- Email: [email protected]
Developing small-molecule libraries for drug discovery
process of being, authorized. However, establishing unequivocally a clinical benefit and transferring from small-scale trials to the industrial development of safe vectors still requires much work (Jean-Franqois Mayaux; RhBne-Poulenc-Rorer, Viny, France). Progress is needed in the development of improved adenovirus vectors that enable the sustained expression ofthe transgene, or improved technologies for the large-scale production of therapeutic DNA cassettes that are devoid of bacterial sequences. It is still difficult to target and regulate the expression of transgenes; however, this is essential for many applications (e.g. in the treatment of hemoglobinopathies). Roger Craig (Therexsys, Keele, UK) indicated that the inclusion of LCR (locuscontrol regulator) elements, which were recently discovered by Frank Grosveld, should allow the persistent,
Meeting report
uniform and position-independent expression of therapeutic genes in the LCR-specific target cells. DNA delivery using nonviral vectors Although most ongoing clinical trials make use of retroviral or adenoviral vectors, nonviral vectors will be preferable in the long term, particularly for direct in viva gene transfer. Jean-FranCois Mayaux and Robert Zaugg (Vital, San Diego, CA, USA) indicated that current research in this field includes screening for improved cationic lipid-based delivery systems and associating these with DNAcompacting agents. Koger Craig and Eric Tomlinson (GeneMedicine, Houston, TX, USA) discussed the possibility that second-generation polymer-based delivery systems will include targeting moieties, DNA-condensing agents, fusogenic peptides and,
Developingsmall-molecule libraries for drug discovery Pharmaceutical research and development is undergoing dramatic change. Laboratories are no longer synthesizing and testing molecules serially, but are applying the rapidly developing techniques of combinatorial chemistry to provide lead compounds for high-throughput screening (HTS) assays. At present, chemistry is the limiting step in the drug discovery process, and efforts are under way to develop larger and more diverse small-molecule libraries to enhance discovery of new leads for drug development. In addition to designing new synthetic pathways for novel pharmacophores, new instrumentation is required to speed up and automate chemical manipulations, as is new computer software for data management. A recent meeting* provided an opportunity to discuss these changes in the pharmaceutical industry, *The meetmg ‘Sol&Phase Synthesis: Developing Small~Molrcule Libraries‘ was held in Coronado, Californu. USA. 1-2 February 1YYh.
-____ ___ ~ TIBTECH APRIL 1996 NOL 14)
Automation for solid-phase synthesis HTS has been integrated into the drug discovery process to speed up the discovery of new lead compounds for pharmaceutical development. In the past, testing novel molecules for biological activity was a time-consuming process, but recent advances in the biological sciences have provided assays that rapidly analyze multiple compounds. In fact, the supply of chemical entities is rapidly depleting and new sources of test substrates are in demand. To meet this need, highthroughput organic synthesis (HTOS) is currently in development. If HTOS can successfully be developed, its use should speed up the process of bringing new phatmaceutical agents to the market by shortening the time at the prediscovery level (i.e. the generation of novel molecules) and at the drug development level (i.e. the synthesis of analogs to discover compounds with improved pharmacokinetic properties). Because provisional
eventually, peptides that allow nuclear transfer in post-mitotic cells (mimicking viruses). It remains to be seen what delays will be encountered and which therapies will be usell. Will these strategies allow the safe and efficient transfer of some or all of these new information-rich drugs at acceptable costs? These questions are the subject of continuous debate by the pharmaceutical industry, by regulatory agencies and by the many scientists involved in this exciting field. The number of multidisciplinary approaches currently being developed and the increasing speed at which applications based on fundamental research into gene expression are being contemplated are striking.
