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Review Series: Nucleic Acids in Medicine

Nucleic acids: In the future, in commerce and inthe clinical laboratory

RAMH.DATAR

INTRODUCTIONThis review series has traversed through five parts so far, whereinwe have covered basic aspects of the impact of molecular biologyresearch on diseases, I and roles of nucleic acids in moleculardiagnosis,' genetic testing and screening,' prognosis" and therapy. 5

This last review in the Series will provide glimpses of what lies inthe future for genetic medicine, and deal with the commercialaspects of this major sector of the biotechnology industry. Finally,the essential requisites for setting up a molecular diagnosticclinical laboratory will be discussed briefly.

The International Human Genome Sequencing Consortiumfound evidence for 29 691 human transcripts." The commercialgenome project of Celera Genomics, Rockville, Maryland, found39 114 genes.' Now, only a few months down the line, thoseestimates have been revised to almost twice the number by at leastone group of workers. Ohio State University (OSU) researchersconsolidated cDNA and EST (expressed sequence tag) transcriptsand protein sequences into an annotated human gene index (OSUdatabase) and arrived at a figure of nearly 75 000 human genes."It will not be long before the efforts in proteomics and trans-criptomics will yield a more accurate number. Concomitantwith these efforts are the high-throughput gene expression profil-ing studies that will yield elucidation of crucial molecular path-ways.

The much touted possibilities in unearthing new drug targetsfuelled much of the excitement surrounding the human genome,and this excitement from the media has led to a flood of funding,both into academia and the biotechnology industry. Partly becauseof this surge in funding from national and international fundingagencies and venture capitalists, and partly because of the healthcare industry requirement, there have been numerous excitingdevelopments in the recent past, some of which seem to bepromising harbingers of an even more electrifying future.

FUTURE OF GENETIC MEDICINE: SOME RECENTADVANCESTechnological advancesDescribed below are some of the novel representative technolo-gies which make high-throughput nucleic acid sample processingpossible in user-friendly formats. While this list is by no meansexhaustive, it should serve to provide an idea of the direction inwhich the field is moving.

Transport, preservation and processing of nucleic acids.FTAR cards are filter paper discs and sheets developed by

Department of Clinical Pathology, University of South ern California, HMR312C, 20 II Zonal Ave, Los Angeles, USA; ramdatar@hotmail.com

© The National Medical Journal of India 2001

researchers at Flinders University in Australia and marketed byInvitrogen Inc., USA. A reincarnation of the famed Guthrie cards(which were a cotton-based matrix), the FfA paper is impreg-nated with proprietary chemicals to lyse cells and entrap DNA.Stored at room temperature, genomic DNA on FfA paper isreported to be stable for at least 7.5 years." After a few washingsteps to remove the stabilizing chemicals and cellular inhibitors ofenzymatic reactions, the captured DNA can be analysed. Since theDNA remains with the paper, the Fl' A card with DNA samples isamenable to automation and offers a compact archival systemcompared to vials or tubes that occupy precious freezer space. Inrecent data from the developers, this technology of nucleic acidsample collection has been shown to be effective even for viralRNA, which allows easy transport, in situ RT-PCR processingand automation. 10 RNAlater, marketed by Ambion Inc., USA, isa reagent designed to maintain RNA integrity in tissues and cellsat ambient temperatures. I I

Nucleic acid sensors. Motorolla Inc., USA, has developedeSensor Clinical Micro Sensors for DNA detection. The technol-ogy relies on bioelectronics, using the complementary bindingproperties of DNA and RNA to assemble an electronic circuitelement. The device creates a detectable electronic signal whenthe target DNA or RNA is present. Capture probes as singlestrands of DNA complementary to a unique region of the targetDNA or RNA sequence are attached to gold-coated electrodes oneach biochip. When a sample containing target DNA is intro-duced into the cartridge, hybridization between the target andcapture probes occurs. Binding of 'signalling probes' with propri-etary electronic labels attached to them, connects the electroniclabels to the surface. This adds a circuit element to the bioelectroniccircuit on that electrode. On application of a slight voltage to thesample, the labels release electrons, producing a characteristicsignal that can be detected through the electrode, indicating thepresence of the target DNA. Currently these devices are availableas multi-chip readers for bench-top use, but the ultimate aim of thecompany is to manufacture handheld devices.

