EP1511856A1 - Procede de sequencage parallele d'un melange d'acide nucleique a l'aide d'un systeme d'ecoulement - Google Patents

Procede de sequencage parallele d'un melange d'acide nucleique a l'aide d'un systeme d'ecoulement

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Publication number
EP1511856A1
EP1511856A1 EP02807482A EP02807482A EP1511856A1 EP 1511856 A1 EP1511856 A1 EP 1511856A1 EP 02807482 A EP02807482 A EP 02807482A EP 02807482 A EP02807482 A EP 02807482A EP 1511856 A1 EP1511856 A1 EP 1511856A1
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EP
European Patent Office
Prior art keywords
nucleic acid
acid molecules
porous support
amplification
carrier
Prior art date
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Application number
EP02807482A
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German (de)
English (en)
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Achim Dr. Fischer
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Individual
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Individual
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Publication of EP1511856A1 publication Critical patent/EP1511856A1/fr
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    • 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
    • C12Q1/6869—Methods for sequencing
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Definitions

  • the invention relates to a method for the simultaneous sequencing of a large number of different nucleic acid molecules, and to a porous carrier which is used in the context of the method.
  • nucleic acids An important method of biological analysis is the sequence analysis of nucleic acids.
  • the exact base sequence of the DNA or RNA molecules of interest is determined here. Knowing this sequence of bases allows, for example, the identification of certain genes or transcripts, i.e. the messenger RNA molecules belonging to these genes, the detection of mutations or polymorphisms, or the identification of organisms or viruses that can be clearly identified by means of certain nucleic acid molecules ,
  • the sequencing of nucleic acids is usually carried out using the chain termination method (Sanger et al. (1977) PNAS 74, 5463-5467).
  • an enzymatic addition of a single strand to the double strand is carried out by extending a "primer” hybridized to said single strand, usually a synthetic oligonucleotide, by adding DNA polymerase and nucleotide building blocks.
  • a "primer” hybridized to said single strand usually a synthetic oligonucleotide
  • a small addition of termination nucleotide building blocks which after their incorporation into the growing strand no longer permit any further extension, leads to the accumulation of partial strands with a known end determined by the respective termination nucleotide.
  • the mixture of strands of different lengths thus obtained is separated by size by means of gel electrophoresis.
  • the nucleotide sequence of the unknown strand can be derived from the resulting band patterns.
  • a major disadvantage of the method mentioned is the required instrumental effort, which limits the achievable throughput of reactions.
  • For each sequencing reaction assuming the use of termination nucleotides labeled with four different fluorophores, at least one track on a flat gel or, when using capillary electrophoresis, at least one capillary is required.
  • the resulting effort is limited to the most modern at the moment commercially available sequencing machines, the number of sequencings that can be processed in parallel to a maximum of 384.
  • the reagents required for each sequencing reaction result in relatively high costs.
  • Another disadvantage is the limitation of the "reading length", ie the number of correctly identifiable bases per sequencing, due to the resolution of the gel system.
  • An alternative method of sequencing is faster and therefore allows the processing of more samples in the same time, but on the other hand is limited to relatively small DNA molecules (for example 40-50 bases).
  • sequencing by hybridization SBH, Sequencing By Hybridization; see Drmanac et al., Science 260 (1993), 1649-1652
  • base sequences are identified by the specific hybridization of unknown samples with known oligonucleotides.
  • said known ohgonucleotides are fixed in a complex arrangement on a support, hybridization with the labeled nucleic acid to be sequenced is carried out, and the hybridizing oligonucleotides are determined.
  • the sequence of the unknown nucleic acid can then be determined from the information about which oligonucleotides have hybridized with the unknown nucleic acid and from their sequence.
  • a disadvantage of the SBH method is the fact that the optimal hybridization conditions for oligonucleotides cannot be predicted exactly and accordingly a large set of oligonucleotides cannot be designed which on the one hand contain all possible sequence variations with their given length and on the other hand require exactly the same hybridization conditions. Thus unspecific hybridization leads to errors in the sequence determination.
  • the SBH method cannot be used for repetitive regions of nucleic acids to be sequenced.
  • Such a strategy for expression analysis consists in the quantification of discrete sequence units. These sequence units can consist of so-called ESTs (expressed sequence tags). If sufficient numbers of clones from cD ⁇ A banks, which come from samples to be compared, are sequenced, identical numbers can be used Sequences are recognized and counted and the relative frequencies of these sequences obtained in the different samples are compared with one another (cf. Lee et al., Proc. Natl. Acad. Sci. USA 92 (1995), 8303-8307). Different relative frequencies of a certain sequence indicate differential expression of the corresponding transcript. However, the method described is very complex since the sequencing of many thousands of clones is already necessary for the quantification of the more common transcripts.
  • a method for the parallel sequencing of nucleic acid tags is disclosed in WO 98/44151.
  • a surface is to be coated with PCR primers, followed by an amplification of nucleic acid molecules to be sequenced on the surface.
  • DNA colonies or “DNA islands” each generate identical molecules, which can subsequently be sequenced.
  • two immobilized PCR primers for amplification which is already known from US Pat. No.
  • a "half-sided" detachment of the nucleic acid molecules is therefore first necessary, ie the separation of one of the two ends from the surface selectively before an analysis can be started.
  • This detachment in turn, which, as described in WO 98/44151, is carried out by incubation with a suitable restriction enzyme is not without problems, since restriction enzymes often cannot completely cut solid-phase-bound nucleic acids
  • Another disadvantage of this method is the fact that individual nucleic acid islands, which are of interest due to their hybridization behavior, for example, cannot be recovered and identified. Furthermore, no possibility for sequencing more than only very short regions (about 15 to 20 bases) of the nucleic acids forming the islands is presented.
  • the known methods for nucleic acid sequencing have one or more of the following disadvantages: They only allow the parallelized passage of individual sequencing reactions to a very limited extent.
  • WO 01/61054 discloses a method for simultaneously carrying out a multiplicity of micro-volume reactions on a substrate which is provided with a multiplicity of holes, the reaction spaces being formed by the holes and the liquid in the reaction spaces being held in the holes by surface tension ,
  • the micro-volume reactions can also be sequencing reactions.
  • cycle sequencing in accordance with the chain termination method generates labeled single-stranded nucleic acids of different lengths, which are separated in size (e.g. by capillary or gel electrophoresis). The sequence is deduced from the sequence of the bands.
  • This method has the disadvantage that a separation step must be carried out in any case in order to obtain the sequence.
  • the labeled nucleic acids are separated by gel electrophoresis, handling problems arise when transferring the reaction volumes to the gel and a problem with the sensitivity of detection as a result of the small recovery volumes. If the separation is to be carried out by capillary electrophoresis, problems with the isolation arise in view of the high voltages. In addition, the correct alignment of the substrate along the field lines is not easy. Finally, the sequencing technology based on Taq polymerase delivers significantly more read errors than other sequencing techniques that do not use heat-stable polymerases.
  • the object of the invention is to provide a method which overcomes the disadvantages of the prior art.
  • the object according to the invention is achieved by a method for highly parallel nucleic acid sequencing, consisting of the steps:
  • step (1) (2) introducing the carrier from step (1) into a flow arrangement, (3) simultaneous determination of at least part of the nucleotide sequence of at least part of the nucleic acid molecules.
  • the porous carrier consists of a solid having cavities, which can be gel-like, but is preferably solid.
  • the cavities can be both liquid-filled and gas-filled, in particular penetrated by the liquid or gaseous phase surrounding the carrier.
  • the cavities can have any regular or irregular shape.
  • the cavities of a porous support can be essentially of the same shape and / or size, but also of different shape and / or size. In any case, it should be an “open-pore” solid, ie the cavities should be able to communicate at least partially with one another and / or with the liquid or gaseous medium surrounding the solid.
  • “Communicating” here means the possibility of mass transfer, for example by diffusion, convection or active transport processes, as can be achieved, for example, by building up a pressure difference.
  • the porous carrier is not one but several solid bodies, for example a packing of solid, identical or non-identical particles which has cavities.
  • the material of the porous support can largely be chosen as long as the requirements for structure (see above), wettability, solvent resistance, temperature resistance, compatibility with the steps to be carried out for nucleic acid sequence determination etc. are met.
  • the porous carrier can consist of a polymer, glass, silicon or another metallic, semi-metallic or non-metallic substance.
  • porous supports from more than one material; for example in the form of the copolymerization of different monomers, by sintering particles consisting of different materials, by coating the cavity walls of a porous solid with one or more other substances or by adding fillers to give a strength-imparting matrix.
  • Most of the porous supports used to achieve the object according to the invention are regularly shaped, in particular flat, preferably plane-parallel, so that a top and a bottom can be assigned to a support.
  • the carrier can have a largely arbitrary thickness, but a thickness between 50 ⁇ m and 20 mm, in particular between 300 ⁇ m and 1 mm, is preferred.
  • porous supports having channels, in particular those supports whose channels are delimited on one side by the top side and on one side by the bottom side of the carrier, in which case the top side and bottom side communicate with one another through at least some of the channels. It is preferred that the channels are substantially parallel to one another and / or run at right angles or approximately at right angles to the top and / or bottom of the carrier, and that the channels are cylindrical and have essentially the same diameter, i.e. from an average diameter of not more than 10% or not more than 50%, for example. The diameter of the channels should generally not exceed 200 ⁇ m.
  • the diameter of the channels is at most 100 ⁇ m, at most 30 ⁇ m, at most 10 ⁇ m, at most 3 ⁇ m, at most 1 ⁇ m or at most 0.3 ⁇ m. In a particularly preferred embodiment, the diameter of the channels is between 0.5 ⁇ m and 50 ⁇ m, in particular between 5 ⁇ m and 25 ⁇ m.
