WO1998018961A1 - Procedes de preparation d'ensembles d'adn monocatenaires - Google Patents

Procedes de preparation d'ensembles d'adn monocatenaires Download PDF

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WO1998018961A1
WO1998018961A1 PCT/GB1997/002961 GB9702961W WO9818961A1 WO 1998018961 A1 WO1998018961 A1 WO 1998018961A1 GB 9702961 W GB9702961 W GB 9702961W WO 9818961 A1 WO9818961 A1 WO 9818961A1
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dna
strand
array
solid support
probe
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PCT/GB1997/002961
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English (en)
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Andrew David Charles
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Zeneca Limited
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Priority to EP97910521A priority Critical patent/EP0935671A1/fr
Priority to JP10520193A priority patent/JP2001502909A/ja
Publication of WO1998018961A1 publication Critical patent/WO1998018961A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • This invention relates to methods for preparing arrays of nucleic acids for use in biological screening procedures such as hybridisation assays, with applications in genetic research and diagnostic applications.
  • arrays of immobilised nucleic acids particularly arrays of DNA
  • arrays consist of a plurality of DNAs organised as a two-dimensional matrix immobilised on an appropriate solid support. Each point in the matrix comprises a DNA element.
  • Each of the DNA elements can be used as a probe to detect complementary sequences in complex mixtures of nucleic acid. This allows parallel determination of the identity and abundance of many DNA species in a single experiment.
  • Such arrays can be formed on porous membranes such as nitrocellulose using a variety of methods.
  • a plurality of DNA samples is transferred to membranes by placing the samples into a manifold consisting of an array of preformed wells applied to the top of the membrane, and drawing the DNA through the membrane using a vacuum.
  • DNA is applied directly to the membrane using an array of pins to transfer DNA onto the membrane surface from DNA samples contained, for example, in the wells of a microtitre plate [Lehrach, H. et ah, "Hybridisation Fingerprinting in Genome Mapping and Sequencing" in "Genome Analysis", Vol. 1, Davies, K. E. and Tilghman S. M. (Eds.), Cold Spring Harbor Laboratory Press, New York, 1990, pp38-82; Nizetic, D. et al., Proceedings of the National Academy of Sciences (USA), 1991, 88, 3233-3237].
  • DNA arrays can be formed on non-porous surfaces such as glass, by either in situ synthesis or direct application.
  • arrays of oligodeoxynucleotides can be assembled by starting with a chemically sensitised glass surface which is protected by a mask, and reacting selected exposed areas with suitably modified nucleotides.
  • masks and nucleotide reagents arrays of synthetic oligodeoxynucleotides of defined sequence can be elaborated at the glass surface [see e.g., Jacobs, J. W. and Fodor, S. P. A., Trends in Biotechnology, 1994, 12, 19-26; Fodor, S. P. A. et al., International Patent Application No.
  • arrays of longer DNA species may be constructed by using robotic micropipetting devices to transfer small, typically nanolitre or smaller quantities of DNA from containers such as 96-well plates to ordered pre-determined positions on a non- porous surface such as a glass microscope slide Each DNA sample is bound at a known position on the microscope slide to constitute one DNA element of the array.
  • robotic micropipetting devices to transfer small, typically nanolitre or smaller quantities of DNA from containers such as 96-well plates to ordered pre-determined positions on a non- porous surface such as a glass microscope slide
  • Each DNA sample is bound at a known position on the microscope slide to constitute one DNA element of the array.
  • a large number of replica slides can be constructed supporting arrays of thousands of individual DNA elements [Schena M. et al., Science, 1995, 270, 467-470; Shalon, T. D. and Brown, P. O., International Patent Application No. WO 95/35505, published 28 December 1995].
  • the DNA samples being transferred to the solid support are typically double-stranded polynucleotide DNA fragments of length greater than 50bp.
  • These DNAs may be obtained from a number of sources, such as cDNA or genomic DNA libraries, and may be of either known or unknown sequence composition.
  • DNA may be coupled to the solid support by a number of techniques.
  • the DNA may be bound to glass through non-covalent electrostatic interactions with a coating film of a polycationic polymer such as poly-L-lysine [see e.g. Shalon, T. D. and Brown, P. O., International Patent Application No. WO 95/35505, published 28 December 1995].
  • a polycationic polymer such as poly-L-lysine
  • DNA can be bound covalently to the solid support.