The second example of an innovative nucleic acid sensor is theInvader system from Third Wave Technologies Inc., USA.Invader assay is a linear signal amplification system for direct,quantitative detection of mRNA in total RNA or cell lysatesamples as well as mutation detection in DNA. Here, two target-specific oligonucleotides (oligos) (Probe and Invader oligos) forman invasive structure by hybridizing in tandem to the target. In thisstructure, the 3'-end of the Invader oligo overlaps the hybridizedtarget-specific region of the Probe by one base. The Probe containsa 5'-flap that does not hybridize to the target. The Cleavase IXenzyme recognizes this structure and cleaves the non-complemen-tary 5'-flap of the Probe, releasing it as a target-specific product

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Q, FREToJiio ~ \

5'Invader_"•.---

TemplateCleavase acts here

Step I Step II Step III

FIGI. Cyclic fluorescent reporting of hybidization events usingInvader Technology: The 'probe' oligonucleotide (oligo) iscomplementary with the primary target sequence (shown inblack) only at its 3' end (shown in blue), while the 5' end ofthe 'probe' -which hangs loose like a 'flap' (shown in red)when hybridized with the primary target-is designed to becomplementary with another synthetic sequence, the 'second-ary target' (shown in purple). An 'Invader' oligo with asequence complementary to the primary template (shown ingreen), has a terminal nucleotide that overlaps and competeswith the nucleotide at the junction of the 5' - 'flap' end and the3' -target-complementary end of the 'probe'. This overlapcauses 'invasion', allowing cleavase enzyme to cleave away theprobe 'flap' (step I). The released 'flap' then 'invades' duplexbetween FRET oligo (shown in turquoise) and the secondarytarget, allowing cleavase to cleave the FRET oligo betweenfluorescently labeled nucleotide and the quencher-labelednucleotide (Step II), generating fluorescence (Step III).

(Fig. 1).The Probe is designed to have a melting temperature closeto the isothermal reaction temperature. Under the assay condi-tions, the Probe cycles on and off the target. This enables multipleisothermal cycles of Probe cleavage per target, amplifying thenumber of released 5'-flaps in a target-specific manner. In thesecondary reaction, each released 5'-flap now acts as an Invaderand forms a structure with both a secondary reaction template(SRT) and a fluorophore-labelled oligo (fluorescence resonanceenergy transfer or FRET oligo). 12 The Clea vase enzyme recognizesthis structure and cleaves the FRET oligo, separating the fluorophore(F) and quencher (Q) to generate a fluorescence signal.

Both the primary and secondary reactions run in the same well.The biplex format of the Invader assay allows simultaneousdetection in the reaction chamber of two mRNA sequences-aspecific target and a housekeeping gene (which acts as an internalcontrol). Two different FRET oligos (each with spectrally distinctfluorophores) are used in this assay. In a recent publication,identification and quantitation directly from total RNA or crudecell lysate samples and detection of changes in gene expressionlevels as small as 1.2-fold or 20% was demonstrated. !3 As few as6000 copies ofRNA can be detected while an analytical sensitivityoffewerthan 100 copies of HIV-1 RNA was demonstrated. SinceInvader assay can detect and quantitate both DNA and RNA, itcan be used for multiple, different applications.

Yet another example is the development of an electronictransponder-based DNA sequence sensor by PharmaSeq Inc.,USA. Microtransponders are cubical radiofrequency transmit-ters. Thousands of oligonucleotide probes of specific sequencecan be attached to the surface of each microtransponder, and theinformation about the probe sequence is stored in the electronicmemory of the microtransponder. After the probe molecules on thesurface of such a microtransponder are allowed to hybridize withfluorescently labelled target molecules from the sample, a dedi-cated read-write scanner detects both the fluorescent signal andthe radiofrequency transmission, signalling both the hybridiza-tion event and the sequence of the probe. When multiple probes

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(and hence multiple transponders) are reacted with a mixture oftarget sequences, the scanner can identify which of the transpon-ders is participating in the hybridization events. This allows highthroughput as well as multiplexing.