  • GCAs glass capillary arrays
  • nonucci et al., Science 258: 783-5 is very particularly preferred (1992)) or porous silicon wafers.
  • the areas from step (1) can comprise one or more cavities, in particular one or more channels.
  • An area is not primarily defined by a property of the porous support itself (such as a special shape etc., although such should not be excluded), but by the inhomogeneous distribution of the immobilized nucleic acid molecules.
  • a certain channel can contain a certain type of nucleic acid molecules which are immobilized on its wall, while the adjacent channels adjacent to this channel do not contain said type of nucleic acid molecules; this channel then forms its own area.
  • a plurality of adjacent, ie adjacent channels can also contain the same type of nucleic acid molecules immobilized along their walls; an area is then formed by the entirety of said channels.
  • nucleic acid molecules immobilized within a region should of course be essentially sequence-identical in order to allow their sequence to be determined; however, nucleic acid molecules of other sequences not participating in the sequence determination process are not harmful. “Identical to the sequence” is understood to mean nucleic acid molecule single strands, their “counter-strands” essentially complementary sequence, and the double strands formed from strand and counter-strand. Furthermore, nucleic acid molecules of only partially identical sequences are not harmful, which may have, for example, at least one identical and at least one non-identical sequence part, as long as the sequence determination predominantly relates to the identical sequence part.
  • the generation of the immobilized nucleic acid molecule-carrying regions is possible by a transfer of preformed nucleic acid molecule solutions to the porous carrier followed by immobilization as well as by a sto-generation of numerous sequence-identical nucleic acid molecules by amplification of one starting molecule at the location of the carrier, in particular within cavities of the carrier Carrier, whereby immobilization can take place during and / or after the amplification.
  • nucleic acid molecule solutions are preferably a collection of solutions of one type of nucleic acid molecule, which can be stored in suitable containers such as microtiter plates.
  • the nucleic acid molecules can have been generated, for example, by processing genomic DNA or by mRNA.
  • genomic DNA is cut with one or more, usually frequently cutting, restriction endonucleases, the resulting fragments are cloned and DNA isolated from the clones (for example phage clones, bacterial clones or yeast clones) or copies produced in vitro therefrom are stored as a “genomic library”
  • mRNA is rewritten into first-strand cDNA, this is converted into double-stranded cDNA, and the procedure is continued as described for genomic DNA.
  • the transfer of the nucleic acid molecule solutions to the porous support can be carried out by using from the state of the art Technique for the production of known DNA arrays, for example with the help of specially shaped needles ("pins"), capillaries or by means of the .H / cyet technique (eg piezo technique or bubble jet technique) suitable liquid volumes (for example between 1 nl and 100 nl) on the surface de s porous carrier can be applied.
  • the applied liquid is usually absorbed by the carrier by capillary action, so that the respective region containing certain nucleic acid molecules is expanded by the expansion of all those cavities of the carrier which were filled by the transferred liquid drop (or possibly several transferred liquid drops) (i.e. the walls thereof) were wetted).
  • said area consists of only a single cavity, for example a single capillary. Most often, however, an area will include several adjacent cavities / capillaries.
  • the immobilized areas are to be generated via amplification of individual nucleic acid molecules to form "clones", that is to say collections of nucleic acid molecules that are essentially sequence-identical, in the areas of the support a suitably dilute solution of the nucleic acid molecule mixture to be amplified is prepared in a suitable amplification mixture which, in addition to the “template” molecules to be amplified, also contains the components required for carrying out the amplification reaction (s), in particular aqueous buffers, nucleotide triphosphates (dNTPs), Ions, at least one polymerase and in many cases amplification primers.
  • dNTPs nucleotide triphosphates
  • Ions at least one polymerase and in many cases amplification primers.
  • the concentration of the template nucleic acid molecules is selected such that there is at most one amplifiable template nucleic acid molecule in the majority of the cavities. In a further preferred embodiment, there are on average a maximum of 0.5 amplifiable nucleic acid molecules in a cavity. In another preferred embodiment, on average there are arithmetically a maximum of 0.2 amplifiable nucleic acid molecules in a cavity. In yet another preferred embodiment, there are on average between about 0.1 and 0.02 amplifiable nucleic acid molecules in a cavity.
  • amplification conditions are set after the amplification solution has been introduced into the cavities of the porous support, copies of the same are produced in those cavities in which an amplifiable template molecule is located.
  • the generation of at least 10 copies of a template molecule is preferred, the generation of at least 10 copies or at least 10 copies being particularly preferred.
  • the amplification can take place by any suitable, isothermal or non-isothermal method such as, for example, PCR, NASBA, RNA amplification, rolling c / rc / e replication or replication using Q beta replicase.
  • voids of the porous support each contain numerous, preferably at least 10, at least 10 7 or at least 10 8 copies of a nucleic acid molecule, different voids typically containing at least partly copies in their sequence of different nucleic acid molecules.
  • the nucleic acid molecules to be amplified can be, for example, restriction fragments obtained from genomic DNA or cDNA, or also unabridged cDNA molecules (so-called fill szze cDNAs). In a preferred embodiment, these restriction segments or molecules are provided at one end or at both ends with a plurality of different molecules common, preferably “universal” primer binding sites common to all molecules.
  • the porous carrier containing the amplification solution can be introduced into a water vapor-saturated atmosphere or brought into contact with a hydrophobic substance such as paraffin or mineral oil.
  • a hydrophobic substance such as paraffin or mineral oil.
  • the porous carrier containing the amplification solution is brought into contact on one side with a suitably temperature-controlled or temperature-controlled surface and overlaid with oil on the other side; then suitable temperature (s) for the amplification is / are set.
  • the porous carrier containing the amplification solution is immersed in a temperature-controlled or temperature-controlled oil bath; the oil is then adjusted to a temperature or temperatures suitable for the amplification. In both cases it is possible to carry out suitable temperature changes, possibly in a cyclical sequence, for the contamination of non-isothermal amplification reactions.
  • the procedure is as in the above embodiment, but the nucleic acid molecules resulting from amplification are not immobilized on the walls of the porous support, but on a preferably planar surface brought into contact with it.
  • the immobilization can be carried out during and / or after the amplification.
  • the porous support is removed from the surface so that the immobilized nucleic acid molecules are present on the surface in the form of defined areas. Said nucleic acid molecules present in regions on the surface are then at least partially sequenced, for example according to one of the procedures mentioned under (a) to (d) below.
  • the said modification is therefore a method for highly parallel nucleic acid sequencing, consisting of the steps:
  • the nucleic acid molecules can be immobilized using methods known from the prior art; it is preferred that the molecules are immobilized at the end, that is to say via their 3 'end or via their 5' end.
  • the immobilization should be irreversible, ie that under the conditions required for determining the nucleotide sequence in step (3) (temperature, ionic strength, enzymatic activity etc.) at most part of the immobilized molecules, preferably at most 10% or at most 50%, of the porous Carrier releases.
  • a detachment of at most 5% or at most 1% of the immobilized nucleic acid molecules is particularly preferred during the determination of the nucleic acid sequence.
  • the immobilization can be mediated via non-covalent interactions, for example the nucleic acid molecules to be immobilized can carry biotm groups.
  • the porous support could be coated with avidin or streptavidin so that the biotin-modified nucleic acid molecules can bind to the coated support.
  • immobilization of the nucleic acid molecules by covalent interactions is preferred.
  • Appropriately modified nucleic acid molecules are mostly used for this purpose, for example nucleic acid molecules which have a 5'- or 3'-terminal amino group (“amino modifier”, cf. Glen Research, Sterling, Virginia 20164: Catalog 2002 p.
  • a terminal thiol group "A terminal phosphate group, an acrydite group, a carboxy-dT group or another reactive group. If desired, there may be any spacer or linker between the immobilizing mediating group and the nucleic acid molecule, for example an oligoethylene glycol spacer or one.
  • cleavable group such as, for example, a dithiol group or a photolytically cleavable nitrobenzyl group, such cleavage groups, if desired, allow suitable immobilization of nucleic acid molecules by detachment from the porous support after setting suitable conditions the groups mediating the immobilization are located in addition to the nucleic acid molecule termini at other locations on the nucleic acid molecules, for example as side chains on the nucleotide bases.
  • the latter would be conceivable, for example, by incorporating aminoallyl-dUTP or biotin-dUTP during the production of the nucleic acid molecules.
  • the carrier and the nucleic acid molecules are first prepared in a suitable manner so that the carrier and the nucleic acid molecules to be immobilized each have a partner of a specific binding pair consisting of two partners, and the nucleic acid molecules can be immobilized by binding the two partners to one another.
  • the carrier and the nucleic acid molecules to be immobilized each have a partner of a specific binding pair consisting of two partners, and the nucleic acid molecules can be immobilized by binding the two partners to one another.
  • further components could also be involved in the binding of the two partners of the binding pair.
  • Numerous methods for immobilizing biomolecules are known from the prior art; Examples are given, for example, in Nucleic Acids Res. 22, 5456-65 (1994) and Nucleic Acids Res. 27, 1970-77 (1999).
  • the density of the nucleic acid molecules on the surface is preferably at least at least 10 molecules / ⁇ m 2 , at least 100 molecules / ⁇ m 2 , at least 1000 molecules / ⁇ m 2 or at least 10,000 molecules / ⁇ m 2 .