  • Arrays of polynucleotide DNA probes immobilised on solid supports can be used to study the composition of complex mixtures of DNA using hybridisation techniques.
  • a complex mixture of labelled cDNA is hybridised to the DNA array under conditions of appropriate stringency, and unbound material is washed away.
  • the array is then scanned using a detection method capable of sensing the remaining bound labelled cDNA, such as a scanning fluorescent microscope.
  • the intensity of the detected signal at any given element in the array is a measure of the concentration of the corresponding complementary cDNA in the original complex mixture [Schena M. et al., Science, 1995, 270, 467-470; Shalon, T. D. and Brown, P. O., International Patent Application No. WO 95/35505, published 28 December 1995; Pinkel, D. et al, International Patent Application No. WO 96/17958, published 13th June 1996].
  • Arrays of immobilised oligonucleotides have been described which use elements containing selected sense, antisense, missense or nonsense sequences at different positions in the array. Such arrays have been used for a number of applications; for example to determine the sequence of DNA [see e.g., Mirzabekov, A. D., Trends in Biotechnology, 1994, 12, 27-32 and references therein; Fodor, S. P. A. et al., International Patent Application No. WO
  • arrays of allele-specific oligonucleotides have been used to detect genetic polymorphisms and determine genotypes [see e.g., Southern, E., European Patent No. 0373203B1, published 31 August 1994; Guo, Z. et al., Nucleic Acids Research, 1994, 22, 5456-546].
  • arrays have limitations when used to probe complex mixtures of labelled polynucleotide DNA targets such as cDNAs, since any given oligonucleotide may hybridise at a particular stringency to sequences in more than one target DNA.
  • arrays of longer non-oligonucleotide probe DNAs provide a much higher specificity for hybridisation to target DNAs.
  • all such arrays to date have incorporated a mixture of both sense and antisense strands of a particular DNA fragment within each DNA element in the array. In cases where it is desired to detect both strands of the corresponding target DNA hybridising to a given DNA element, this is not a problem.
  • direct detection of a first-strand labelled cDNA target requires only a sense strand DNA probe.
  • the presence of the unwanted sense or antisense strand of the probe within each DNA element will reduce signal sensitivity by reducing the number of probe sites available for target hybridisation. It may also increase background signals by hybridising to non-specific target DNAs.
  • a method for preparing an array of single-stranded DNA immobilised on a solid support comprises (i) providing samples of double-stranded DNA chemically modified on the sense or antisense strand for attachment to the solid support, and (ii) linking the DNAs to the solid support and, before or after step (ii), removing the non-modified strand whereby an array of single-stranded DNA is provided on the solid support.
  • a second chemical modification is provided on the strand that is not to be bound to the solid support.
  • the purpose of this second chemical modification is to assist in either the separation of the two strands or the selective degradation of the unwanted strand.
  • the single-stranded DNA preferably comprises DNA molecules containing more than 75 nucleotides such as more than 100 nucleotides or more than 200 nucleotides. Preferred ranges of nucleotides include 100-10,000; 200-10,000 and 300-10,000.
  • the samples of double-stranded DNA chemically modified on the sense and/or antisense strand are conveniently provided by extension of chemically modified primer(s).
  • primer(s) are preferably used as polymerase chain reaction (PCR) primers, whereby the desired chemical modification(s) are selectively incorporated into the sense and/or antisense strands of the double-stranded DNA.
  • the primer(s) may be modified at any convenient position(s). Modif ⁇ cation(s) are preferably made to the 5' nucleotide of the primer at either the 5' phosphate, the 5' deoxyribose group or the 5' base (adenine, guanine, thymidine or cytosine).
  • the modification involves the addition of a chemical functionality for binding to the solid surface, together with an optional spacer group of appropriate length to improve the accessibility of the probe DNA to the target nucleic acid. Both covalent and non-covalent binding may be used.
  • the chemical functionality may direct non-covalent binding to the solid surface, for example a biotin moiety which will interact with a streptavidin coating on the solid surface.
  • the chemical functionality may covalently link the selected DNA strand to the solid surface.
  • Chemical modification of the DNA may be performed in one or more steps.
  • each DNA probe may be separated either prior to or after arraying onto the solid support.
  • the separation may involve physical denaturation of the probes using for example heat or alkali, or the enzymic degradation of the unwanted strand for example using an appropriate exonuclease, or a combination of both methods.
