Nothing Special   »   [go: up one dir, main page]

WO1996027025A1 - Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire - Google Patents

Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire Download PDF

Info

Publication number
WO1996027025A1
WO1996027025A1 PCT/US1996/002342 US9602342W WO9627025A1 WO 1996027025 A1 WO1996027025 A1 WO 1996027025A1 US 9602342 W US9602342 W US 9602342W WO 9627025 A1 WO9627025 A1 WO 9627025A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
molecules
methods
molecule
particles
Prior art date
Application number
PCT/US1996/002342
Other languages
English (en)
Inventor
Ely Michael Rabani
Original Assignee
Ely Michael Rabani
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ely Michael Rabani filed Critical Ely Michael Rabani
Priority to AU51716/96A priority Critical patent/AU5171696A/en
Publication of WO1996027025A1 publication Critical patent/WO1996027025A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the invention relates to the massively parallel single molecule
  • first complex molecules which may be diverse, and second single or plural probing molecules, which may or may not be diverse, with applications to biology, biotechnology, pharmacology, immunology, the novel field of cybernetic immunology, molecular evolution, cybernetic molecular evolution, genomics, comparative genomics, enzymology, clinical enzymology, pathology, medical research, and clinical medicine.
  • the present invention has applications in the area of polynucleotide sequence determination, including DNA sequencing.
  • Mapping techniques include restriction enzyme analysis of genetic material, and the hybridization and detection of specific oligonucleotides which test for the presence or absence of particular alleles or loci, and may further be used to gain spatial information about the occurrence of their targets when appropriate analytic techniques are subsequently applied. Note that such characterizations presently are methodologically and operationally distinct from other processes comprehended within the biotechnological and related arts.
  • a modified nucleotide compound possessing two properties particularly useful for purposes of the present invention has been described by N.
  • This compound is 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate
  • This nucleotide bears a 3' protecting group linked via an ester function which should be susceptible to hydrolysis by appropriate chemical treatments.
  • the protecting moiety is suitable for photoactivation, and this property was utilized by those investigators to probe the structure of mitochondrial F 1 -ATPase, indicating that this analog will interact properly with at least some enzymes. Under appropriate circumstances, the protecting moiety may also serve as a label.
  • B Canard and R. S. Sarfati 2 have described similar nucleotides, here comprising all four nucleobases, with chemically removable 3'-hydroxyl protecting groups.
  • Said protecting groups comprise various fluorescent dye moieties.
  • These investigators have shown that these compounds may be added to appropriately primed polynucleotides by polymerases according to Watson-Crick base-pairing rules, and serve to terminate chain elongation in a manner which may be reversed by removal of said protecting groups by appropriate chemical treatments, admitting resumption of polymerization.
  • These workers propose that such compounds may form the basis of a novel sequencing methodology availing stepping control by means of said removable protecting groups and detection of labels following their release from the nascent strand by appropriate chemical treatment.
  • polynucleotide sequencing methodologies which depend on the addition of a polymerization terminating labeled nucleotide to a primed or elongated daughter strand on a polynucleotide sample with template dependent polynucleotide polymerases.
  • Most, but not all, of these methods (referred to herein as previously disclosed base-addition sequencing schemes) avail nucleotide triphosphate monomers with some base-specific label which may be removed by some deprotection treatment. It must be emphasized that all of these other previously disclosed base-addition sequencing schemes examine not single molecules individually but rather large homogeneous populations of substantially identical molecules, wherein the observed signal used to identify label type originates from the totality of such a population of molecules rather than an individual molecule. It must be further
  • composition and structure but to populations comprising millions or more molecules of identical structure.
  • a careful reading of these prior disclosures reveals that these investigators are not working with samples consisting of single molecules but rather with samples comprising a plurality of identical molecules.
  • these investigators do not (as is consistent with conventional usage) explicitly note this point, they take measures which would apply only to samples of pluralities of identical molecules, and do not take measures associated with working with single molecules.
  • nucleotide triphosphates comprising removable fluorescent 3' protecting groups.
  • the present invention provides methods of detection and discrimination which address the complexity found in biological systems, though they may further be applied to non-natural systems including but not limited to mimetics. Much of this complexity derives from combinations or
  • polydeoxyribonucleic acids and polyribonucleic acids or the twenty common amino acids found in polypeptides and proteins.
  • Combinatorial chemistry, affinity characterization, therapeutic synthetic immunochemistry, pharmacology and drug development, in vi tro evolution and other fields concerned with the elaboration of a diverse population of molecules, their characterization according to desired properties, and recovery or identification of molecules displaying suitable characteristics may be favorably improved by the availability of methods which permit the introduction of and both qualitative and quantitative characterization of kinetic and equilibrium properties of molecular recognition and binding phenomena, particularly where such parameters may be used as selective constraints.
  • the present invention approaches the vastness of biological complexity through massive parallelism, which may conveniently be attained through various single molecule examination (SME) methods variously referred to heretofore as single molecule detection (SMD) 3 , single molecule
  • SMV single molecule visualization
  • SMS single molecule spectroscopy
  • composition of complex molecules including co-polymers of natural or of synthetic origin
  • determinations of interactions between large numbers of molecules may be applied to genome-scale sequencing methods.
  • the latter case may be applied to rapid determination of molecular complementarity, with applications in (biological or non-biological) affinity characterization, immulogical study, clinical pathology, molecular evolution (e. g. m vi tro evolution), and the
  • equilibria may be examined.
  • Kinetics may be examined by observing the rates of occupation of appropriate sites or diverse populations thereof by some homogenous or heterogeneous sample, and the rates of vacancy formation from occupied sites.
  • Equilibria constants may be determined by observing the proportion (number of occupied sites divided by number of total sites) of sites occupied under equilibrium conditions, with greater quantitative confidence yielded by, for example, examining more binding sites.
  • Priming which may be random or non-random, is effected by any of a variety of methods, most of which are obvious to those skilled in the relevant arts Genome sequencing applications availing of enzymatic polymerization's and corresponding embodiments of the present invention, rely upon control over polymerization rate and nucleotide incorporation specificity, consistent with the well-known Watson-Crick base pairing rules which may to enforced (upon single nucleotides in a processive manner, as conditions permit) by the use of DNA polymerases or analogs thereof, in combination with repeatable single molecule detection applied to a large population diverse molecules.
  • a sequencing cycle comprises the steps of: (1.) polymerizing one or less nucleotides, which carry some removable or neutralizable molecular label and may optionally be reversibly 3' protecting (or otherwise protected in any manner which modulates polymerization rd'' onto each sample molecule at the primer or at subsequent extensions the and in opposition to (and pairing with) a single unique, base of the template polynucleotide strand; (2.) optionally washing away any unreacted labeled nucleotides; (3.) detecting, by either direct or indirect methods said labeled nucleotides incorporated into said sample molecules, in a manner which repeatably associates information obtained about the type of label observed with the unique identity of the template molecule under observation, which may be uniquely distinguished by a variety of methods (which include: a mappable location of immobilization of the sample template molecule on a substrate surface; a mappable location of
  • the sequence determination applications of the present inveition enjoys substantial advantages deriving from sample manipulation in the single-molecule-regime.
  • Working instead in the distinct single-molecule- regime rather than with populations of identical molecules provides substantial advantages of parallelism, facility of use and implementation (including automated implementation,) and operability Among these are unanticipated advantages: (1) because a single molecule is necessarily monodisperse, failure of a molecule to undergo addition in a cycle does not cause a loss of sample monodispersion (i.e.
  • samples comprising multiple identical molecules may thus take on non-identical lengths, complicating data collection and analysis;
  • samples comprising a plurality of individually distinct single molecules (species) may be handled unitarily without requiring any handling measures to keep distinct molecules apart, providing a large reduction in manipulations required on a per-species basis and not requiring the use of many separate, parallel fluid handling steps or means;
  • inadvertent multiple base additions are more readily detected and their extent is more readily quantified because these changes in quantity are large compared to the signal expected from the incorporation of a single base (i.e. single label) into a single molecular species;
  • deprotection or delabeling failures may also be readily detected and noted for the correct single molecule, such that addition failure, the presence of a label, or overlabeling in the
  • oversampling i.e. the examination of some multiple (j) of the number (m) of sample molecules suggested by combinatoric computations to be minimally sufficient for full alignment of data from a sample of a given complexity.
  • oversampling redundancy will increase the confidence interval for accuracy of collected data and reduce the likelihood of artifacts arising from sequence duplications which may occur in any given sample.
  • Oversampling redundancy may be availed to increase data confidence by providing the opportunity to score and match multiple occurrences of the same sequence segment and thus detect and eliminate erroneous sequence segment information by virtue of its less frequent occurrence
  • Erroneous sequence segment information may arise for instance by nucleotide incorporation errors which are an inevitable feature of polymerization with polymerases having a characteristic fidelity, i.e. displaying a characteristic nucleotide misincorporation rate.
  • Such methods will be particularly useful where polynucleotide polymerases fidelity would otherwise be unacceptably low. It should be noted that an error rate of one percent or more has been deemed conventionally acceptable for genome lnformatic purposes.
  • known molecules having sequences that are highly unrelated to the sample may be included as internal controls to monitor the efficiency and accuracy of a particular sequence collection process; such internal control sequences will present negligibly small overhead because molecular parallelism may easily accommodate any such comparatively small increase in sample complexity, even though it might be considered large with respect to pre-existing methods.
  • data alignment may be performed in tandem or parallel with later cycles and may be monitored by appropriate computational algorithms for data quality and confidence of sequence information, and cycling may continue till desired criteria are satisfied.
  • Computer, microprocessor, electronic or other automated control of instrumentation, including fluidics and robotics for the manipulation of samples, and the automated effectuation of the various methods of the present invention, all according to parameterized algorithms, may be accomplished by means obvious from the present disclosure to those skilled in the relevant arts (e.g. fluidics, robotics, electronics, microelectronics, computer science and engineering, and mechanical engineering).
  • Concurrent data alignment and monitoring will permit modifications of the sequencing cycle described above, such as dynamic adjustment of polymerization reaction conditions and durations, label removal or neutralization procedure parameters, polymerization deprotection conditions, and any other desired parameter, so as to permit optimization of procedures and results.
  • polynucleotides may be examined. Where single stranded polynucleotide molecules are preferred, second strands may be removed by performing said immobilization so as to only involve only one strand in covalent linkage with said surface and then performing a denaturation of the sample with washing. Priming means required by any particular enzyme must then be provided, usually by hybridization of a complementary oligo- or
  • polynucleotide to the sample template molecules, though other means are possible.
  • Other methods which will be obvious to those skilled in the arts of recombinant DNA technology may also be employed to yield immobilized or otherwise uniquely identifiable single stranded polynucleotide samples Where double stranded molecules are preferred, said second strands may be treated with an appropriate exonuclease under appropriate conditions and for an appropriate lengths of time to provide a good distribution of lengths of said second strands such that the termini of the undegraded portions of said second strands provide convenient priming for enzymatic nucleotide polymerization (i.e. DNA directed DNA synthesis or DNA
