- Email: [email protected]
Molecular genetic applications of streptavidin-coated manifold supports
Biomolecular Engineering 16 (1999) 105 – 111 www.elsevier.com/locate/geneanabioeng
Molecular genetic applications of streptavidin-coated manifold supports Gisela Barbany a,b,1, Anette Hagberg a,2, Erik Waldenstro¨m a,b,1, Ulf Landegren a,1,* a
Department of Medical Genetics, Box 589, Uppsala Biomedical Centre, Uni6ersity of Uppsala, S-751 23 Uppsala, Sweden b Department of Internal Medicine, Uppsala Uni6ersity Hospital, Uppsala, Sweden
Abstract Practical problems of handling large numbers of samples limit the application of molecular genetic procedures in clinical settings and in research. In the present review we describe a multipronged manifold support, coated with streptavidin, that offers distinct advantages in preparative and diagnostic applications. In order to increase the surface available on the manifold, porous Sepharose particles conjugated with streptavidin were attached to the plastic support. This procedure increased the surface by almost three orders of magnitude, permitting sufficient streptavidin to be coupled to the support for most routine applications. The manifold supports have been used for sample preparation and in a number of genetic assays, including allele discrimination assays and DNA sequencing. In all these assay formats the manifold supports allow large numbers of samples to be processed in parallel. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Solid phase; Solid support; Genetic analysis; Streptavidin; mRNA isolation; Sequencing; Allele discrimination; Sample preparation; Capture PCR
1. Introduction Information about genetic alterations leading to disease is accumulating rapidly. As a consequence, more and more molecular genetic tests are becoming available to detect alterations associated with pathology. However, most of these genetic analyses are time-consuming and cumbersome to perform, limiting their applicability in clinical practice and in research. Strategies are sought to simplify protocols for genetic testing, in order to allow the analysis of large numbers of samples. Solid phase methods have proven valuable in immunoassays, and more recently also in molecular genetic procedures. Immobilization of DNA molecules to supports via the biotin – avidin or streptavidin link is * Corresponding author. Tel.: +46-18-4714910; fax: + 46-18526849. E-mail address: [email protected] (U. Landegren1) 1 Current address: Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden. 2 Current address: Department of Genetics and Pathology, Rudbeck Laboratory, Se-75185 Uppsala, Sweden.
particularly useful since the bond can be established rapidly and it remains stable under extreme physical conditions, including temperatures and pH that denature DNA molecules into single strands [1]. Different solid phases such as wells from microtiter plates, polystyrene and Sepharose beads, and paramagnetic particles have all been conjugated with streptavidin and used for affinity capture of biotinylated DNA [1]. We have developed a streptavidin-coated manifold support that offers particular advantages for processing sets of samples. Manifold supports, useful for molecular genetic reactions, are configured as a set of projections that fit individual wells of a reaction plate. The use of such supports to immobilize and manipulate nucleic acids can simplify many aspects of gene analysis. The supports make it possible to handle large number of samples simultaneously and they permit transfer of samples between consecutive reaction steps with minimal pipetting, thus reducing the risk of sample mix-up and saving considerable time. In the present review we describe the construction of manifold supports covered with streptavidin, and we exemplify several applications of the supports.
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1.1. Coupling of strepta6idin to manifold supports The prongs of manifold supports offer a limited area for binding streptavidin. A simple protocol was therefore designed to expand this surface area in order to increase the binding capacity of the manifolds. Streptavidin was first conjugated to porous Sepharose particles that were then attached to the manifold. The procedure has been described in detail elsewhere [2]. Briefly, Sepharose particles (HiTrap, NSH-activated HP Sepharose, Pharmacia Biotech) were washed in ice-cold 1 mM HCl, followed by a solution of 1 M NaCl and 0.4 M NaCO3 buffered to pH 8.3. The particles were then transferred to 5 ml of the above buffer, containing 10 mg of streptavidin. After 1 h incubation the particles were blocked in 0.1 M ethanolamine buffer for 15 min. The streptavidin-conjugated Sepharose particles were then washed in 0.1 M
acetate buffer pH 4.0 with 1 M NaCl, and either used immediately or stored in 0.05 M Tris–HCl pH 7.3 with 0.02% sodium azide. The Sepharose surface should not be allowed to dry during this process. Conjugated particles were filtered, washed with distilled water, dried with methanol, and then equilibrated with triethylamine, to obtain a slurry of about 50% (v/v) particles ready to be attached to the supports. We have been using commercially available polystyrene supports (Falcon, Nunc) as well as a cone-shaped prototype (Pharmacia Biotech) that fits the conical wells of microtiter plates used for PCR (Falcon, Costar). The supports were washed with ethanol for 20 min in an ultrasonic bath, and the particles were then grafted onto the tips of the support by submerging these in the slurry, followed by evaporation of triethylamine in air. Loosely bound particles were removed by rinsing the supports in distilled water. This procedure increases the surface area available for coupling by a factor of at least 800. In this manner large amounts of streptavidin are bound to the supports. Fig. 1 shows two scanning electron micrographs of the surface of the support at different magnifications, revealing the partially embedded particles. Variants of the described procedure can be used to conjugate a wide variety of molecules to supports via porous particles.
1.2. Binding capacity and binding kinetics of strepta6idin-coupled supports Fig. 1. Scanning electron micrographs at two different magnifications of the surface of a solid support with avidin-coated Sepharose particles attached. The scale bars represent 100 and 10 mm, respectively.
Fig. 2. Binding capacity of the support for biotin. The effect of increasing concentrations of biotin on the immobilization of a biotinylated reporter oligonucleotide was analyzed. The results are expressed in counts per second. The mean and standard deviation of triplicate samples are shown.