Development of integrated nucleic acid analysis devices.Advances from distant fields such as computer hardware develop-ment, robotics and nanotechnology are impacting moleculardiag-nostics in important ways. A variety of microfabrication technolo-gies such as photolithography, micropatterning, microjet printing,light-directed chemical synthesis, laser stereochemical etching,and microcontact printing are being used to construct nucleic acidmicrochips. The new microchip-based analytical devices areexpected to successfully address the problems of contamination,low throughput and high cost. 14 A typical biochemical analysisinvolves four steps: (i) sample preparation; (ii) biochemical reac-tion; (iii) detection; and (iv) data acquisition and interpretation.While a number of biotechnology companies are concentratingtheir efforts on miniaturizing and automating each of these stepsseparately, there are also ongoing efforts to combine all the fouraspects on one platform (,lab-on-chip' concept). Thus, for ex-ample, Bums et al:" have developed a device that uses micro-fabricated fluidic channels, heaters, temperature sensors, andfluorescence detectors to analyse nanolitre-size DNA samples.This device is capable of mixing measured volumes of aqueousreagent and DNA-containing solutions, amplifying or digestingthe DNA to form discrete products, and separating and detectingthese products by microelectrophoresis, all operating as a singleclosed system. The authors claim that the system has the potentialfor assembly into complex, low-power, integrated analysis sys-tems at low unit cost. Such portable devices can facilitate rapidDNA analysis in medical diagnostics.

LabCard, a technology marketed by Aclara Biosciences Inc.,USA, is a microfluidic device. Here, an electrokinetic pump(which functions upon principles based on electro-osmosis andelectrophoresis) separates species according to their size andcharge on the LabCard. Movement of as little as picolitre volumesof fluids through interconnected networks of microscopic chan-nels allows a variety of laboratory tasks on one single plasticmicrofluidic platform. Thus, mixing, incubation, measuring (me-tering), dilution, purification, capture, concentration, injection,separation and detection are all carried out in a high-throughputmanner, allowing researchers to rapidly perform numerous bio-chemical measurements in a miniaturized and automated for-mat. 16 A similar electro kinesis principle is used in the Labchiptechnology of Caliper Technologies Inc., USA.

Geniom, a high-throughput system marketed by Febit, Ger-many, is another such example. The system combines all processsteps from production of biochips to hybridization, detection andanalysis in one single instrumentthe size of a computer tower. Theuser is supposed to load a DNA sequence of interest, enterparameters in a software interface, add fluorescently labelledDNA probes of interest, press 'run' and pick up the digitizedresults on the screen for analysis. The entire process is expected totake a few hours, with neither set-up time nor significant hands-on time. At the heart of the system is the micro-optical DNAprocessor, a micro-channel glass structure that serves as a reactionchamber for both in situ DNA synthesis and target-probe hybrid-ization. Thousands of oligonucleotides can be synthesized simul-taneously within a few hours and the probe-target DNA hybrid-ization is performed in minutes. A cooled CCD-chip cameradetects the fluorescent signals.

Microarray product development is proceeding with one un-

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derlying principle:Jast is notJast enough. Integrated microfluidicdevices are at the forefront of these developments. It is expectedthat DNA chips and integrated processing will penetrate theemerging point-of-care market within a decade, if not earlier.

Diagnostic mutation screeningCancer is a disease of the genome and well understood from agenomics point of view. New methods are being devised fordetecting and identifying genomic markers associated with cancersuch as mutations in K-ras, APC, specific microsatellite instabil-ity, etc. for colorectal cancer. This technology, expected to beavailable soon, will be applicable to colorectal, breast, prostateand lung cancers, and help to diagnose existing disease at itsearliest stage. A positive finding in the colorectal cancer test, forinstance, would immediately direct the individual to a confirma-tory and potentially therapeutic follow up colonoscopy.