patents are filed at the time that lead compounds are discovered, using HTOS to advance the drug development process will allow more time for the final pharmaceutical product to be on the market while it is covered by patent. Thus, the use of HTOS should provide a higher return on marketable drugs for pharmaceutical firms (John P. Devlin; ARRT International, New Milford, CT, USA). Efficient time-management during the planning of organic syntheses will also extend the period of time during which the marketed product is covered by patent. Combinatorial chemistry requires that chemists design syntheses around a library of molecules, not around specific compounds. A software package currently being developed provides chemists with a user-friendly and highly interactive electronic reaction retrieval system (Guenter Grethe; MDL Information Systems, San Leandro, CA, USA); the Solid-Phase Reactions Database Organic (SPORE) will provide searchable information that is designed to enable synthetic chemists to plan reactions more efficiently. Another software product being developed provides organic chemists with a tool for designing reaction pathways, as well as for making predictions about the properties of members
CopyrIght 0 1996, Elswer Science Ltd. All rights reserved. 0167 - 7799/96/$15.00
PII: SO167-7799(96)30006-l
of the proposed small-molecule library (Ferenc Darvas; ComGenex, Budapest, Hungary). The heterogeneous reaction conditions intrinsic to solid-phase chemistry have prevented traditional analytical methods from being used. At present, destructive methods are used to monitor reaction progress: molecules of interest are cleaved from the resin and analyzed by standard procedures. Keith Russell (Zeneca Pharmaceuticals, Wilmington, DE, USA) discussed recent advances in quantitative infrared (IR) spectroscopy. Spectra obtained from single resin beads by IR microscopy can be quantified if the polystyrene backbone is used as an internal standard. Although the signal that is obtained is weak, if deuterium-tagged compounds are used, the signal obtained from the tagged groups can be isolated from those of other functional groups. Several examples were presented that indicate that the development of a fast, nondestructive method of monitoring reactions on solid supports is possible. The solid-phase synthesis of smallmolecule libraries can be simplified using automated laboratory workstations that enable the reactants, solvents, temperature and reaction atmosphere to be precisely controlled. A modular approach allows individual synthetic operations, such as resin dispensing, reactant preparation and chemical reactions, to be performed with existing instrumentation (James Harness; Bohdan Automation, Mundelein, IL, USA). However, details of a&inclusive workstations were also presented. Chemical manipulation can be achieved using two independent robotic arms (Mark Peterson; Advanced ChemTech, Louisville, KY, USA), or using a fluid-delivery device in a closed system without robotics (Joel Martin; Argonaut Technologies, San Carlos, CA, USA). One conclusion reached during the panel discussion was that there is probably not a single, best automated synthesizer, but that different workstations may be required to meet the specific applications of various laboratories. Linkers, supports and alternatives Several examples of new smallmolecule libraries constructed using novel linkage strategies were presented. Palladium(O)-mediated chemistry can be used for the syn-
thesis of 1%26-membered macrocyclic structures, heterocycles and substituted biaryl structures from solid-phase carbobenzyloxy chloride and imidazole equivalents (James Hauske; Versicor, Marlborough, MA, USA). This method produces higher yields of macrocyclic molecules than can be obtained using solution-phase chemistry because the polymer-bound molecules are segregated from each other. A siliconbased polymer linkage enables the synthesis of biphenyl derivatives, also through a palladium-catalyzed reaction (Balan Chenera; SmithKline Beecham, King of Prussia, PA, USA). The arylsilane linker can be made on the 50-gram scale from inexpensive, commercially available starting materials. Alternative methodologies for the synthesis of small-molecule libraries were discussed. If polyethylene glyco1 (PEG) is used as a polymeric support, familiar homogeneous reaction conditions can be employed, but product purification can be carried out using simple polymer precipitation, filtration, and washing (Kim Janda; The Scripps Research Institute, La Jolla, CA, USA). By combining the positive aspects of classical solution-phase chemistry and solid-phase synthesis, novel smalmolecule libraries can be characterized using traditional analytical techniques such as thin-layer chromatography and proton nuclear magnetic resonance (NMR) spectroscopy. The design of dipeptidomimetic and iminodiacetic anhydride templates has enabled solution-phase chemistry to replace polymer-based purification in the synthesis of smallmolecule libraries isolated from byproducts and excess reagents by either acid/base dissolution or washings (Christine Tarby; CombiChem, San Diego, CA, USA). Segregating reactions in a matrix format has allowed libraries to be synthesized in relatively high yields with >85% purity, as assessedby NMR. Heterocycles and phosphoramidate libraries New strategies for the production of highly functionalized pharmacophores for drug screening were discussed at the meeting. A one-pot, four-component condensation reaction (the Ugi reaction) has been adapted for solid-phase synthesis to yield several templates for library production (Adnan Mjalli; Ontogen,
