Universal and flexible DNA microarray approach: arrayed primer extension
2
Poster abstracts 62 nucleic acids, such as DNA, RNA and PNA, proteins, such as antibodies, peptides and lectins, as well as other classes of biomolecules such as carbohydrates and lipids. The ‘2 x 96’ XNA format, as well as the ‘2 x 384’ format under develop- ment, provide a flexible strategy for constructing custom 'user-defined' arrays. Use of the microscope slide footprint and the standard well spacing simplifies pro- cessing by allowing the use of a wide range of commercially available manual and robotic, handling and deposition equipment for slides and microtiter plates. XNA is compatible with most detection schemes including radiometric, fluorescence and chemiluminescence. Model hybridisation and immunoassay data will be pre- sented. XNA was originally developed as a tool for studying biological self-assembly processes. Biology uses nanometer sized units, that is to say proteins, carbohy- drates, lipids, etc. to build everything from viruses to elephants. In this context, developmental biology defines the frontier in biosciences. If we are to understand the molecular basis of developmental processes, then we must learn to fabricate coatings capable of mimicking biological surfaces in relevant dimensions, i.e. nanometers, and in relevant environments, i.e. aqueous, as tools for studying these (self-) assembly processes. XNA technology provides a tool for ‘reverse engi- neering’ self-assembly processes that will serve as the basis for the development of a new generation of diagnostic bioassays. Meltzer, Paul An altered gene expression profile is associated with tumour vasculogenesis by human melanoma cells Paul S. Meltzer 1 , Andrew J. Maniotis 2 , Robert Folberg 3,4 , Angela Hess 2 , Elisabeth A. Seftor 2 , Lynn M.G. Gardner 3 , Jacob Pe’er 5 , Jeffrey M. Trent 1 & Mary J.C. Hendrix 2 1 Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, Maryland, USA Departments of 2 Anatomy and Cell Biology, 3 Ophthalmology and 4 Pathology, The University of Iowa College of Medicine, Iowa City, Iowa, USA 5 Hadassah University Hospital, Jerusalem, Israel We have recently observed that tissue sections from uveal melanoma and metasta- tic cutaneous melanoma generally lack evidence of significant necrosis and con- tain patterned networks of interconnected loops of extracellular matrix. Similar networks are formed in tissue cultures of highly invasive primary and metastatic melanoma. These networks, which are not formed by melanocytes or poorly inva- sive melanomas, have the properties of vascular channels and suggest that certain tumours may have an intrinsic ability to undergo vasculogenesis without a contri- bution from host endothelial cells. Previous studies have identified abnormal co- expression in aggressive melanoma cells of intermediate filaments (an intercon- verted phenotype), suggesting reversion to a pluripotent cell. We sought to extend the definition of this phenotype at the gene expression level by cDNA microarray analysis. Comparison of aggressive melanomas that form vascular channels with less aggressive cells that do not exhibit this phenotype reveal differences in their gene expression profile. The specific genes expressed in the highly invasive melanoma cultures include those encoding extracellular matrix proteins, sig- nalling and regulatory molecules which are highly consistent with the vasculo- genic phenotype exhibited by these cells. The results of this study support the con- cept that some tumours generate vascular channels independently of convention- al angiogenesis and have important implications for the development of thera- peutic interventions directed at this novel property of aggressive melanomas. Menzel, Rolf EDDS, an Escherichia coli-based system with a variety applications amenable to miniaturization Rolf Menzel Small Molecule Therapeutics, Inc., 11 Deer Park Drive, Monmouth Junction, New Jersey 08852, USA The miniaturization of molecular biology technologies into formats reminiscent of electronic ‘chips’ has proceeded rapidly, with nucleic acids allowing high- throughput DNA and RNA analysis. This has ushered in new eras in both biology and commerce with potentials yet to be fully appreciated and realized. Similar developments will be possible in other biological areas, as appropriate tools are adapted to formats amenable to miniaturization. Microchips with fluid-handling capabilities will allow for the growth of organisms in micro-formats. Whole- cell–based approaches will permit complex biological endpoints to be assayed. Escherichia coli provides a robust platform for a variety of assays that cover a diverse collection of biological endpoints, and we have developed a number of E. coli-based assays. Our most advanced platform is EDDS (E. coli dimer detection system). EDDS is a protein-protein interaction system (in some ways analogous to the yeast two-hybrid system) that is uniquely able to address membrane protein interactions. This presentation describes the principles behind EDDS and several of its implementations. We have developed applications of EDDS that include small molecule drug discovery, receptor-ligand interactions and matching, and proteomic applications that generate reagents useful in the description of gene function. EDDS and other microbial models that express mammalian genes pro- vide surrogate systems for the development of many assays with diverse biologi- cal endpoints. Microbes are more robust and economical than mammalian cells