  • RNA directed RNA synthesis or transcription DNA directed RNA synthesis or transcription
  • RNA directed DNA synthesis or reverse transcription DNA directed RNA synthesis or reverse transcription
  • polynucleotide sequencing methods of the present invention represent the converse of conventional enzymatic and chemical sequencing methods in that those conventional methods rely upon the production of multiple homogeneous sub-populations of DNA molecules which together comprise a nested set, and the detection of each of such sub-population (with deviant chain terminator misincorporation molecules arising with significantly lower frequency and thus constituting a poorly detected population), while the present invention relies on alignment of information from a highly inhomogeneous population molecules and repeatable detection of single molecules.
  • each species yields information about only one base at one position within the sample sequence, while with the methods of the present invention, each individual sample template molecule may yield information about the identity of several bases.
  • some effort has been expended in increasing the number of bases yielding information per sample i.e. lengthening the linear sequence information obtained from any one segment of a sample, which is substantially frustrated by the inherent limitations of electrophoretic separation and particularly gel
  • instrumentation or robotics may perform each relevant step, and that the ensemble of such instrumentation may readily be integrated to form a coordinated system, for example matching throughput at different stages by adjusting the parallelism of appropriate stages
  • throughput and data accuracy are tradeoffs, but may individually vastly exceed any such measures attainable with conventional methods.
  • nucleotide shall comprehend both the standard four deoxyribonucleotides or the standard four ribonucleotides, as well as any variations thereupon which pair with similar or other bases according to definable pairing rules.
  • nucleotide analog shall refer to any naturally occurring or synthetic variation thereupon which may be utilized within the unity of the present invention to gain sequence information about a given polynucleotide sample.
  • nucleotides are all 5 triphosphates
  • Phosphorothioate derivatives of nucleotides may be employed interchangeably with these without departing from the essence of the present invention.
  • Said phosphorothioates provide the additional advantage upon polymerization of yielding polynucleotide backbones which are less susceptible to the intrinsic exonuclease activity of various polynucleotide polymerases Alternatively, polymerases with no detectable intrinsic exonuclease activity, or with no detectable 5' to 3' exonuclease activity, may be favorably used.
  • polymerase (as well as the term transcriptase) shall refer to any molecule or complex capable of enforcing fidelity of pairing on single nucleotides at a structurally defined site of a template polynucleotide molecule, whether or not said any molecule or complex itself catalyses the addition of said single nucleotide to said structurally defined site or not, and whether said any molecule or complex is a naturally occurring enzyme or ribozyme or sythetically derived catalyst such as an abzyme with the appropriate catalytic specificity or an artificial receptor molecule or any purely synthetic assemblage capable of enforcing high fidelity of comonomer association (e.g. base pairing) according to well defined rules.
  • microscopy methods such as video microscopy including confocal fluorescence microscopy with or without enhancement, and with or without variations incorporated into the present invention; near field scanning optical microscopy (NFSOM) 8 and variations thereof; contact and non-contact varieties of scanning force microscopy (SFM; also termed atomic force microscopy [AFM]) 9 and variations thereof; other scanning probe microscopies including scanning tunneling microscopy (STM), scanning tunneling spectroscopy (STS) 10 , and so-called field emission mode STM (which is more accurately described as microscopy by field emission from a scanned conductive probe, or scanning field emission microscopy, SFEM, because no tunneling actually occurs) 11 Any
  • optical detection methods employing optoelectronic array devices (OADs), such as spatial light modulators (SLMs), laser diode arrays (LDAs), light-emitting diode arrays, or charge coupled photo-diode arrays (conventionally termed CCDs), in combination with appropriately high sensitivity detection methods, may also be employed, particularly with samples immobilized such that the maximal proportion of pixel elements of said array will be involved with the detection of a signal from exactly one sample molecule CCD and SLM array device are presently available at pixel densities of approximately 10 5 to 10 6 per cm 2 LDAs of comparable density are currently under development. Device level constraints upon parallelism will thus be significant , but may be overcome by increasing the data obtained per molecule (i. e.
  • Such devices may be employed remotely, i.e. in some arrangement where light passes through the sample under study and is detected by some apparatus involving said array devices, or in close or direct contact with said sample, as for instance, polynucleotides have been immobilized to
  • molecules may be labeled by a first label, for example with a particular fluorescent dye incorporated by nick translation, in a manner identifying a portion of the molecule near the site of
  • Conditions such as solution viscosity, sample molecule diffusion rate, sample molecule concentration, sample dimensions, etc., may be optimized to reduce the occurrence of such unacceptable proximity, and oversampling methods described in other portions of the present disclosure may be applied to preclude this form of error from degrading final data quality These methods may be applied to either immobilized or unimmobilized sample molecules.
  • Light microscopic visualization represents a particularly convenient and technically simple detection and unique molecule localization method.
  • a visualization method of particular interest for purposes of the present invention in higher performance or more demanding applications is video enhanced confocal fluorescence microscopy (VECFM) 12 , preferably utilizing optics well matched to the refractive index of the reaction or detection medium.
  • VCFM video enhanced confocal fluorescence microscopy
  • microscopy based detection method must be sufficiently convenient, capable of use with a stepper translated or otherwise translatable sample, not destructive of the sample, and capable of detection of any labeling methodology to be used with it
  • microscopy methodologies not yet developed may readily be employed with the present invention.
  • microscopy and corresponding apparata shall comprehend any miniaturized or microfabricated microscopy devices or other comparable integrated detection means.
  • VECFM which is particularly suited for SMD and SMV relies upon selective fluorescent excitation of an appropriate dye molecule label (or of molecules within a sample with appropriate fluorescent properties independent of labeling) in some sample by means of some tightly defined beam, with dimensions at or near the resolution limit of the apparatus, of an appropriate frequency, or of parametrically controllable frequency, where said beam is caused to scan in a controlled manner through the sample region within the visual field
  • This microscopy including numerous variations, may be termed either scanned beam confocal microscopy or steered beam con focal microscopy (in either case, SBCM).
  • Scanning of said beam through the sample within the visual field may be accomplished by introducing said beam into the optical path of the VECFM via mobile mirrors which may effect said controlled scanning, or by first producing said beam with a pinhole which is itself scanned, before deflection towards the sample via said mirrors, which in the present case may be fixed in position, through the use of pinholes in a rotating disk arranged in one or more spiral arms to effect an approximately rastering illumination of the sample as said disk rotates, or by other means which will be obvious to those skilled in the design of optical instrumentation and microscopy.
  • An optical splitter may then redirect a fraction of the light transmitted from the sample through the objective lens, and direct it through a narrow bandwidth, high transmissiveness filter which ma, be specific for a fixed or for a parametrically
  • controllable variable frequency to uniquely select the appropriate fluorescent emission frequency
  • a highly sensitive photodetector which may record either intensity as intensity information or as the number of photons detected per unit time, as a function of the region being subjected to fluorescence exiting illumination or being distinctly observed (see below).
  • the entire sample of visual field may be subjected to illumination by an appropriate excitation frequency, and a pinhole scanned through the portion of the output of said optical splitter, such that light passing through said pinhole will reach said highly sensitive
  • an SLM may be used in place of said pinhole (in either configuration), and fluorescent excitatory illumination may be either broadly distributed or scanned.
  • sensitive photodetection may be accomplished with a highly sensitive CCD, and fluorescent excitatory illumination may be either broadly distributed or scanned.
  • CCD sensitivity approaching single photon detection is technically possible though is not practical for high volume
  • said scanned beam may originate from a laser diode array device or a light emitting diode array device, where only one of, or a contiguous group of elements of, such an array is active at any particular time so as to produce a particular beam, and the group of active elements of said such an array is changed as a function of time to effect scanning of the sample by the coordinated activation and deactivation of the plural beams thus produced.
  • spatial information is gained about any particular fluorescent emission, and this may then be combined with other visual information obtained via the same VECFM apparatus.
  • This information may then be subjected, for instance to averaging or other computations to determine the relationship between the location of the molecule within the beam and the intensity observed, and finally that information used to estimate the intensity which would be observed when such a calibration sample molecule is in the precise center of the beam.
  • This information may then be used in image enhancement of unknown samples. Note, however, that localization to below optical resolution limitations is distinct from increasing the resolution capability for two nearby objects.
  • a beam of predetermined frequency for instance delimited and scanned by means of a pinhole as described above, may be used to selectively modify a particular sample molecule.
  • a beam of predetermined frequency may be used to effect the photobleaching of the labeling moiety on a particular sample molecule, to selectively remove a photocleavable protecting group on a particular sample molecule, to selectively remove a moiety joined to a sample molecule by a photocleavable linker, or selectively control any photochemical reactions in a highly localized but non-invasive manner
  • implementations permitting variations of illumination frequency and/or variations of the frequency or frequencies selected by filters for detection purposes constitute microspectroscopy or
  • microfluorimetry and may be applied to any of the various light
  • Applicable methods include those described by S P A Fodor et al effect micropatterned surface immobilization and controlled synthesis of polypeptides and polynucleotides those described by M. Hegner et al. 14 : effect the end-wise immobilization of terminally thiol modified double helical DNA molecules to a gold coated surface, or those methods recently used by L. Finzi and J. Gelles 15 to effect end-wise attachment of DNA molecules to an antibody coated glass surface Many alternative methods will be obvious to those skilled in the relevant arts.
  • DNA from a cosmid library which may have been prepared from total genomic material, from a cDNA library derived from a particular tissue type, from a cosmid library which may have been prepared for a single chromosome or group of chromosomes or particular chromosome segments, or directly purified genomic DNA or directly purified RNA from a particular cell type, etc. may be subjected to fragmentation. Physical methods such as shearing with a hypodermic apparatus may be suitable. Where the sample is in the form of duplex DNA, it may be treated with restriction enzymes, which preferably restrict either 6- or 4-base recognition sequences, so as to produce sample molecules of mean length of either 4 kilobases or 256 bases, respectively. Such lengths are sufficiently short to yield a high number density of sample molecules Said sample molecules may then be
  • any matrix used in the present invention must admit the sufflciently rapid transport or diffusion of reaqents enzymes and buffers as required by the particular embodiment.
  • a sample For detection an discrimination within a volume, whether for matrix immobilized samples or diffusion constrained free molecules in solution especially where fluorescent labeling of one form or another has been employed, a sample may be examined by microscopy with reconstruction of three-dimensional spatial information by scanning the focal plane through the depth of the sample and collecting image data at appropriate intervals. Such methods of three-dimensional reconstruction are well known within the art of microscopy.
  • optical means such as moving slits or SLMs or laser diode arrays may be employed to selectively illuminate a particular region, preferably a single plane (of thickness similar to the wavelength of light employed or feature size of integrated device means employed), to examine a particular subset of sample template molecules and labels associated with them, providing spatial reconstructability of the data thus collected.
  • Volume distributed samples may also be examined with methods closely analogous to those recommended for three dimensional optical mass data storage, for instance, by Sadik Esener in U.S. Patent Number 5,325,324.
  • labels requiring excitation by photons of two distinct frequencies for photoemission may be employed.