  • each probe comprising at least 75 nucleotides and immobilised on a solid support. More conveniently each probe comprises at least 100 or 200 nucleotides, such as at least 500 or 1 ,000 nucleotides. A particular range is 300-10,000 nucleotides. A particular advantage of such an array is that the sequence of the probe DNAs may be unknown.
  • Each probe DNA may be the sense or antisense strand for a given gene sequence.
  • every probe DNA in the array is antisense strand DNA or every probe DNA in the array is sense strand DNA.
  • the array conveniently comprises at least 10 DNA elements, such as at least 100 elements. Further convenient arrays comprise at least 1,000, 10,000 or 100,000 DNA elements.
  • the invention also provides a method whereby such selected single-strand arrays can be used to provide a quantitative estimate of the abundance of individual mRNAs or their corresponding first strand cDNAs within a complex mixture of such derived from a biological sample comprising a single cell type or a mixed population of cell types.
  • the abundance is determined by measuring the amount of hybridisation between single-strand probe DNA at each element in the array and its complementary strand within the complex mixture of mRNA or cDNA.
  • a label is incorporated into the mRNA or cDNA molecules in the complex mixture, for example a fluorescent nucleotide.
  • mRNAs are isolated from cells and either directly labelled in vitro or, in a preferred embodiment, converted into first strand cDNAs, in which case the label is introduced on modified nucleotide which is incorporated into the single-strand cDNAs by reverse transcriptase.
  • the abundance of any given cDNA species within the population of single-strand cDNAs generated by reverse transcriptase is taken to represent the abundance of the corresponding mRNA within the biological sample.
  • the labelled cDNAs are hybridised to selected single-strand arrays which contain pairs of elements in which either the sense or antisense strand of each of the polynucleotide probes is immobilised at each element.
  • the amount of immobilised DNA present at each element in the array is controlled such that it is considerably greater than the amount of the corresponding target mRNA or cDNA within the sample applied to the array. Under such conditions, the amount of labelled target nucleic acid (mRNA or cDNA) that remains bound to each element under the hybridisation conditions employed will represent the concentration of each mRNA or cDNA in the original sample.
  • the bound target nucleic acid can be determined using an appropriate detection system capable of measuring the label carried on the target nucleic acid; e.g., a scanning fluorescence microscope [see e.g., Schena M. et al., Science, 1995, 270, 467-470].
  • the abundance of a particular cDNA may be quantified by comparing the intensity of the specific hybridisation signal, such as fluorescence intensity, at a given sense element to the non-specific hybridisation determined by the signal obtained at its corresponding antisense element. In this way, a precise quantitation of the absolute abundance of multiple cDNAs can be obtained within a single experiment.
  • Such single-strand arrays can be readily used to quantify the abundance of single- strand nucleic acid species such as mRNAs or their corresponding first-strand cDNAs in a variety of cell types or populations.
  • the abundance information thus obtained can be used to draw up a quantitative transcript profile describing the expression of a large number of genes within any given cell type or cell population. This information can be used to determine for example which genes are differentially expressed in diseased versus normal tissue, or treated versus untreated tissue, and hence provide valuable information in diagnosing and monitoring disease processes, and in research to identify new treatments to restore the healthy state.
  • animal As used herein, the term "animal" is used in its broadest sense to include all members of the animal kingdom.
  • biological sample encompasses any cell or tissue in any state from any organism which may be selected to provide a source of target nucleic acids.
  • disease or “diseased state” refer to any condition which deviates from the normal or standardised healthy state in an organism of the same species in terms of differential expression of the organism's genes.
  • a disease state can be any illness or disorder of genetic or environmental origin which is characterised or may be described by the expression of genes which are either (i) normally silent in the healthy organism but activated in the diseased state as a cause of or in response to the disease, or (ii) normally expressed within some standard range in the healthy organism but over- or under-expressed in the diseased state as a cause of or in response to the disease.
  • the terms "element” or “DNA element” refer to a number of immobilised DNA molecules, which may be either single-stranded or double-stranded, bound to a solid support at a specific physical location which defines one point within a 2- dimensional matrix constructed from a plurality of such elements.