Carlsbad, CA, USA). Carboxylic acids, isocyanides, &mines and aldehydes form the basis of a diverse collection of molecules that can be further modified to produce small, ring heterocycles such as imidazoles, oxazoles and lactams. In addition, the ‘tea bag’ method of solid-phase synthesis (in which the resin is enclosed in a porous polypropylene bag to facilitate manipulations) can be used to produce heterocyclic small-molecule libraries (John Kiely; Torrey Pines Institute for Molecular Studies, San Diego, CA, USA). Polymerbound amines are cyclized (through imine intermediates) to quinoline and isoquinoline libraries that can be characterized in pools by mass spectrometry. Further derivatization of these libraries yields new libraries of additional diversity and biological activity. Several reactions, such as 1,3 dipolar cycle-additions, monoreductive amination of dialdehydes and unsymmetrical benzoin condensations, have been shown to produce higher yields by solid-phase synthesis than by solution-phase chemistry (John Nuss; Chiron, Emeryville, CA, USA). These reactions have been used to create novel heterocyclic libraries by a dentrimeric approach. Fathi (PharmaGenics, Reza Allendale, NJ, USA) gave details of how a DNA synthesizer has been used to create combinatorial libraries of phosphoramidates; by incorporating a range of diols and amines it has been possible to produce a diverse library, which could be monitored using phosphorus NMR. Normand Hebert (Isis Pharmaceuticals, Carlsbad, CA, USA) discussed another approach that uses aminodiols to yield a collection of molecules in which the functional groups are positioned in various orientations; computer analysis has verified that there is very little conformational overlap of functional groups. Libraries can be synthesized using an automated solidphase synthesizer that carries out up to 96 independent reactions in an inert gas atmosphere. Combinatorial library strategies and programs The meeting concluded with several more examples of combinatorial libraries that have been created using solid-phase synthesis (Joseph Hogan, Jr; ArQule, Medford, MA, USA). The synthesis of small bioactive molecules and scaffolds has yielded libraries of inhibitors of proteolytic TIBTECH APRIL 1996 WL 14)
enzymes (Dinesh Pate]; A@max Research Institute, Santa Clara, CA, USA) Novel nonpeptidic libraries have been screened for binding to streptavidin (Viktor Krchnak; Selectide, Tucson, AZ, USA), and a multipin method of organic synthesis on solid support has been applied to
Workshop
the area ofreaction optimization, and has led to the production of p-turn mimics (Andrew Bray; Chiron Mimotopes, Victoria, Clayton, Australia), Further innovations in the synthesis of combinatorial small-molecule libraries should soon provide new
Multi-mutation screening using PCR and ligation - principles and applications Many genetic analysis applications rely on the discrimination of singlebase differences in sequence, or alleles that can have a profound effect on the function of the encoded protein. In humans, for example, such differences can lead to a loss of function of a protein or enzyme that is required for normal cell function or for growth control, resulting in an inherited disease or disease prediposition. They can also be the basis for differentiation between a pathogenic or antibiotic-resistant microorganism and its non-pathogenic counterpart. The ability to analyze a number of these traits simultaneously through multiplexing assayswill make genetic analysis more cost-effective, and will lead to more-widespread testing. Detecting inherited disease Multiple mutations must be screened to obtain high detection rates for many diseases. Diseases such as cystic fibrosis and inherited cancers have been shown to arise from any one of many potential mutations scattered throughout their respective genes. To date, over 400 diseasecausing mutations have been reported for cystic fibrosis’, 36 mutations have been reported for the inherited BRCA- 1 breast cancer gene’, and over 280 mutations have been reported in the p.53 oncogene, which is associated with -50% of all cancers”. Large-scale multiplex analysis is required for investigating these types ofgenetic problems, which rely on discriminating single-base differences at a multitude of loci that are sometimes closely spaced. We have reviewed a number of potential
I IBTECH APRIL l996 (VOL 14)