  • the related methods of illuminating an entire plane of a sample with one of said distinct frequencies may be availed as a mechanism for imaging with spatial reconstructability. Immobilization via concatenation;
  • extended linear molecules such as polynucleotides
  • immobilization of said extended linear molecules may be conveniently effected by their concatenation with second extended linear molecules which are likewise conveniently circularized by appropriate treatments (which will again generally be obvious to those skilled in the relevant arts) bearing chemical properties (i.e. functional groups such as thiols or affinity moieties such as biotin) favorable for convenient, specific immobilization to a surface, matrix or other solid support.
  • said second extended linear molecules are favorably bound (with methods which will generally be obvious to those skilled in the relevant arts) at a predetermined location along their length to some protein, which may be an enzyme such as a polymerase, before
  • oligonucleotide or polynucleotide with or without ligation thereof, or some appropriate multifuctional binding protein or receptor.
  • said second extended linear molecule is bound to said enzyme; said protein is caused to bind to said first extended linear molecule (which may be circularized either in a prior or subsequent step), said second extended linear molecule to which said protein has been bound is caused to circularize by
  • first extended linear molecule is at this stage linear, it is caused to circularize. Without any special measures, there is a fifty percent chance that such a process will result in concatenation of the first extended linear molecule with the second extended linear molecule Numerous methods, such as size separation followed by retention by immobilization, may be used to purify the resulting desired concatenate. Where said second extended linear molecule was chosen to be relatively short, such an assemblage will provide for the retention of said first extended linear molecule, now in concatenated circular form, in proximity to said protein, with specific immobilization or convenient immobilizability.
  • said protein and said first extended linear molecule now in concatenated circular form have a high effective concentration with respect to eachother upon dissociation, and said protein and said first extended linear molecule now in concatenated circular form will not interact with the molecules of other such assemblages when said assemblages are at sufficiently low density or said second extended linear molecule now in concatenated circular form is particularly short (i. e. effectively shackles said first extended linear molecule now in
  • Such an immobilization scheme will be particularly desirable in for example, sequencing applications of the present invention where a polymerase must perform a cycle in which it binds modifies and releases a sample molecule at a high rate.
  • a polymerase must perform a cycle in which it binds modifies and releases a sample molecule at a high rate.
  • a particular instance in w hich such desirabi l ity obta ins is for sampl es to be anal yzed with long sequence segments (e.g. hundreds or thousands of bases) where dissociation of the polymerase is necessary to permit either 3 hydroxy deprotection (e. g.
  • sample molecule density may be overcome, with the new limit being that imposed by the detection method, thus increasing sample density and in some
  • the parallelism that thence may readily be achieved with detection methods such as microscopy. It is therefore feasible, with such assemblages, to collect sequence data dynamically from each molecule at a rate approaching the limits imposed by the slower of: the characteristic nucleotide incorporation rate of the polymerase; or, the diffusion rate limit of nucleotide association with the nucleotide binding site of the polymerase (divided by four) when nucleotides are at a sufficiently low concentration that their presence as labeled but free molecules in the detection field does not interfere with the detection (which may be time averaged according to the particular instrumentation used) of incorporated labeled nucleotides, which concentration will be dependent in part on the geometry of the liquid volume, or, the maximum rate of single label detection (but note that such a rate need not be low because detection rate will increase for multimeric labels, which may be employed).
  • Such an immobilization method will favorably be employed for embodiments locating sample molecules on or near the surface of a CCD or SLM. Note that kinetic control of polymerization rate (and hence stepping rate, e g by adjusting nucleotide concentration) is also enhanced by the use of such a
  • One such random immobilization method may avail of the invention of N.C. Seeman described in U.S. Patent Number 5,278 051 which provides a process for the construction of complex geometrical objects These methods may be applied to the production of regular two- and three-dimensional molecular lattices from polynucleotide compositions.
  • the process of this invention may be extended by the incorporation of appropriate affinity groups at predetermined locations within the objects, which for present purposes may favorably be small ligands such as biotin or digoxigeinn, which may then be used as the target for a sample molecule which has been terminally labeled by a similar small ligand which has subsequently been bound by (an excess of) an appropriate multimeric receptor..
  • Said multimeric receptor will then recognize and bind the complementary small molecule ligand incorporated into the structure of said lattice, and thus effect sample molecule immobilization according to the non-random pattern predetermined by the precise structure of said lattice and the precise distribution of ligands thereupon..
  • the objects provided by the invention of N.C. Seeman comprise polynucleotide structures, care must be taken in using such a sample substrate with the methods of the present invention to ensure that said objects will be stable to all treatments which are to be applied to sample molecules, including denaturation, exonucleolytic degradation, primer hybridization, exposure to active polymerases, etc.,.
  • constraints may be met by effecting topological closure of all strands such that no free polynucleotide terminus is carried on such a lattice, and no denaturation procedures will result in matrix dissociation; the methods of the invention of N.C. Seeman may be availed in a manner meeting these constrains..
  • the extremities of these lattices may be bound to solid supports which are then positioned so as to apply tensile stresses to said molecular lattices which will enforce constraints limiting flexural internal degrees of freedom and enforcing substantial spatial regularity on sample molecule distributions.
  • said appropriate affinity groups incorporated (directly or, by conjugation or other methods, indirectly) at appropriate sites in a lattice may be chosen so as to interact directly with polynucleotide sample molecules in a sequence dependent or independent manner.
  • Sequence dependent affinity binding may be effected with oligonucleotides or analogs thereof capable of forming double-, triple- or quadruple helices with said sample polynucleotides, ribozymes, or sequence dependent binding proteins including but not limited to: transcriptional activators (e.g. TATA- Binding Protein), enhancers and repressors, integrases; restriction enzymes; replicator proteins (e.g.
  • DnaA DNA repair proteins
  • anti-polynucleotide antibodies e. g. snRNPs
  • RNA binding proteins all under conditions permitting desired selectivity, specificity or stringency but, where appropriate, preventing polynucleotide cleavage or degradation.
  • sequence specific binding is desired, and hierarchially prepared lattices are used, the distribution of particular specificities may be controlled by the staged incorporation of said affinity groups at various hierachial levels of the synthetic procedure This will permit classification of sequence data according to the location of the sample template molecule from which it is obtained in the lattice (i.e. on the surface or within the matrix).
  • Sequence independent binding of polynucleotides may be effected by the use of proteins such as RecA, histones, U1, etc., Repeatable identification of unimmobilized molecules:
  • molecules may be perceptibly labeled, for example by perceptible microscopic beads or the incorporation of a first fluorescent label, and tracked by the use of image analysis
  • nucleotide labeling does not necessitate the use of large beads or other complexes for detection Instead, single or oligomeric fluorescent labeling moieties, or enzymatic label affinity conjugation are preferred, such that labels may be removed without greatly disturbing the trajectory of said respective sample molecules.
  • Either the direct colocalization (to within the resolution of the imaging method) of nucleotide label with said first fluorescent label or reductions in the Brownian motion of said nucleotide label sufficiently near (e.g. closest to) said first fluorescent label may be exploited in the detection of nucleotide label incorporation.
  • Labels may be visually discriminatable, or may be diverse affinity labels or combinations thereof. Labels of this type may conveniently be random combinations of some basis set of distinct labels, formed for example, by a random coupling or polymerization of such labeling moieties to a defined chemical site provided by chemical modification of sample molecules.
  • Visual labeling may be accomplished by the use of a sufficient number of distinguishable fluorescent dye molecules, or other visual labels, such that the presence or absence of association of any one of said
  • distinguishable fluorescent dye molecules may comprise the state of a bit in a binary code. Such labeling is similar to the combinatorial encoding described by S. Brenner and R. A. Lerner 18 , but differs in that:
  • perceptible labels may be used for encoding; labels need not be genetic material or linear copolymers, where only unique identiflability is required, the label moiety employed for encoding may be synthesized separately and possibly randomly, and bound possibly randomly with sample molecules; the information contained by each labeling moiety need not depend on its precise spatial association with sample molecules, or its location within a sequence, only its sufficient proximity; and, because of such modes of independence between the encoding, which serves here only for purposes of unique labeling, difficulties which may arise for particular orthogonal polymerization chemistries of different copolymer types may be avoided either by separate synthesis.
  • biopolymers and possibly for specifically encoded libraries the use of specific enzymes which may for example ligate polynucleotides or polypeptides, may be used to specifically control reactions and prevent polymerizations of one biopolymer from affecting a second, linked biopolymer.
  • specific enzymes which may for example ligate polynucleotides or polypeptides, may be used to specifically control reactions and prevent polymerizations of one biopolymer from affecting a second, linked biopolymer.
  • moieties different from biologically occurring comonomers may be used as encoding label moieties, via functionalization of appropriate biopolymer segments with such moieties, in synthetic manners which will be obvious to those skilled in the relevant arts, or may be used, similarly, as constituents the random library thus encoded.
  • detectable multiplications of other photolabels may be used to effect higher modulo coding of labels.
  • the labeling methods of the present invention suggest a convenient solution to the problem recognized by Brenner and Lerner 19 , as limiting the facility of their encoding system, i. e. the requirement of separate distinct comonomer (or co-oligomer) type addition steps for each polymer type.
  • This prevents the use of highly random (but step-controlled) synthetic preparation of such encoded libraries, because the information encoded is realized by individual preparative synthetic steps, i.e. all of the information content of the encoding is conferred upon these compounds by the intervention or agency of a chemist (or automated systems) at each step.
  • Such encoded libraries of either the sequence encoded or modulo encoded types, including compounds comprising more than two polymer types, may be prepared with the following stepped random method in one container (with or without the favorable use of solid phase synthetic methodologies).
  • random refers to the mixture of two or more multimacromonomers in each addition step, such that addition to all compounds under preparation will occur in a random manner within the reaction mixture, in a manner weighted according to the relative
  • Such multimacromonomers may also be used in more directly controlled addition schemes with advantages which will be obvious to those skilled in the relevant arts.
  • Multimacromonomers comprising two or more monomer (or macromonomer) types (e. g. comprising an amino acid monomer and a trinucleotide oligomer, or an amino acid monomer, a trinucleotide oligomer and a fluorescent or affinity labeling moiety) may be prepared by joining some or all of said two or more monomer (or macromonomer) types by cleavable linkers such as those described in other sections of the present disclosure.
  • monomer (or macromonomer) types e. g. comprising an amino acid monomer and a trinucleotide oligomer, or an amino acid monomer, a trinucleotide oligomer and a fluorescent or affinity labeling moiety
  • each multimacromonomer may be added to compounds under synthesis by addition of one of the monomer or macromonomer types to the corresponding polymer or macropolymer types of said compounds under synthesis by appropriate polymer synthesis chemistry followed by addition of some or all of each of the remaining monomer or macromonomer types to the respective corresponding polymer or macropolymer types of said compounds under synthesis by appropriate polymer synthesis chemistry Control over the details of such additions may be effected by control over, for example, removal of distinct protecting groups from distinct polymer or macropolymer types of said compounds under synthesis by appropriate polymer synthesis chemistry.
  • Linkers or specific linker branches may be cleaved at appropriate steps or after synthesis has otherwise been completed