and with their simple growth requirements will be amenable to the development of chip-based assays. A nanoliter of culture media can contain over 10 3 bacterium that can be engineered to provide signals that could be based on a wide range of complex biological endpoints. Metspalu, Andres Universal and flexible DNA microarray approach: arrayed primer extension Andres Metspalu 1,2 , Neeme Tõnisson 1,3 , Elin Lõhmussaar 1 , Kristiina Tensing 1 , Jana Zernant 2 & Ants Kurg 1,2 1 Institute of Molecular and Cell Biology, University of Tartu 2 Estonian Biocentre; and 3 Asper Ltd., Tartu, Estonia We describe an integrated genetic testing system consisting of DNA oligonu- cleotide array production, template preparation, multiplex primer extension on the array, fluorescence imaging equipment and data analysis. DNA oligonucleotide (ON) arrays are produced by spotting presynthesized 25-mer ON with a 5´ amino group on the glass microscope slide. This approach involves a number of poten- tial problems, such as silanization of the slides, ON selection and immobilization. As a matter of fact, these steps should be more efficient and reproducible. Currently, the spacer separating ON from the glass surface is 2–18 carbons, but longer linker arms seem to be better. Two DNA strands have been analysed simul- taneously according to our protocol. The double-stranded DNA template prepara- tion can be described as an one-tube reaction with minimal number of pipetting steps without centrifugation or ethanol precipitation. Arrayed primer extension (APEX) is carried out as a single-step reaction simultaneously with all four dye terminator nucleotides (fluoresceine, Cy3, Texas Red and Cy5) in the presence of © 1999 Nature America Inc. • http://genetics.nature.com © 1999 Nature America Inc. • http://genetics.nature.com
Transcript of Universal and flexible DNA microarray approach: arrayed primer extension
Poster abstracts
62
nucleic acids, such as DNA, RNA and PNA, proteins, such as antibodies, peptidesand lectins, as well as other classes of biomolecules such as carbohydrates andlipids. The ‘2 x 96’ XNA format, as well as the ‘2 x 384’ format under develop-ment, provide a flexible strategy for constructing custom 'user-defined' arrays.Use of the microscope slide footprint and the standard well spacing simplifies pro-cessing by allowing the use of a wide range of commercially available manual androbotic, handling and deposition equipment for slides and microtiter plates. XNAis compatible with most detection schemes including radiometric, fluorescenceand chemiluminescence. Model hybridisation and immunoassay data will be pre-sented.
XNA was originally developed as a tool for studying biological self-assemblyprocesses. Biology uses nanometer sized units, that is to say proteins, carbohy-drates, lipids, etc. to build everything from viruses to elephants. In this context,developmental biology defines the frontier in biosciences. If we are to understandthe molecular basis of developmental processes, then we must learn to fabricatecoatings capable of mimicking biological surfaces in relevant dimensions, i.e.nanometers, and in relevant environments, i.e. aqueous, as tools for studying these(self-) assembly processes. XNA technology provides a tool for ‘reverse engi-neering’ self-assembly processes that will serve as the basis for the developmentof a new generation of diagnostic bioassays.
Meltzer, Paul
An altered gene expression profile isassociated with tumour vasculogenesis
by human melanoma cells
Paul S. Meltzer1, Andrew J. Maniotis2, Robert Folberg3,4, AngelaHess2, Elisabeth A. Seftor2, Lynn M.G. Gardner3, Jacob Pe’er5,
Jeffrey M. Trent1 & Mary J.C. Hendrix2
1Cancer Genetics Branch, National Human Genome Research Institute,Bethesda, Maryland, USA
Departments of 2Anatomy and Cell Biology, 3Ophthalmology and 4Pathology,The University of Iowa College of Medicine, Iowa City, Iowa, USA
5Hadassah University Hospital, Jerusalem, Israel
We have recently observed that tissue sections from uveal melanoma and metasta-tic cutaneous melanoma generally lack evidence of significant necrosis and con-tain patterned networks of interconnected loops of extracellular matrix. Similarnetworks are formed in tissue cultures of highly invasive primary and metastaticmelanoma. These networks, which are not formed by melanocytes or poorly inva-sive melanomas, have the properties of vascular channels and suggest that certaintumours may have an intrinsic ability to undergo vasculogenesis without a contri-bution from host endothelial cells. Previous studies have identified abnormal co-expression in aggressive melanoma cells of intermediate filaments (an intercon-verted phenotype), suggesting reversion to a pluripotent cell. We sought to extendthe definition of this phenotype at the gene expression level by cDNA microarrayanalysis. Comparison of aggressive melanomas that form vascular channels withless aggressive cells that do not exhibit this phenotype reveal differences in theirgene expression profile. The specific genes expressed in the highly invasivemelanoma cultures include those encoding extracellular matrix proteins, sig-nalling and regulatory molecules which are highly consistent with the vasculo-genic phenotype exhibited by these cells. The results of this study support the con-cept that some tumours generate vascular channels independently of convention-al angiogenesis and have important implications for the development of thera-peutic interventions directed at this novel property of aggressive melanomas.