approaches to providing a solution to this problem. At present, the most promising approach is a combination of multiplex PCR DNA amplification and multiplex ligation assay detection. Present status of testing The majority of DNA-analysis techniques begin with amplification of the target sequence using the polymerase chain reaction (PCR). Three classes of assay dominate the tield of allelic discrimination. Solid-phase probe-target hybridization of PCR products can be carried out in microtiter-plate wells or as spots on membranes; signals from different alleles in the same sample can be differentiated using the location in the microtiter plate or on the membrane. Allele-specific PCR normally requires size-dependent separation, but allelic PCR products are often the same size and can only be differentiated by separating the allelic PCR primers into two different reactions and analyzing them on a gel4,s. Alternatively, allelic PCR products can be differentiated by attaching different fluorescent dyes to the competing allelic primers”. However, the application of this technique to complex genetic analysis is limited by the small number of dyes (four) that can be spectrally resolved using existing electrophoresis instrumentation. Small allelic changes (single-base insertions, deletions or substitutions) can also be analyzed using the oligonucleotide ligation assay (OLA; Fig. 1; Ref. 7) or the ligase chain reaction (LCR; Ref. 8). Ligation
assays provide a higher degree of specificity than it is possible to obtain using traditional DNA-probe assays because the two probes must anneal to positions on the target DNA that are immediately adjacent co one another; they must form a perfect match at the nick site before they can be joined by DNA ligase”. If one of the probe pair is labeled with a separate capture molecule (‘hook’) and the other with a ‘label’, the probes can easily be differentiated from one another, and from the bifunctionally labeled ligation product, using either solid-phase-capture technologylo~t’ or electrophoresis8,ia. OLA is typically used to detect single-base changes in an abundant or pi-e-amplified target sequence. LCR is the amplification counterpart of OLA and, through the use of two sets of probes, can be used to obtain exponential signalamplification as well as single-base discrimination. Until recently, no method had been adapted to the practical needs of large-scale multiplex analysis. Solid-phase assays hybridization require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to maintain the stringency needed for single-nucleotide mismatch discrimination. Multiplexing this approach has proved difficult because optimal hybridization conditions vary greatly among probe sequences. The multiplexing limitations of allele-specific PCR that are discussed above also apply to multiplex allelic discrimination by the TaqManTM assay13.‘“. This PCR-based assay system uses a fluorogenic probe that binds to an intervening sequence between the PCR primers. During primer extension, the TaqMan probe is hydrolyzed by the 5’ to 3’ exonuclease activity ofDNA polymerase, liberating the fluorescent dye from its corresponding quencher. The capacity of this assay to be multiplexed is limited by the number of dyes that can be spectrally resolved.
CopyrIght 0 1996, Elsevler Science Ltd. All rights reserved. 0167
7799/96/$15.00
PII: SO167-7799(96110014-7
Meeting report
uniform and position-independent expression of therapeutic genes in the LCR-specific target cells. DNA delivery using nonviral vectors Although most ongoing clinical trials make use of retroviral or adenoviral vectors, nonviral vectors will be preferable in the long term, particularly for direct in viva gene transfer. Jean-FranCois Mayaux and Robert Zaugg (Vital, San Diego, CA, USA) indicated that current research in this field includes screening for improved cationic lipid-based delivery systems and associating these with DNAcompacting agents. Koger Craig and Eric Tomlinson (GeneMedicine, Houston, TX, USA) discussed the possibility that second-generation polymer-based delivery systems will include targeting moieties, DNA-condensing agents, fusogenic peptides and,
Developingsmall-molecule libraries for drug discovery Pharmaceutical research and development is undergoing dramatic change. Laboratories are no longer synthesizing and testing molecules serially, but are applying the rapidly developing techniques of combinatorial chemistry to provide lead compounds for high-throughput screening (HTS) assays. At present, chemistry is the limiting step in the drug discovery process, and efforts are under way to develop larger and more diverse small-molecule libraries to enhance discovery of new leads for drug development. In addition to designing new synthetic pathways for novel pharmacophores, new instrumentation is required to speed up and automate chemical manipulations, as is new computer software for data management. A recent meeting* provided an opportunity to discuss these changes in the pharmaceutical industry, *The meetmg ‘Sol&Phase Synthesis: Developing Small~Molrcule Libraries‘ was held in Coronado, Californu. USA. 1-2 February 1YYh.