  • correspondence between the composition of each polymer or macropolymer type comprised within each molecule of the compound under synthesis (which final composition may vary widely from molecule to molecule of the compound under synthesis, but strictly observe the correspondence between composition of some or all of each of the polymers or macropolymers comprised within each molecule of the compound under synthesis) is provided by the communication of the distinct monomer or macromonomer types comprised within each multimacromonomer.
  • the first bond formed between a first monomer or first macromonomer of a multimacromonomer and a molecule of the compound under synthesis will thus ensure that other monomer or macromonomer types of the multimacromonomer which will be added at the respective multimacromonomer addition stage will correspond to the identity of the first monomer or first macromonomer thus added.
  • correspondence of some or all of each of the polymer or macropolymer types of final compounds is enforced (by the communication effected by, for example, linkers) even where the composition of some or all of the polymer or macropolymer types is respectively random.
  • such linkers (which may be multiply branched, each of such branches possibly comprising cleavable groups susceptible to distinct cleaving treatments) are held in communication with some or all of the two or more distinct monomer or macromonomer types (which are added to the compounds under synthesis with distinct and mutually non-interfering addition or polymerization, deprotection and/or activation chemistries, termed "orthogonal" chemistries in the respective art) by attachment to the protecting groups used to effect the stepping of additions of each such multimacromonomer.
  • Said diverse affinity labels may be used in conjunction with multiple affinity separation paths and nucleotide label detection that associates the detected said nucleotide label with the resolved location of the respective affinity labeled sample molecule, thus accomplishing the required assignment of detection and discrimination of the appropriate nucleotide label precisely to the correct respective sample molecule.
  • said diverse affinity labels may be added to sample molecules so as to be independently recognizable by appropriate receptor molecules or other affinity means, each complementarity type of which is respectively labeled with some distinct independently perceptible label.
  • Such labeling methods permit the processing of samples in fluid flow based apparata without the loss of single molecule identiflability or assignability of results
  • manipulation with a laser trap as for instance described by T.T. Perkins et al,, 20, 2 1 may be employed with such uniquely labeled molecules.
  • a synthesis directing informational molecule (favorably DNA or RNA) with the correspondingly synthesized one or more functional molecules (generally a polypeptide) may be effected by the in vi vo coupling or otherwise compartmentally enforced unique one-to-one corresponding coupling of said informational and said functional molecule.
  • a particularly convenient instance of such a molecules comprises the fused expression of said functional molecule or molecules as segments of the terminal proteins of the informational molecules (i, e, DNA) of various virus (e.g. adenovirus) or bacteriophage (e g PRD1 or phi29) genomes.
  • said functional molecules may be fused with some molecule which associates in a specific manner with said terminal proteins, and which has sufficient opportunity during its in vi vo synthesis, without or preferably with concurrent viral or bacteriophage replication, to associate with the terminal protein of the genomic material which determines the composition of said functional molecules, such that upon purification or lysis functional molecules remain in communication with the genetic material that determines their composition Because biosynthesis of functional and informational moieties may favorably occur within the confines of a single cell, cross-coupling of inappropriate molecules may be readily avoided.
  • polypeptide and polynucleotide moieties may be effected with some intermediate snRNP or snRNP-like moiety, where such an intermediate moiety may be targeted on the one hand by an appropriate affinity characteristic of one or more
  • Such complexes comprising an intermediate snRNP or snRNP-like moiety may also favorably be formed within the confines of a single cell.
  • Such polynucleotide-polypeptide chimera, or other molecule types comprising thus communicating and informationally corresponding chimera
  • polypeptide moiety has further been subjected to post-translational modification such as specific glycosylation and has been associated by some method to the respective genetic material determining its composition, for example by the sorting of individual cells carrying said genetic material in the form of a DNA vector with terminal proteins and expressing and processing said polypeptide, into distinct wells or vessels followed by disruption of membranes such that terminal proteins fused with peptides having affinity for the particular polypeptide of interest may come into contact with the processed polypeptide of interest, comprising a method for the molecular evolution of multiple-biopolymer containing macromolecules), which may be termed phenogenocouples, may be used as sample molecules with the broad methods of the present invention to effect the affinity characterization (including either or both equilibrium and kinetic characterization of molecular recognition including catalytic recognition and catalysis) of functional moieties and then the affinity characterization (including either or both equilibrium and kinetic characterization of molecular recognition including catalytic recognition and catalysis) of functional moieties and then the affinity characterization (including
  • Selected informational molecules may be selectively replicated or transcribed by activatable (e g photodeprotectable and especially 3' hydroxyl photodeprotectable) primers with appropriate complementarity to some region which bounds the informational content specifying said functional molecule or molecules.
  • activatable e g photodeprotectable and especially 3' hydroxyl photodeprotectable
  • immobilization of a sample to be subjected to such manipulations may be effected so as to comprise some photolabile linkage, which may then be subjected to selective photodegradation to effect specific release
  • informational molecules which carry the relevant genetic component of a phenogenocouple may thus be released by either of these methods either singly, or as the population of multiple such molecules simultaneously copied or otherwise released according to the pattern of deprotection.
  • successive generations of molecules need only be related informationally, by analysis of composition of one generation, by, for example, the massively parallel characterization methods of the present invention, followed by de novo synthesis of molecules carrying the desired complexity and diversity of the succeeding generation. This is a
  • Released molecules may then be recovered for subsequent amplification, mutation and subsequent rounds of selection by similar or other methods, as will be obvious to those skilled in the art of m vi tro molecular
  • post transcriptionally modified polypeptide moieties or other phenogenocouples may also be selected and otherwise subjected to in vi tro evolution by conventional means as well as by the massively parallel examination and modification methods of the present invention.
  • Detection methods for the present invention may favorably exploit fluorescent labeling techniques.
  • Genome sequencing applications of the present invention may thus avail of established fluorescent modification and detection methods.
  • Other applications of the present invention may also benefit from the application of fluorescence modification and detection methods.
  • nucleotide triphosphate compounds and analogs thereof. Many such compounds are acceptable substrates for polynucleotide polymerase molecules. These compounds have therefore proven suitable for use in various electrophoresis based DNA sequencing methodologies utilizing fluorescence detection, as well as in other applications such as chromatin mapping. There are therefore various compounds comprising a fluorescent dye moiety and a nucleotide triphosphate moiety commercially available.
  • Affinity labels Other single molecule detection methods have availed of compounds having well studied affinity interactions with other molecules, such as receptor-ligand interactions.
  • Genome sequencing applications of the present invention may thus avail of established affinity labeling and detection methods.
  • Other applications of the present invention my also benefit from the application of affinity labeling and detection methods.
  • affinity labels may be used to bind a microscopic colloid or bead which has been modified with an appropriate complementary affinity group such as a receptor.
  • Bead types include polymeric spheres of micron or submicron dimensions, metallic colloids such as colloidal gold, silica beads and magnetic beads.
  • polymer beads include dyes or liquid crystal molecules as side chains or within polymeric backbones and these may facilitate optical detection methods. Attachment of appropriate receptor or affinity molecules to the surfaces of such beads yields a reagent suitable for the detection of an affinity labeled molecule.
  • One such detection scheme was utilized by Finzi and Gelles, 23 albeit for different purposes. Multimeric labels:
  • labeled reagents may comprise multiple occurrences of said labeling moiety in a manner that does not interfere with the corresponding molecular recognition and monomer addition processes, to increase the likelihood of correct signal amplification of any labeled molecule.
  • the ordinary single biotin moiety attached to a nucleotide by a linker may be replaced with a polymer having multiple biotin moieties as side chains, such that the likelihood of a streptavidin molecule interacting with this multimeric affinity label is increased.
  • Fluorescent labels may similarly multiplied, as may any other labeling moieties. Measures must be taken in the design and synthesis of such multimerically labeled reagents to ensure that solubility is retained. This may be accomplished by choosing a highly soluble polymer as the backbone carrying said labeling moieties comprising the multimeric label.
  • Any compound capable of serving as an initiator for some aqueous polymerization may also serve as a labeling moiety.
  • This initiator nucleates the formation of a perceptible polymer attached to the sample molecule.
  • a perceptible polymer may, for example, comprise multiple fluorescent moieties, or simple effect a local change in transmitance of light or a local change of refractive index. After detection has been accomplished, said perceptible polymer is degraded or otherwise removed from the sample molecule.
  • Such polymerizations may be self-limiting, as is the case for some dendrimeric polymers.
  • polymerization is caused to occur in a step after the labeled nucleotide is added to the sample molecule, and must proceed via a chemistry that leaves the sample molecule in tact
  • Labeling moieties are favorably in communication with or coupled to nucleotides via a linker of sufficient length to ensure that the presence of said labeling moieties on said nucleotides will not interfere with the action of a polymerase enzyme on said nucleotides
  • Linkers will also necessarily be of some minimal length when stepping control is effected through the use of various preformed enzyme-nucleotide complexes (as described below).
  • nucleotides requires the elimination of said accompanying label This may favorably be accomplished through the cleavage of said linkers which have been designed and synthesized to admit of cleavage by treatments which will not degrade or otherwise modify the relevant state or information content of sample molecules.
  • said linker may include along its length one or more ester linkages, which will be susceptible to hydrolysis, which may be
  • Linkages comprising disulfide bonds within their length have been developed to provide for cleavability 24 reagents comprising such linkages are commercially available 25 and have been used to modify nucleotides 2 ⁇ in a manner which may be conveniently reversed by treatment with mild reducing agents such as dithiothreitol Cleavable linkages may be provided so as to minimize the portion of the linker which remains on the sample molecule.
  • mild reducing agents such as dithiothreitol Cleavable linkages
  • biotin derived nucleotides frequently contain, along the linker joining said biotin moiety to said nucleotide moiety, one or more ester or amide bonds, which is susceptible to cleavage by various chemical treatments.
  • cleavage of a labeling moiety may also be effected by the disruption of some affinity interaction which effects the communication between said labeling moiety and the nucleotide moiety.
  • moieties joined by non-covalent associations may, for example, be
  • Photocleavable moieties may also comprise an intermediate portion of linkers joining labeling moieties to nucleotide moieties, such that upon photocleavage of said photocleavable moieties, communication between the termini of said linker is disrupted and the label moiety is liberated from the nucleotide moiety. Because photodeprotection or photocleavage reactions 27 generally proceed quite rapidly, with appropriate detection and photoexcitation means, detection, label removal and nucleotide
  • incorporation rates per sample molecule may approach the limit imposed by any particular polymerase enzyme and the processivity of said enzyme. Long linkers with photocleavable termini have been synthesized 28
  • compounds which thermally degrade into two or more portions may comprise an intermediate portion of such linkers, such that thermal cycling may be employed to effect linker cleavage.
  • thermolabile linkers
  • Fluorescent labels of nucleotides may be neutralized by photobleaching, such that while some product of said fluorescent label may remain in communication with the sample molecule (e g the daughter strand of a polynucleotide being sequenced) it will no longer provide a signal sufficiently strong to interfere with the detection and discrimination of subsequently added labels.
  • affinity labels with appropriate photochemical properties may be subjected to photochemical modification rendering them inert to binding, generally subsequent to dissociation of the corresponding receptor by appropriate means.
  • affinity labels fluorescent labels or any other labeling moieties