Menzel, Rolf
EDDS, an Escherichia coli-based systemwith a variety applications amenable to
miniaturization
Rolf Menzel
Small Molecule Therapeutics, Inc., 11 Deer Park Drive, Monmouth Junction, New Jersey 08852, USA
The miniaturization of molecular biology technologies into formats reminiscentof electronic ‘chips’ has proceeded rapidly, with nucleic acids allowing high-throughput DNA and RNA analysis. This has ushered in new eras in both biologyand commerce with potentials yet to be fully appreciated and realized. Similardevelopments will be possible in other biological areas, as appropriate tools areadapted to formats amenable to miniaturization. Microchips with fluid-handlingcapabilities will allow for the growth of organisms in micro-formats. Whole-cell–based approaches will permit complex biological endpoints to be assayed.Escherichia coli provides a robust platform for a variety of assays that cover adiverse collection of biological endpoints, and we have developed a number of E.coli-based assays. Our most advanced platform is EDDS (E. coli dimer detectionsystem). EDDS is a protein-protein interaction system (in some ways analogousto the yeast two-hybrid system) that is uniquely able to address membrane proteininteractions. This presentation describes the principles behind EDDS and severalof its implementations. We have developed applications of EDDS that includesmall molecule drug discovery, receptor-ligand interactions and matching, andproteomic applications that generate reagents useful in the description of genefunction. EDDS and other microbial models that express mammalian genes pro-vide surrogate systems for the development of many assays with diverse biologi-cal endpoints. Microbes are more robust and economical than mammalian cellsand with their simple growth requirements will be amenable to the developmentof chip-based assays. A nanoliter of culture media can contain over 103 bacteriumthat can be engineered to provide signals that could be based on a wide range ofcomplex biological endpoints.
Metspalu, Andres
Universal and flexible DNA microarrayapproach: arrayed primer extension
Andres Metspalu1,2, Neeme Tõnisson1,3, Elin Lõhmussaar1, Kristiina Tensing1, Jana Zernant2 & Ants Kurg1,2
1Institute of Molecular and Cell Biology, University of Tartu 2Estonian Biocentre; and
3Asper Ltd., Tartu, Estonia
We describe an integrated genetic testing system consisting of DNA oligonu-cleotide array production, template preparation, multiplex primer extension on thearray, fluorescence imaging equipment and data analysis. DNA oligonucleotide(ON) arrays are produced by spotting presynthesized 25-mer ON with a 5´ aminogroup on the glass microscope slide. This approach involves a number of poten-tial problems, such as silanization of the slides, ON selection and immobilization.As a matter of fact, these steps should be more efficient and reproducible.Currently, the spacer separating ON from the glass surface is 2–18 carbons, butlonger linker arms seem to be better. Two DNA strands have been analysed simul-taneously according to our protocol. The double-stranded DNA template prepara-tion can be described as an one-tube reaction with minimal number of pipettingsteps without centrifugation or ethanol precipitation. Arrayed primer extension(APEX) is carried out as a single-step reaction simultaneously with all four dyeterminator nucleotides (fluoresceine, Cy3, Texas Red and Cy5) in the presence of
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the DNA polymerase (Thermo Sequenase). For this purpose, a special four-colourinstrument, FD-003, was developed at Asper Ltd. The latter uses a total internalreflection fluorescence (TIRF)-based excitation mechanism combined with fourlasers, filter wheel and scientific grade CCD camera. Image acquisition is fast(0.5–2 min for 4-colour) and automatic, followed by software analysis to convertthe fluorescence information into DNA sequence data. Signal-to-noise ratio is atleast 30:1, allowing comfortable analysis of heterozygous positions in DNA. Thissystem is used for mutation detection in ß-globin, BRCA1 and BRCA2 genes,resequencing of p53 gene fragments, SNP testing and gene expression usingmtComplex I genes.
Mills, Jr, Allen
Interconversion of electrical and DNAdata for molecular computation
Allen P. Mills Jr & Bernard Yurke
Bell Labs, Lucent Technologies, 600 Mountain Avenue, Murray Hill, New Jersey 07974, USA
To make practical use of the large-scale computing possibilities associated with amolecular computer based on DNA hybridization 1 it will be necessary to quicklyconvert data back and forth from electrical to molecular form. In a DNA comput-er configured as an analogue neural network2 we will let a subset of all single-stranded DNA q-mers Ei be in 1:1 correspondence with the basis vectors ei, I =1,2,...,m in the abstract m-dimensional vector space used to represent the network.The concentration [Ei] of strands with index i in a DNA sample representing a vec-tor V = i Vi ei is proportional to the amplitude Vi of the i-th component of the vec-tor.