To investigate the binding capacity of the support, a europium-labeled biotinylated oligonucleotide was constructed by adding a series of europium chelates to the 3% end of a 5%-biotinylated oligonucleotide [3]. Binding to the support was measured by incubating individual prongs with 60 fmol of the europium- and biotin-labeled oligonucleotide with increasing amounts of free biotin in 1 M NaCl, 100 mM Tris–HCl pH 7.5 and 0.1% Triton X-100. The support was subsequently washed and incubated in a fluorescence enhancement solution (Wallac), whereupon europium ions were detected by time-resolved flourometry in a DELFIA plate reader fluorometer [3]. Fig. 2 shows a representative experiment, indicating that 12.5 pmol of free biotin were required to reduce the binding of the indicator oligonucleotide by one half. The biotin binding capacity of the manifolds with avidin-conjugated Sepharose particles has been compared to that of manifolds and two types of microtiter plates, passively coated with avidin. In all cases it was found that the biotin binding capacity of the passively coated surfaces was far lower than that of the supports with immobilized avidinmodified particles [2]. Experiments were performed to study the rate at which biotinylated oligonucleotides could be bound to
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Fig. 3. RNA isolation followed by reverse transcription and PCR: (i) configuration of a streptavidin-coupled Sepharose-coated support; (ii) hybridization to the RNA directly from the cell lysate; (iii) cDNA synthesis; and (iv) PCR amplification.
the support. A 10 min incubation on a shaking platform at room temperature was sufficient to immobilize most or all of the oligonucleotides added, and to saturate the binding capacity of the support [2].
2. Applications of streptavidin-coated manifold supports
2.1. mRNA isolation and cDNA amplification mRNA purification protocols involve many processing steps and frequently require the use of hazardous chemicals [4,5]. We have developed a method to specifically bind the RNA of interest directly from a cell lysate onto a manifold support, and to subsequently transfer the isolated sequences to reverse transcription reactions and PCR (Hagberg et al., unpublished). A
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biotinylated oligonucleotide, complementary to the mRNA of interest, is bound to the support via a streptavidin-biotin interaction and it is then allowed to hybridize to RNA from the cell lysate. Once hybridized to the support the RNA and subsequent reaction intermediates are conveniently transferred through the different enzymatic and washing steps with the aid of the manifold (Fig. 3). We have used this method to isolate and amplify by RT-PCR the bcr/abl fusion mRNA characteristic of chronic myelogenous leukemia (CML) (Barbany et al., unpublished). In these experiments 10 pmol of the biotinylated capture oligonucleotide, complementary to 40 bases of the abl mRNA, were incubated with individual prongs of the support in 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2.5 M NaCl. The cells were lysed in a buffer containing 500 mM LiCl, 1% LiDS 100 mM Tris–HCl pH 8.0, 10 mM EDTA, and 5 mM DTT. Lysates corresponding to 105 cells were diluted 1:10 in H2O and incubated together with the support. The supports were extensively washed before being transferred to a new set of wells, containing the first strand cDNA synthesis mixture. After synthesis was completed the manifold was washed once in 1×PCR buffer and then transferred to wells containing the appropriate amplification primers in standard PCR mixes. The support was removed from the microtiter plate after 2 cycles. The brc/abl mRNA was amplified with a nested set of primers. Fig. 4 shows the amplification products obtained from the cell line K562 (lane 1), which is positive for the fusion mRNA, and from three different CML patients, two of them positive (lanes 3 and 4) and one below detection limit (lane 2). The cell line BSM was used as a negative control (lane 5). By combining the described RT-PCR with a homogeneous detection reaction such as the Taqman assay [6] a simple and rapid quantitative detection of malignant cells is possible. Manifold supports can also be used with oligo-d(T) coupled to the prongs. In this manner total poly(A)+ RNA is isolated by hybridization to the manifold. The method greatly simplifies mRNA isolation and cDNA amplification from many samples, and it should be useful in mutation detection and gene expression studies.
2.2. Capture PCR
Fig. 4. Ethidium bromide-stained agarose gel showing the correct amplification product from K562 cells (lane 1), three different chronic myelogenous leukemia patients (lanes 2–4). The BSM cell line was included as a negative control (lane 5).
Capture PCR was developed as a method to amplify and isolate DNA fragments when sequence information is available from only one end of the fragment of interest [7]. With this method a DNA fragment can be exponentially amplified using sequence information from only one side of the amplified fragment, but with similar specificity as in a conventional PCR. In this procedure total genomic DNA is first restriction digested and a double stranded oligonucleotide linker is
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Fig. 5. Capture PCR: (i) digestion of genomic DNA; (ii) ligation of the linker to the restricted ends; (iii) extension from a 5%-biotinylated primer; (iv) immobilization on the streptavidin-coated support and (v) amplification with a second specific primer together with the linker oligonucleotide.
ligated to the restricted ends. After ligation primer extensions are performed using a biotinylated sequencespecific primer. Extended primers from many separate genomic samples and restriction digestions are individually captured onto the prongs of a streptavidin-coated manifold support and used as templates in a subsequent PCR. In this amplification reaction a second sequencespecific primer, hybridizing downstream of the first one, amplifies the fragment of interest together with the linker oligonucleotide, serving as a non-specific amplification primer (Fig. 5). By capturing the primer extension product on the support, the complexity of the DNA sample is greatly reduced before the amplification step. The identity of the amplification product can be
Fig. 6. Manifold-assisted sequencing: (i) binding of biotin-labeled PCR products to the support; (ii) denaturation of complementary strands; (iii) annealing of the fluorescein-labeled sequencing primer; (iv) extension reactions in the presence of dideoxynucleotides; and (v) transfer of reaction products to the gel using the manifold support. The sequencing reaction products are released from the supports by being denatured in a layer of formamide, added to the top of the gel.
confirmed using a third downstream amplification primer together with the linker primer. This inner PCR can also be used to prepare the amplification products for solid phase sequencing (see below) by using a biotinylated version of the linker for subsequent immobilization of the amplification products on a support. By further incorporating a 5%-sequence representing the M13 sequencing primer in the sequence-specific amplification primer, the amplicons can be analyzed by fluorescent sequencing using a standard sequencing primer [8]. For a given DNA sample, for example a source of human genomic DNA, restriction digestion with a particular enzyme and linker ligation need only be performed once. In this way a library of DNA fragments is created with the appropriate linker ligated to the restricted ends. This library can then be used with any primer set to generate a specific human DNA segment by capture PCR. In our laboratory this technique has been used to isolate ends of YAC clones [7,9] to determine the breakpoints of a 5-kb deletion in the amelogenin gene using sequence information near one of the breakpoints [10], to extend partial cDNA sequences, and to isolate homologs of a gene for a peptide hormone from more than 30 different species (Lagerstro¨m-Ferme´r unpublished results).