  • Immobilization of nucleic acid molecules generated by amplification can take place during or only after the amplification. Immobilization is possible during the amplification, for example, in that in an amplification method based on primer extension such as PCR, in addition to the primers present in solution, primers immobilized on the inner walls of the cavities (or even exclusively immobilized primers are used, for example in WO) 96/04404), which then likewise hybridize with single-stranded template molecules and can subsequently be incorporated by primer extension. Accordingly, in the sense of the present invention, the immobilization of a nucleic acid molecule in solution can also be the synthesis of a complementary complementary strand. Molecule can be understood by extension of a primer hybridized to the dissolved molecule, ie a "description" of the molecule from the liquid to the solid phase.
  • the flow arrangement from step 2 is characterized by two spaces which are connected to one another via the porous support, so that liquids can flow from one of the spaces through the porous support into the other of the two spaces.
  • An additional feature of the arrangement can consist in a means for building up a pressure difference between the two spaces, so that an active delivery of liquids is possible.
  • Liquids here are solutions, mostly aqueous solutions, which in particular contain the reagents required for the nucleotide sequence determination in step (3).
  • the flow arrangement is designed in such a way that it is possible to observe processes which include the nucleic acid molecules immobilized on the porous support.
  • the apparatus used in the method according to the invention is at least one detection device which allows said registration, in particular an optical detection device which is capable of detecting light in the infrared, visible and / or ultraviolet range.
  • the detection device can, for example, be one of the known confocal microscopy device. In an alternative embodiment, it is a CCD camera with an optical system, which allows an observation of the processes on or allowed inside the porous support with sufficient resolution.
  • the surface of the porous support imaged by a pixel of the CCD camera preferably has an edge length of at most 100 ⁇ m, particularly preferably an edge length of at most 10 ⁇ m or at most 2 ⁇ m.
  • a further feature of the apparatus used to carry out the method according to the invention is at least one source of radiation suitable for fluorescence excitation, preferably a source of monochromatic light, in particular a laser.
  • the simultaneous determination of at least a part of the nucleotide sequence of the immobilized nucleic acid molecules can be carried out in any manner, but preferably by step-by-step strand formation or strand degradation.
  • Step by step means that the nucleic acid strands changed in the course of the sequencing are simultaneously extended or shortened by the same amount, that is to say by a known number of nucleotides, preferably by exactly one nucleotide in each case.
  • the nature of the respective nucleotide or the sequence of nucleotides becomes in each step for the nucleic acid molecules immobilized in several, preferably all or largely all, areas of the porous carrier.
  • the nucleic acid molecules can be sequenced, for example, in the following ways:
  • nucleotide triphosphates ("ordinary" nucleotides, dNTPs) with determination of reaction by-products
  • dNTPs nucleotide triphosphates
  • a partial nucleic acid double strand containing the nucleic acid strand to be sequenced is incubated under conditions which are favored by a polymerase-catalyzed replenishment reaction, each with a type of labeled nucleotide (eg labeled dATP). After washing away non-incorporated nucleotides, it is determined by detecting the marking whether or how many nucleotides have been incorporated (eg lxA, 2xA, etc.). In the next step, incubation and detection is carried out with a second labeled nucleotide type (eg labeled dCTP), then the same is carried out with a third (e.g.
  • a second labeled nucleotide type eg labeled dCTP
  • the cycle begins again with the addition of labeled nucleotide of the first kind.
  • the signal strengths measured in the course of a detection result in each case from the sum of the signal strength from the nucleotide incorporation of the last nucleotide incorporation carried out and all nucleotide incorporations previously carried out.
  • the marking of the incorporated nucleotides is deleted at suitable times, for example after each addition of nucleotides, after each cycle consisting of the successive addition of all four different nucleotides or one or more repetitions thereof.
  • This is preferably done by removing or changing the marking group or the marking groups.
  • the labeling group can be bound to the corresponding nucleotide via a chemically, photochemically or enzymatically cleavable spacer, for example a spacer containing a disulfide group or a nitrobenzyl group.
  • One way of changing the marking group would be, for example, to bleach a fluorescent dye, which would be possible, for example, by sufficiently intense laser radiation.
  • procedure (c) over (b) is that only one part of the incorporated nucleotides, ideally only the last incorporated nucleotide, is determined in one measurement, without a signal background due to the previously incorporated nucleotides, which often is in the multiple of the signal of interest, must be taken into account.
  • nucleotide-wise extension of nucleic acid strands is achieved through the use of nucleotide triphosphates reversibly blocked on their 3 'OH group, which can be incorporated by polymerases into a growing DNA double strand, but after their incorporation act as chain extension terminators. If the blocking group is removed, a free 3 'OH group is restored so that a next nucleotide can be incorporated.
  • nucleic acid molecules in step (3) are to be sequenced according to procedure (a), (b), (c) or (d), at least some of the nucleic acid molecules will generally be at least partially in a single-stranded state.
  • a so-called sequencing primer is generally required, that is to say an oligo- or polynucleotide which can hybridize with the nucleic acid strand to be sequenced and is present in the hybridized state in this way. that it can be extended at its 3 'end by means of a DNA polymerase, the complementary counter-strand to the region to be sequenced being synthesized.
  • the immobilized nucleic acid molecule is optionally converted into the single-stranded state, if appropriate, by removing the opposite strand, and then a suitable sequencing primer, which is at least partially complementary to the nucleic acid molecule, and which has a 3 ′ end that can be extended by means of a polymerase, with the nucleic acid molecule hybridized.
  • a suitable sequencing primer which is at least partially complementary to the nucleic acid molecule, and which has a 3 ′ end that can be extended by means of a polymerase, with the nucleic acid molecule hybridized.
  • the nucleic acid molecules to be immobilized on the porous support and to be sequenced in step (3) are preferably at one end, preferably the other end protruding into the solution space after immobilization, with a further refolded or capable of refolding. or double-stranded nucleic acid molecule such as a partially self-complementary oligonucleotide.
  • a “masked hairpin”, that is to say a double-stranded nucleic acid molecule containing an inverted repetition, can also be attached to the double-stranded nucleic acid molecule before the immobilization.
  • the nucleic acid molecule to be sequenced can remain on then "fold back" this counter strand attached with its 5 'end and extend it at its free 3' end by means of a polymerase.
  • the invention is characterized in more detail by the following description.
  • the invention particularly relates to a method for parallel
  • Nucleic acid sequencing comprising the steps of: (i) providing a monolithic porous support comprising at least two
  • porous support having at least two distinguishable locations which have nucleic acids of different sequences
  • step (ii) providing a solution containing one or more nucleotide compounds selected from mono- and oligonucleotides, (iii) introducing the solution from step (ii) into the sample chambers of the porous support, the binding of the nucleotide compounds to the single-stranded
  • Sections of the immobilized nucleic acids and thus the indirect binding to the porous carrier is effected, (iv) detection of the amount and / or identity of the nucleotide compounds indirectly bound to the porous carrier via the immobilized nucleic acids at the at least two distinguishable locations of the porous carrier.
  • Steps (ii) to (iv) can be repeated one or more times, with sequence information being obtained with each cycle.
  • the solution from step (ii) is introduced into the sample chambers of the porous support in step (iii) by generating a flow of the solution from step (ii) through the sample chambers.
  • step (iv) the detection is carried out as to whether nucleotide compounds are (indirectly) bound to the porous support. If the solution in step (ii) contains several nucleotide compounds, then in step (iv) it is checked which nucleotide compounds are involved, that is to say their identity is ascertained. It is usually necessary to measure the amount of nucleotide compounds bound in order to distinguish significant signals from the background. Under certain conditions, it is advisable measure the amount more accurately.
  • step (iii) This is the case if, under certain circumstances, several nucleotide compounds can be bound to the single-stranded sections of the immobilized nucleic acids in step (iii) and this allows conclusions to be drawn about the sequence, as in the case of sequencing by enzymatic beach extension with nucleotides without a chain termination group.
  • the porous carrier consists of a solid having cavities, which can be gel-like, but is preferably solid.
  • the cavities can be both liquid-filled and gas-filled, in particular penetrated by the liquid or gaseous phase surrounding the carrier.
  • the cavities can have any regular or irregular shape.
  • the cavities of a porous support can be essentially of the same shape and / or size, but also of different shape and / or size.
  • the cavities preferably have a dimension such that when they are gas-filled they can absorb aqueous solutions by capillary forces or, when they are liquid-filled, they can hold the liquids.
  • the cavities should be able to communicate at least partially with the liquid or gaseous medium surrounding the solid; it is also conceivable that the cavities also communicate with one another.
  • “Communicating” here means the possibility of mass transfer, for example by diffusion, convection or active transport processes, as can be achieved, for example, by building up a pressure difference.
  • the porous carrier is a coherent solid, a monolith, for example a packing of solid, identical or non-identical particles or capillaries which are firmly, in particular covalently, connected.
  • the material of the porous support can largely be chosen as long as the requirements for structure (see above), wettability, solvent resistance, temperature resistance, compatibility with the steps to be carried out for nucleic acid sequence determination etc. are met.
  • the porous support can consist of a polymer, for example a copolymer of different monomers, of glass, of silicon or another metallic, semi-metallic or non-metallic substance.
  • porous supports from more than one material; for example by sintering particles consisting of different materials, by coating the cavity walls of a porous support with one or more other ones Substances or by adding fillers to a strength-imparting matrix.
  • the porous supports used to achieve the object according to the invention are regularly shaped, in particular flat, preferably plane-parallel.
  • the porous support generally has opposite, that is to say non-adjacent, preferably substantially mutually parallel surfaces, namely a first and a second side, which preferably represent the top and the bottom of the support and are preferred, but not necessarily flat
  • the carrier can have a largely arbitrary thickness, but a thickness between 50 ⁇ m and 20 mm, in particular between 300 ⁇ m and 1 mm, is preferred.