  • EST or Expressed Sequence Tag refers to a partial DNA or cDNA sequence, typically of between 50 and 500 sequential nucleotides, obtained from a genomie or cDNA library prepared from a selected cell, cell type, tissue or tissue type, organ or organism which longer sequence corresponds to an mRNA of a gene found in that library [cf. Adams, M. D. et al., Science, 1991, 252. 1651-1656 and International Application No. PCT/US92/05222, published 7 January 1993].
  • An EST is generally DNA.
  • the term "gene” refers to the genomie nucleotide sequence from which a cDNA sequence is derived.
  • immobilised refers to the attachment of probe DNA to a solid support.
  • the attachment may be of a covalent or non-covalent nature and will depend on the nature of the solid support being used.
  • the term "insert” refers to any DNA sequence incorporated within a vector using methods of molecular biology available to anyone ordinarily skilled in the art.
  • oligonucleotide refers to a molecule containing up to 50 nucleotides, but more typically 20 nucleotides of either DNA or RNA.
  • organism includes without limitation, microbes, plants and animals.
  • probe means a DNA species immobilised to a solid support within a DNA element.
  • solid support refers to any known substrate which is useful for the immobilisation of probe DNA by any available method to enable detectable hybridisation of the immobilised oligonucleotides or polynucleotide DNA sequences to other polynucleotide sequences in a sample.
  • useful solid supports include, but are not limited to, paper, nitrocellulose, myelin, glass, silica, nylon, plastics such as polyethylene, polypropylene or polystyrene, or other solid material.
  • solid support can refer to gels constructed from such materials as agarose, polyacrylamide, polysaccharide or proteins, which may themselves be overlaid on a further solid surface such as glass or metal, to provide mechanical strength, electrical conductivity or other desired physical property.
  • solid support is porous, the term “solid support” refers without distinction to a range of pore sizes, depending upon the nature of the system.
  • the term "surface” means any generally two-dimensional structure on a solid support to which the desired probe DNA is attached or immobilised.
  • the term “target” refers to any complex mixture of nucleic acid or any individual component thereof which can be labelled such as to permit its detection by anyone ordinarily skilled in the art.
  • vector means a DNA sequence capable of maintenance and replication within a host organism.
  • vector includes, but is not limited to, plasmids such at pBluescript (Stratagene Inc., La Jolla, CA) or bacteriophages such as Lambda UniZAP (Stratagene).
  • the DNA used to generate probes for subsequent arraying may be obtained from a large number of sources.
  • DNA fragments may be obtained from a random selection of clones from a DNA library prepared from the organism of interest. In the case of animals such as man or rodents, these clones would preferably be obtained from one or more cDNA libraries.
  • the fragments may also be selected from collections of clones which have been characterised to some extent, for example by partial sequence analysis of the insert DNA or by mapping of the insert DNA to particular chromosomal loci.
  • Such clones may include, but are not limited to the I.M.A.G.E Consortium collection of clones isolated from human or rodent cDNA libraries and characterised by the generation of one or more ESTs for each clone [Lennon, G. et al., Genomics, 1996, 33, 151-152]. In the case of probes derived from bacterial genes, genomie DNA libraries may also be used.
  • Each clone consists of a polynucleotide DNA fragment inserted at a known site within a suitable vector.
  • the vector may for example be a plasmid vector such as pBluescript (Stratagene) or a bacteriophage vector such as Lambda UniZAP (Stratagene).
  • Individual bacterial clones from selected DNA libraries are cultured in the appropriate liquid medium using standard techniques. A small sample of each culture is used as a source of template DNA for subsequent amplification by PCR, using oligonucleotide primers complementary to the vector sequences immediately flanking the insert DNA sequence. In this way it is not necessary to know the sequence of the polynucleotide DNA fragment comprising the insert in order to practise the invention.
  • the primer used to direct DNA-polymerase dependent synthesis of the selected strand contains a first chemical modification which will be used to couple that strand to the solid support.
  • a first chemical modification which will be used to couple that strand to the solid support.
  • the chemical modification will be incorporated into the 5' nucleotide of the primer at either the 5' phosphate, the 5' deoxyribose group or the 5' base (adenine, guanine, thymidine or cytosine) during synthesis of the oligonucleotide.
  • the modification may also be made at other positions within the 5' primer sequence.