-____ ___ ~ TIBTECH APRIL 1996 NOL 14)
Automation for solid-phase synthesis HTS has been integrated into the drug discovery process to speed up the discovery of new lead compounds for pharmaceutical development. In the past, testing novel molecules for biological activity was a time-consuming process, but recent advances in the biological sciences have provided assays that rapidly analyze multiple compounds. In fact, the supply of chemical entities is rapidly depleting and new sources of test substrates are in demand. To meet this need, highthroughput organic synthesis (HTOS) is currently in development. If HTOS can successfully be developed, its use should speed up the process of bringing new phatmaceutical agents to the market by shortening the time at the prediscovery level (i.e. the generation of novel molecules) and at the drug development level (i.e. the synthesis of analogs to discover compounds with improved pharmacokinetic properties). Because provisional
eventually, peptides that allow nuclear transfer in post-mitotic cells (mimicking viruses). It remains to be seen what delays will be encountered and which therapies will be usell. Will these strategies allow the safe and efficient transfer of some or all of these new information-rich drugs at acceptable costs? These questions are the subject of continuous debate by the pharmaceutical industry, by regulatory agencies and by the many scientists involved in this exciting field. The number of multidisciplinary approaches currently being developed and the increasing speed at which applications based on fundamental research into gene expression are being contemplated are striking.
patents are filed at the time that lead compounds are discovered, using HTOS to advance the drug development process will allow more time for the final pharmaceutical product to be on the market while it is covered by patent. Thus, the use of HTOS should provide a higher return on marketable drugs for pharmaceutical firms (John P. Devlin; ARRT International, New Milford, CT, USA). Efficient time-management during the planning of organic syntheses will also extend the period of time during which the marketed product is covered by patent. Combinatorial chemistry requires that chemists design syntheses around a library of molecules, not around specific compounds. A software package currently being developed provides chemists with a user-friendly and highly interactive electronic reaction retrieval system (Guenter Grethe; MDL Information Systems, San Leandro, CA, USA); the Solid-Phase Reactions Database Organic (SPORE) will provide searchable information that is designed to enable synthetic chemists to plan reactions more efficiently. Another software product being developed provides organic chemists with a tool for designing reaction pathways, as well as for making predictions about the properties of members
CopyrIght 0 1996, Elswer Science Ltd. All rights reserved. 0167 - 7799/96/$15.00
PII: SO167-7799(96)30006-l
of the proposed small-molecule library (Ferenc Darvas; ComGenex, Budapest, Hungary). The heterogeneous reaction conditions intrinsic to solid-phase chemistry have prevented traditional analytical methods from being used. At present, destructive methods are used to monitor reaction progress: molecules of interest are cleaved from the resin and analyzed by standard procedures. Keith Russell (Zeneca Pharmaceuticals, Wilmington, DE, USA) discussed recent advances in quantitative infrared (IR) spectroscopy. Spectra obtained from single resin beads by IR microscopy can be quantified if the polystyrene backbone is used as an internal standard. Although the signal that is obtained is weak, if deuterium-tagged compounds are used, the signal obtained from the tagged groups can be isolated from those of other functional groups. Several examples were presented that indicate that the development of a fast, nondestructive method of monitoring reactions on solid supports is possible. The solid-phase synthesis of smallmolecule libraries can be simplified using automated laboratory workstations that enable the reactants, solvents, temperature and reaction atmosphere to be precisely controlled. A modular approach allows individual synthetic operations, such as resin dispensing, reactant preparation and chemical reactions, to be performed with existing instrumentation (James Harness; Bohdan Automation, Mundelein, IL, USA). However, details of a&inclusive workstations were also presented. Chemical manipulation can be achieved using two independent robotic arms (Mark Peterson; Advanced ChemTech, Louisville, KY, USA), or using a fluid-delivery device in a closed system without robotics (Joel Martin; Argonaut Technologies, San Carlos, CA, USA). One conclusion reached during the panel discussion was that there is probably not a single, best automated synthesizer, but that different workstations may be required to meet the specific applications of various laboratories. Linkers, supports and alternatives Several examples of new smallmolecule libraries constructed using novel linkage strategies were presented. Palladium(O)-mediated chemistry can be used for the syn-