  • the porous carrier has at least two sample chambers, preferably more than 100, more than 10, more than 10 or 10, more than 10, in particular more than 10, which are formed by the cavities.
  • a sample chamber extends through the entire porous support, i.e. it penetrates the porous support from the outer surface to the outer surface, and has one or more surfaces in its lumen and at least one inlet and one outlet opening, preferably one inlet and at least one or more Exit openings or one exit opening each and at least one or more entry openings.
  • the sample chamber has dimensions such that the content, when it is liquid, is held in the sample chamber by capillary forces.
  • the sample chambers preferably have a regular, preferably round or hexagonal cross-section along their axis. It is preferred that the sample chambers have essentially the same diameter, that is to say they do not deviate from an average diameter by more than 50%, in particular not more than 10%. However, the cross-section can also be irregular, as will be the case with a porous carrier which has been produced by sintering processes. It is not excluded that different sample chambers are connected. This is particularly the case with porous supports that have been produced by sintering processes. The use of such carriers in the context of the invention is also possible. In this case there is a network that makes it difficult to distinguish between individual sample chambers. It should be noted here that the mass transport along the axis of a sample chamber is greater than the mass transport between two different chambers. The ratio of mass transfer (through diffusion and ____, __ ,,
  • step (iii)) along the axis of a sample chamber for mass transport between two different chambers at least 10, preferably at least 100, especially at least 1000.
  • the sample chambers run essentially rectilinearly and are oriented in the porous carrier in such a way that they have a preferred direction, which facilitates the generation of a flow through the sample chambers in step (iii), since this ensures that with a flow through a large number of sample chambers evenly.
  • An axis of the sample chamber is defined by two points, the center of the inlet and outlet opening of a sample chamber. If a sample chamber has several entrance or exit openings, it has accordingly several axes.
  • the axes of the sample chambers preferably form an angle of less than 30 °, in particular less than 15 °, especially less than 5 °, an essentially parallel alignment being most preferred
  • the nucleic acid molecules within a sample chamber represent a sequencing sample.
  • the nucleic acid molecules are preferably compartmentalized in the porous carrier, that is to say the nucleic acid molecules immobilized within a sample chamber preferably have essentially the same sequence with respect to the single-stranded section, they are preferably sequence-identical in the context of the definition below.
  • sequence-identical is defined below and does not mean in the context of the invention that the sequences must be exactly the same, even if this will usually apply.
  • sequence-identical takes account of the fact that nucleic acid molecules which are produced enzymatically have, often as a result of incorrect reproduction of a common template nucleic acid molecule, have sequence errors which represent a deviation from the sequence identity, but are so rare that they do not prevent the sequence determination.
  • nucleic acid molecules of other sequences not participating in the sequence determination process are not harmful, that is, they are disregarded when assessing whether sequence identity is present.
  • deviations in the sequence of nucleic acid molecules with only partially identical sequences can be disregarded, which may have, for example, at least one identical and at least one non-identical sequence part, as long as the sequence determination (predominantly) relates to the identical sequence part.
  • the sequence determination takes place within the framework of the Invention preferred by the method of enzymatic strand extension. In the method of enzymatic strand extension, the sequence determination relates only to the sequence section which connects 3 'to the section to which the sequencing primer used for priming the polymerase binds, provided that intermolecular priming is used.
  • the nucleic acid only takes part in the sequence determination if it is able to form such a hairpin.
  • sequence determination according to the method of the enzymatic strand extension only affects the section of a nucleic acid which extends 3'-wards over the number of bases from the boundary between the double-stranded and single-stranded section on the nucleic acid molecule, as corresponds to the maximum reading length.
  • the distinguishable locations in step (i) thus preferably have nucleic acids with single-stranded sections of different sequences.
  • the term "distinguishable” refers to the detection that takes place in step (iv), the spatial resolution of which must allow the identification of distinguishable locations.
  • the locations in the sense of the invention have a two-dimensional or three-dimensional extent.
  • the distinguishable locations preferably each comprise at least one sample chamber (more precisely its surface (s)) on the porous support, but can also comprise several sample chambers.
  • the sample chambers are each formed by at least one cavity on the porous support.
  • the sample chambers are designed as channels.
  • the channels can be capillaries.
  • the channels are open to form at least one entrance and one exit opening to the first and to the second side of the carrier, so that both sides of the Carrier, for example top and bottom, through which channels communicate with each other. If the two sides of the carrier are top and bottom, then the channels are bounded on one side by the top and on one side by the bottom of the carrier. Channels ending blindly in the porous support do not correspond to the above definition, so that the following explanations do not refer to such channels. However, their presence in the porous carrier is not excluded.
  • channels also includes those which are connected to one another, that is to say they communicate with one another. This can lead to a distinguishable location being formed by a plurality of channels which each have their own entrance and exit openings and are connected to one another. Although this leads to a reduction in the resolution, that is to say the maximum number of distinguishable locations which, according to step (i), can be present on a unit area of the porous carrier. However, this is not detrimental as long as the diameter of the channels is sufficiently small that, despite the possibility of communication between some channels, sufficient resolution is achieved.
  • porous support the channels of which are partially connected to one another, is the porous support sold by PamGene called the PamChip TM array (PamGene, Burgemeester Loeffplein 70 A, 5211 RX's-Hertogenbosch, NL).
  • the channels run essentially rectilinearly and are oriented in the porous carrier in such a way that they have a preferred direction, which facilitates the generation of a flow through the channels in step (iii), since this ensures that with a single flow can flow through a large number of channels evenly.
  • the axes of the channels preferably form an angle of less than 30 °, in particular less than 15 °, especially less than 5 °, an essentially parallel alignment being most preferred.
  • the channels run at right angles or approximately at right angles to the first and / or second side of the carrier, which preferably represent the upper or lower side of the porous carrier.
  • the channels are cylindrical or polygonal, especially hexagonal. It is further preferred that they be of substantially the same diameter have, i.e. deviate from an average diameter of not more than 50%, in particular not more than 10%.
  • the diameter of the channels should generally not exceed 200 ⁇ m. In preferred embodiments, the diameter of the channels is at most 100 ⁇ m, at most 30 ⁇ m, at most 10 ⁇ m, at most 3 ⁇ m, at most 1 ⁇ m or at most 0.3 ⁇ m. In a particularly preferred embodiment, the diameter of the channels is between 0.5 ⁇ m and 50 ⁇ m, in particular between 5 ⁇ m and 25 ⁇ m.
  • the substrates disclosed in WO 99/34920 and WO 00/56456, to which reference is hereby made, can also be used as supports.
  • the use of so-called “glass capillary arrays"("GCAs”; Burle Electro-Optics, Inc., Sturbridge, MA, USA) or of “nanochannel glass” (Tonucci et al., Science 258: 783-5) is very particularly preferred (1992)) or porous silicon wafers.
  • step (i) comprises the steps (i-aO) providing a monolithic porous carrier, comprising at least two sample chambers which extend through the porous carrier, which have at least one inlet and one outlet opening and one or have multiple surfaces, (i-al) impregnating the porous support with a nucleic acid solution which contains at least two nucleic acid molecules of different sequence, so that at least two sample chambers are filled with the nucleic acid solution, (i-a2) amplifying the nucleic acid molecules in the sample chambers, ( i-a3) immobilization of the nucleic acid molecules on the surfaces of the sample chambers of the porous support, steps (i-a2) and (i-a3) being able to take place simultaneously,
  • step (i-a4) Transfer of the nucleic acid molecules into a state in which they have a single-stranded section, step (i-a4) also being able to take place before step (i-a3) or simultaneously with step (i-a3).
  • step (i-a4) the nucleic acid molecules preferably have both a single-stranded and a double-stranded section.
  • step (i) comprises the steps
  • (i-bO) providing a monolithic porous support, comprising at least two sample chambers, which extend through the porous support, which have at least one inlet and one outlet opening and which have one or more surfaces,
  • nucleic acid molecules of different sequences in each case at different locations on the porous support, so that at least two sample chambers are filled with the nucleic acid solutions,
  • Sample chambers of the porous support, (i-b3) bringing the nucleic acid molecules into a state in which they have a single-stranded section, step (i-b3) also before step (i-b2) or step (i-bl) or simultaneously with one of these steps can take place and can be omitted if the nucleic acid molecules in step (i-bl) have a single-stranded section.
  • the nucleic acid molecules in step (i-b3) preferably have both a single-stranded and a double-stranded section.
  • step (i-bl) is preferably carried out using needles, capillaries or by means of the ink jet technique.
  • the immobilized nucleic acid molecules that are located in the regions or, according to another version, in the channels can be generated by one of two measures:
  • the porous support is impregnated with a solution which contains at least two nucleic acid molecules of different sequence, that is to say a mixture of different nucleic acids, which is followed by an amplification of the nucleic acids.
  • a suitably dilute solution of the mixture is first prepared and the components which are necessary for successful amplification are added.
  • This amplification solution then contains, in addition to the nucleic acid molecules to be amplified (“template” molecules), in particular aqueous buffers, nucleotide triphosphates (dNTPs), ions, at least one polymerase and, depending on the chosen amplification method, also amplification primers.
  • template molecules in particular aqueous buffers, nucleotide triphosphates (dNTPs), ions, at least one polymerase and, depending on the chosen amplification method, also amplification primers.
  • the solution is brought into contact with the porous support in such a way that the sample chambers of the support are partially or completely filled with solution.
  • the concentration of the amplifiable nucleic acids is preferably selected such that there is at most one (amplifiable) nucleic acid molecule in the majority of the sample chambers (or channels).
