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DNA duplication trick may lead to faster testing

By raiding nature's tool cabinet, researchers have developed a potentially faster and more practical version of the polymerase chain reaction (PCR), itself a foundation of modern genetics.

The breakthrough, called helicase-dependent amplification (HDA), could result in small, hand-held devices which enable doctors to test blood samples directly in the surgery and forensic teams to detect a suspect's DNA at a crime scene.

PCR enables detection of tiny traces of DNA through a reaction which creates multiple copies of a single strand of target DNA until enough is available to show up in a detector.

As well as revolutionising crime forensics, PCR is now used routinely in hospitals to diagnose disease, in paternity cases that rely on genetic fingerprinting, in archaeology, and in all branches of biological and genetics research.

Thermal recycling

But it remains cumbersome and time-consuming because of the need to uncoil double-stranded DNA with heat. To make multiple copies of a target strand of DNA, the sample has to be repeatedly heated up to break the DNA, then cooled to allow more to form. But this "thermal recycling" requires energy, time and equipment that can only be used in a lab setting.

HDA could be about to change that because it relies on a technique which uncoils DNA the way nature does - with enzymes called helicases. "We have hundreds of different helicases in our own cells," explains Huimin Kong, head of the team which developed the new technique at New England Biolabs, a company in Beverly, Massachusetts.

All organisms need them for uncoiling DNA when cells replicate, repair DNA or recombine it. Given that their uncoiling function has been known since the 1970s, Kong is amazed that no-one had previously tried using helicases as a replacement for heat in PCR. But no rival groups appeared to have attempted it when Kong's group applied for a patent on HDA 18 months ago.

In their experiments, Kong and his colleagues successfully multiplied strands of DNA with a helicase from the bacterium, Escherichia coli, instead of thermal recycling. The entire process took place at the same temperature, 37 oC.

Target strands

As with conventional PCR, Kong had to add "primers", slugs of DNA which bind to pre-selected target sequences in the uncoiled target strands, and which serve as the starting point for the strand to be copied. He also had to add polymerase, the enzyme which actually copies the strands.

In conventional PCR, newly formed strands recombine with each other to re-form DNA, which then has to be uncoiled again by raising the temperature to allow yet more to be made. But the helicase does the job instead, without reheating.

"It worked like a dream," says Kong, whose team proved that HDR can detect microbial DNA in simulated blood samples. But his team is working to improve the system. At present, it can only detect relatively small amounts of DNA, up to about 200 base pairs, compared with PCR.

Kong is confident of overcoming these limitations, and of finding helicases which work at room temperature. He is also confident that he can get the technique to give answers within 15 minutes compared with the 30 to 60 minutes it takes for PCR.

"The feature in greatest need of tweaking is the reaction time," agrees Mark Griep, an expert in helicases at the University of Nebraska-Lincoln.

Journal reference: EMBO reports (DOI: 10.1038/sj.embor.7400200)



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