  • chemical modification appropriate to the chemistries of said labels which effects a change or reduction in the detectable signal provided by said label may be availed to prevent interference of said labels with similar or distinct labels subsequently added to sample molecules or complexes thereof.
  • spirobenzopyran which have labile, structurally and photochemically distinct but interconvertible isomers
  • an excited state of such a moiety may be used as a means of detection.
  • chemical modification of one or another state of such interconvertible molecules may then neutralize it.
  • activation may cause such a label to convert to some unstable but discernible state, which then irreversibly degrades according to characterizable kinetics.
  • Such molecules must be chosen so as to remain in said discernible state for a sufficient time period to permit detection, but reliably degrade (to completion for a population of such molecules) within a practical time period.
  • Agents which specifically inhibit binding reactions may be identified rapidly through the detection of molecules, of a diverse library each molecular species of which is uniquely labeled, not bound by particles of some sample which may comprise many different species, in the presence of test reagent, which is labeled, and permitted to associate with said sample (preferably during a preincubation step before the addition of said diverse library to said sample,) in analogy to blocking antibody assays Results are compared to those obtained with an aliquot of said diverse library and another portion of the same said sample. Such an assay may be performed for increasing concentrations of said test reagent. Enzymatically enforced associations at defined molecular sites:
  • Methods are provided to enforce highly specific associations and reactions, including molecular recognition processes, on individual sample molecules or on populations and subpopulations of sample molecules. These are described for genome sequencing applications, but the methods included thereunder have broad applicability, including to any molecular affinity interaction.
  • Various methods may be used to accomplish the controlled addition of monomers, including nucleotides and especially labeled or protected nucleotides, to the daughter strand of a sample template molecule.
  • Means of slowing the time required for the addition of a single nucleotide to a sample molecule will circumvent the requirement of stepping control. This will be particularly applicable for detection mechanisms not requiring separate manipulation steps (such as the separate association of beads to affinity labeled sample molecules).
  • neutralizable fluorescent labels may be added to appropriately primed sample template molecules in the presence of polymerases, at low
  • concentrations Said concentrations must be sufficiently low that two nucleotides are not added to the sample molecule in less than the time required to accomplish the detection of the first such addition. Because all labels are present in the observation field, detection is accomplished through the observation of the reduction of the Brownian motion of a fluorescent moiety due to its addition to the sample molecule, in close analogy to the experiments of Finzi and Gelles 30 , but it will be noted the change in mobility is much larger in the present case. Alternatively detection may be understood to depend on an increase in the net residence of some fluorescent moiety within a defined region or the occupancy of a region, above the occupancy arising from the background of unbound labeled nucleotides.
  • Such detection is preferably conducted with a scanning excitation bear fluorescence confocal microscopic method as described above, or with a scanning detection light path, as also described above.
  • nucleotide concentration are chosen such that on average less than one labeled nucleotide will be present within the area
  • a light pulse of appropriate frequency passing through, for example, the pinhole which effects the scanning of the excitation beam may be used to photobleach of photocleave the fluorescent label from the sample molecule after it has been detected to have been added to the sample molecule, without the appreciable accumulation of incidentally unlabeled nucleotides.
  • an SLM may be used to spatially control illumination of the sample by an appropriate frequency of light to effect photochemical unlabeling, and thus permit the simultaneous unlabeling of multiple sample molecules.
  • This method may be understood as concentration modulated control of the kinetics of polymerization processivity, which is used to facilitate direct observation of successive addition of individual (labeled) nucleotides, with controlled unlabeling Scanning rate and other instrumentation dependent parameters will influence optimal conditions and concentrations.
  • concentration modulated control of the kinetics of polymerization processivity which is used to facilitate direct observation of successive addition of individual (labeled) nucleotides, with controlled unlabeling Scanning rate and other instrumentation dependent parameters will influence optimal conditions and concentrations.
  • direct observation of the addition of comonomers is dynamically observed, and sequence information for the respective sample molecule may be reconstructed accordingly.
  • a simple method to effect adequate stepping control for sequencing applications of the present invention relies on equilibrium control.
  • nucleotides which are labeled
  • nucleotides are limiting, and there is a relative excess of sample molecules.
  • Exonuclease activity intrinsic to most polynucleotide polymerases is circumvented by the use of alpha-phosphorothioate nucleotides (which are appropriately labeled) which are resistant to such degradation, in this method
  • alpha-phosphorothioate nucleotides which are appropriately labeled
  • a thirty-three-fold excess of sample molecules relative to labeled complementary nucleotides per cycle may be chosen
  • Polymerase molecules are preferably provided in excess of sample template molecules. Each sample molecule has a three percent chance of undergoing a single nucleotide addition. Nucleotides are rapidly depleted. Any sample molecule which has undergone one nucleotide addition has a further three percent chance, or in total approximately a 0.1% chance of undergoing a second nucleotide addition. For a sequencing segment run of 20 bases per sample molecule each segment will experience an error contribution of (20) (0.1%) or 2% from multiple additions within a cycle.
  • Such erroneous segment data will be conspicuous when oversampling is performed due to the correspondingly low frequency with which it occurs.
  • the binomial distribution indicates that there is approximately a 94..2% chance that three or more segments including a particular base contain correct data regarding that base. Any specific individual data error is highly unlikely to occur more than once for fivefold oversampling. Note that in practice such
  • x ⁇ 1 there is a probability p equal to x that a particular sample molecule will experience the addition of at least one nucleotide, and a probability p k that any sample molecule will experience at least k nucleotide additions within the same sequencing cycle.
  • p 2 The probability (d) of such a multiple incorporation error occurring within the sequence segment data obtained from a particular sample molecule in a sequencing run of n bases will be less than 2(n) (p 2 ).
  • the net sequence information per sample molecule obtained per sequencing cycle will be x bases, and the net sequence information for a sample with N molecules will be (x) (N) bases, which will be large for large N.
  • Stepping control may favorably be applied to any polymerization process useful within the scope of the present invention, including both genome sequencing and affinity characterization applications.
  • Template directed polymerization depends on the processive addition of comonomers at the terminus of a growing daughter strand as specified by the respective complementary base of the parent template strand.
  • Complementarity may be enforced through molecular recognition of said complementarity of protected analogs of said comonomers with the
  • chain Numerous monomers which may thus be added but do not provide an appropriate chemical functional group for subsequent elongation of the polynucleotide strand to which they have been enzymatically added are known within the relevant arts, and are generally referred to as chain
  • photochemically modified particularly in a manner not disrupting the sample molecule, to a form which may support subsequent addition of comonomers in the usual manner, may be employed to effect controlled stepping of polymerization addition.
  • Removable protecting groups are particularly advantageous for the genome sequencing applications of the present invention because they may be utilized to permit and ensure that exactly one nucleotide is added to a sample molecule per sequencing cycle. This will permit an even greater rate of data accumulation than may be achieved by equilibrium control methods, with which only a fraction of the sample molecule population per cycle yields data.
  • Photoremovable protecting groups may be used to gain similar advantage but further permit controlled spatial localization of deprotection.
  • photodeprotection reactions 32 generally proceed rapidly, with appropriate detection and photoexcitation means processivity and nucleotide
  • incorporation rates per sample molecule may approach the limit imposed by any particular polymerase enzyme.
  • Nucleotide analogs comprising such removable protecting groups preferably further comprise labeling moieties.
  • a particularly convenient category of such compounds comprises a labeling moiety or multimer thereof in communication with the nucleotide moiety exclusively through said removable protecting group. For such compounds, removal of said removable protecting group will simultaneously effect removal of said labeling moiety. Simultaneous removal of both protecting moiety and labeling moiety will conveniently prepare a sample molecule for the next sequencing cycle in a single step.
  • Such protecting groups should therefore be compatible with either naturally occurring or genetically modified polymerases.
  • primers comprising a photodeprotectable 3' hydroxyl terminus may provide for the selective polymerization of a polynucleotide moiety selectable by control over illumination of the appropriate region of the sample.
  • a polynucleotide moiety to which such a primer is hybridized and then selectively deprotected may thus be subjected to amplification techniques such as PCR in a selectable manner
  • modified primers shall simply be referred to as photoactivatable primers.
  • the 3' deprotectable nucleotides employed in some variations on the present invention may also find other uses in molecular biology and biotechnology. They may be used as chain terminators in conventional enzymatic sequencing methods. If such manipulations are performed any species terminating in a particular base may be extracted from the resolution medium (conventionally polyacrylamide gel), deprotected and then subjected to other manipulations requiring an active 3' hydroxyl group, such as ligation..
  • deprotectable compounds may serve as a convenient stepping control means for polymerization. Included among such
  • deprotectable nucleotides are nucleotides with photocleavable protecting groups, including those which reside on the 3' hydroxyl of a nucleotide. Label encoding and labeling methods for data collection:
  • the most rudimentary encoding system will be a one-bit binary labeling system, consisting of only one label moiety type, indicating whether or not an association of only one resolvable type occurred during the preceding association step.
  • sample template molecules bearing appropriate primers with an appropriate polymerase and limiting quantities of only one labeled nucleotide (and no unlabeled nucleotides) such that this monomer will be added only if the template molecule has the complementary base in the template position immediately 5' to the base opposite the 3' terminal base of the primer, and no monomers will be added otherwise, sample molecules are then washed to remove any remaining free nucleotides; the sample is then exposed to excess quantities of streptavidin modified fluorescently labeled beads for a sufficient length of time to ensure that all biotin moieties are bound by said labeled beads, and then all unbound beads are washed away, detection is then performed and data recorded linkers are then cleaved.
  • Said sub-cycle is repeated for the remaining three nucleotides, to constitute a cycle which successively tests for the presence in the sample template molecule of each type of base immediately 5' to the base opposite the 3' terminal base of the primer. If a sample molecule does not bind any label through such a cycle, then it was most likely "missed” due to the limiting concentration of nucleotides used to effect stepping of polymerization. If a sample molecule is labeled multiply during such a cycle, then the respective subsequent bases are detected as occurring in the template according to the pattern of labeling.
  • a somewhat more efficient encoding system is provided if two distinct labeling moieties may be availed.
  • Each nucleotide will be indicated by the presence or absence of each of the two moiety types, as a binary code.
  • the moieties may, for instance be biotin (B) and digoxigenin (D).
  • B biotin
  • D digoxigenin
  • Either two perceptibly distinct bead types may be used for simultaneous detection, provided distinct affinity labels are sufficiently well separated by extended linkers for simultaneous binding, or a single bead type with two distinct receptor molecules may be used in two separate binding and release cycles, in which case the release of one bead type will have to leave the remaining affinity moiety bound to sample molecules.
  • nucleotides of each of the four types distinctly labeled with a fluorescent dye moiety may be used with fluorescence detection means, and a sequencing cycle consisting of only one sub-cycle
  • four antibodies or four other appropriate receptor molecules or affinity reagents which each bind each of the four distinct dye moieties may be bound to each of four perceptibly distinct beads.
  • nucleotides may each be labeled with some distinct combination of multiple dye moieties, again encoding a unique binary label.
