The second annual IBC ‘Nucleic Acid Detection and Screening Technologies’conference (Tucson, Arizona; 23–25April 2001) featured 16 scheduled pre-sentations in the categories of detection enhancement, applications, and DNAand protein arrays. In addition, severalposters were selected for oral pres-entation. Highlights of ten of the excel-lent presentations will be covered here,with an emphasis on emerging newtechnologies; a common theme at themeeting being miniaturization, speedand parallelism.

Technological advancesThe plenary address, entitled ‘Newtechnologies for deciphering the genome’,was delivered by Lloyd M. Smith(University of Wisconsin, Madison, WI,USA). Smith described his laboratory’sinterest in MS and DNA arrays, and alsohis involvement with Third WaveTechnologies (Madison, WI, USA) andthe company’s Cleavase technology.The difficulty in using single nucleotidepolymorphisms (SNPs) as genetic markersfor widespread screening were high-lighted, because of the cost and compli-cations involving amplification (i.e. PCR)and multiplexing. Because of the limitedvisible spectrum, in which only 5–6 distinct fluorophores can be resolved,MS presents an appealing alternativewith potentially much greater multi-plexing capacity because of the widemass-range. SNP-typing with a SurfaceInvader™ assay (Third Wave Technologies)can potentially accommodate up to 106

SNPs in a single step and opens up thepossibility of large-scale population SNP

typing without PCR. Smith also de-scribed limitations with matrix-assistedlaser desorption ionization mass spec-trometry (MALDI-MS), whereby the signal intensity decreases as a function ofthe nucleic-acid length. A new techniqueof charge-reduction electrospray MS(CREMS), however, allows significant im-provement in signal uniformity across a broad mass-range. In particular, a new instrument design implements apiezo drop-on-demand sample delivery,whereby spectra are collected from single 100 pl drops and provide a50–100-fold increase in signal intensity.

Probes and genotypingTim Tieman (Clinical Microsensors,Pasadena, CA, USA) contrasted DNA‘discovery’ activities where thousands ofassays are necessary, cost per datapointis a key factor and comprehensive cover-age is vital, with DNA ‘diagnostic’ activi-ties where only tens of assays are run,panel cost is more important than data-point cost and the market is heavily regulated, to illustrate the distinctionsbetween these two businesses. The com-pany’s eSensor™ platform is a homo-geneous electrochemical detectionscheme applicable to any amplified nucleic-acid target, and gives a digitalreadout without interference from cont-aminants (e.g. buffers, proteins and reaction components). The current inte-grated system accommodates 48 chips,each with 36 distinct sensors. Two DNAprobes are used for each target: an immobilized capture probe and a fer-rocene-labeled signaling probe. The immobilized probe is connected to an

electrode through phenyl acetylene‘wires’ embedded in a self-assembledmonolayer. An insulating monolayerprotects the electrode from interferents.A genotyping example was shown thatused two types of ferrocene labels, eachwith a different redox potential. Otherexamples included SNP detection, genedetection and quantitative genotypingof HIV, where <10% wildtype sequencewas readily measured (conventional geltechniques only detect 30% differencesin sequence).

The Multiplexed Molecular Profilingassay (MMP) was described by MatthewRounseville (High-Throughput Genomics,Tucson, AZ, USA); this assay handles 16 RNA targets from 96 samples with material from as few as 1000 cells (notarget amplification is needed). Each microtiter well contains a 4 × 4 array ofimmobilized capture probes. A key com-ponent of the nuclease protection assayis the combined lysis and hybridizationbuffer. The process involves lysis and solution-hybridization with DNA protec-tion probes, S1 nuclease digestion, basehydrolysis and transfer to a functional-ized MMP 96-well plate, followed by horseradish peroxidase chemilumin-escence detection. Four weeks areneeded to completely set up the assay.The conversion of RNA to DNA is stoi-chiometric and signal attenuation can beachieved to widen the dynamic range.Examples of MMP in drug developmentinclude monitoring drug effects, dose responses and compound screening. AnELISA example was shown to demon-strate possible proteomic applications ofthe MMP assay.