Genetic testingWe are rapidly advancing to the postgenomic era in which geneticinformation will have to be examined in multiple health caresituations throughout the lives of individuals. Currently, newbornbabies can be screened for treatable genetic diseases such asphenylketonuria. In the not-so-distant future, children at high riskfor coronary artery disease will be identified and (if researcherslike W. French Anderson could convince the Recombinant Advi-sory Committee of the utility and safety of in utero gene therapy)treated to prevent changes in their vascular walls during adult-hood. Parents will have the option to be told their carrier status formany recessive diseases before they decide to start a family. Formiddle-aged and older populations, we will be able to determinerisk profiles for numerous late-onset diseases, preferably beforethe appearance of symptoms, which at least could be partlyprevented through dietary or pharmaceutical interventions. In thenear future, the monitoring of individual drug response profileswith DNA tests throughout life will be standard practice. Soon,genetic testing will comprise a wide spectrum of different analyseswith a host of consequences for individuals and their families-an issue worth emphasizing when explaining genetic testing to thepublic.

Rational therapeuticsWe have seen earlier that cancer cells have altered signal transduc-tion pathways. IThese include protein kinases (such as Src proteinkinase, Abl protein kinase and Raf protein kinase), G proteins(such as a-subunit protein gsp), transcription factors (such asAP-l and myc) , nuclear and growth factor receptors (such asthyroid hormone receptor erbA, epidermal growth factor receptorerb B, nerve growth factor receptor trk, insulin receptor ros andplatelet -deri ved growth factor receptor kit). Src kinase, the first ofthe superfamily of protein tyrosine kinases to be identified andcharacterized, has a 3-dimensional (3-D) structure that includestwo non-catalytic domains (SH3 and SH2, which are shared bymany other signal transduction proteins dysregulated in cancer),and a tyrosine kinase catalytic domain. Similar 3-D structuralstudies have been carried out on a number of other oncogenicproteins such as ras (which is known to be mutated in about 25%of human tumours and as many as half of colon cancers), p53(mutated in 50% of human cancers) and Rb. The knowledge of 3-D structure is leading to rational drug design. Thus, HER-21neu(erb B2, commonly over-expressed in 30% of human breastcancers) has been specifically targeted by the humanized mono-clonal antibody trastuzumab (Herceptin, marketed by Genentech

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Inc., USA), which binds to the extracellular domain of the HER-2 receptor. A number of epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors have already progressed to humanclinical testing (ZD-1839, OSI-774 and PD-183805), with ZD-1839 showing promising anti tumour activity against non-smallcell lung cancer and head/neck cancer. Exceptional results havealso been in human clinical testing for STI-571 (Gleevec, aproduct of Novartis Inc., USA), an inhibitor of bcr-abl tyrosinekinase, which is known to be the hallmark aberration in chronicmyeloid leukaemia (CML). The success with structurally specificrecombinant antibodies and peptidomimetic drugs has led toclinical trials with the additional weapons of the 'brave newmedicine', which will include small-molecule synthetic drugs,antisense RNA, ribozymes, peptide nucleic acids and aptamers;some of which were briefly discussed in the last review in thisSeries.' Telomerase, a ribonucleoprotein reverse transcriptaseenzyme which uses its internal RNA component to synthesizetelomeric DNA repeat sequence (TTAGGG)n directly onto theends of chromosomes, is expressed in most advanced cancers.Methods for telomerase inhibition using small molecules such asmodified oligonucleotides or peptide nucleic acids may thereforehave therapeutic potential for cancer. 17 (There are potential risksin the use of such therapy that must be considered, such as theeffects of inhibitors on telomerase-expressing stem cells. How-ever, this approach is likely to be less toxic than conventionalchemotherapy. )

In silico drug designThis concept involves molecular modelling to computationallyvisualize, analyse and probe the 3-D structures of therapeutictarget molecules. Information gleaned from such analyses is thenused to generate or select appropriate cognate ligand molecules asdrugs, either from natural, synthetic or combinatorial resources.Since highly powerful and fast programmes are used to generatemolecular models for targets and drugs, this approach is expectedto yield rationally acting drugs much faster (and at a much lessercost) than the conventional pharmaceutical approach. The leadcompounds so designed will neutralize their targets with highspecificity and least toxicity.