  • a DNA sample is prepared by shearing or digestion at a first sequence with a first restriction enzyme producing a 3' overhang terminus to some appropriate known size distribution and labeled with a digoxigenin bearing nucleotide by the action of terminal deoxynucleotidyl transferase. After such digoxigenin labeling, said DNA sample is then subjected to random internal cleavage, for example by shearing so as to produce a population of molecules with an average length half that produced in the previous sizing step, or digestion with a second restriction enzyme recognizing a distinct, second recognition sequence.
  • Sample molecules of said sample are then bound at some convenient surface density to a transparent surface modified with a monolayer or a sub-monolayer density of anti-digoxigenin antibody.
  • Said sample molecules which will thus be bound to said transparent surface by the 3' termini of one strand, are then subjected to treatment by a 3' to 5 ' exonuclease, which will only act at the 3' terminus which does not bear the digoxigenin moiety due to the hindrance of this latter 3' terminus by its interaction with the surface, preferably not to completion of digestion of susceptible strands.
  • primed DNA sample template molecules bound to a transparent surface in an end-wise manner are prepared.
  • nucleotide labeling affinity moiety in a manner similar to the example provided for one-bit binary labeling systems, utilizing for example each of the four nucleotides derrivatized to effect communication of said nucleotides with a biotin moiety via a chemically cleavable linker such as those described by S.W. Ruby et al 34 polymerization directed by the template provided by each involved DNA sample template molecule is effected with an appropriate DNA polymerase lacking a 3' to 5' exonuclease activity, such as Sequenase 2.0, 35 with only one nucleotide type present during each polymerization step sub-cycle, at sufficiently low
  • Bead labeled molecules may then be observed by a video microscope and the position of said bead labeled molecules within a sample ma be recorded by image analysis of digital images thus obtained in a manner similar to that used by Finzi and Gelles 36 Dithiothreitol or other reagents capable of cleaving said linker holding said biotin in
  • the same subcycle (comprising polymerization, bead association, video microscopic examination, bead and label cleavage and removal by washing, and optionally a bead removal confirmation video microscopic examination step) is then repeated in succession for each of the three remaining nucleotide types, to complete a full base sequencing cycle (which as noted may yield information about more than one base location for some template molecules according to the sequence composition and the order of sub-cycles, and no information for other sample template molecules) Multiple said base sequence cycles are repeated until enough data have been accumulated relative to the total complexity of the initial DNA sample. Recorded data are then used to reconstruct sequence information for a segment of each sample template molecule, and segment sequence data are then aligned by appropriate computational algorithms.
  • this embodiment avails only existing and generally available materials and devices, relies on relatively simple manipulations which are known to be highly reproducible according to their general use in the relevant fields, but due to the novel process of the present invention may yield genome sequence information far more rapidly and inexpensively than highly complex robotic instruments with sequencing methods utilizing electrophoretic separation.
  • microscopic detection may be performed with a computer controlled stepable sample stage to effect the automated examination of large surface areas and hence very large numbers of sample molecules.
  • the transparent substrate providing the surface for immobilization may be that of a spooled film, which may be advanced at an appropriate rate before the objective of said video microscope of the present embodiment.
  • said film may be circular, and continuously advanced through multiple video microscope apparata and wells effecting polymerization sub-cycles, all in appropriate order such that benefit of full pipelining of each step may be enjoyed.
  • the construction of such instrumentation and rudimentary robotic actuation systems will be straightforward to those skilled in the relevant engineering arts.
  • Sequence determination may additionally effected by the random immobilization at some appropriate density of appropriately prepared and primed sample molecules on the surface of a transparent film, and stepwise polymerization with some appropriate polymerase, of all four nucleotides, all of which are protected at the 3'-hydroxyl with a photolabile (and hence photoremovable) protecting group in communication with labeling moieties which distinctly correspond to each nucleoside base type of the respective nucleotide.
  • Label incorporation is detected, for example by the scanned beam light microscopic methods of the present invention, or with highly sensitive CCDs, and assigned to the spatial region occupied by a particular molecule. Said film is translated appropriately such that the full complexity of the sample may be examined after each polymerization cycle.
  • Data are recorded electronically and according to the molecule for which they are obtained. Illumination of the sample with an appropriate frequency and intensity of light to effect 3'-hydroxy deprotection and hence also labeling moiety removal is performed, and a wash step is performed to remove freed label Such polymerization, detection and deprotection cycles are repeated until the sample is sufficiently well characterized.
  • Methods of the present invention may be combined with the immobilization of highly diverse libraries of binding specificities with either encoding labels or phenogenocouples, which may therefore be characterized
  • a polynucleotide binding dye e.g. ethidium bromide, DAPI
  • some general label such as a polynucleotide binding dye (e.g. ethidium bromide, DAPI) or some
  • chromophorigenic or photoemissive or labeled competitive inhibitor analog reagent detecting some metabolically fundamental reaction such as ATP hydrolysis, or the presence enzymes catalyzing said metabolically
  • Pathogens containing polynucleotides or capable of said metabolically fundamental reaction may thus be detected.
  • the profile of any sample type from an individual organism according to such an assay may be monitored over time, and a profile is preferably obtained for a state of presumed health for comparison to samples
  • Samples of similar type may also be compared across populations and subpopulations, and the profile of these samples also correlated with state of health of the respective individuals (cross-sectional comparison)
  • immunoglobulin specificities comprises a large number of immunoglobulin specificities Libraries comprising immunoglobulin specificities may include such specificities in the form of immunoglobulins expressed on bacteriophages viruses, or in the form of the phenogenocouples of the present invention
  • Banks comprising all of the specificities of a library may be maintaines as monoclones, and upon detection of a pathogen in association with one of more binding specificity contained in some library and the identification and/or characterization of said one or more binding specificity, an all in of the respective said monoclone, from one of said banks, may be provide: to the organism
  • Such analysis and provision of one or more monoclones be automated and controlled by algorithms
  • enzymes contained within some sample may be analyzed accordinq to their binding probability, binding duration or dissociation rate, and conformational of phosphorylation or other status.
  • assays may favorably be performed by the methods of the present invention, with immobilized libraries which may include competitive inhibitors, and with pre- or post-binding labeling of sample enzymes by encoded label antibodies, to permit classification of sample enzyme type on a molecule by molecule basis, which classification data may be combined with the data obtained in this assay.
  • each oligonucleotide, of known sequence, to be used as a specific gene probe is synthesized with some perceptible encoded label, as described above, where the codes assigned to the sequence of said each oligonucleotide are known (due to the synthetic scheme by which they are produced and concurrently labeled).
  • sample polynucleotide molecules which either have previously been or will subsequently be immobilized, or may otherwise be separated from probe oligonucleotides, and the presence or absence of said each oligonucleotide in the sample polynucleotide
  • the complexity of the polynucleotide sample is not too large, and the population made up of said oligonucleotides is sufficiently large and complex, preferably exhaustively enumerating all possible oligonucleotides of the respective and sufficiently long length, and provided hybridization may be sufficiently stringent, which stringency is affected by a large number of known factors but also has sequence dependent components, information about the binding of said each oligonucleotide, which may be related to the respective known sequence and by Watson-Crick pairing rules to the respective sample polynucleotide sequence segment (or by identity with the strand complementary to the strand to which said each
  • oligonucleotide may thus be obtained.
  • alignment of such data may yield information about the sequence of the sample.
  • the methods of the present invention further provide for the quantitation of such oligonucleotide hybridization by way of counting the number of times a particular perceptible encoded label is retained by a said polynucleotide sample, which may be availed both in the monitoring and correcting of errors and in the modulation of binding (hybridization) conditions.
  • probing may be accomplished by oligomeric sequences immobilized in some known configuration, for example by spatially patterned methods such as those of S P A Fodor et al 37 or by the lattices produced hierarchially by the method of N.C Seeman noted above but comprising an ordered array (the order of which is predetermined by the incorporation or association of single stranded oligonucleotides or other single stranded termini of known sequence into or with modular components used to build up said lattices) of short single stranded regions of known sequence and preferably one free terminus (so as not to hinder conformational changes required for hybridization), but detected by the methods of the present invention, where sample polynucleotides are labeled with some appropriate discernible label, such as the dye YOYO-1, to facilitate the detection of their presence in association with each of said oligomeric sequences.
  • some appropriate discernible label such as the dye YOYO-1
  • a yet further variation for effecting the spatially predetermined distribution of for example and exhaustively enumerated population of single stranded oligonucleotides may be effected by the used of the methods of N.C. Seeman to produce a uniform two dimensional lattice with a repeating pattern of short single stranded sequences with photoprotected termini for example all of the 256 possible 4-mers. Such a lattice ma have a periodicity substantially smaller than the wavelength of 'isible light.
  • Said short single stranded sequences mav be comprise some synthetic backbone so as to be resistant to enzymatic cleavage which backbone preferably also is non-ionic (for example, of alkyl or beta-cyanoethyl derivation peptide-nucleic-acid composition, or methylphosphonate composition) so as to denature from a complementary sequence only at markedly elevated temperatures relative to ordinary oligonucleotides
  • a pattern of oligonucleotide complexity may be distributed in a predetermined manner below the resolution of light directed patterning. Light patterning techniques may then be availed to spatially direct the photodeprotection of said short single stranded sequences at lower resolution.
  • Such light directed syntheses are preferably terminated with some comonomer which will prevent exonucleolytic degradation of said short single stranded sequences, or all of said short single stranded sequences are of a polarity opposite to that specified by the exonuclease to be subsequently used.
  • patterning resolution is not limited by the properties of light, but may avail of the convenience of light directed patterning at lower resolutions.
  • Hybridized molecules are treated mildly with a single strand specific nuclease, followed by an exonuclease, to degrade or by the same process to free those regions which are not bound to the probing said short single stranded sequences
  • Label incorporated into the nick translation products of said polynucleotide sample is then detected and spatially mapped by the methods of the present invention, and binding is thus scored according to the known probing said short single stranded sequences.
  • This method thus avails the molecular parallelism made possible by the molecular recognition, high density and high resolution detection methods availed with the present invention.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des moyens pour effectuer des caractérisations parallèles à grande échelle de molécules complexes et de phénomènes de reconnaissance moléculaire avec parallélisme et redondance acquis par des procédés d'examen de molécules isolées. Parmi les applications, on peut citer le séquençage ultra-rapide de génomes, la caractérisation par affinité, la caractérisation d'agents pathogènes et les déterminations à usage clinique, ainsi que le développement/réalisation de systèmes immunitaires cybernétiques. L'invention concerne également de nouveaux procédés d'examen et de manipulation de molécules isolées, en particulier des moyens et des procédés de microscopie avec balayage par un faisceau de lumière et des moyens de détection utilisant un système de dispositifs optoélectroniques. Différents appareils de commande de vitesse, tels que des commandes pas-à-pas pour différentes réactions, sont combinés avec la reconnaissance moléculaire, l'amplification de signaux et les procédés d'examen de molécules isolées. Une commande interne dans les échantillons, des commandes de manipulations à réaction dynamique basées sur des algorithmes, et la redondance d'échantillons, peuvent être utilisées pour obtenir un degré de précision arbitrairement choisi pour les résultats finaux.
PCT/US1996/002342 1995-02-27 1996-02-21 Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire WO1996027025A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU51716/96A AU5171696A (en) 1995-02-27 1996-02-21 Device, compounds, algorithms, and methods of molecular characterization and manipulation with molecular parallelism