DDT Vol. 6, No. 13 July 2001 updateconference

1359-6446/01/$ – see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(01)01858-X 667

New technologies in nucleic aciddetection and screening Robert G. Kuimelis, Phylos, 128 Spring Street, Lexington, MA 02421 USA, tel: +1 781 862 6400, fax: +1 781 402 8800, e-mail: rkuimelis@Phylos.com

Bead technologies and DNA detection James Jacobson (Luminex, Austin, TX,USA) described ‘analyte profiling’ withthe LabMap suspension array system,which uses derivatizable color-codedmicrospheres, a flow-analyzer and ahigh-speed digital signal processor, aswell as the necessary hardware, softwareand reagents. Currently, 100 uniquelycolored beads are available, and 1,000are expected to be available soon. Eachnew color allows introduction of yet an-other level of multiplexing. Hybridizationkinetics for the 5.6 µm diameter beadsare similar to solution hybridization and,therefore, assay times are significantly reduced compared with conventionalmicroarrays. Several examples wereshown for SNP detection [e.g. Factor Vand cystic fibrosis (CF)] and expressionprofiling. In the case of CF, only a smallvolume (a few µl) of patient sampleyielded results, in <15 min, using a 5-plexassay that covers the majority of mu-tations that cause CF in North America.The suspension arrays allow simple assayreconfiguration and elaboration, in con-trast to a conventional two-dimensional(2D) microarray.

Details of a universal electrocatalyticDNA detector based on ruthenium-mediated guanine oxidation were given(Natasha Popovich, Xanthon, ResearchTriangle Park, NC, UCA). This approachdoes not require labels or target ampli-fication, and sample preparation is mini-mal. In fact, assays can be performed directly from cell lysates. The electrodeshave been miniaturized to 200 µm, whichstill produces nA currents and allows con-ventional potentiostat designs to be used,thus simplifying the instrumentation. Ina microplate format, each well containsup to seven electrodes bearing immobi-lized DNA capture probes (or analogueDNA capture probes with enhanced bio-physical properties) on an engineered sur-face. The prototype instrument can reada complete 96-well plate (672 electrodes)in five minutes, and its associated softwareproduces easy-to-understand results.

The randomly self-assembled BeadArraytechnology was explained by KevinGunderson (Illumina, San Diego, CA,USA) in which 5 µm diameter beads fitinto the etched wells of a drawn optical-fiber bundle. The individual optical fibersbecome light pipes with a density of up tofour million individual capillaries percm2, which far exceeds the density ofconventional DNA microarrays. Total in-ternal reflection within each light pipeminimizes crosstalk and ensures efficientsignal transfer. A key challenge is decod-ing the random array after assemblywith encoded beads. Beads can be encoded with dyes or with DNA (i.e. optically- or sequence-encoded). In thecase of DNA encoded beads, sequential hybridization allows binary sorting. A prototype assay model consists of 2,000bead types with a 50,000-core fiber bun-dle (1 mm in diameter), which allows for20-fold redundancy. Examples of appli-cation of this technology included SNPgenotyping with ZipCode arrays andOligonucleotide Ligation Assays (OLA),splice variant profiling and expressionprofiling.

ApplicationsPaul Rochelle (Metropolitan WaterDistrict of Southern California, La Verne,CA, USA) discussed the water industry’srequirement for rapid methods ofpathogen and polymorphism detectionand determination of viability, infectivityand virulence; these rapid methods do not yet exist. In Southern California,0.5 billion gallons of water are treatedwithin 2 h and transported to the furthest customer in <24 h. However,microbial analysis requires 28 h for bac-teria, 3–5 days for protozoa and 14–48days for viruses. Further challenges include extremely dilute samples con-founded by numerous inhibitors to PCR.In the USA alone, 80 million cases of illness are attributed to contaminatedfood and/or water each year. Worldwide,a child dies every eight seconds becauseof microbial contaminants in water, and

half of the world’s diseases are transmit-ted by water. Clearly, the challenge exists to create rapid tools for pathogendetection in water supplies. Ideally, theindustry needs a ‘Star-Trek Tricorder’,which gives an instantaneous and com-prehensive analysis. Until these devicesare available, microarray technologiesoffer the most promise.

Spyro Mousses (National Institutes of Health, Cancer Genetics Branch,Bethesda, MD, USA) presented work on tissue microarrays where up to 1,000tissue samples are placed on a micro-scope slide for the in situ analysis of morphology, nucleic acid [by in situ hybridization (ISH)] and protein [by immunohistochemistry (IHC)] at the cellular level. Tissue microarrays enablethe determination of the cellular locali-zation of targets and also integrate DNA, RNA and protein information for a comprehensive molecular profile.Applications were shown for heregulin(HER-2) and S6 kinase frequency andtheir clinicopathological associations inbreast cancer.