Suppose that input data are presented in the form of images of nn = m pixels.The images may be flashed on a DNA microplate3 whose pixels match the imagepixels and each of which is covered with unique DNA strands Ei attached byhybridization to complementary anchored strands. The image flash causes localheating4 that melts some of the double-stranded DNA. The liberated single strand-ed DNA is subsequently collected to yield a solution containing a concentrationof Ei molecules proportional to the image intensity at the i-th pixel for I = 1,2,...,m.The DNA image solution may be used for DNA computation and may be recon-verted to electrical form at any time, that is “read out”, by allowing a portion ofthe solution to rehybridize with a single-stranded microarray. The amount ofduplex DNA at each pixel may be determined by fluorescence analysis in theusual fashion to reconstitute the original image.
1. Adleman, L.M. Science 266, 1021-1024 (1994).2. Mills, A.P. Jr, Yurke, B. & Platzman, P.M. “DNA analog vector algebra and physical constraints on large-
scale DNA-based neural network computation”, Proc. of Fifth DIMACS conference on DNA computing,Cambridge, MA, June 14-15, (1999).
3. Fodor, S.P.A et al. Science 251, 767-773 (1991).4. Edman, et al. Nucleic Acids Res. 25, 4907-4914 (1998).
Mir, Kalim U.
What length of probe is optimal formicroarrays?
Kalim U. Mir
University of Oxford, Biochemistry, South Parks Rd, Oxford, OX1 3QU, UK
DNA microarrays, comprising spotted PCR products of 0.6Kb or above, havebecome a major platform for functional genomics. It is considered that arrayperformance could be improved by turning to oligonucleotides instead ofcDNAs and by controlling the site of attachment. Furthermore, to bring the
approach within the reach of methods for combinatorial synthesis, the length ofthe probe needs to be minimised.
However, under the low stringency conditions required for short nucleic acidprobes to form stable interaction, the longer single-stranded targets are able toengage in secondary structure. This can mask the desired interaction between tar-get and probe.
We find that the extent of interaction of target with short complementaryprobes varies sharply even at adjacent positions.The factors that affect hybridisa-tion have been studied using combinatorial arrays and the well-characterised yeasttRNAphe as a model. We find that heteroduplexes giving high hybridisation yieldincorporate target single- and double-stranded portions and that specific structur-al features may increase or decrease yield.
We have conducted studies to find the optimal length of probe for achievingefficient hybridisation across all target sites in nucleic acids. A combinatorial“scanning” array was synthesised in situ comprising a set of probes which rangedin length from 1 to 76 nucleotides—a length that is within the constraints for effi-cient oligonucleotide synthesis.
We find that the effects of secondary structure can be overcome with probesbeyond a specific length when hybridisation is performed within a specific tem-perature range and in an appropriate buffer.
DNA has a negatively charged backbone that requires cations to form aduplex. We have also studied an alternative to DNA, the Peptide Nucleic Acids(PNA) which have a neutral backbone. An array of PNA probes complementaryto tRNAphe was used to study the hybridisation behaviour of PNA in comparisonto DNA and to see if hybridisation could be enhanced (in collaboration with J.Hoheisel, S. Wuertz, S. Matysiak).
Our work has demonstrated that the molecular basis of target-probe interac-tions can be exquisitely studied using combinatorial arrays and that this is animportant resource for developing and optimising applications of array tech-nologies.
Montgomery, Don
Very large-scale arrays of biomolecules
Don Montgomery
CombiMatrix Corporation, 887 Mitten Road, Suite 200, Burlingame, California 94010, USA
ArrayChips are semiconductor devices that enable rapid and flexible fabricationof very large-scale arrays of biomolecules. Array fabrication is controlled by elec-tronic hardware on the chip and is software configurable. This feature enablesvery rapid turnaround times of new array designs for testing and optimization.Biomolecules in the array are synthesized in situ or immobilized at selectedaddressable locations. Synthesis occurs directly within the interstices of a hydrat-ed and highly porous membrane that overlays the ArrayChip. Synthesis is accom-plished in situ using reagents that are generated electrochemically at individuallyaddressable microelectrodes on ArrayChips. We have developed technologies forin situ synthesis of arrays of oligonucleotides, peptides and small molecules.ArrayChips obviate any need for microfluidics or other unconventional semicon-ductor hardware. First-generation ArrayChips have microelectrode array densitiesof over 1,000 individually addressable sites per cm2. Second-generation hardwaredesigns have over 500,000 individually addressable sites per cm2.
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