2.3. Sequencing Since the advent of DNA sequencing methods in the mid seventies [11,12] and automated, real-time detection of gel-separated products of sequencing reactions in the mid eighties [13,14], efforts have been made to simplify the handling of sequencing reactions. An important improvement consisted in the immobilisation of biotin-labeled PCR products to magnetic beads, coated with streptavidin for subsequent solid phase sequencing [15,16]. A further step was taken when Lagerkvist et al. constructed a manifold solid support consisting of flat plastic prongs grafted with Sepharose particles that were covalently coated with avidin or streptavidin [8]. The increased area allowed for binding sufficient biotinylated PCR products to perform sequencing reactions. The streptavidin-coated devices, shaped like sequencing combs, are suitable for handling many sequencing reactions in parallel, with little increase in effort over the handling of a single one; the samples are conveniently transferred through all processing steps (binding of biotinylated PCR products; denaturation; annealing of fluorescent primer; primer extension; and loading of samples onto the gel of an automated laser fluorescent DNA sequencer) (Fig. 6). Pipetting is greatly reduced compared to ordinary techniques and the process time is shorter.
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Fig. 7. Results from an oligonucleotide ligation assay. A group of 22 patients with known abnormalities of a1-antitrypsin were screened for the presence of the Z mutation using the ligase-based assay. The europium and terbium fluorescence readings from individual DNA samples are indicated as circles. Three clearly resolved clusters of data points can be distinguished corresponding to samples amplified from individuals homozygous for the normal sequence (open circles), individuals homozygous for the mutant sequence (filled circles) and from heterozygous individuals (shaded circles).
The solid phase sequencing method using plastic combs has been successfully applied for instance to detect mutations in the entire coding sequence of the p53 gene in breast cancer tumors from 316 patients [17]. We have used it to analyze the coding sequence for the copper transporting P-type ATPase ATP7B that is mutated in Wilson’s disease [18]. The 21 exons were amplified by PCR from genomic DNA with one biotinylated primer and one primer with the M13 universal primer sequence added as a 5%-tag. The PCR products were used as templates for solid phase sequencing with a handling time of just 1 h per set of 100 templates. Twenty-two families were included in the study and out of the expected total of 44 mutations, 40 were found among these patients. Reagents for the method are now commercially available as the AutoLoad Solid Phase Sequencing Kit from Pharmacia Biotech.
2.4. Oligonucleotide ligation assay The manifolds can be used in an oligonucleotide ligation assay (OLA) to detect sequence variations in the genome [19]. The method takes advantage of the inability of oligonucleotides to serve as substrates for enzymatic ligation, when the ends to be joined are mismatched to their target sequences [20 – 22]. By using a manifold support to perform the steps of the reaction, known mutations can be screened for in large numbers of samples. The supports used in this type of assay fit
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into the wells of a microtiter plate, making it possible to simultaneously process 96 DNA samples. After DNA amplification, the reactions are diluted with water and heat-denatured before a ligation mixture is added. This mixture contains T4 DNA ligase, buffer, ATP, and a set of 3 oligonucleotides that are designed based on the sequence surrounding the mutation site. Two of them differ at their 3% end depending on whether they are specific for the normal or the mutant sequence. Their 5% ends are differentially labeled with two lanthanide chelates (europium for the normal and terbium for the mutant sequence). The third oligonucleotide is 5%-phosphorylated and biotinylated at its 3% end, and it hybridizes immediately downstream of the lanthanide-labeled oligonucleotides. Once ligation is completed the products are trapped on the streptavidincoated multipronged support and following denaturing washes the two types of lanthanide ions are detected through time-resolved fluorometry. The OLA method has been used to analyze a group of patients with a1-antitrypsin deficiency and their relatives [19]. Fig. 7 shows the genotypes of 22 individuals that were analyzed for the presence of the Z mutation in the a1-antitrypsin gene, altering a single nucleotide position. Three clearly resolved point clusters are seen. Samples derived from individuals homozygous for the normal sequence result in strong europium signals and individuals homozygous for the mutant sequence exhibit strong terbium signals. Samples from heterozygous carriers support ligation of both europiumand terbium-labeled probes and in the diagram the symbols fall along a diagonal.
2.5. Minisequencing The minisequencing reaction, as oligonucleotide ligation, allows detection of single nucleotide variations in amplified DNA fragments [23,24]. The method makes use of a DNA polymerase to specifically extend the 3% end of a primer that anneals immediately upstream of a variable nucleotide position. This results in the incorporation of a labeled nucleotide, complementary to the template at the variable site. The identity of the nucleotide serves as an indicator of the nucleotide present at the variable site of the template [24]. In combination with streptavidin-coated sequencing combs the minisequencing method has been used to detect multiple polymorphic nucleotide positions in a single reaction. Following PCR amplification, the biotinylated products are captured on manifold supports and the strands denatured. This step efficiently removes nucleotide triphosphates and amplification primers before the minisequencing reaction, and renders the template strands accessible for hybridization to the sequencing primers. The minisequencing reactions are carried out by immersing the immobilized templates in 4 different
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wells, each containing a set of extension primers, a DNA polymerase, one fluorescein-labeled dideoxynucleotide triphosphate and the corresponding three other unlabeled dideoxynucleotides. After the extension reaction the manifolds are used to load the samples on an automated DNA sequencer (ALFexpress, Pharmacia Biotech). The detection primers are designed to have distinct lengths by adding a nucleotide tail at their 5% ends. In this way the length of the detection primer defines the site of variation and the incorporated nucleotide reveals the polymorphic nucleotide (Fig. 8). The results from the gel electrophoresis are interpreted with the aid of the ALF Fragment Manager software (Pharmacia Biotech). This method has been successfully applied to type HLA-DQA1 and HLA DRB1 genes [25]. The PCR amplification primers were designed to amplify all alleles equally in the DQA1 and DRB1 loci. Nine different polymorphic nucleotide positions were studied simultaneously by using primers that differed in size by increments of three bases. This technique makes it feasible to analyze multiple polymorphic nucleotides simultaneously in large numbers of samples.