PharmacogenomicsPharmacokinetics, simply put, is the study of the effect of thebody acting on drugs (i.e. drug metabolism), while pharmaco-dynamics is the study of drugs acting on the body (therapeuticefficacy). While so far most of the drugs developed traditionallyand marketed were chosen to have high therapeutic efficacy, it isbecoming obvious that differential drug metabolism is a result ofgenotypic differences in patients and can cause significant drug-related morbidity or even mortality, besides being therapeuti-cally ineffective. Individual variation in response to drugs is asubstantial clinical problem, which includes failure to respondto a drug, adverse drug reactions and undesirable drug-druginteractions when several drugs are taken concomitantly." Animportant proportion of this individuality in drug response isinherited. The research area that defines the genetic basis forvariability in drug response is known as pharmacogenetics.Pharmacogenomics, on the other hand, is a relatively moreglobal field, which uses knowledge from genetic variability atthe levels of single nucleotide polymorphism (SNP), themicrosatellite repeats and inter-individual variations due totranscription and translation (all resulting as a fall-out from dataof genomics) to rationally develop newer drugs. Once pharma-

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cologists have developed a new drug, it is tested in humans bythe process of 'clinical testing'. Clinical testing to ensuresafety and efficacy is long and tedious. On an average, it requires12 years and US$ 500 million for each drug before it can reachthe shelves of pharmacies. Pharmacogenomics can reduce thetime and cost of clinical testing. To understand how, one mustlook at the current modus operandus of clinical trials. Clinicaltesting can proceed only when trials in humans give clearanswers. As of now, drug companies abandon work on a drug ifthe toxicity and efficacy studies in the patient populations testedyield an unsatisfactory answer (either a 'no' or an 'unclear'answer). Considering that the companies study large numbers ofpeople in their experiments, the genetic variability of drugmetabolizing enzyme genes often results in a 'no' or 'unclear'answer. Redirecting efforts to the development of a newer andbetter candidate drug comes at hefty costs, both in terms of timeand money. Since a person's genetic profile can affect theirresponse to a drug, pharmacogenomics can permit the use of anappropriate smaller patient cohort, allowing studies to be con-ducted more rapidly and fruitfully.

With their development based upon such genetic testing, moredrugs will survive the clinical testing process and these willprobably work more efficiently because they will be targetedprimarily to persons whose genetic profile indicates success.While there could be some people with genetic profiles that maynot be agreeable to any of the available drugs, 'no drug' may stillbe a better option than 'wrong drug'. Personalized therapeuticswill thus be an integrative approach of 'right target, right drugand the right patient' .

Pharmaceutical companies are realizing the benefits ofpharmacogenomics and some big ones are developing 'in-house'facilities to incorporate the 'new' genetic elements into theirclinical trials. But the rapid fluxes in the nature of functionalgenomics and proteomics technologies often makes them unfit forassimilation into existing pharmaceutical companies. Also, need-less to say, identification of the important genetic profiles for anyuntested drug could be time-consuming and a diversionary ex-pense, which smaller companies can do without. This has resultedin the creation of a market for platform technology companies thatfocus on technology development and validation for functionalgenomics and proteomics. These companies began to emerge asgenomics firms in the early 1990s and offer their expertise to thepharmaceutical industry to permit faster drug discovery anddevelopment. The realization by the pharmaceutical industry thatit makes good sense to collaborate with the biotechnology industryhas resulted in numerous new alliances over the last few years,with the total value of these new deals growing to billions ofdollars.

Molecular profilingHigh-throughput analysis of gene expression in a multiplex fash-ion can provide molecular profiles for the cells in a diseased organor tissue. Such profiles can allow distinction between differentstates of a given disease, or can distinguish between differentdiseases. One recent example of each of these possibilities is givenbelow.

Measurement of a molecular marker-methylation of theGSTPI gene-may have the potential to aid in the detection ofearly-stage prostate cancer. Methylation of the GSTPI gene,which encodes a detoxification enzyme, occurs in precursor le-sions of the prostate but is rare in benign prostate hyperplasia.'?The authors used a quantitative real-time methylation-specific

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PCR to determine the ratio of methylated GSTPI to that of areference gene, MYOD 1.The medianratios of methylated GSTPlIMYODI were 0 for benign prostate hyperplasia (BPH) patients,1.4for prostatic intraepithelialneoplasia (PIN) patients, and 250.8for patients with clinically localized prostate cancer. Such mea-surement has a potential to augment the standard screening tech-niques currently used (such as prostate-specific antigen) to assessprostate tissue, although larger clinical trials are required.