  • Non-amplifiable nucleic acids, especially primers, are disregarded. In this way, distinct locations are formed on the porous support, which have nucleic acids of different sequences.
  • double-stranded nucleic acids are converted into a state in which they have a single-stranded section. The transfer of the nucleic acid molecules into a state in which they have a single-stranded section is usually carried out by denaturing.
  • the distinguishable locations of the porous support which have nucleic acids of different sequences are generally those which have nucleic acids with single-stranded sections of different sequences. This means that the sequence differences usually concern the single-stranded section. This applies in particular to the sequencing process of the enzymatic strand extension.
  • the distinguishability of the locations results here from the fact that they lie on different sample chambers of the porous support and can thus be distinguished from one another in the detection. It follows from what has been said that the distinguishable locations on the porous carrier are arranged in a random arrangement (statistically).
  • amplification of this molecule to numerous copies then takes place within those sample chambers which contain an (amplifiable) nucleic acid molecule.
  • the production of at least 10 6 copies of a nucleic acid molecule is preferred, the production of at least 10 7 copies or at least 10 8 copies being particularly preferred.
  • the amplification can take place by any suitable, isothermal or non-isothermal method such as, for example, PCR, NASBA, RNA amplification, rolling circle replication or replication using Q beta replicase, with PCR being preferred.
  • the sample chambers of the porous support in which an amplification took place each contain numerous, preferably at least 10 6 , at least 10 7 or at least 10 8 copies of a nucleic acid molecule, different sample chambers typically containing at least partially copies of different nucleic acid molecules.
  • the nucleic acid molecules to be amplified in step (i-a2) can represent, for example, restriction fragments obtained from genomic DNA or cDNA or also unabridged cDNA molecules (so-called full-size cDNAs).
  • these restriction fragments or molecules are provided at one end or at both ends with a plurality of different molecules common, preferably "universal" primer binding sites common to all molecules.
  • This can be done by cloning into a suitable vector, but also by attaching "linkers", ie double-stranded DNA molecules with a length of, for example, between 15 bp and 50 bp.
  • linkers ie double-stranded DNA molecules with a length of, for example, between 15 bp and 50 bp.
  • the porous carrier containing the amplification solution can be introduced into an atmosphere saturated with water vapor or brought into contact with a hydrophobic substance such as paraffin or mineral oil.
  • the carrier on or off can be brought into contact on both sides with surfaces that are firmly sealed. These could consist of glass, metal or a polymer, for example.
  • the porous carrier containing the amplification solution is brought into contact on one side with a suitably temperature-controlled or temperature-controlled surface and overlaid with oil on the other side; then suitable temperature (s) for the amplification is / are set.
  • the porous carrier containing the amplification solution is immersed in a temperature-controlled or temperature-controlled oil bath; the oil is then adjusted to a temperature or temperatures suitable for the amplification. In both cases it is possible to carry out suitable temperature changes, possibly in a cyclical sequence, for carrying out non-isothermal amplification reactions.
  • the nucleic acid molecules in step (i-a3) can be immobilized during the amplification in step (i-a2) or after the amplification.
  • the amplification by PCR and the immobilization during the amplification takes place in that primers are immobilized on the surfaces of the sample chambers, which primers participate in the PCR reaction.
  • one primer or both primers of a pair of primers is immobilized on the surfaces of the sample chambers. If only one primer of a pair of primers is immobilized, the other primer of the pair of primers is in the respective sample chamber in free solution, that is to say not immobilized. Only a portion of the primers of a pair of primers can be immobilized, that is to say that a percentage of a primer of a pair of primers is immobilized, but the other portion is not.
  • a single nucleic acid solution from step (i-bl) preferably contains only sequence-identical nucleic acid molecules of the aforementioned definition.
  • a single nucleic acid solution from step (i-bl) contains only nucleic acid molecules with the same sequence.
  • the nucleic acid molecules transferred to the support are optionally first amplified there and then only subsequently immobilized or initially immobilized and optionally subsequently amplified.
  • the amplification is optional. Whether amplification makes sense depends on the amount of nucleic acid in the individual nucleic acid molecule solutions applied to the support.
  • the nucleic acid solutions in step (i-bl) can be entire collections of solutions of sequence-identical nucleic acid molecules.
  • the solutions are brought into contact with the porous support in such a way that the nucleic acid solutions, which each contain nucleic acid molecules of different sequences, are not mixed as possible.
  • regions of nucleic acid molecules of one type which can comprise one or more sample chambers, form on the porous support.
  • distinct locations are formed on the porous support, which have nucleic acids of different sequences.
  • the nucleic acids are brought into a state in which they have a single-stranded section.
  • the distinguishable locations of the porous support which have nucleic acids of different sequences are generally those which have nucleic acids with single-stranded sections of different sequences. This means that the sequence differences usually concern the single-stranded section. This applies in particular to the sequencing process of the enzymatic strand extension. It follows from what has been said that the distinguishable locations on the porous support are not statistically arranged in the case of measure (i-b). Rather, the location of each distinguishable location can be selected.
  • the nucleic acid solutions in step (i-bl) can be stored in suitable vessels such as microtiter plates.
  • the nucleic acid molecules can have been generated, for example, by processing genomic DNA or by mRNA.
  • genomic DNA is cut with one or more, mostly frequently cutting, restriction endonucleases, the resulting fragments are cloned and DNA isolated from the clones (for example phage clones, bacterial clones or yeast clones) or copies thereof which have been produced in vitro are stored as a "genomic library" ,
  • mRNA is rewritten into first-strand cDNA, this is converted into double-stranded cDNA, and the procedure is continued as described for genomic DNA, wherein the fragmentation with restriction endonucleases can be omitted.
  • the nucleic acid solutions can be transferred to the porous support in step (i-bl) by using methods known from the prior art for the production of DNA arrays, for example with the aid of specially shaped needles (“pins”), capillaries or by means of suitable ink volumes (for example between 1 nl and 100 nl) are applied to the surface of the porous support using the ink jet technique (for example piezo technique or bubble jet technique).
  • the liquid which is preferably applied in the form of one or more drops of liquid to a location on the porous support, is absorbed by the support, as a rule by capillary action, so that an area or distinguishable location forms where the nucleic acid molecules have the same sequence or are sequence-identical within the framework of the Definition are.
  • the area or distinguishable location of identical or sequence-identical nucleic acids extends to all sample chambers of the carrier which were filled by the liquid drop or drops during the transfer of a nucleic acid solution.
  • the area or distinguishable location of sequence-identical nucleic acids then comprises only a single sample chamber, for example a single capillary.
  • the nucleic acid molecules can be immobilized in step (i-a3) or (i-b2) by means of methods known from the prior art; it is preferred that the molecules are immobilized at the end, that is to say via their 3 'end or via their 5' end.
  • the immobilization is said to be irreversible, ie that under the conditions required for determining the nucleotide sequence (temperature, ionic strength, enzymatic activity etc.) at most part of the immobilized molecules, preferably at most 10% or at most 50%, detaches from the porous support.
  • a detachment of at most 5% or at most 1% of the immobilized is particularly preferred Nucleic acid molecules during the determination of the nucleic acid sequence.
  • the immobilization can be mediated via non-covalent interactions, for example the nucleic acid molecules to be immobilized can carry biotm groups.
  • the porous support could be coated with avidin or streptavidin so that the biotin-modified nucleic acid molecules can bind to the coated support.
  • immobilization of the nucleic acid molecules by covalent interactions is preferred.
  • appropriately modified nucleic acid molecules are mostly used, for example nucleic acid molecules which have a 5'- or 3'-terminal amino group ("amino modifier", cf. Glen Research, Sterling, Virginia 20164: Catalog 2002 p.
  • a terminal thiol group contain a terminal phosphate group, an acrydite group, a carboxy-dT group or another reactive group.
  • any spacer or linker for example an oligoethylene glycol spacer or a cleavable group such as a dithiol group or a photolytically cleavable nitrobenzyl group, may be located between the reactive group mediating the immobilization and the nucleic acid molecule. If desired, such cleavable groups allow recovery of immobilized nucleic acid molecules by detachment from the porous support after setting suitable conditions.
  • the groups mediating the immobilization can also be located at other locations on the nucleic acid molecules, for example as side chains on the nucleotide bases. The latter would be conceivable, for example, by incorporating aminoallyl-dUTP or biotin-dUTP during the production of the nucleic acid molecules.
  • the carrier and nucleic acid molecules are first prepared in a suitable manner, so that the carrier and nucleic acid molecules to be immobilized each have a partner of a specific binding pair consisting of two partners, and the nucleic acid molecules can be immobilized by binding both partners to one another.
  • the density of the nucleic acid molecules on the surface is preferably at least at least 10 molecules / ⁇ m 2 , at least 100 molecules / ⁇ m 2 , at least 1000 molecules / ⁇ m 2 or at least 10,000 molecules / ⁇ m 2 .
  • usable fluorescent dyes reference is made to the catalog from Molecular Probes, Eugene, OR, USA, 6th edition 1996.
  • the immobilization of nucleic acid molecules generated by amplification in step (i-a3) or in step (i-a2), if an amplification takes place in measure (ib), can take place during or only after the amplification, as described above under measure (i- a ) has already been mentioned. Immobilization is possible during the amplification, for example, in that in an amplification method based on primer extension such as PCR, in addition to the primers present in solution, primers immobilized on the inner walls of the cavities (or even exclusively immobilized primers are used, for example in WO) 96/04404), which then likewise hybridize with single-stranded “template” molecules and can subsequently be incorporated by primer extension.