thesis of 1%26-membered macrocyclic structures, heterocycles and substituted biaryl structures from solid-phase carbobenzyloxy chloride and imidazole equivalents (James Hauske; Versicor, Marlborough, MA, USA). This method produces higher yields of macrocyclic molecules than can be obtained using solution-phase chemistry because the polymer-bound molecules are segregated from each other. A siliconbased polymer linkage enables the synthesis of biphenyl derivatives, also through a palladium-catalyzed reaction (Balan Chenera; SmithKline Beecham, King of Prussia, PA, USA). The arylsilane linker can be made on the 50-gram scale from inexpensive, commercially available starting materials. Alternative methodologies for the synthesis of small-molecule libraries were discussed. If polyethylene glyco1 (PEG) is used as a polymeric support, familiar homogeneous reaction conditions can be employed, but product purification can be carried out using simple polymer precipitation, filtration, and washing (Kim Janda; The Scripps Research Institute, La Jolla, CA, USA). By combining the positive aspects of classical solution-phase chemistry and solid-phase synthesis, novel smalmolecule libraries can be characterized using traditional analytical techniques such as thin-layer chromatography and proton nuclear magnetic resonance (NMR) spectroscopy. The design of dipeptidomimetic and iminodiacetic anhydride templates has enabled solution-phase chemistry to replace polymer-based purification in the synthesis of smallmolecule libraries isolated from byproducts and excess reagents by either acid/base dissolution or washings (Christine Tarby; CombiChem, San Diego, CA, USA). Segregating reactions in a matrix format has allowed libraries to be synthesized in relatively high yields with >85% purity, as assessedby NMR. Heterocycles and phosphoramidate libraries New strategies for the production of highly functionalized pharmacophores for drug screening were discussed at the meeting. A one-pot, four-component condensation reaction (the Ugi reaction) has been adapted for solid-phase synthesis to yield several templates for library production (Adnan Mjalli; Ontogen,
Carlsbad, CA, USA). Carboxylic acids, isocyanides, &mines and aldehydes form the basis of a diverse collection of molecules that can be further modified to produce small, ring heterocycles such as imidazoles, oxazoles and lactams. In addition, the ‘tea bag’ method of solid-phase synthesis (in which the resin is enclosed in a porous polypropylene bag to facilitate manipulations) can be used to produce heterocyclic small-molecule libraries (John Kiely; Torrey Pines Institute for Molecular Studies, San Diego, CA, USA). Polymerbound amines are cyclized (through imine intermediates) to quinoline and isoquinoline libraries that can be characterized in pools by mass spectrometry. Further derivatization of these libraries yields new libraries of additional diversity and biological activity. Several reactions, such as 1,3 dipolar cycle-additions, monoreductive amination of dialdehydes and unsymmetrical benzoin condensations, have been shown to produce higher yields by solid-phase synthesis than by solution-phase chemistry (John Nuss; Chiron, Emeryville, CA, USA). These reactions have been used to create novel heterocyclic libraries by a dentrimeric approach. Fathi (PharmaGenics, Reza Allendale, NJ, USA) gave details of how a DNA synthesizer has been used to create combinatorial libraries of phosphoramidates; by incorporating a range of diols and amines it has been possible to produce a diverse library, which could be monitored using phosphorus NMR. Normand Hebert (Isis Pharmaceuticals, Carlsbad, CA, USA) discussed another approach that uses aminodiols to yield a collection of molecules in which the functional groups are positioned in various orientations; computer analysis has verified that there is very little conformational overlap of functional groups. Libraries can be synthesized using an automated solidphase synthesizer that carries out up to 96 independent reactions in an inert gas atmosphere. Combinatorial library strategies and programs The meeting concluded with several more examples of combinatorial libraries that have been created using solid-phase synthesis (Joseph Hogan, Jr; ArQule, Medford, MA, USA). The synthesis of small bioactive molecules and scaffolds has yielded libraries of inhibitors of proteolytic TIBTECH APRIL 1996 WL 14)
enzymes (Dinesh Pate]; A@max Research Institute, Santa Clara, CA, USA) Novel nonpeptidic libraries have been screened for binding to streptavidin (Viktor Krchnak; Selectide, Tucson, AZ, USA), and a multipin method of organic synthesis on solid support has been applied to
Workshop
the area ofreaction optimization, and has led to the production of p-turn mimics (Andrew Bray; Chiron Mimotopes, Victoria, Clayton, Australia), Further innovations in the synthesis of combinatorial small-molecule libraries should soon provide new