2.6. Multicolor detection with lanthanides In addition to the molecular genetic tests mentioned, the manifolds have also been successfully used in an immunological assay. An avidin-coated manifold support was used to detect specific IgE in allergic individuals [26]. The allergen, in this case soy protein extract, was biotinylated and subsequently bound to the avidincoupled support. Manifolds with immobilized antigen were incubated in serum samples, and subsequently with a Fab% fragment of a mouse monoclonal antibody conjugated with europium chelates. The fluorescence from the europium ions was recorded in time-resolved mode [3]. Attaching the antigen directly to the manifold support resulted in higher sensitivity compared to the use of antigen immobilized via an avidin-biotin interaction. The assay was compared to the Pharmacia CAP system for specific IgE detection and it was concluded
that the binding characteristics of the manifolds and the cellulose matrix were quite similar. In time-resolved detection of lanthanides, several colors are available, permitting the simultaneous analysis of different substances in the same sample, e.g. different antibody classes with similar specificities or different nucleic acid sequences. Samiotaki et al. described a method to simultaneously analyze amplification products for the presence of any of seven different DNA sequences using a manifold support [27]. The multicolor-typing system was applied to the identification of human papilloma virus (HPV) types. PCR primers were designed to anneal to conserved regions in the genome. Biotinylated amplification products were immobilized on streptavidin-conjugated manifold supports, and after denaturing washes a mixture of seven different HPV type-specific probes were allowed to hybridize to the bound sequence. These seven type-specific oligonucleotides had been labeled with of 1, 2 or 3 of the lanthanide ions europium, terbium and samarium. The predefined combinations exhibit distinct fluorescence that can be detected by time-resolved fluorometry. An additional feature of the method is that probes can be removed from the bound targets by denaturing washes, followed by rehybridization of the sample with a new set of seven different oligonucleotides. This procedure can be repeated at least twice without significant loss of signal intensity, allowing hybridization with a total of 21 probes.
3. Summary Information on specific genetic alterations in the human genome and their role in disease has accumulated over the past few years with the development of increasingly efficient molecular techniques. As a consequence there is an increasing need for genetic assay formats that permit analysis of large numbers of samples. One important trend in molecular genetics is the immobilization of large sets of probes in arrays, re-
Fig. 8. Minisequencing protocol: (i) binding of biotin-labeled PCR products to the support; (ii) denaturation of complementary strands; (iii) annealing of primers, (iv) extension with fluorescence-labeled dideoxynucleotides; and (v) analysis by separation in an automated DNA sequencer.
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ferred to as DNA chips. These types of devices are useful for the simultaneous analysis of multiple sequences in a single sample. In this review we describe a streptavidin-coated manifold support that is suitable to analyze large numbers of samples in parallel. Streptavidin-conjugated supports have proven very useful due to the stability of the biotin-streptavidin interaction, but as shown herein the manifold supports can also advantageously be coated with a variety of proteins or nucleic acids. Manifold supports could also be coated with a DNA-binding matrix, simplifying genomic DNA preparation (Parik et al. unpublished). Even larger numbers of individual samples could be handled simultaneously as assay plates with up to 864 wells are available. By reducing the size of the prongs not only may many more reactions be handled in parallel but also less reagents and sample material are required. It is important that the sample handling device is compatible with downstream analytical instruments. In conclusion, the important goal of parallel handling of large sets of samples may be met using streptavidin-conjugated manifold supports, as described herein. The supports allow the user to go seemlessly from sample preparation, over sequential molecular genetic reactions, to the loading of reaction products in analysis instruments such as automated gel readers or quantitative fluorometers. Excellent sample tracking is possible and hands-on time is minimized. A wide variety of assays can be adapted to a manifold format, and future developments may permit far greater numbers of samples to be processed, facilitating large-scale research and diagnostic applications.
Acknowledgements Our work reported herein has been supported by grants from the Beijer Foundation, the Swedish Research Council for Engineering Sciences and from the Swedish Cancer Society.