  • immobilization of the nucleic acid molecules takes place by means of primers immobilized on surfaces of the sample chambers, immobilization of only one primer of a primer pair is advantageous , because in this way only one strand of nucleic acid is immobilized by a double-stranded nucleic acid molecule, so that the separation of the non-immobilized strand of nucleic acid is facilitated, so that nucleic acid molecule ule can be provided with a single-stranded section.
  • immobilization of a nucleic acid molecule in solution can also be understood to mean the synthesis of a complementary strand molecule which is complementary thereto by extension of an immobilized primer hybridized to the dissolved molecule, that is to say a "transcription" of the molecule from the liquid to the solid phase.
  • a preferred embodiment of the invention relates to a method for parallel nucleic acid sequencing by enzymatic strand extension, wherein in step (i) the nucleic acid molecules have a double-stranded and a single-stranded section and in step (ii) the nucleotide compounds are nucleotides and the solution has a strand-extending enzyme in addition to the nucleotides and in step (iv) the enzyme forms the hydrogen bonds to the nucleotides binds single-stranded sections of the immobilized nucleic acid molecules and thus indirectly binds to the porous support and incorporates them into the immobilized nucleic acid molecules at the boundary between the double-stranded and single-stranded section.
  • This preferred embodiment of the method according to the invention for parallel nucleic acid sequencing by enzymatic strand extension thus comprises the following steps (i) providing a monolithic porous support, comprising at least two sample chambers which extend through the porous support and which have at least one
  • step (iii) introducing the solution from step (ii) into the sample chambers of the porous support, the enzyme binding the nucleotides to the single-stranded sections of the immobilized nucleic acid molecules with the formation of hydrogen bonds and thus indirectly binding to the porous support and at the boundary between double-stranded and incorporates single-stranded section into the immobilized nucleic acid molecules, (iv) detection of the amount and / or identity of the nucleotides indirectly bound to the porous support via the immobilized nucleic acids at the at least two distinguishable locations of the porous support.
  • step (ii) and the subsequent steps are repeated, each with a different nucleotide than in the previous step (ii).
  • the nucleic acid molecules are preferably sequenced by enzymatic strand extension using one of the methods now to be described.
  • step (ii) contains only one of the four nucleotides dATP, dGTP, dCTP and dTTP and step (iv) comprises the detection of the amount of the immobilized nucleic acids indirectly on the porous support bound nucleotides by determining the amount of reaction by-products formed and step (iv) is followed by a step (v) which comprises the removal of unincorporated nucleotides and optionally reaction by-products from the porous support if step (ii) and the subsequent steps are repeated , each with a different nucleotide than in the previous step (ii).
  • the cavities or channels of the porous support are flowed through with the flow arrangement by a solution which contains a certain deoxynucleoside triphosphate, for example dATP or its thio-analogue dATP S, and further components necessary for strand extension, such as the polymerase and optionally, in each sequencing cycle Contain buffers, ions, etc.
  • a solution which contains a certain deoxynucleoside triphosphate for example dATP or its thio-analogue dATP S
  • further components necessary for strand extension such as the polymerase and optionally, in each sequencing cycle Contain buffers, ions, etc.
  • the amount of pyrophosphate released in the sample chambers when a certain nucleotide flows through them corresponds to the amount of ATP formed. This is determined spatially resolved luminometrically.
  • the locations in the porous support at which a nucleotide was incorporated into the growing nucleic acid strand can thus be determined.
  • a second specific nucleotide triphosphate for example dCTP
  • dCTP a second specific nucleotide triphosphate
  • Unincorporated dCTP is removed, usually by flowing a wash solution through the sample chambers of the porous support, and the procedure described above is repeated with a third and fourth nucleotide triphosphate.
  • a next reaction cycle consisting of the time-delayed addition of a nucleotide triphosphate, the measurement of the amount of ATP formed and the removal of unincorporated nucleotide triphosphate is carried out and this is repeated as often as desired.
  • a nucleotide triphosphate it can be determined from the relative signal strengths whether one or more identical nucleotide bases were incorporated into the growing strand in the course of the strand extension, so that the sequence and strength of the spatially resolved chemiluminescence signals obtained result in the base sequence of the nucleic acid strand, of the template can be reconstructed at the respective location of the porous support. In this way, the sequences of the nucleic acids can be identified at different locations on the porous support and the base sequence in the sequenced sequence section can be determined.
  • step (ii) contains only one of the four nucleotides dATP, dGTP, dCTP and dTTP, which are each marked by a labeling group
  • step (iii) is followed by a step (iii b) which comprises the removal of unincorporated nucleotides from the porous support and in step (iv) the amount and / or identity of the nucleotides indirectly bound to the porous support via the immobilized nucleic acids is determined by determining the amount and / or identity of the marker groups bound on the support at at least two distinguishable locations of the porous support.
  • step (ii) and the subsequent steps are repeated, each with a different nucleotide than in the previous step (ii).
  • a condition which favors the nucleic acid molecule to be sequenced is incubated under a polymerase-catalyzed replenishment reaction with in each case one type of labeled nucleotide, ie with labeled dATP, dCTP or dGTP or dTTP.
  • a polymerase-catalyzed replenishment reaction with in each case one type of labeled nucleotide, ie with labeled dATP, dCTP or dGTP or dTTP.
  • the signal strengths measured in the course of a detection result in each case from the sum of the signal strength from the nucleotide incorporation of the last nucleotide incorporation carried out and all nucleotide incorporations previously carried out.
  • a nucleotide triphosphate it can be determined from the relative signal strengths whether one or more identical nucleotide bases were incorporated into the growing strand in the course of the strand extension in one cycle, so that the sequence and strength of the spatially resolved marker signals obtained result in the base sequence of the nucleic acid molecule can be reconstructed at the respective location of the porous support (i.e. spatially resolved). In this way, different areas or distinguishable locations of sequence-identical nucleic acids on the porous support can be identified and the base sequence in the sequenced sequence section can be determined.
  • the nucleotides are reversibly marked and the marking of already incorporated nucleotides is deleted in order to reduce the signal background after one or more passes through steps (ii) to (iv).
  • method (C) the incorporation of reversibly labeled nucleotides
  • the procedure is as in method (B), except that at appropriate times, for example after each addition of nucleotides or after each cycle, one consists of the successive addition of all four different nucleotides or one - or repeated repetition of the cycle, the marking of the incorporated nucleotides deleted. This is preferably done by removing or changing the marking group or the marking groups.
  • the marker group can have a chemically, photochemically or enzymatically cleavable spacers, such as a spacer containing a disulfide group or a nitrobenzyl group, can be bound to the corresponding nucleotide which is cleaved photochemically or chemically. If the enzymatic cleavage is selected, this is preferably done by flowing through the cavities or channels of the porous support, with the aid of the flow arrangement with a solution which contains the cleaving enzyme. Attachment of the labeling group to the nucleobase of the nucleotide is very suitable.
  • the photochemical cleavage takes place by excitation of the cleavable group with light of suitable intensity and wavelength, for example with the aid of a laser.
  • One way of changing the marking group would be, for example, to bleach a fluorescent dye, which would be possible, for example, by sufficiently intense laser radiation.
  • the advantage of method (C) over (B) is that only one part of the incorporated nucleotides, ideally only the last incorporated nucleotide, is determined during a measurement without a signal background due to the previously incorporated nucleotides, which often is in the multiple of the signal of interest, must be taken into account.
  • the solution from step (ii) contains one or more nucleotides selected from dATP, dGTP, dCTP and dTTP, the nucleotides having a removable group which leads to chain termination and has a labeling group
  • Step (iii) is followed by a step (iii-b) which comprises the removal of unincorporated nucleotides from the porous support and in step (iv) the amount of nucleotides indirectly bound to the porous support via the immobilized nucleic acids is detected by Determination of the amount of the label groups bound on the support at at least two distinguishable locations of the porous support and between step (iii-b) and step (iv) or between step (iv) and step (v) step (vi) is carried out, which the Removal of the chain terminating group from nucleotides bound to the porous supports.
  • This preferred embodiment of the method according to the invention for parallel nucleic acid sequencing by enzymatic strand extension thus comprises the steps: (i) providing a monolithic porous support comprising at least two
  • Sample chambers which extend through the porous support, which have at least one inlet and one outlet opening and which one or more
  • nucleotides selected from dATP, dGTP, dCTP and dTTP where the nucleotides have a removable group which leads to chain termination and are labeled with a labeling group, (iii) introducing the solution from step (ii) into the sample chambers of the porous support the enzyme forming the hydrogen bonds
  • the removable bar that leads to the chain break is also the marking bar and the order of steps (iv) and (v) is not interchanged.
  • the sequencing according to method (D) by incorporating reversible break-off nucleotides can be carried out, for example, as described in US Pat. No. 5,302,509, US Pat. No. 5,798,210 or also in WO 01/48184, to which reference is made in full.
  • method (D) method then both labeled nucleotides as described under method (B) and reversibly labeled nucleotides as described under method (C) can be used, although the nucleotides have the additional property that they have a functional group have, which leads to chain termination, but which can be removed.
  • nucleotide-wise extension of nucleic acid strands is achieved by using nucleotide triphosphates which are reversibly blocked on their 3'-OH group and which polymerases can incorporate into a growing DNA double strand, but after their incorporation as chain extension terminators Act. If the blocking group is removed, a free 3 'OH group is restored so that a next nucleotide can be incorporated.
  • Canard and Sarfati (Gene 148, 1-6 [1994]) describe reversibly blocked nucleotide triphosphates, which can be identified on the basis of a fluorescent label of the reversible protective group after their incorporation.
