640 | SEPTEMBER 2004 | VOLUME 5 www.nature.com/reviews/genetics

R E S E A R C H H I G H L I G H T S

Modern genetics relies so heavily on PCRthat it is hard to imagine laboratory lifewithout it. However, although PCR wasindisputably one of the biggestbreakthroughs in genetics — transformingfields from forensic science to diseasediagnosis — the thermal cycling at the heartof this technique makes it hungry for energy,time and special equipment. By using abacterial helicase instead of heat to denaturethe template, Huimin Kong and colleagueshave now overcome the limitations of PCR bydevising a means to amplify DNA at a singletemperature. This opens the door to thedevelopment of quick, hand-held diagnosticdevices that could move DNA analysis fromthe bench into the field.

The new amplification technique, calledhelicase-dependent amplification (HDA),mimics a process that occurs in nature. Ahelicase — in this case the Escherichia coliUvrD helicase — is first used to separate thedsDNA template molecules. The separatedstrands are then coated with ssDNA bindingproteins to keep them apart, allowing primers

to anneal and be extended by a polymerase.The reaction occurs at 37°C, from start tofinish, and can achieve over a million-foldamplification of genomic DNA. It is alsosensitive enough to pick up DNA from intactcells and to detect pathogen DNA in blood.

HDA is not the first isothermal reaction tobe proposed, but the alternatives involvemore complex reactions and rely on aninitial denaturing step that requires heat.Essential tweaks are now being carried outto improve the efficiency of HDA. Byexperimenting with the reactioncomponents — nature has hundreds ofhelicases to choose from — and theirconcentrations, Kong and colleagues hope tobring reaction times down to 15 minutes.Machine-free DNA amplification ‘while-you-wait’ might be just around the corner.

Tanita Casci

References and linksORIGINAL RESEARCH PAPER Vincent, M., Xu, Y. & Kong,H. Helicase-dependent isothermal DNA amplification. EMBOReports 9 July 2004 (doi:10.1038/sj.embor.7400200)WEB SITENew England Biolabs: http://www.neb.com

No-bake recipe for DNA

T E C H N O LO G Y

or worms are maintained for many generationsin a benign environment. Such assays are longand tedious: for one collection, scientists evenseparated each worm in each generation fromeveryone else to avoid any selection pressureinduced by the stressful dating scene, andinstead, self-fertilize.

Previous studies have used fitness-basedassays to assess the number of mutations thatdeveloped in each line, yielding an indirect esti-mate of mutation rate.For a more direct estimate¸Denver and colleagues have now revisited a col-lection of these special worm lines and seq-uenced more than 4 Mb of loci scatteredaround the genome. In a recent paper in Nature,they report the total haploid genomic mutationrate to be approximately 2.1 mutations pergenome per generation — an estimate that is anorder of magnitude higher than previous bestguesses, and 2 orders higher than the indirectestimate from the same collection (althoughthe previous estimates referred to deleteriousmutation rate, whereas these authors estimate

total mutation rate). Not only that, but the morefrequently observed mutations were insertions,in contrast to reports based on pseudogenesthat indicated that most naturally occurringmutations in worms are deletions.

So, with one study, Denver and colleaguesprompt the entire field to rethink the process ofmutation over time and our measurement of it.It does not take long for this to generate contro-versy: in a thought-provoking News and Viewspiece, Rosenberg and Hastings speculate on themechanisms at work in the Denver study. Eitherprevious estimates were wrong because theyonly detected mutations that produce pheno-types, as Denver et al. would suggest, or the newstudy uses methods that push the worms todevelop more mutations even in the absence ofdeleterious selection, maybe even through trig-gering stress-response pathways. Future studiesmight have to bring the worms out of their poshretirement to settle the question.

Chris Gunter, Senior Editor, Nature

References and linksORIGINAL RESEARCH PAPER Denver, D. R. et al.High mutation rate and predominance of insertions in theCaenorhabditis elegans nuclear genome. Nature 430, 679–682 (2004)FURTHER READING Rosenberg, S. M. & Hastings, P. J.Worming into genetic instability. Nature 430, 625–626 (2004) WEB SITENemATOL: http://nematol.unh.edu/index.php

Mutation rate, whether per locus or genome-wide, features as a variable in many calculationsthat underlie evolutionary genetic hypotheses.But a true experimental estimate of mutationrate is difficult to obtain. To this end, severalgroups have used ‘mutation-accumulation’lines in both Drosophila melangaster andCaenorhabditis elegans : in an attempt to mini-mize selection pressure, genetically identical flies

Mutations: more common than you thought

G E N O M E E V O L U T I O N

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