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References [1] Diamandis EP, Christopoulos TK. Clin Chem 1991;37:625–36. [2] Parik J, Kwiatkowski M, Lagerkvist A, Lagerstro¨m-Ferme´r M, Samiotaki M, Stewart J, Glad G, Mendel-Hartvig M, Landegren U. Anal Biochem 1993;211:144 – 50. [3] Hemmila¨ I, Dakubu S, Mukkala V-M, Siitari H, Lo¨vgren T. Anal Biochem 1984;137:335 – 43. [4] Chomczynski P, Sacchi N. Anal Biochem 1987;162:156–9. [5] Chirgwin J, Aeyble A, McDonald R, Rutter W. Biochemistry 1979;18:5294 – 99. [6] Livak KJ, Flood SJA, Marmaro J, Giusti W, Deetz K. PCR Methods Appl 1995;4:357 – 62. [7] Lagerstro¨m M, Parik J, Malmgren H, Strewart J, Petersson U, Landegren U. PCR Methods Appl 1991;1:111 – 9. [8] Lagerkvist A, Stewart J, Lagerstro¨m-Ferme´r M, Landegren U. Proc Natl Acad Sci USA 1994;91:2245 – 9. [9] Palmieri G, Romano G, Casamassimi A, D’Urso M, Little RD, Abidi FD, Schlessinger D, Lagerstro¨m M, Malmgren H, SteenBondeson ML, Petersson U, Landegren U. Genomics 1993;16:586 – 92. [10] Lagerstro¨m-Ferme´r M, Petersson U, Landegren U. Genomics 1993;17:89 – 92. [11] Sanger F, Nicklen S, Coulson AR. Proc Natl Acad Sci USA 1977;74:5463 – 7. [12] Maxam AM, Gilbert W. Proc Natl Acad Sci USA 1977;74:560–4. [13] Ansorge W, Sproat B, Stegeman J, Schwager CJ. Biochem Methods 1986;13:315 – 23. [14] Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB, Hood LE. Nature 1986;321:674–9. [15] Hultman T, Sta˚hl S, Hornes E, Uhle´n M. Nucl Acids Res 1989;17:4937 – 46. [16] Syva¨nen AC, Aalto-Setala K, Kontula K, So¨derlund H. FEBS Lett 1989;258:71 – 4. [17] Bergh J, Norberg T, Sjo¨gren S, Lindgren A, Holmberg L. Nature Med 1995;1:1029 – 34. [18] Waldenstro¨m E, Lagerkvist A, Dahlman T, Westermark K, Landegren U. Genomics 1996;37:303 – 9. [19] Samiotaki M, Kwiatkowski M, Parik J, Landegren U. Genomics 1994;20:238 – 42. [20] Landegren U, Kaiser R, Sanders J, Hood L. Science 1988;241:1077– 80. [21] Alves AM, Carr FJ. Nucl Acid Res 1988;16:8723 – 30. [22] Barany F. Proc Natl Acad Sci USA 1991;88:189 – 93. [23] Sokolov BP. Nucl Acid Res 1990;18:3671. [24] Syva¨nen AC, Aalto-Seta¨la¨ K, Harju L, Kontula K, So¨derlund H. Genomics 1990;12:590 – 5. [25] Pastinen T, Partanen J, Syva¨nen AC. Clin Chem 1996;42:1391–7. [26] Kwiatkowski M, Parik J, Landegren UJ. Immunol Methods 1994;168:137 – 43. [27] Samiotaki M, Kwiatkowski M, Ylitalo N, Landegren U. Anal Biochem 1997;253:156 – 61.
Molecular genetic applications of streptavidin-coated manifold supports Gisela Barbany a,b,1, Anette Hagberg a,2, Erik Waldenstro¨m a,b,1, Ulf Landegren a,1,* a
Department of Medical Genetics, Box 589, Uppsala Biomedical Centre, Uni6ersity of Uppsala, S-751 23 Uppsala, Sweden b Department of Internal Medicine, Uppsala Uni6ersity Hospital, Uppsala, Sweden
Abstract Practical problems of handling large numbers of samples limit the application of molecular genetic procedures in clinical settings and in research. In the present review we describe a multipronged manifold support, coated with streptavidin, that offers distinct advantages in preparative and diagnostic applications. In order to increase the surface available on the manifold, porous Sepharose particles conjugated with streptavidin were attached to the plastic support. This procedure increased the surface by almost three orders of magnitude, permitting sufficient streptavidin to be coupled to the support for most routine applications. The manifold supports have been used for sample preparation and in a number of genetic assays, including allele discrimination assays and DNA sequencing. In all these assay formats the manifold supports allow large numbers of samples to be processed in parallel. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Solid phase; Solid support; Genetic analysis; Streptavidin; mRNA isolation; Sequencing; Allele discrimination; Sample preparation; Capture PCR
1. Introduction Information about genetic alterations leading to disease is accumulating rapidly. As a consequence, more and more molecular genetic tests are becoming available to detect alterations associated with pathology. However, most of these genetic analyses are time-consuming and cumbersome to perform, limiting their applicability in clinical practice and in research. Strategies are sought to simplify protocols for genetic testing, in order to allow the analysis of large numbers of samples. Solid phase methods have proven valuable in immunoassays, and more recently also in molecular genetic procedures. Immobilization of DNA molecules to supports via the biotin – avidin or streptavidin link is * Corresponding author. Tel.: +46-18-4714910; fax: + 46-18526849. E-mail address: [email protected] (U. Landegren1) 1 Current address: Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden. 2 Current address: Department of Genetics and Pathology, Rudbeck Laboratory, Se-75185 Uppsala, Sweden.
particularly useful since the bond can be established rapidly and it remains stable under extreme physical conditions, including temperatures and pH that denature DNA molecules into single strands [1]. Different solid phases such as wells from microtiter plates, polystyrene and Sepharose beads, and paramagnetic particles have all been conjugated with streptavidin and used for affinity capture of biotinylated DNA [1]. We have developed a streptavidin-coated manifold support that offers particular advantages for processing sets of samples. Manifold supports, useful for molecular genetic reactions, are configured as a set of projections that fit individual wells of a reaction plate. The use of such supports to immobilize and manipulate nucleic acids can simplify many aspects of gene analysis. The supports make it possible to handle large number of samples simultaneously and they permit transfer of samples between consecutive reaction steps with minimal pipetting, thus reducing the risk of sample mix-up and saving considerable time. In the present review we describe the construction of manifold supports covered with streptavidin, and we exemplify several applications of the supports.
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1.1. Coupling of strepta6idin to manifold supports The prongs of manifold supports offer a limited area for binding streptavidin. A simple protocol was therefore designed to expand this surface area in order to increase the binding capacity of the manifolds. Streptavidin was first conjugated to porous Sepharose particles that were then attached to the manifold. The procedure has been described in detail elsewhere [2]. Briefly, Sepharose particles (HiTrap, NSH-activated HP Sepharose, Pharmacia Biotech) were washed in ice-cold 1 mM HCl, followed by a solution of 1 M NaCl and 0.4 M NaCO3 buffered to pH 8.3. The particles were then transferred to 5 ml of the above buffer, containing 10 mg of streptavidin. After 1 h incubation the particles were blocked in 0.1 M ethanolamine buffer for 15 min. The streptavidin-conjugated Sepharose particles were then washed in 0.1 M
acetate buffer pH 4.0 with 1 M NaCl, and either used immediately or stored in 0.05 M Tris–HCl pH 7.3 with 0.02% sodium azide. The Sepharose surface should not be allowed to dry during this process. Conjugated particles were filtered, washed with distilled water, dried with methanol, and then equilibrated with triethylamine, to obtain a slurry of about 50% (v/v) particles ready to be attached to the supports. We have been using commercially available polystyrene supports (Falcon, Nunc) as well as a cone-shaped prototype (Pharmacia Biotech) that fits the conical wells of microtiter plates used for PCR (Falcon, Costar). The supports were washed with ethanol for 20 min in an ultrasonic bath, and the particles were then grafted onto the tips of the support by submerging these in the slurry, followed by evaporation of triethylamine in air. Loosely bound particles were removed by rinsing the supports in distilled water. This procedure increases the surface area available for coupling by a factor of at least 800. In this manner large amounts of streptavidin are bound to the supports. Fig. 1 shows two scanning electron micrographs of the surface of the support at different magnifications, revealing the partially embedded particles. Variants of the described procedure can be used to conjugate a wide variety of molecules to supports via porous particles.