  • the modification comprises a chemical functionality for binding to the solid surface, together with a spacer group of appropriate length to improve the accessibility of the probe to the target nucleic acid [see e.g., Maskos, U. and Southern, E. M., Nucleic Acids Research. 1992, 20, 1679-1684 for a discussion of factors influencing linker design].
  • the chemical functionality may direct non-covalent binding to the solid surface, for example a biotin moiety which will interact with a streptavidin coating on the solid surface.
  • the chemical functionality may covalently couple the selected DNA strand to the solid surface.
  • the spacer group may be, for example, a long-chain hydrocarbon of general formula -(CX 2 )n- where X may be H or F and n is generally 6-20.
  • the primer used to direct the DNA-polymerase dependent synthesis of the sense strand contains a first chemical modification which will be used to couple that strand to the solid support.
  • the corresponding antisense strand primer is either unmodified, or contains a second chemical modification, different from that used in the sense primer.
  • the modification carried on the antisense strand is designed to facilitate subsequent strand separation, as described below.
  • the antisense strand primer could contain either a 5' phosphate or a 5' biotinylated nucleotide derivative.
  • the primer used to direct the DNA- 5 polymerase dependent synthesis of the antisense strand contains a first chemical modification which will be used to couple that strand to the solid support.
  • the corresponding sense strand primer is either unmodified, or contains a second chemical modification different from that used in the antisense primer.
  • the modification carried on the sense strand is designed to facilitate subsequent strand separation, 0 as described below.
  • the antisense strand primer contains a 5' 6- aminohexyl-phosphodiester group
  • the sense strand primer could contain either a 5' phosphate or a 5' biotinylated nucleotide derivative.
  • PCR reactions are carried out on DNA obtained from individual clones to obtain the desired number of 5' modified polynucleotide DNA fragments that are to be used as probes in
  • the PCR products prepared using modified sense or antisense primers may be separated into sense and antisense strands in two ways.
  • the two strands are separated prior to arraying onto the solid support.
  • One desirable method to achieve this is to generate PCR products in which the unwanted strand contains one or more biotinylated nucleotides at the 5' end [Guo, Z. et al., Nucleic Acids Research. 1994, 22, 5456-
  • the PCR product is bound to streptavidin-coated agarose beads which may be washed to remove other reagents such as salts, primers and free nucleotides, and the two strands then separated by treatment with 0.1N NaOH for 10 minutes.
  • the beads containing the bound unwanted strand are removed by centrifugation and the supernatant containing the desired non-biotinylated strand is decanted and neutralised to pH7.0 with HC1.
  • This strand is then arrayed and bound to the solid support through the functionality it carries at its 5' end; for example a 5' aminohexyl-phosphodiester group which will couple to glass activated with 1,4- phenylene di-isothiocyanate (DITC).
  • DITC 1,4- phenylene di-isothiocyanate
  • the double-stranded PCR product is arrayed first and the chosen strand coupled to the solid support using the desired chemistry incorporated into the appropriate PCR primer.
  • a strand containing a 5' aminohexyl-phosphodiester group can be coupled to DITC-activated glass.
  • the unwanted strand will be unable to couple to the solid support because it has been generated using a PCR primer which lacks a 5 ' amino group.
  • the arrayed double-stranded probe is then denatured, for example using a bath containing 0.1N NaOH, and the unwanted strand washed off.
  • the solid support is then placed in a neutralising bath at pH7.0 to generate the selected strand array.
  • the double-stranded PCR product is arrayed first and the chosen strand linked to the solid support using the desired chemistry incorporated into the PCR primer for that strand.
  • the unwanted strand is synthesised using a PCR primer which carries an unmodified 5' phosphate group.
  • This strand is then enzymically degraded using a 5' -3' exonuclease, for example lambda exonuclease which cannot attack 5' ends unless they carry a 5' terminal phosphate group ["Current Protocols in Molecular Biology", Ausubel, F. M. et al. (Eds.), Green/Wiley, New York, 1995, ppl5.2.5].
  • the unwanted strand may be removed by a combination of enzymic degradation followed by alkaline denaturation, washing and neutralisation. This combination is particularly effective for probes derived from polynucleotide DNA fragments of lengths approaching lOkb.