Another use of molecular profiles is their ability to deduce the'molecular signature' of tissues. Thus, for example, Giordano etal. have recently demonstrated that the organ-specific gene ex-pression profiles can accurately classify tumours," thus providingan important adjunct to histopathology.

It is thus becoming obvious that rational drug development inmodem molecular medicine, based upon molecular profiling andstructural analysis of targets, will essentially be ajoint endeavourbetween molecular biologists, pharmacologists, chemists, physi-cists, computational experts and physicians. Ultimately, all this isexpected to lead to the development of 'smarter' drugs andindividualized medical care that is rationally designed to suit thepatient's specific genetic make-up. For physicians, it should notbe difficult to explain the utility of rational therapy to patientsbecause genotypic treatment is only an extension of phenotypic(symptomatic) therapy, which has been practised for centuries.

COMMERCIAL ASPECTS OF MOLECULAR MEDICINEWorldwide, the pharmaceutical industry is a 250 billion dollarbehemoth, and it caters to an even larger health care industry.There is a growing realization that the genotype-based inter-individual differences resulting in drug-related toxicities canlead to retraction of drugs from markets and cost billions ofdollars worth of research and development efforts. Therefore,genotyping and rational drug development are likely to benefitthe industry. Corporate demands and shareholders are pushingthe research into high-throughput genomics, which would speedup the analyses. Molecular profiles can allow the definition offewer molecular targets, which span the crossroads in differentcellular networks. Inhibition or neutralization of multiple cellu-lar pathways at such points would require fewer drugs. There-fore, profiling analysis will be another cost-saving benefit for thepharmaceutical industry.

Besides the health care industry itself, there is also anothercurrently small but potent force-that of the ultimate consumersof this industry, the patients and their families: more people aregetting onto the internet, resulting in more public awareness ofproblems with drugs and options. This will lead to the demand forbetter drugs and therapy.

Genomics is quietly transforming the pharmaceutical industry.Companies are moving from drug discovery and developmentbased on medicinal chemistry to rational design and developmentof drugs based on information provided by genomics. These newmethods, which are replacing the traditional trial-and-error dis-covery methods, are receiving considerable attention in the phar-maceutical industry and media. The new methods focus on com-binatorial chemical syntheses based upon data from high-through-put genomic analyses, and have already produced agents that haveentered clinical trials. As a result of the new technologies, costsand development times are falling, and knowledge about each newagent's mode of action has increased. New screening technologiessuch as DNA microarrays, will affect pharmaceutical discoverysignificantly. The worldwide market for microarrays, scanners,arrayers and microfluidics is projected to approach US$ 1 billion

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by 2001. Approximately 60% of the market is thoughtto be in theUSA, 30% in Europe and the remaining in the Far East. All thosewho work in drug discovery, laboratory testing, medical diagnos-tics, point-of-care device development, or the pharmaceuticalindustry, are making conscious efforts to incorporate the high-throughput micro arrays or their variations into their work areas.

Nucleic acid testingWith reports of substantial human genome sequencing, discoveryof the number of human disease genes will increase. The worldmarket for molecular diagnostic and cytogenetic disease testingprocedures was estimated at US$ 66.3 million in the year 2000.21

With an estimated average annual growth rate of 10.3% during the5-year forecast period, this combined worldwide market wasexpected to reach US$ 108.3 million by the year 2005. The USdomestic market share, which currently represents nearly 65% ofthe total, was expected to reach US$ 69.3 million by the year 2005.The report also estimated that by the year 2005, internationally,molecular methods will represent 11.9% of the overall geneticdisease testing application market, and grow to US$ 12.9 millionin 2005. It is predicted that a wide variety of technologies will beused to develop DNA diagnostic genetic disease tests, and findingcost-effective diagnostic tests will be the focus. The sales ofreagents and appliances that are used in polymerase chain reaction(PCR) technology, which has dominated the genetic disease DNAdiagnostic market, was projected to be around US$ 606.6 millionby the year 2005.