1.2. Binding capacity and binding kinetics of strepta6idin-coupled supports Fig. 1. Scanning electron micrographs at two different magnifications of the surface of a solid support with avidin-coated Sepharose particles attached. The scale bars represent 100 and 10 mm, respectively.
Fig. 2. Binding capacity of the support for biotin. The effect of increasing concentrations of biotin on the immobilization of a biotinylated reporter oligonucleotide was analyzed. The results are expressed in counts per second. The mean and standard deviation of triplicate samples are shown.
To investigate the binding capacity of the support, a europium-labeled biotinylated oligonucleotide was constructed by adding a series of europium chelates to the 3% end of a 5%-biotinylated oligonucleotide [3]. Binding to the support was measured by incubating individual prongs with 60 fmol of the europium- and biotin-labeled oligonucleotide with increasing amounts of free biotin in 1 M NaCl, 100 mM Tris–HCl pH 7.5 and 0.1% Triton X-100. The support was subsequently washed and incubated in a fluorescence enhancement solution (Wallac), whereupon europium ions were detected by time-resolved flourometry in a DELFIA plate reader fluorometer [3]. Fig. 2 shows a representative experiment, indicating that 12.5 pmol of free biotin were required to reduce the binding of the indicator oligonucleotide by one half. The biotin binding capacity of the manifolds with avidin-conjugated Sepharose particles has been compared to that of manifolds and two types of microtiter plates, passively coated with avidin. In all cases it was found that the biotin binding capacity of the passively coated surfaces was far lower than that of the supports with immobilized avidinmodified particles [2]. Experiments were performed to study the rate at which biotinylated oligonucleotides could be bound to
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Fig. 3. RNA isolation followed by reverse transcription and PCR: (i) configuration of a streptavidin-coupled Sepharose-coated support; (ii) hybridization to the RNA directly from the cell lysate; (iii) cDNA synthesis; and (iv) PCR amplification.
the support. A 10 min incubation on a shaking platform at room temperature was sufficient to immobilize most or all of the oligonucleotides added, and to saturate the binding capacity of the support [2].
2. Applications of streptavidin-coated manifold supports
2.1. mRNA isolation and cDNA amplification mRNA purification protocols involve many processing steps and frequently require the use of hazardous chemicals [4,5]. We have developed a method to specifically bind the RNA of interest directly from a cell lysate onto a manifold support, and to subsequently transfer the isolated sequences to reverse transcription reactions and PCR (Hagberg et al., unpublished). A
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biotinylated oligonucleotide, complementary to the mRNA of interest, is bound to the support via a streptavidin-biotin interaction and it is then allowed to hybridize to RNA from the cell lysate. Once hybridized to the support the RNA and subsequent reaction intermediates are conveniently transferred through the different enzymatic and washing steps with the aid of the manifold (Fig. 3). We have used this method to isolate and amplify by RT-PCR the bcr/abl fusion mRNA characteristic of chronic myelogenous leukemia (CML) (Barbany et al., unpublished). In these experiments 10 pmol of the biotinylated capture oligonucleotide, complementary to 40 bases of the abl mRNA, were incubated with individual prongs of the support in 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2.5 M NaCl. The cells were lysed in a buffer containing 500 mM LiCl, 1% LiDS 100 mM Tris–HCl pH 8.0, 10 mM EDTA, and 5 mM DTT. Lysates corresponding to 105 cells were diluted 1:10 in H2O and incubated together with the support. The supports were extensively washed before being transferred to a new set of wells, containing the first strand cDNA synthesis mixture. After synthesis was completed the manifold was washed once in 1×PCR buffer and then transferred to wells containing the appropriate amplification primers in standard PCR mixes. The support was removed from the microtiter plate after 2 cycles. The brc/abl mRNA was amplified with a nested set of primers. Fig. 4 shows the amplification products obtained from the cell line K562 (lane 1), which is positive for the fusion mRNA, and from three different CML patients, two of them positive (lanes 3 and 4) and one below detection limit (lane 2). The cell line BSM was used as a negative control (lane 5). By combining the described RT-PCR with a homogeneous detection reaction such as the Taqman assay [6] a simple and rapid quantitative detection of malignant cells is possible. Manifold supports can also be used with oligo-d(T) coupled to the prongs. In this manner total poly(A)+ RNA is isolated by hybridization to the manifold. The method greatly simplifies mRNA isolation and cDNA amplification from many samples, and it should be useful in mutation detection and gene expression studies.
2.2. Capture PCR
Fig. 4. Ethidium bromide-stained agarose gel showing the correct amplification product from K562 cells (lane 1), three different chronic myelogenous leukemia patients (lanes 2–4). The BSM cell line was included as a negative control (lane 5).