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US39430795A 1995-02-27 1995-02-27
US08/394,307 1995-02-27

Publications (1)

Publication Number Publication Date
WO1996027025A1 true WO1996027025A1 (fr) 1996-09-06

Family

ID=23558396

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/002342 WO1996027025A1 (fr) 1995-02-27 1996-02-21 Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire

Country Status (2)

Country Link
AU (1) AU5171696A (fr)
WO (1) WO1996027025A1 (fr)

Cited By (127)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044152A1 (fr) * 1997-04-01 1998-10-08 Glaxo Group Limited Methode de sequençage d'acide nucleique
WO2000012759A1 (fr) * 1998-08-26 2000-03-09 Alpha Innotech Corporation Systeme de detection sur biopuce
US6124092A (en) * 1996-10-04 2000-09-26 The Perkin-Elmer Corporation Multiplex polynucleotide capture methods and compositions
DE19952160A1 (de) * 1999-10-29 2001-05-03 Markus Sauer Verfahren, Vorrichtung und farbstoffmarkiertes Peptid zum Nachweis eines Moleküls, sowie Verfahren zur Herstellung eines farbstoffmarkierten Peptids und dessen Verwendung
WO2001057248A2 (fr) * 2000-02-01 2001-08-09 Solexa Ltd. Groupes de polynucleotides et leur utilisation dans le sequencage
WO2001057249A1 (fr) * 2000-02-02 2001-08-09 Solexa Ltd. Synthese de reseaux moleculaires a adressage spatial
EP1130389A1 (fr) * 2000-02-29 2001-09-05 Horiba, Ltd. Capteur CCD chimique du typ reconnaissance moleculaire
WO2002002813A2 (fr) * 2000-07-05 2002-01-10 Amersham Biosiences Uk Ltd. Procede de sequencement
WO2002061127A2 (fr) * 2001-01-30 2002-08-08 Solexa Ltd. Jeux de polynucleotides et utilisation de ceux-ci en analyse genomique
WO2002074988A2 (fr) * 2001-03-16 2002-09-26 The Chancellor, Master And Scholars Of The University Of Oxford Series de molecules et procedes d'utilisation
AU770831B2 (en) * 1998-07-30 2004-03-04 Solexa Ltd. Arrayed biomolecules and their use in sequencing
EP1407046A1 (fr) * 2001-06-29 2004-04-14 Agilent Technologies Inc. Procede de sequen age d'adn au moyen d'etiquettes clivables
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
WO2005040425A2 (fr) * 2003-10-20 2005-05-06 Isis Innovation Ltd Methodes de sequençage de polymeres en parallele
US7033764B2 (en) 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7037687B2 (en) 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US20080199874A1 (en) * 2007-02-16 2008-08-21 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle dna
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
WO2009007294A1 (fr) 2007-07-06 2009-01-15 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Ensemble d'électrodes à membranes
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US7541444B2 (en) 2002-08-23 2009-06-02 Illumina Cambridge Limited Modified nucleotides
US7563574B2 (en) 2006-03-31 2009-07-21 Pacific Biosciences Of California, Inc. Methods, systems and compositions for monitoring enzyme activity and applications thereof
US7592435B2 (en) 2005-08-19 2009-09-22 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US7626704B2 (en) 2006-02-13 2009-12-01 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7630073B2 (en) 2006-02-13 2009-12-08 Pacific Biosciences Of California Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US20100081140A1 (en) * 2008-08-08 2010-04-01 President And Fellows Of Harvard College Chemically cleavable phosphoramidite linkers for sequencing by ligation
US7692783B2 (en) 2006-02-13 2010-04-06 Pacific Biosciences Of California Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7763423B2 (en) 2005-09-30 2010-07-27 Pacific Biosciences Of California, Inc. Substrates having low density reactive groups for monitoring enzyme activity
US7772384B2 (en) 2001-12-04 2010-08-10 Illumina Cambridge Limited Labelled nucleotides
US7805081B2 (en) 2005-08-11 2010-09-28 Pacific Biosciences Of California, Inc. Methods and systems for monitoring multiple optical signals from a single source
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US7820983B2 (en) 2006-09-01 2010-10-26 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
EP2256478A2 (fr) 2004-05-06 2010-12-01 CLONDIAG GmbH Dispositif et procede pour de detecter des interactions moleculaires
EP2266699A1 (fr) 2003-04-02 2010-12-29 CLONDIAG GmbH Appareil pour l'amplification et la détection d'acide nucléique
US7887753B2 (en) 2000-11-16 2011-02-15 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7901889B2 (en) 2007-07-26 2011-03-08 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
EP2327797A1 (fr) * 1997-04-01 2011-06-01 Illumina Cambridge Limited Methode de séquençage d'acides nucléiques
EP2330215A1 (fr) 2005-11-04 2011-06-08 CLONDIAG GmbH Procédé et dispositif de détection d'interactions moléculaires
US7960116B2 (en) 2007-09-28 2011-06-14 Pacific Biosciences Of California, Inc. Nucleic acid sequencing methods and systems
US20110151449A1 (en) * 2003-11-12 2011-06-23 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US7972820B2 (en) 2000-12-08 2011-07-05 Illumina Cambridge Limited Isothermal amplification of nucleic acids on a solid support
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
US7993895B2 (en) 2005-12-02 2011-08-09 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions
US8003330B2 (en) 2007-09-28 2011-08-23 Pacific Biosciences Of California, Inc. Error-free amplification of DNA for clonal sequencing
US8053192B2 (en) 2007-02-02 2011-11-08 Illumina Cambridge Ltd. Methods for indexing samples and sequencing multiple polynucleotide templates
US8143030B2 (en) 2008-09-24 2012-03-27 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8153375B2 (en) 2008-03-28 2012-04-10 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
US8168139B2 (en) 2002-06-24 2012-05-01 Fluidigm Corporation Recirculating fluidic network and methods for using the same
US8182994B2 (en) 2009-09-15 2012-05-22 Illumina Cambridge Limited Centroid markers for image analysis of high denisty clusters in complex polynucleotide sequencing
US8192961B2 (en) 1998-12-14 2012-06-05 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US8193123B2 (en) 2006-03-30 2012-06-05 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US8198023B2 (en) 2008-08-05 2012-06-12 Pacific Biosciences Of California, Inc. Prevention and alleviation of steric hindrance during single molecule nucleic acid synthesis by a polymerase
US8207509B2 (en) 2006-09-01 2012-06-26 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8236499B2 (en) 2008-03-28 2012-08-07 Pacific Biosciences Of California, Inc. Methods and compositions for nucleic acid sample preparation
US8252910B2 (en) 2008-11-19 2012-08-28 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US8252911B2 (en) 2008-02-12 2012-08-28 Pacific Biosciences Of California, Inc. Compositions and methods for use in analytical reactions
US8263364B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US8274040B2 (en) 2008-09-16 2012-09-25 Pacific Biosciences Of California, Inc. Substrates and optical system having at least one optical waveguide, at least one nanometer-scale aperture and at least one lens array and methods of use thereof
US8318094B1 (en) 2010-06-18 2012-11-27 Pacific Biosciences Of California, Inc. Substrate analysis systems
EP2548647A1 (fr) 2006-10-20 2013-01-23 CLONDIAG GmbH Dispositifs et méthodes de dosage destinés à la détection de substances á analyser
US8370079B2 (en) 2008-11-20 2013-02-05 Pacific Biosciences Of California, Inc. Algorithms for sequence determination
US8383369B2 (en) 2008-09-24 2013-02-26 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8399188B2 (en) 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US8465922B2 (en) 2010-08-26 2013-06-18 Pacific Biosciences Of California, Inc. Methods and systems for monitoring reactions
US8481264B2 (en) 2008-09-19 2013-07-09 Pacific Biosciences Of California, Inc. Immobilized nucleic acid complexes for sequence analysis
US8501406B1 (en) 2009-07-14 2013-08-06 Pacific Biosciences Of California, Inc. Selectively functionalized arrays
US8501405B2 (en) 2009-04-27 2013-08-06 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8518643B2 (en) 2010-02-04 2013-08-27 Pacific Biosciences Of California, Inc. Method to improve single molecule analyses
US8603741B2 (en) 2010-02-18 2013-12-10 Pacific Biosciences Of California, Inc. Single molecule sequencing with two distinct chemistry steps
US8628940B2 (en) 2008-09-24 2014-01-14 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
US8658364B2 (en) 2011-03-23 2014-02-25 Pacific Biosciences Of California, Inc. Isolation of polymerase-nucleic acid complexes
US8728764B2 (en) 2008-10-02 2014-05-20 Illumina Cambridge Limited Nucleic acid sample enrichment for sequencing applications
US8795961B2 (en) 2008-09-05 2014-08-05 Pacific Biosciences Of California, Inc. Preparations, compositions, and methods for nucleic acid sequencing
US8802424B2 (en) 2008-01-10 2014-08-12 Pacific Biosciences Of California, Inc. Methods and systems for analysis of fluorescent reactions with modulated excitation
US8834797B2 (en) 2008-04-04 2014-09-16 Life Technologies Corporation Scanning system and method for imaging and sequencing
US8834847B2 (en) 2010-08-12 2014-09-16 Pacific Biosciences Of California, Inc. Photodamage mitigation compounds and systems
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US8921046B2 (en) 2008-09-19 2014-12-30 Pacific Biosciences Of California, Inc. Nucleic acid sequence analysis
US8993230B2 (en) 2008-12-04 2015-03-31 Pacific Biosciences of Californ, Inc. Asynchronous sequencing of biological polymers
US9062091B2 (en) 2012-02-15 2015-06-23 Pacific Biosciences Of California, Inc. Polymerase enzyme substrates with protein shield
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9127314B2 (en) 2002-08-23 2015-09-08 Illumina Cambridge Limited Labelled nucleotides
US9133511B2 (en) 2000-10-06 2015-09-15 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9169515B2 (en) 2010-02-19 2015-10-27 Life Technologies Corporation Methods and systems for nucleic acid sequencing validation, calibration and normalization
US9175348B2 (en) 2012-04-24 2015-11-03 Pacific Biosciences Of California, Inc. Identification of 5-methyl-C in nucleic acid templates
US9175341B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
US9175338B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9212394B2 (en) 2013-08-19 2015-12-15 Singular Bio, Inc. Assays for single molecule detection and use thereof
US9238836B2 (en) 2012-03-30 2016-01-19 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing modified nucleic acids
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9255292B2 (en) 2005-10-31 2016-02-09 The Trustees Of Columbia University In The City Of New York Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods
US9297042B2 (en) 2005-10-31 2016-03-29 The Trustees Of Columbia University In The City Of New York Chemically cleavable 3′-O-allyl-dNTP-allyl-fluorophore fluorescent nucleotide analogues and related methods
US20160222441A1 (en) * 2002-12-20 2016-08-04 Callida Genomics Materials and Methods Relating to Nano-Tags and Nano-Barcodes
US9416414B2 (en) 2013-10-24 2016-08-16 Pacific Biosciences Of California, Inc. Delaying real-time sequencing
EP2119722B1 (fr) 2002-08-23 2016-10-26 Illumina Cambridge Limited Nucléotides étiquetées
US9528151B2 (en) 2006-12-01 2016-12-27 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US9593373B2 (en) 2013-03-15 2017-03-14 Illumina Cambridge Limited Modified nucleosides or nucleotides
US9611510B2 (en) 2011-04-06 2017-04-04 The University Of Chicago Composition and methods related to modification of 5-methylcytosine (5-mC)
US9624539B2 (en) 2011-05-23 2017-04-18 The Trustees Of Columbia University In The City Of New York DNA sequencing by synthesis using Raman and infrared spectroscopy detection
WO2017087823A1 (fr) 2015-11-18 2017-05-26 Mir Kalim U Séquençage super-résolution
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US9909177B2 (en) 2005-06-21 2018-03-06 The Trustees Of Columbia University In The City Of New York Pyrosequencing methods and related compositions
US9957291B2 (en) 2013-08-05 2018-05-01 Pacific Biosciences Of California, Inc. Protected fluorescent reagent compounds
US10150872B2 (en) 2015-02-04 2018-12-11 Pacific Biosciences Of California, Inc. Multimeric protected fluorescent reagents
US10273537B2 (en) 2011-10-14 2019-04-30 Pacific Biosciences Of California, Inc. Real-time redox sequencing methods
US10302972B2 (en) 2015-01-23 2019-05-28 Pacific Biosciences Of California, Inc. Waveguide transmission
US10450560B2 (en) 2002-09-12 2019-10-22 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
EP3572789A1 (fr) 2005-11-04 2019-11-27 Alere Technologies GmbH Dispositif et procédé de détection de particules
US10648026B2 (en) 2013-03-15 2020-05-12 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
US10669299B2 (en) 2015-11-20 2020-06-02 Pacific Biosciences Of California, Inc. Protected dye-labeled reagents
US10676788B2 (en) 2015-11-20 2020-06-09 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
US10781483B2 (en) 2015-11-20 2020-09-22 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
US10995111B2 (en) 2003-08-22 2021-05-04 Illumina Cambridge Limited Labelled nucleotides
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
US11739371B2 (en) 2015-02-18 2023-08-29 Invitae Corporation Arrays for single molecule detection and use thereof
US11787831B2 (en) 2020-06-22 2023-10-17 Illumina Cambridge Limited Nucleosides and nucleotides with 3′ acetal blocking group
US11844666B2 (en) 2008-12-11 2023-12-19 Pacific Biosciences Of California, Inc. Classification of nucleic acid templates

