concentration is just right. PNA’s electronic neutrality also makes the reaction less sensitive to pH levels. Thus preparatory steps to get salt and pH levels just right are no longer needed, and the samples can be much less pure. A sample requiring PCR amplification is commonly treated by using DNA purification kits, which remove extraneous proteins and other material that might interfere with the amplification process. The chimeric primers don't need that step. The group tested samples with 100 milligrams of soil to a milliliter of water, in which all-DNA primers don't work at all, and found the chimeric primer produced results almost as good as in a pristine sample. This insensitivity of the process to sample purity increases testing speed while cutting costs. The process also worked in a drop of blood only slightly diluted with water; such a sample would stymie normal PCR because of salts and other components in the blood. Moreover, the PNA renders the primer unrecognizable to DNases and proteinases—that is, the enzymes that break down natural DNA and proteins. Again, it's the nonstandard backbone that accounts for the difference. With fewer molecules attacking the primer in the blood sample, more of it survives to find the target DNA, making the test more sensitive. In tests, the group found that, depending on the type of sample, their PNA primers cut prep time approximately in half and cut costs by two-thirds.

The PNA system has appeal for military and homeland security uses, but it could also be developed for food safety and environmental testing, or even medical diagnostics, the researchers say. They're hoping for funding to test the PNA method for other types of targets. For instance, they haven't yet shown that it can work in the reverse-transcription PCR assays required to detect RNA. But they see no reason that a chimeric primer wouldn't work there, too. "If you could do that, you could do HIV detection in blood without sample preparation," says Bortolin, who is currently a principal research scientist at Syracuse Research Corp. "That would be tremendously useful." It could mean a test in a doctor's office could detect actual virus in a few minutes, whereas today's quick tests can find only antibodies.


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