Capture PCR was developed as a method to amplify and isolate DNA fragments when sequence information is available from only one end of the fragment of interest [7]. With this method a DNA fragment can be exponentially amplified using sequence information from only one side of the amplified fragment, but with similar specificity as in a conventional PCR. In this procedure total genomic DNA is first restriction digested and a double stranded oligonucleotide linker is
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Fig. 5. Capture PCR: (i) digestion of genomic DNA; (ii) ligation of the linker to the restricted ends; (iii) extension from a 5%-biotinylated primer; (iv) immobilization on the streptavidin-coated support and (v) amplification with a second specific primer together with the linker oligonucleotide.
ligated to the restricted ends. After ligation primer extensions are performed using a biotinylated sequencespecific primer. Extended primers from many separate genomic samples and restriction digestions are individually captured onto the prongs of a streptavidin-coated manifold support and used as templates in a subsequent PCR. In this amplification reaction a second sequencespecific primer, hybridizing downstream of the first one, amplifies the fragment of interest together with the linker oligonucleotide, serving as a non-specific amplification primer (Fig. 5). By capturing the primer extension product on the support, the complexity of the DNA sample is greatly reduced before the amplification step. The identity of the amplification product can be
Fig. 6. Manifold-assisted sequencing: (i) binding of biotin-labeled PCR products to the support; (ii) denaturation of complementary strands; (iii) annealing of the fluorescein-labeled sequencing primer; (iv) extension reactions in the presence of dideoxynucleotides; and (v) transfer of reaction products to the gel using the manifold support. The sequencing reaction products are released from the supports by being denatured in a layer of formamide, added to the top of the gel.
confirmed using a third downstream amplification primer together with the linker primer. This inner PCR can also be used to prepare the amplification products for solid phase sequencing (see below) by using a biotinylated version of the linker for subsequent immobilization of the amplification products on a support. By further incorporating a 5%-sequence representing the M13 sequencing primer in the sequence-specific amplification primer, the amplicons can be analyzed by fluorescent sequencing using a standard sequencing primer [8]. For a given DNA sample, for example a source of human genomic DNA, restriction digestion with a particular enzyme and linker ligation need only be performed once. In this way a library of DNA fragments is created with the appropriate linker ligated to the restricted ends. This library can then be used with any primer set to generate a specific human DNA segment by capture PCR. In our laboratory this technique has been used to isolate ends of YAC clones [7,9] to determine the breakpoints of a 5-kb deletion in the amelogenin gene using sequence information near one of the breakpoints [10], to extend partial cDNA sequences, and to isolate homologs of a gene for a peptide hormone from more than 30 different species (Lagerstro¨m-Ferme´r unpublished results).
2.3. Sequencing Since the advent of DNA sequencing methods in the mid seventies [11,12] and automated, real-time detection of gel-separated products of sequencing reactions in the mid eighties [13,14], efforts have been made to simplify the handling of sequencing reactions. An important improvement consisted in the immobilisation of biotin-labeled PCR products to magnetic beads, coated with streptavidin for subsequent solid phase sequencing [15,16]. A further step was taken when Lagerkvist et al. constructed a manifold solid support consisting of flat plastic prongs grafted with Sepharose particles that were covalently coated with avidin or streptavidin [8]. The increased area allowed for binding sufficient biotinylated PCR products to perform sequencing reactions. The streptavidin-coated devices, shaped like sequencing combs, are suitable for handling many sequencing reactions in parallel, with little increase in effort over the handling of a single one; the samples are conveniently transferred through all processing steps (binding of biotinylated PCR products; denaturation; annealing of fluorescent primer; primer extension; and loading of samples onto the gel of an automated laser fluorescent DNA sequencer) (Fig. 6). Pipetting is greatly reduced compared to ordinary techniques and the process time is shorter.
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Fig. 7. Results from an oligonucleotide ligation assay. A group of 22 patients with known abnormalities of a1-antitrypsin were screened for the presence of the Z mutation using the ligase-based assay. The europium and terbium fluorescence readings from individual DNA samples are indicated as circles. Three clearly resolved clusters of data points can be distinguished corresponding to samples amplified from individuals homozygous for the normal sequence (open circles), individuals homozygous for the mutant sequence (filled circles) and from heterozygous individuals (shaded circles).
The solid phase sequencing method using plastic combs has been successfully applied for instance to detect mutations in the entire coding sequence of the p53 gene in breast cancer tumors from 316 patients [17]. We have used it to analyze the coding sequence for the copper transporting P-type ATPase ATP7B that is mutated in Wilson’s disease [18]. The 21 exons were amplified by PCR from genomic DNA with one biotinylated primer and one primer with the M13 universal primer sequence added as a 5%-tag. The PCR products were used as templates for solid phase sequencing with a handling time of just 1 h per set of 100 templates. Twenty-two families were included in the study and out of the expected total of 44 mutations, 40 were found among these patients. Reagents for the method are now commercially available as the AutoLoad Solid Phase Sequencing Kit from Pharmacia Biotech.
2.4. Oligonucleotide ligation assay The manifolds can be used in an oligonucleotide ligation assay (OLA) to detect sequence variations in the genome [19]. The method takes advantage of the inability of oligonucleotides to serve as substrates for enzymatic ligation, when the ends to be joined are mismatched to their target sequences [20 – 22]. By using a manifold support to perform the steps of the reaction, known mutations can be screened for in large numbers of samples. The supports used in this type of assay fit
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into the wells of a microtiter plate, making it possible to simultaneously process 96 DNA samples. After DNA amplification, the reactions are diluted with water and heat-denatured before a ligation mixture is added. This mixture contains T4 DNA ligase, buffer, ATP, and a set of 3 oligonucleotides that are designed based on the sequence surrounding the mutation site. Two of them differ at their 3% end depending on whether they are specific for the normal or the mutant sequence. Their 5% ends are differentially labeled with two lanthanide chelates (europium for the normal and terbium for the mutant sequence). The third oligonucleotide is 5%-phosphorylated and biotinylated at its 3% end, and it hybridizes immediately downstream of the lanthanide-labeled oligonucleotides. Once ligation is completed the products are trapped on the streptavidincoated multipronged support and following denaturing washes the two types of lanthanide ions are detected through time-resolved fluorometry. The OLA method has been used to analyze a group of patients with a1-antitrypsin deficiency and their relatives [19]. Fig. 7 shows the genotypes of 22 individuals that were analyzed for the presence of the Z mutation in the a1-antitrypsin gene, altering a single nucleotide position. Three clearly resolved point clusters are seen. Samples derived from individuals homozygous for the normal sequence result in strong europium signals and individuals homozygous for the mutant sequence exhibit strong terbium signals. Samples from heterozygous carriers support ligation of both europiumand terbium-labeled probes and in the diagram the symbols fall along a diagonal.