  • sample chambers of the porous carrier are flowed through in each sequencing cycle by a solution which contains a certain deoxynucleoside triphosphate or several, in particular all four
  • the non-incorporated nucleotides are usually removed by flowing a washing solution through the porous support and determining the location on the porous support at which a certain nucleotide has been incorporated in a locally resolved manner. In this way, the incorporation of a single nucleotide can be tracked per sequencing cycle, the information for all 4 nucleotide types being able to be obtained simultaneously if the solution with which the sample chambers of the porous support were flowing contained all four nucleotides.
  • nucleotides it is possible to offer all four types of nucleotides at the same time because, due to their function as chain terminators, only one nucleotide can be inserted per cycle until the blocking group is split off again, which can be done chemically, photochemically or enzymatically (see WO 01/48184) and before the spatially resolved determination of the location on the porous support on which a specific nucleotide has been incorporated or can take place afterwards.
  • the markings can be removed after one or more sequencing cycles, depending on the background that still appears tolerable, which depends, among other things, on the reading length.
  • the removable group that leads to the chain termination also represents the marking group, because the marking group therefore does not have to be deleted or removed separately. In this case, the order of steps (iv) and (v) is not reversed.
  • the described methods for sequencing the nucleic acid molecules by enzymatic strand extension (A) to (D) are based on the use of enzymes, usually DNA polymerases, which have a DNA single strand complementary to the DNA strand on which the DNA single strand is bound to extend. This presupposes that the nucleic acid molecules in step (i) have a single-stranded and a double-stranded section. If the nucleic acids are not present from the start, this state is brought about in steps (i-a4) and (i-b3).
  • immobilization of only one primer of a pair of primers is advantageous because in this way only one strand of nucleic acid is immobilized from a double-stranded nucleic acid molecule, so that the separation of the non-immobilized strand of nucleic acid is facilitated, thereby providing nucleic acid molecules with a single-stranded section.
  • a sequencing primer is used, i.e. an oligo- or polynucleotide which can hybridize with the nucleic acid strand to be sequenced and is present in the hybridized state in such a way that it can be extended by means of a DNA polymerase at its 3 'end, the complementary counter strand to the region to be sequenced being synthesized (intermolecular priming of the polymerase).
  • an immobilized double-stranded nucleic acid molecule optionally by completely or partially removing the counter strand into a state in which it has a single-stranded section, and then a suitable, at least partially complementary to the nucleic acid molecule sequencing primer, which has a 3 'end extendable by means of a polymerase, is hybridized with the nucleic acid molecule , so that the nucleic acid molecule is now in a state in which it has a single-stranded and a double-stranded section.
  • a primer in the course of the amplification of the nucleic acid molecules by PCR, a primer is used which has self-complementary regions (see WO 01/48184, page 9, first indent), so that the nucleic acid molecules amplified by PCR after partial or complete denaturation with partial or complete removal of the opposite strand, fold back to form a hairpin, creating single-stranded and double-stranded sections.
  • a hairpin structure can be attached to a nucleic acid molecule which has a single-stranded section, likewise producing single-stranded and double-stranded sections (intramolecular priming of the polymerase).
  • a "masked hairpin”, ie a double-stranded nucleic acid molecule containing an inverted repeat, can also be attached to the double-stranded nucleic acid molecule before immobilization. If, after immobilization, one of the two strands is removed by denaturation, the counter strand remaining on the nucleic acid molecule to be sequenced and attached to it with its 5 'end can then "fold back", producing single-stranded and double-stranded sections, and at its free 3' end be extended by means of a polymerase (see WO 01/48184, page 9, second indent).
  • the sequencing can also be carried out by other methods such as SBH (SBH, Sequencing By Hybridization; see Drmanac et al., Science 260 (1993), 1649-1652).
  • SBH Sequencing By Hybridization
  • a solution containing labeled oligonucleotides of known sequence and possibly other compounds which correctly hybridize the oligonucleotides with it flow through the sample chambers of the porous support at each sequencing cycle ensures complementary sequence segments on the nucleic acid molecules to be sequenced. If the labels are different, then the solution can contain as many types of oligonucleotides that differ in their sequence as can be distinguished on the basis of the labels.
  • Another embodiment of the invention thus relates to a method for parallel nucleic acid sequencing by hybridization, the nucleotide compounds in step (ii) being one or more oligonucleotides which have a labeling group and which in step (iii) form hydrogen bonds with the immobilized nucleic acid molecules hybridize so that the oligonucleotides are bound to the porous support and in step (iii) the amount of the oligonucleotides bound to the porous support is determined by determining the amount of the marker groups bound to the support at at least two distinguishable locations on the porous support.
  • step (iii) introducing the solution from step (ii) into the sample chambers of the porous support, the oligonucleotides being bound to the single-stranded sections of the immobilized nucleic acid molecules and thus being indirectly bound to the porous support, with the formation of hydrogen bonds;
  • Steps (ii) through (iv) can be repeated, with sequence information being obtained with each cycle.
  • the invention further relates to a monolithic porous support having at least two sample chambers which extend through the porous support, which have at least one inlet and one outlet opening and which have one or more surfaces to which nucleic acid molecules are immobilized.
  • the nucleic acid molecules which are immobilized on the porous support preferably have a single-stranded section and on the porous support there are at least two distinguishable locations which have nucleic acids of different sequences.
  • the invention further relates to a monolithic porous carrier, comprising at least two sample chambers which extend through the porous carrier, which have at least one inlet and one outlet opening and which have one or more surfaces, the surfaces bearing a coating which is used to immobilize Nucleic acids is suitable.
  • a further solution to the problem consists in a method for parallel nucleic acid sequencing, which comprises the steps:
  • a monolithic porous support comprising at least two liquid-filled sample chambers, which extend through the porous support and which have at least one entrance and one exit opening, the porous support having at least two distinguishable locations which have nucleic acids of different sequences, ( vii) amplifying the nucleic acid molecules in the sample chambers, (viii) providing a surface,
  • step (ix) contacting the surface from step (viii) with the porous support with formation of a liquid film between the surface and the sample chambers, (x) Immobilization of the nucleic acids from step (ix) on the surface to form at least two distinguishable locations on the surface which have nucleic acids of different sequences, steps (vii), (viii) (ix) and (x) being able to take place simultaneously , (xi) converting the nucleic acids on the surface into a state in which they have a single-stranded section, this step also being able to take place between steps (vii) and (x) or simultaneously with these steps,
  • step (xii) providing a solution which contains one or more nucleotide compounds selected from mono- and oligonucleotides, (xiii) contacting the solution from step (xii) with the nucleic acids on the surface from step (xi), the binding of the nucleotide compounds to the single-stranded sections of the immobilized nucleic acids and thus the indirect binding to the surface is effected,
  • Step (vi) the steps (vi-aO) providing a monolithic porous support comprising at least two sample chambers which extend through the porous support and which have at least one entrance and one exit opening,
  • step (vi) comprises the steps
  • vi-bO providing a monolithic porous support, comprising at least two sample chambers, which extend through the porous support and which have at least one entrance and one exit opening, (vi-bl) transfer of at least two nucleic acid solutions, each containing nucleic acid molecules of different sequences, to different locations on the porous support, so that at least two sample chambers are filled with the nucleic acid solutions, so that the porous support subsequently has at least two distinguishable locations that
  • the monolithic porous carrier from step (vi) is the porous carrier from step (i), which likewise has at least two sample chambers which extend through the porous carrier and which have at least one inlet and one outlet opening.
  • the sample chambers are filled with liquid, since otherwise neither the amplification in step (vii) nor the formation of a liquid film takes place in step (viii).
  • the porous support therefore has at least two distinguishable locations which have nucleic acids of different sequences. What was said under step (i) applies to the concept of places.
  • step (ix) the locations are transferred to a surface, projected, so to speak, onto a plane formed by the surface.
  • nucleic acids in step (vi) do not have to have single-stranded sections and also need not be immobilized.
  • the latter is not excluded as long as the mobilization is reversible or only affects a single strand of a nucleic acid duplex, so that at least the opposite strand in question can be detached by denaturation and transferred in step (ix).
  • the amplification takes place as described under (ib) and in particular with the aid of PCR.
  • a primer of a pair of primers or a portion of this primer could also be immobilized on the surfaces of the sample chambers.
  • a surface is provided in step (viii). This is the accessible surface of a body made of plastic, metal, glass, silicon or similar suitable materials, which allow the immobilization of nucleic acids and, if necessary, are appropriately functionalized.
  • the surface can have a swellable layer, for example made of polysaccharides, poly sugar alcohols or swellable ones Silicates.
  • the surface is a porous support as defined in step (vi).
  • Step (ix) comprises contacting the surface from step (viii) with the porous support. This can be done by simply placing the surface on the porous support. Since the sample chambers are filled with liquid, a liquid film is formed between the surface and the sample chambers. In the context of the invention, the term “liquid-filled” means the partial or complete filling of the lumen of a sample chamber. As a result of this measure, after immobilization in the subsequent step, at least two distinguishable sites are formed on the surface which have nucleic acids of different sequences. The nucleic acids of different sequences are transferred to the surface by diffusion or convection.
  • Step (ix) results in a projection of the distinguishable locations of the porous support onto a plane on the surface, whereby different locations on the surface are obtained after immobilization.
  • step (i-a3) or (i-b2) for the immobilization of the nucleic acid molecules in step (x).
  • the transfer of the nucleic acids in step (xi) into a state in which they have a single-stranded section is generally carried out by completely or partially denaturing a nucleic acid double strand and, if appropriate, removing the opposite strand. This step can also take place between steps (vii) and (x) or simultaneously with these steps. However, the transfer should be easier if the nucleic acids are already immobilized. Steps (i-a4) and (i-b3) can be referred to.