Protein arrays were introduced as toolsto explore the human proteome by IanHumphery-Smith (Glaucus Proteomics,Utrecht, The Netherlands). Key chal-lenges for proteomics include small orhydrophobic proteins, or those presentin low abundance. 2D electrophoresisappears to be inadequate to cope withmany of these challenges, in addition tothe technique’s inability to display theentire proteome. Antibody arrays presenta viable alternative approach but requirethe parallel generation and screening of large numbers of new antibodies in an extremely high-throughput mode. Inparticular, the new antibodies must bescreened for cross-reactivity by emulat-ing the antigenic diversity encoded by a genome. Recombinant proteins mustbe carefully purified and properly im-mobilized to a suitable surface. The absence of a PCR equivalent for proteinsis a major complication of this tech-nique, which also needs to incorporate

update conference DDT Vol. 6, No. 13 July 2001

668

reproducibility. Of significant impor-tance is the ease of replication, qualityassurance and scalability.

PROfusionTM technologyRobert Kuimelis (Phylos, Lexington, MA,USA) described PROfusionTM technology,which covalently joins proteins to theirencoding mRNA during in vitro trans-lation, thus effectively linking phenotypeand genotype. Massive protein libraries(1013 members) based on an 8.5 kDasingle-domain antibody-mimic scaffoldwere subjected to directed protein evolution using PROfusionTM technologycoupled with the PCR amplification ofenriched binding sequences. After 8–10rounds of selection and enrichment, afunctional screen identified numerous

high-affinity and specific binders againstnumerous protein targets. These binderswere immobilized to a solid-phase in an oriented manner to generate affinityprotein microarrays. These protein mi-croarrays were stable for at least eightweeks, functioned in human serum, anddetected subnanomolar levels of proteintarget. An example was shown using an immobilized binder in a microarrayformat to selectively capture a cytokinefrom serum followed by label-free detection using matrix-assisted laser desorption ionization time-of-flight MS(MALDI-TOF MS). The entire process ofmRNA–protein fusion formation, in vitroselection, screening and protein produc-tion is currently being automated and‘industrialized’ so that antibody-mimics

can be prepared against a vast numberof protein targets both quickly and cost-effectively.

ConclusionNucleic acid amplification, detection andscreening is central to all molecular biological research. Emerging technol-ogies are driven by the field’s need forfaster, easier, cheaper and more robustapproaches. The Tucson meeting show-cased the most recent advances in the field, primarily from the corporatesector, and emphasized real-world appli-cations of these new technologies. Othertopics presented at the meeting in-cluded sample preparation and nucleicacid purification, DNA probe design andbioinformatics.

DDT Vol. 6, No. 13 July 2001 updatebook

1359-6446/01/$ – see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(01)01861-X 669

AppliedBiocatalysis in SpecialtyChemicalsand Pharma-ceuticals

by Saha, B.C. andDemirjian, D.C., eds,

ACS/Oxford University Press, 2000, 292 pages, hardback, £79.50, ISBN 0-8412-3679-8

The past several years have seen thecontinuing expansion of appliedbiocatalysis. As a result, more than twodozen commercial processes for thesynthesis of fine chemicals andpharmaceuticals now use biologicalcatalysts. The expanding role ofbiocatalysis has been clearly reflected bythe growing number of publications onthis subject – it has been estimated that15% of papers on asymmetric synthesisuse biological catalysts. This wealth ofinformation was aptly summarized intwo recent books: Biocatalysis for Fine

Chemical Synthesis (published by J. Wiley& Sons in 1999) and IndustrialBiotransformations – A ComprehensiveHandbook (published by Weinheim:Wiley-VCH in 2000).

The book being reviewed here isbased on a recent symposium onAdvances in Applied Biocatalysis held atthe 217th National Meeting of theAmerican Chemical Society in Anaheim inMarch 1999. The book represents acompilation of 16 manuscripts organizedinto three sections: biocatalyst discovery,characterization and engineering;applications: specialty chemicals; andapplications: pharmaceuticals.

ContentIt has been widely recognized that thecurrent expansion of industrialbiocatalysis is attributed to recentadvancements in molecular biology, HTS and bioengineering. This notion iswell reflected in the first section of thebook. The screening of large collectionsof isolated enzymes or biocatalyticlibraries requires the development of

new and efficient HTS methods. InChapter 3, Moris-Varas and co-authorspresent an effective tool for the rapidevaluation of enantioselectivity ofhydrolytic enzymes. The method,developed for screening of a library oflipases against numerous substrates in amicroplate format, is a general one andis applicable to screening of otherhydrolases.

Directed evolution is undoubtedly themost powerful technique for improvingthe functional characteristics of anenzyme. The advantage of directedevolution over the more traditionalprotein engineering techniques is that itallows many characteristics of a proteinto be improved without any knowledgeof the protein’s structure, function ormechanism of action. Chapter 6 of thebook describes the application of thistechnique to β-glucosidase, lysozyme,xylanase and aminopeptidase resultingin the generation of mutants withimproved thermostability.

Metabolic engineering has beensuccessfully applied to the production