2.5. Minisequencing The minisequencing reaction, as oligonucleotide ligation, allows detection of single nucleotide variations in amplified DNA fragments [23,24]. The method makes use of a DNA polymerase to specifically extend the 3% end of a primer that anneals immediately upstream of a variable nucleotide position. This results in the incorporation of a labeled nucleotide, complementary to the template at the variable site. The identity of the nucleotide serves as an indicator of the nucleotide present at the variable site of the template [24]. In combination with streptavidin-coated sequencing combs the minisequencing method has been used to detect multiple polymorphic nucleotide positions in a single reaction. Following PCR amplification, the biotinylated products are captured on manifold supports and the strands denatured. This step efficiently removes nucleotide triphosphates and amplification primers before the minisequencing reaction, and renders the template strands accessible for hybridization to the sequencing primers. The minisequencing reactions are carried out by immersing the immobilized templates in 4 different
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wells, each containing a set of extension primers, a DNA polymerase, one fluorescein-labeled dideoxynucleotide triphosphate and the corresponding three other unlabeled dideoxynucleotides. After the extension reaction the manifolds are used to load the samples on an automated DNA sequencer (ALFexpress, Pharmacia Biotech). The detection primers are designed to have distinct lengths by adding a nucleotide tail at their 5% ends. In this way the length of the detection primer defines the site of variation and the incorporated nucleotide reveals the polymorphic nucleotide (Fig. 8). The results from the gel electrophoresis are interpreted with the aid of the ALF Fragment Manager software (Pharmacia Biotech). This method has been successfully applied to type HLA-DQA1 and HLA DRB1 genes [25]. The PCR amplification primers were designed to amplify all alleles equally in the DQA1 and DRB1 loci. Nine different polymorphic nucleotide positions were studied simultaneously by using primers that differed in size by increments of three bases. This technique makes it feasible to analyze multiple polymorphic nucleotides simultaneously in large numbers of samples.
2.6. Multicolor detection with lanthanides In addition to the molecular genetic tests mentioned, the manifolds have also been successfully used in an immunological assay. An avidin-coated manifold support was used to detect specific IgE in allergic individuals [26]. The allergen, in this case soy protein extract, was biotinylated and subsequently bound to the avidincoupled support. Manifolds with immobilized antigen were incubated in serum samples, and subsequently with a Fab% fragment of a mouse monoclonal antibody conjugated with europium chelates. The fluorescence from the europium ions was recorded in time-resolved mode [3]. Attaching the antigen directly to the manifold support resulted in higher sensitivity compared to the use of antigen immobilized via an avidin-biotin interaction. The assay was compared to the Pharmacia CAP system for specific IgE detection and it was concluded
that the binding characteristics of the manifolds and the cellulose matrix were quite similar. In time-resolved detection of lanthanides, several colors are available, permitting the simultaneous analysis of different substances in the same sample, e.g. different antibody classes with similar specificities or different nucleic acid sequences. Samiotaki et al. described a method to simultaneously analyze amplification products for the presence of any of seven different DNA sequences using a manifold support [27]. The multicolor-typing system was applied to the identification of human papilloma virus (HPV) types. PCR primers were designed to anneal to conserved regions in the genome. Biotinylated amplification products were immobilized on streptavidin-conjugated manifold supports, and after denaturing washes a mixture of seven different HPV type-specific probes were allowed to hybridize to the bound sequence. These seven type-specific oligonucleotides had been labeled with of 1, 2 or 3 of the lanthanide ions europium, terbium and samarium. The predefined combinations exhibit distinct fluorescence that can be detected by time-resolved fluorometry. An additional feature of the method is that probes can be removed from the bound targets by denaturing washes, followed by rehybridization of the sample with a new set of seven different oligonucleotides. This procedure can be repeated at least twice without significant loss of signal intensity, allowing hybridization with a total of 21 probes.
3. Summary Information on specific genetic alterations in the human genome and their role in disease has accumulated over the past few years with the development of increasingly efficient molecular techniques. As a consequence there is an increasing need for genetic assay formats that permit analysis of large numbers of samples. One important trend in molecular genetics is the immobilization of large sets of probes in arrays, re-
Fig. 8. Minisequencing protocol: (i) binding of biotin-labeled PCR products to the support; (ii) denaturation of complementary strands; (iii) annealing of primers, (iv) extension with fluorescence-labeled dideoxynucleotides; and (v) analysis by separation in an automated DNA sequencer.
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ferred to as DNA chips. These types of devices are useful for the simultaneous analysis of multiple sequences in a single sample. In this review we describe a streptavidin-coated manifold support that is suitable to analyze large numbers of samples in parallel. Streptavidin-conjugated supports have proven very useful due to the stability of the biotin-streptavidin interaction, but as shown herein the manifold supports can also advantageously be coated with a variety of proteins or nucleic acids. Manifold supports could also be coated with a DNA-binding matrix, simplifying genomic DNA preparation (Parik et al. unpublished). Even larger numbers of individual samples could be handled simultaneously as assay plates with up to 864 wells are available. By reducing the size of the prongs not only may many more reactions be handled in parallel but also less reagents and sample material are required. It is important that the sample handling device is compatible with downstream analytical instruments. In conclusion, the important goal of parallel handling of large sets of samples may be met using streptavidin-conjugated manifold supports, as described herein. The supports allow the user to go seemlessly from sample preparation, over sequential molecular genetic reactions, to the loading of reaction products in analysis instruments such as automated gel readers or quantitative fluorometers. Excellent sample tracking is possible and hands-on time is minimized. A wide variety of assays can be adapted to a manifold format, and future developments may permit far greater numbers of samples to be processed, facilitating large-scale research and diagnostic applications.
Acknowledgements Our work reported herein has been supported by grants from the Beijer Foundation, the Swedish Research Council for Engineering Sciences and from the Swedish Cancer Society.
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