Abbott Laboratories, USA, Becton Dickinson Inc., USA, ChironCorporation, USA, Digene Corporation, USA, Gen-Probe, Inc.,USA, Organon Teknika Corporation, Netherlands, and RocheDiagnostics, Inc., USA, are some of the major biotechnologycompanies in this sector. Their target markets include the viralload monitoring market, infectious disease diagnostic systemsmarket and food-borne pathogen detection market.

MOLECULAR MEDICINE LABORATORY SET-UPWhile clinicians in increasing numbers will soon be expected bytheir patients to be aware of the molecular basis of diseases, asubstantial majority of them-especially the clinical patholo-gists-will also be required to have the know-how of setting upand successfully implementing various molecular tests in clinicallaboratories.

Every new molecular diagnostic laboratory director will haveto know how to:

1. avail of New Laboratory Start-up Programme for purchase ofequipment, chemicals and kits (vendors such as Fisher/ VWRhave such programmes);

2. design utilitarian laboratory spaces;3. ensure that the major equipments are available on-site or at

least on-campus with collaboration;4. develop contracts for maintenance and repair of equipment

with a capable local company;5. obtain appropriate biosafety, chemical safety and radiation

safety licences; and6. have an easy access to computers with internet connectivity,

reference management software, literature and sequence data-base search software, PCR primer design software and sub-scriptions to electronic medical research journals.

Standard operating procedures1. Diagnostic and prognostic molecular biology primarily em-

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ploy the principles of nucleic acid amplification and/or hy-bridization, although other alternatives are also emerging asmentioned above. Nucleic acid amplification methods aregenerally classified in one of three groups: target, probe orsignal amplification. Among the various limitations of PCR,one that must be kept in mind is the serious concern aboutcontamination of specimens with either post-amplificationproducts from previous analyses, or contamination of nega-tive specimens with control and/or positive specimens pre-pared at the same workstation. Such contamination can beprevented by meticulous technique, separation of work areas,use of containment devices, dedicated equipment, post-PCRtemplate inactivation protocols (photochemical, enzymatic,and chemical techniques), as well as aggressive environmen-tal decontamination using bleach and ultraviolet irradiation.

2. Specimens with suspected infectious material such as virusesmust be handled according to proper safety precautions.Typically, specimens should be received and accessioned inan area of the facility that is isolated from testing areas. Frozenspecimens should be kept frozen at -20 °C to -70 oC,avoidingrepeated freezing and thawing of clinical specimens. Other-wise, this can have deleterious effects on the ability to recoveramplifiable nucleic acid from some organisms, especiallylabile RNA from retroviruses. Refrigerated or frozen speci-mens must be kept in a designated refrigerator or freezer. Thekits and reagents should be stored separately.

3. Each reagent must be carefully quality controlled to ensuremaximum and reproducible assay performance. Stock solu-tions must always be prepared in an area separate from thespecimen processing area, and pre-amplification materialsare never prepared in a post-amplification area. As many ofthe individual reagent components as is feasible should becombined into master mixes. This minimizes the number ofpipetting steps, thereby decreasing the potential for volumeerrors, contamination and mistakes due to the exclusion of anecessary reagent. Master mixes must be used only until theearliest expiry date of one of the components.

4. Commercially obtained oligonucleotide primers must haveappropriate documentation of purity. High-performance liq-uid chromotography (HPLC) data from the vendor can be usedto ensure full-length primer sequences. All other reagentsmust have appropriate Certificates of Analysis. Probe orprimer sequence characteristics must be documented to per-mit proper interpretation and troubleshooting of test results, ifany.

5. Positive-control stocks and aliquots of low-copy numberworking dilutions should be prepared in an area physicallyseparate from that used for pre- or post-amplification tasks.

6. Material safety data sheets (MSDS) for all hazardous andorganic reagents used in preparative procedures (such asphenol, chloroform, etc.) must be maintained in a secure yetaccessible location.