Multi-mutation screening using PCR and ligation - principles and applications Many genetic analysis applications rely on the discrimination of singlebase differences in sequence, or alleles that can have a profound effect on the function of the encoded protein. In humans, for example, such differences can lead to a loss of function of a protein or enzyme that is required for normal cell function or for growth control, resulting in an inherited disease or disease prediposition. They can also be the basis for differentiation between a pathogenic or antibiotic-resistant microorganism and its non-pathogenic counterpart. The ability to analyze a number of these traits simultaneously through multiplexing assayswill make genetic analysis more cost-effective, and will lead to more-widespread testing. Detecting inherited disease Multiple mutations must be screened to obtain high detection rates for many diseases. Diseases such as cystic fibrosis and inherited cancers have been shown to arise from any one of many potential mutations scattered throughout their respective genes. To date, over 400 diseasecausing mutations have been reported for cystic fibrosis’, 36 mutations have been reported for the inherited BRCA- 1 breast cancer gene’, and over 280 mutations have been reported in the p.53 oncogene, which is associated with -50% of all cancers”. Large-scale multiplex analysis is required for investigating these types ofgenetic problems, which rely on discriminating single-base differences at a multitude of loci that are sometimes closely spaced. We have reviewed a number of potential
I IBTECH APRIL l996 (VOL 14)
approaches to providing a solution to this problem. At present, the most promising approach is a combination of multiplex PCR DNA amplification and multiplex ligation assay detection. Present status of testing The majority of DNA-analysis techniques begin with amplification of the target sequence using the polymerase chain reaction (PCR). Three classes of assay dominate the tield of allelic discrimination. Solid-phase probe-target hybridization of PCR products can be carried out in microtiter-plate wells or as spots on membranes; signals from different alleles in the same sample can be differentiated using the location in the microtiter plate or on the membrane. Allele-specific PCR normally requires size-dependent separation, but allelic PCR products are often the same size and can only be differentiated by separating the allelic PCR primers into two different reactions and analyzing them on a gel4,s. Alternatively, allelic PCR products can be differentiated by attaching different fluorescent dyes to the competing allelic primers”. However, the application of this technique to complex genetic analysis is limited by the small number of dyes (four) that can be spectrally resolved using existing electrophoresis instrumentation. Small allelic changes (single-base insertions, deletions or substitutions) can also be analyzed using the oligonucleotide ligation assay (OLA; Fig. 1; Ref. 7) or the ligase chain reaction (LCR; Ref. 8). Ligation
assays provide a higher degree of specificity than it is possible to obtain using traditional DNA-probe assays because the two probes must anneal to positions on the target DNA that are immediately adjacent co one another; they must form a perfect match at the nick site before they can be joined by DNA ligase”. If one of the probe pair is labeled with a separate capture molecule (‘hook’) and the other with a ‘label’, the probes can easily be differentiated from one another, and from the bifunctionally labeled ligation product, using either solid-phase-capture technologylo~t’ or electrophoresis8,ia. OLA is typically used to detect single-base changes in an abundant or pi-e-amplified target sequence. LCR is the amplification counterpart of OLA and, through the use of two sets of probes, can be used to obtain exponential signalamplification as well as single-base discrimination. Until recently, no method had been adapted to the practical needs of large-scale multiplex analysis. Solid-phase assays hybridization require multiple liquid-handling steps, and some incubation and wash temperatures must be carefully controlled to maintain the stringency needed for single-nucleotide mismatch discrimination. Multiplexing this approach has proved difficult because optimal hybridization conditions vary greatly among probe sequences. The multiplexing limitations of allele-specific PCR that are discussed above also apply to multiplex allelic discrimination by the TaqManTM assay13.‘“. This PCR-based assay system uses a fluorogenic probe that binds to an intervening sequence between the PCR primers. During primer extension, the TaqMan probe is hydrolyzed by the 5’ to 3’ exonuclease activity ofDNA polymerase, liberating the fluorescent dye from its corresponding quencher. The capacity of this assay to be multiplexed is limited by the number of dyes that can be spectrally resolved.
CopyrIght 0 1996, Elsevler Science Ltd. All rights reserved. 0167
7799/96/$15.00
PII: SO167-7799(96110014-7