  • Covalently coupling the probe DNA to the solid support at each elemental position through a 5' chemical linker has several advantages. It ensures a robust linkage of DNA to the solid surface which will be resistant to chemical degradation during storage and subsequent procedures, with consequent loss of signal. Importantly, it also provides the maximal amount of single-strand probe DNA which is free to hybridise to target DNA sequences. This is an important advantage over methods that rely on non-specific electrostatic interactions, such as the binding of probe DNA to poly-L-lysine coated slides, where portions of the DNA probe are complexed with the poly-L-lysine and therefore not available for hybridisation to target DNA.
  • the quantity of single-strand DNA that is arrayed at each element in the array and is free to hybridise to target nucleic acid will vary according to the nature of the solid surface and the chemistry used to link the probes to the solid surface. However it will be present in sufficient quantity to ensure that it is always in excess relative to the concentration of its corresponding labelled target nucleic acid in the sample to be analysed. In this way, the intensity of the resultant hybridisation signal will be proportional to the amount of target nucleic acid present in the biological sample.
  • sense strand arrays which comprise a plurality of DNA elements comprising sense strands immobilised on a single solid surface, where the strands in each element are derived from a different polynucleotide DNA fragments and are prepared according to the method described above.
  • antisense strand arrays which comprise a plurality of DNA elements comprising antisense strands immobilised on a single solid surface, where the strands in each element are derived from a different polynucleotide DNA fragment and are prepared according to the method described above
  • mixed arrays can be constructed containing pairs of elements comprising either the sense or antisense strand of a given DNA.
  • the pairs of elements do not necessarily have to be arrayed side-by-side within the array.
  • the precise disposition of the two types of element, either within the same array or on different arrays will depend on the precise application for which they are intended.
  • Selected single-strand arrays generated as described above may be hybridised to a sample containing a plurality of single-strand target nucleic acids, either mRNAs or preferably, first strand cDNAs that have been isolated from a chosen biological sample and labelled by any of the techniques known to one ordinarily skilled in the art, such as radiolabelling, fluorescent labelling or ehemiluminescent labelling [see e.g., Schena M. et al., Science, 1995, 270, 467-470].
  • specific hybridisation is measured using an array of elements comprising antisense single-strand probes, and nonspecific hybridisation is measured using an array of the corresponding sense single-strand probes.
  • Hybridisation conditions at the solid support will depend on the nature of the support and the arrayed DNA, but may be defined and optimised using a number of methodologies available to one ordinarily skilled in the art [see e.g., Sambrook, J. et al., "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989].
  • hybridisation takes place under stringent conditions, i.e. those which reveal nucleic acid identities of greater than 95%. However, if desired, other less stringent hybridisation conditions may be selected.
  • Hybridisation of a particular nucleic acid species is detected by measuring the strength of the signal from the labelled target nucleic acid that remains bound its cognate element in the array after washing the array at the particular stringency chosen for the application.
  • the absolute abundance of a particular single-strand nucleic acid species (be it mRNA or first strand cDNA) in a plurality of nucleic acids may be determined by subtracting the signal at the element in the array corresponding to the non-specific hybridisation from the signal at the element in the array affording the specific hybridisation signal for that particular nucleic acid species.
  • a particular mRNA is altered in some condition, for example a diseased state compared to the normal state
  • identical arrays are hybridised to labelled samples of target nucleic acids isolated from the diseased and normal biological samples. Differences in the measured abundance can be used to indicate which genes may be involved in the cause, maintenance or progression of the chosen diseased state.
  • the same approach can be used to follow the effects of drug treatment or other investigation of or manipulation of a set of cells or an organism on the expression levels of the genes within the biological sample.
  • Array elements were selected from a set of clones isolated from a human liver cDNA library containing cDNA inserts cloned unidirectionally into a pBluescript vector (Stratagene) between the EcoRI and Xhol sites, such that the 3' end of the insert DNA, including the 5 polyA tail, is located immediately adjacent to the Xhol site.
  • the library was maintained in E. coli strain SOLRTM (Stratagene). The average insert size was 1.5kb. Randomly selected clones were transferred to a 96-deep well microtitre plate and grown in L-broth supplemented with 100 micrograms/ml ampicillin.
  • the sense primer complementary to pBluescript sequences 5' to the EcoRI site was synthesised with a 6-aminohexyl-phosphodiester at its 5' end.
  • the antisense primer complementary to pBluescript sequences 3' to the Xhol site was biotinylated at its 5' end according to standard procedures [Agrawal, S. et al., Nucleic Acids Research, 1986, 14, 6227-
  • PCR reactions with the modified primers were performed directly on small volumes (typically ⁇ 1 microlitre of overnight culture) of the bacterial cultures in a 96-well thermocycler in final reaction volume of 70 microlitres.