  • nucleic acids are preferably brought into a state in which they have a single-stranded section.
  • steps (xii), (xiii) and (xiv) can be referred to analogously, although in step (xiii) the liquid provided in the previous step is only included the surface is brought into contact. However, this does not rule out that the solution is introduced into sample chambers, if the surface is formed by a porous support which has sample chambers.
  • Steps (xii) to (xiv) can be repeated one or more times, with sequence information being obtained with each cycle.
  • step (xiv) detection is carried out as to whether nucleotide compounds are (indirectly) bound to the surface. If the solution in step (ii) contains several nucleotide compounds, then in step (xiv) it is checked which nucleotide compounds are involved, that is to say their identity is ascertained. It is usually necessary to measure the amount of nucleotide compounds bound in order to distinguish significant signals from the background. Under certain conditions, it is advisable to measure the quantity more precisely.
  • step (xiii) This is the case if, under certain circumstances, several nucleotide compounds can be bound to the single-stranded sections of the immobilized nucleic acids in step (xiii) and this allows conclusions to be drawn about the sequence, as in the case of sequencing by enzymatic beach extension with nucleotides without a chain-terminating group.
  • steps (xii), (xiii) and (xiv) are implemented as follows:
  • step (xii) providing a solution which has one or more nucleotides and a strand-extending enzyme, (xiii) bringing the solution from step (xii) into contact with the nucleic acids on the surface from step (xi), the enzyme forming the nucleotides with the formation of hydrogen bonds to the single-stranded
  • step (xii) and the subsequent steps are repeated, each with a different nucleotide than in the previous step (ii).
  • the sequencing of the nucleic acid molecules by enzymatic strand extension is preferably carried out by a method that will now be described.
  • step (xii) contains only one of the four nucleotides dATP, dGTP, dCTP and dTTP, which are each marked by a marking group and step (xiii) is followed by a step (xiii-b ), which includes the removal of unincorporated nucleotides from the surface and in step (xiv) the amount and / or identity of the nucleotides indirectly bound to the surface via the immobilized nucleic acids is determined by determining the amount and / or identity of the nucleotides Marking groups bound to the surface in at least two distinguishable locations on the surface.
  • step (xii) and the subsequent steps are repeated, each with a different nucleotide than in the previous step (xii).
  • the nucleotides are reversibly marked and the marking of already incorporated nucleotides is deleted in order to reduce the background signal after one or more steps through (xii) to (xiv).
  • steps (xii), (xiii) and (xiv) are implemented as follows:
  • step (xiii) contacting the solution from step (xii) with the nucleic acids on the surface from step (xi), the enzyme forming Hydrogen bonds which bind the nucleotides to the single-stranded sections of the immobilized nucleic acid molecules and thus indirectly binds to the surface and incorporates them into the immobilized nucleic acid molecules at the boundary between the double-stranded and single-stranded section, (xiii-b) removal of unincorporated nucleotides from the surface,
  • nucleic acids indirectly bound to the surface by nucleotides
  • the removable bar that leads to the chain termination is also the marking bar and the order of steps (xiv) and (xv) is not interchanged.
  • steps (xii), (xiii) and (xiv) are implemented as follows:
  • FIG. 1 shows a porous carrier for performing the method according to the invention
  • 2 shows the immobilization of nucleic acids in channels of the porous support
  • 3 shows the amplification of a suitably diluted solution of nucleic acid molecules in the channels of the porous support
  • 4 shows a flow arrangement for carrying out the method according to the invention
  • FIG. 5 shows a possible functional structure for the flow arrangement from FIG. 4
  • FIG. 6 shows the sequencing of immobilized nucleic acid molecules using reversible
  • Abbrachnukleotide; 7 shows the simultaneous sequencing of the immobilized nucleic acid molecules in several different areas of the porous support.
  • Fig. 1 shows a porous support for performing the method according to the invention, in detail
  • a device suitable for transferring nucleic acid solutions to the porous support such as a needle, a capillary or a nozzle,
  • 3 shows a section of the porous carrier, 4 solution of nucleic acid molecules taken up by channels of the carrier mediated by capillary forces,
  • 11 represents nucleic acid molecules of the same type immobilized on the wall of the channel.
  • FIG. 6 shows channels in which no amplification of nucleic acid molecules took place.
  • Fig. 4 shows a flow arrangement for carrying out the method according to the invention with Fig. 4a: flow arrangement after insertion of the porous support and Fig. 4b: flow arrangement in operation. Show in detail
  • FIG. 5 shows a possible functional fracture for the flow arrangement from FIG. 4.
  • FIG. 7 shows the simultaneous sequencing of the immobilized nucleic acid molecules in several different areas of the porous support, with 1 a section of the support containing different areas, the signals obtained during the sequencing of a first base being identified in the figure by different filling patterns, 2 one the same Areas of the carrier which contain areas, the area at
  • FIG. 5 shows a superimposition of the results obtained in the sequencing of the first to the nth base for all the regions detected
  • FIG. 6 shows the sequencing results obtained in (5) for the first to the nth base of the nucleic acid molecules immobilized in the regions detected.

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Abstract

L'invention concerne un procédé de séquençage parallèle d'acide nucléique comprenant les étapes suivantes : (1) préparer un support poreux comportant des zones dans lesquelles se trouvent des molécules d'acide nucléique immobilisées ; (2) insérer le support obtenu à l'étape (1) dans un système d'écoulement ; (3) déterminer simultanément au moins une partie de la séquence nucléotide d'au moins une partie de la molécule d'acide nucléique.
EP02807482A 2002-05-29 2002-08-09 Procede de sequencage parallele d'un melange d'acide nucleique a l'aide d'un systeme d'ecoulement Withdrawn EP1511856A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10224339A DE10224339A1 (de) 2002-05-29 2002-05-29 Verfahren zur hochparallelen Nukleinsäuresequenzierung
DE10224339 2002-05-29
PCT/EP2002/008918 WO2003102231A1 (fr) 2002-05-29 2002-08-09 Procede de sequençage parallele d'un melange d'acide nucleique a l'aide d'un systeme d'ecoulement

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EP (1) EP1511856A1 (fr)
JP (1) JP2005527242A (fr)
AU (1) AU2002333379A1 (fr)
CA (1) CA2487534A1 (fr)
DE (1) DE10224339A1 (fr)
WO (1) WO2003102231A1 (fr)

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DK1907583T4 (da) 2005-06-15 2020-01-27 Complete Genomics Inc Enkeltmolekyle-arrays til genetisk og kemisk analyse
EP2546360A1 (fr) * 2005-10-07 2013-01-16 Callida Genomics, Inc. Réseaux de molécules simples auto-assemblées et leurs utilisations
US7960104B2 (en) 2005-10-07 2011-06-14 Callida Genomics, Inc. Self-assembled single molecule arrays and uses thereof
SG10201405158QA (en) 2006-02-24 2014-10-30 Callida Genomics Inc High throughput genome sequencing on dna arrays
EP1994180A4 (fr) 2006-02-24 2009-11-25 Callida Genomics Inc Séquençage génomique à haut débit sur des puces à adn
DE102006033875A1 (de) * 2006-07-21 2008-01-31 Siemens Ag Analysesystem basierend auf porösem Material für hochparallele Einzelzelldetektion
US8592150B2 (en) 2007-12-05 2013-11-26 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
US9524369B2 (en) 2009-06-15 2016-12-20 Complete Genomics, Inc. Processing and analysis of complex nucleic acid sequence data
CA2856163C (fr) 2011-10-28 2019-05-07 Illumina, Inc. Systeme et procede de fabrication de micropuces
DE102017218849A1 (de) * 2017-10-23 2019-04-25 Robert Bosch Gmbh Reaktionsträger für eine mikrofluidische Vorrichtung und Verfahren zur Bestimmung einer Nukleotidsequenz
WO2019191613A1 (fr) * 2018-03-30 2019-10-03 Arizona Board Of Regents On Behalf Of The University Of Arizona Appareil de dosage moléculaire à écoulement vertical

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JP3488465B2 (ja) * 1993-10-28 2004-01-19 ヒューストン・アドバンスド・リサーチ・センター 結合反応を別々に検出する微細加工フロースルー多孔性装置
SE9402518D0 (sv) * 1994-07-18 1994-07-18 Pharmacia Biotech Ab Processing system
US5641658A (en) * 1994-08-03 1997-06-24 Mosaic Technologies, Inc. Method for performing amplification of nucleic acid with two primers bound to a single solid support
FR2726286B1 (fr) * 1994-10-28 1997-01-17 Genset Sa Procede d'amplification d'acides nucleiques en phase solide et trousse de reactifs utile pour la mise en oeuvre de ce procede
US6156502A (en) * 1995-12-21 2000-12-05 Beattie; Kenneth Loren Arbitrary sequence oligonucleotide fingerprinting
WO2001048184A2 (fr) * 1999-12-23 2001-07-05 Axaron Bioscience Ag Procede de sequençage parallele d'un melange d'acides nucleiques sur une surface
US7332271B2 (en) * 2000-02-18 2008-02-19 Board Of Trustees Of The Leland Stanford Junior University Apparatus and methods for parallel processing of micro-volume liquid reactions
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CA2487534A1 (fr) 2003-12-11
AU2002333379A1 (en) 2003-12-19
WO2003102231A1 (fr) 2003-12-11
JP2005527242A (ja) 2005-09-15
DE10224339A1 (de) 2003-12-11
US20080038718A1 (en) 2008-02-14

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