7. Sufficiency of reagents should be visually confirmed prior tosetting up assays. Mixing of reagents from different kits (evenfrom the same vendors) should be avoided since lot-to-Iotvariations can result in assay failures.

8. Any safety precautions for equipments or reagents (UVtransilluminators, ethidium bromide, etc.) must be rigorouslyfollowed. All electrical equipment must be checked for propergrounding and electrical safety. Use of frost-free freezersshould be avoided since temperature fluctuations lead toreagent degradation. Temperature settings for various ther-

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mal instruments must be adhered to. The typical tolerancelimits for some of these are as follows:

a. Incubators: Set temperature at ±2 °Cb. Water baths and heating blocks: Set temperature at ±1 -c.c. Refrigerators: 4 °C to 8°Cd. Standard laboratory freezers: -20 °C±2 °Ce. Ultra low freezers: ±1O °C, but below -60°C

9. All quality controls and sample tests must be documented.Any adjustments in temperature settings, reagent changes,etc. should be recorded. Formal reporting must follow afterany steps are taken to solve the problem. A corrective actionform must document steps taken to resolve the problem. If anerror trend is identified with either an equipment or a reagentsystem, documentation is necessary to avoid the same prob-lem in future.

10. There must be clear personnel identified for responsibilitiessuch as equipment certification and maintenance, reagentinventory, etc.

SYNTHESIS OF ROLE OF NUCLEIC ACIDS IN THE NEWMEDICINEIn this Series, we have covered various roles that nucleic acidshave come to occupy in modern medicine. Thus far, we havediscussed the various aspects of gene expression regulation,control of cell cycle and apoptosis, molecular aberrations that leadto anomalies in these vital cellular functions in disease states andmolecular biology tools and technologies to detect these aberra-tions.' We explained how different molecular diagnostic assayformats are applied to various infectious and genetic diseases andcancers.' We also examined the technologies for genetic testingand screening and their ethical, legal and social implications.'This was followed by a discussion of the variety of ways in whichmolecular techniques can be applied to monitor disease progres-sion.' Subsequently, we covered numerous aspects of gene-basedtherapies, including treatment of issues regarding gene deliverymethodologies, tissue-specific therapeutic gene expression,antisense therapeutics, clinical trials for genetic therapy andfuture directions for genetic medicine for diseases as diverse ascancers, hypertension, spinal cord injury and AIDS.s

In the first Review, I had quoted from the fiction writer DonDeLillo (Underworld): 'You didn't see because you don't knowhow to look. And you don't know how to look, because you don'tknow the names.' I had commented that the exciting researchfindings in molecular biology of diseases have helped us 'know thenames', which, in turn, is letting us 'see'. Now, at the conclusionof this Series, it can be said that what we can 'see' in terms ofmolecular pathology of diseases, can be used to 'do' things;namely, devising better ways to diagnose, prognosticate and treatdiseases.

While novel assays and technologies are reforming diagnosisand prognosis, therapeutics is being transformed by designing theright drugs for patients using knowledge from functional genomics.Functional genomics and proteomics are revolutionizing drugdiscovery. The discovery of new genes and proteins and theircharacterization in terms of disease involvement is proceeding atan accelerating pace, promising a large number of new targetswith which highly specific and effective therapeutics can be

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developed. Automated, high-throughput technologies are beingused to characterize and validate potential target genes andproteins. Rapid elucidation of the biochemical pathways con-trolled by these target genes and proteins is allowing appropriatedrug design and selection of therapeutic intervention.

While all these developments take place simultaneously onvarious fronts, it is essential that the medical research communitydoes not lose its one element of focus: the patient, and evolves intoa society that respects reason while acting as a catalyst forprogress. This will include examining a host of implicationsregarding genetic medicine (ethical, legal and social) and devel-oping acceptability for the modern form of medicine throughpublic education-all leading to improved health. Equally impor-tant, it is imperative that the savings accrued due to the high-throughput technological developments in genomics andtranscriptomics (which will drive down the cost of drug develop-ment for the pharmaceutical industry) are passed on to the patientsand result in more economical treatment for their ailments.

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