  • Each of the PCR products was purified using a QIAquickTM PCR purification kit (Qiagen Inc., Chatsworth, CA).
  • the PCR reactions were carried out as described above using an antisense primer with a 5' 6-aminohexyl-phosphodiester group and a
  • biotinylated sense primer 25 biotinylated sense primer.
  • the resulting products were purified and strand-separated as described above.
  • Glass slides containing arrays of paired elements comprising sense and antisense probes are hybridised with first-strand cDNA prepared by reverse transcription of polyA mRNA isolated from HepG2 cells and labelled with the fluorescent nucleotide analogue dCTP-Cy5 (Amersham International, Chalfont, UK) essentially as described by Schena et al.
  • the labelled cDNA (5 micrograms in 7.5 microlitres) is denatured at 95°C. 2.5 microlitres of concentrated hybridisation solution (5 x SSC, 0.1%

Abstract

L'invention concerne un procédé de préparation d'un ensemble d'ADN monocaténaires, immobilisés sur un support solide. Ledit procédé consiste (i) à fournir des échantillons d'ADN bicaténaires, qui sont chimiquement modifiés sur le brin sens ou le brin antisens afin d'être fixés sur ledit support solide, puis (ii) à lier les ADN audit support solide et, avant ou après l'étape (ii), à extraire le brin non modifié, un ensemble d'ADN monocaténaires étant fourni sur ledit support solide.
PCT/GB1997/002961 1996-10-31 1997-10-28 Procedes de preparation d'ensembles d'adn monocatenaires WO1998018961A1 (fr)

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EP97910521A EP0935671A1 (fr) 1996-10-31 1997-10-28 Procedes de preparation d'ensembles d'adn monocatenaires
JP10520193A JP2001502909A (ja) 1996-10-31 1997-10-28 一本鎖ｄｎａアレイの調製方法

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6077674A (en) * 1999-10-27 2000-06-20 Agilent Technologies Inc. Method of producing oligonucleotide arrays with features of high purity
WO2003012124A2 (fr) * 2001-07-31 2003-02-13 New York Institute Of Technology Methodes pour immobiliser des molecules sur une phase solide et leurs utilisations
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US7563573B2 (en) 1999-02-26 2009-07-21 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Method for detecting radiation exposure
US6077674A (en) * 1999-10-27 2000-06-20 Agilent Technologies Inc. Method of producing oligonucleotide arrays with features of high purity
JP2003515112A (ja) * 1999-11-15 2003-04-22 クロンテック・ラボラトリーズ・インコーポレーテッド 長鎖オリゴヌクレオチドアレイ
WO2003012124A2 (fr) * 2001-07-31 2003-02-13 New York Institute Of Technology Methodes pour immobiliser des molecules sur une phase solide et leurs utilisations
WO2003012124A3 (fr) * 2001-07-31 2003-11-06 New York Inst Techn Methodes pour immobiliser des molecules sur une phase solide et leurs utilisations
US10876971B2 (en) 2010-10-29 2020-12-29 President And Fellows Of Harvard College Nucleic acid nanostructure barcode probes
US10024796B2 (en) 2010-10-29 2018-07-17 President And Fellows Of Harvard College Nucleic acid nanostructure barcode probes
WO2015017586A1 (fr) * 2013-07-30 2015-02-05 President And Fellows Of Harvard College Imagerie à très haute résolution et imagerie reposant sur l'adn quantitatif
US11536715B2 (en) 2013-07-30 2022-12-27 President And Fellows Of Harvard College Quantitative DNA-based imaging and super-resolution imaging
US11198900B2 (en) 2014-12-06 2021-12-14 Children's Medical Center Corporation Nucleic acid-based linkers for detecting and measuring interactions
US11396650B2 (en) 2015-06-02 2022-07-26 Children's Medical Center Corporation Nucleic acid complexes for screening barcoded compounds
US11092606B2 (en) 2015-08-07 2021-08-17 President And Fellows Of Harvard College Super resolution imaging of protein-protein interactions
US11713483B2 (en) 2016-02-09 2023-08-01 Children's Medical Center Corporation Method for detection of analytes via polymer complexes
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