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (fr) * 1989-10-26 1991-05-16 Sri International Sequençage d'adn

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006678A1 (fr) * 1989-10-26 1991-05-16 Sri International Sequençage d'adn

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JAPAN J. APPL. PHYS., Volume 33, No. 3A, issued March 1994, M. ISHIKAWA et al., "Single-Molecule Detection by Laser-Induced Fluorescence Technique with a Positive-Sensitive Photon-Counting Apparatus", pages 1571-1576. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 91, issued June 1994, M. EIGEN et al., "Sorting Single Molecules: Application to Diagnostics and Evolutionary Biotechnology", pages 5740-5747. *
SCIENCE, Volume 264, issued 06 May 1994, PERKINS et al., "Relaxation of a Single DNA Molecule Observed by Optical Microscopy", pages 822-825. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 257, No. 6, issued 25 March 1982, N. WILLIAMS et al., "Exploring the Adenine Nucleotide Binding Sites on Mitochondrial F1-ATPase with a New Photoaffinity Probe, 3'-o-(4-Benzoyl)Benzoyl Adenosine 5'-Triphospate", pages 2834-2841. *

Cited By (372)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6124092A (en) * 1996-10-04 2000-09-26 The Perkin-Elmer Corporation Multiplex polynucleotide capture methods and compositions
US6514699B1 (en) 1996-10-04 2003-02-04 Pe Corporation (Ny) Multiplex polynucleotide capture methods and compositions
US8143008B2 (en) 1997-04-01 2012-03-27 Illumina, Inc. Method of nucleic acid amplification
EP2327797A1 (fr) * 1997-04-01 2011-06-01 Illumina Cambridge Limited Methode de séquençage d'acides nucléiques
US7985565B2 (en) 1997-04-01 2011-07-26 Illumina, Inc. Method of nucleic acid amplification
WO1998044152A1 (fr) * 1997-04-01 1998-10-08 Glaxo Group Limited Methode de sequençage d'acide nucleique
US9902951B2 (en) 1997-04-01 2018-02-27 Illumina, Inc. Method of nucleic acid amplification
US9593328B2 (en) 1997-04-01 2017-03-14 Illumina, Inc. Method of nucleic acid amplification
US8993271B2 (en) 1997-04-01 2015-03-31 Illumina, Inc. Method of nucleic acid amplification
EP1498494A3 (fr) * 1997-04-01 2007-06-20 Solexa Ltd. Procédé de séquencage d'acides nucléiques
EP3034626A1 (fr) * 1997-04-01 2016-06-22 Illumina Cambridge Limited Methode de séquencage d'acide nucléique
EP1498494A2 (fr) 1997-04-01 2005-01-19 Solexa Ltd. Procédé de séquencage d'acides nucléiques
US8476044B2 (en) 1997-04-01 2013-07-02 Illumina, Inc. Method of nucleic acid amplification
US7037687B2 (en) 1998-05-01 2006-05-02 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US8263364B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7645596B2 (en) 1998-05-01 2010-01-12 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9957561B2 (en) 1998-05-01 2018-05-01 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US10214774B2 (en) 1998-05-01 2019-02-26 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US8263365B2 (en) 1998-05-01 2012-09-11 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9458500B2 (en) 1998-05-01 2016-10-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9540689B2 (en) 1998-05-01 2017-01-10 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9212393B2 (en) 1998-05-01 2015-12-15 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9725764B2 (en) 1998-05-01 2017-08-08 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US10208341B2 (en) 1998-05-01 2019-02-19 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US9096898B2 (en) 1998-05-01 2015-08-04 Life Technologies Corporation Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US7232656B2 (en) * 1998-07-30 2007-06-19 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
AU770831B2 (en) * 1998-07-30 2004-03-04 Solexa Ltd. Arrayed biomolecules and their use in sequencing
US6271042B1 (en) 1998-08-26 2001-08-07 Alpha Innotech Corporation Biochip detection system
WO2000012759A1 (fr) * 1998-08-26 2000-03-09 Alpha Innotech Corporation Systeme de detection sur biopuce
US9845501B2 (en) 1998-12-14 2017-12-19 Pacific of Biosciences of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US8192961B2 (en) 1998-12-14 2012-06-05 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US8980584B2 (en) 1998-12-14 2015-03-17 Pacific Biosciences Of California, Inc. System and methods for nucleic acid sequencing of single molecules by polymerase synthesis
US7052847B2 (en) 1999-05-19 2006-05-30 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7056661B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7943305B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation High speed nucleic acid sequencing
US7943307B2 (en) 1999-05-19 2011-05-17 Cornell Research Foundation Methods for analyzing nucleic acid sequences
US7056676B2 (en) 1999-05-19 2006-06-06 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7361466B2 (en) 1999-05-19 2008-04-22 Cornell Research Foundation, Inc. Nucleic acid analysis using terminal-phosphate-labeled nucleotides
US7033764B2 (en) 1999-05-19 2006-04-25 Cornell Research Foundation, Inc. Method for sequencing nucleic acid molecules
US7416844B2 (en) 1999-05-19 2008-08-26 Cornell Research Foundation, Inc. Composition for nucleic acid sequencing
US7485424B2 (en) 1999-05-19 2009-02-03 Cornell Research Foundation, Inc. Labeled nucleotide phosphate (NP) probes
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
US6818395B1 (en) 1999-06-28 2004-11-16 California Institute Of Technology Methods and apparatus for analyzing polynucleotide sequences
DE19952160A1 (de) * 1999-10-29 2001-05-03 Markus Sauer Verfahren, Vorrichtung und farbstoffmarkiertes Peptid zum Nachweis eines Moleküls, sowie Verfahren zur Herstellung eines farbstoffmarkierten Peptids und dessen Verwendung
WO2001057248A3 (fr) * 2000-02-01 2002-02-14 Solexa Ltd Groupes de polynucleotides et leur utilisation dans le sequencage
WO2001057248A2 (fr) * 2000-02-01 2001-08-09 Solexa Ltd. Groupes de polynucleotides et leur utilisation dans le sequencage
WO2001057249A1 (fr) * 2000-02-02 2001-08-09 Solexa Ltd. Synthese de reseaux moleculaires a adressage spatial
US7384737B2 (en) 2000-02-02 2008-06-10 Solexa Limited Synthesis of spatially addressed molecular arrays
EP1130389A1 (fr) * 2000-02-29 2001-09-05 Horiba, Ltd. Capteur CCD chimique du typ reconnaissance moleculaire
US6824660B2 (en) 2000-02-29 2004-11-30 Horiba, Ltd. Molecular recognition type chemical CCD
US9926521B2 (en) 2000-06-27 2018-03-27 Fluidigm Corporation Microfluidic particle-analysis systems
WO2002002813A2 (fr) * 2000-07-05 2002-01-10 Amersham Biosiences Uk Ltd. Procede de sequencement
WO2002002813A3 (fr) * 2000-07-05 2003-03-13 Amersham Biosiences Uk Ltd Procede de sequencement
US7056670B2 (en) 2000-07-05 2006-06-06 Ge Healthcare Uk Limited Sequencing method
US10648028B2 (en) 2000-10-06 2020-05-12 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10570446B2 (en) 2000-10-06 2020-02-25 The Trustee Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10669577B2 (en) 2000-10-06 2020-06-02 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10457984B2 (en) 2000-10-06 2019-10-29 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9719139B2 (en) 2000-10-06 2017-08-01 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10435742B2 (en) 2000-10-06 2019-10-08 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9718852B2 (en) 2000-10-06 2017-08-01 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9725480B2 (en) 2000-10-06 2017-08-08 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10428380B2 (en) 2000-10-06 2019-10-01 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9133511B2 (en) 2000-10-06 2015-09-15 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10669582B2 (en) 2000-10-06 2020-06-02 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10577652B2 (en) 2000-10-06 2020-03-03 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10662472B2 (en) 2000-10-06 2020-05-26 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10407459B2 (en) 2000-10-06 2019-09-10 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10407458B2 (en) 2000-10-06 2019-09-10 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US10633700B2 (en) 2000-10-06 2020-04-28 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US9868985B2 (en) 2000-10-06 2018-01-16 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US8673645B2 (en) 2000-11-16 2014-03-18 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US10509018B2 (en) 2000-11-16 2019-12-17 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US7887753B2 (en) 2000-11-16 2011-02-15 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8273574B2 (en) 2000-11-16 2012-09-25 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US9176137B2 (en) 2000-11-16 2015-11-03 California Institute Of Technology Apparatus and methods for conducting assays and high throughput screening
US8455258B2 (en) 2000-11-16 2013-06-04 California Insitute Of Technology Apparatus and methods for conducting assays and high throughput screening
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US7972820B2 (en) 2000-12-08 2011-07-05 Illumina Cambridge Limited Isothermal amplification of nucleic acids on a solid support
GB2389904A (en) * 2001-01-30 2003-12-24 Solexa Ltd Arrayed polynucleotides and their use in genome analysis
WO2002061127A3 (fr) * 2001-01-30 2003-07-24 Solexa Ltd Jeux de polynucleotides et utilisation de ceux-ci en analyse genomique
WO2002061127A2 (fr) * 2001-01-30 2002-08-08 Solexa Ltd. Jeux de polynucleotides et utilisation de ceux-ci en analyse genomique
US9376677B2 (en) 2001-03-16 2016-06-28 Singular Bio, Inc. Arrays and methods of use
WO2002074988A2 (fr) * 2001-03-16 2002-09-26 The Chancellor, Master And Scholars Of The University Of Oxford Series de molecules et procedes d'utilisation
US9556429B2 (en) 2001-03-16 2017-01-31 Singular Bio, Inc. Arrays and methods of use
US9481883B2 (en) 2001-03-16 2016-11-01 Singular Bio, Inc. Arrays and methods of use
US9057730B2 (en) 2001-03-16 2015-06-16 Singular Bio, Inc. Arrays and methods of use
EP2465943A3 (fr) * 2001-03-16 2012-10-03 Kalim Mir Affichage de polymère linéaire
WO2002074988A3 (fr) * 2001-03-16 2003-08-14 Chancellor Master And Scholars Series de molecules et procedes d'utilisation
US7833708B2 (en) 2001-04-06 2010-11-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8486636B2 (en) 2001-04-06 2013-07-16 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US8936764B2 (en) 2001-04-06 2015-01-20 California Institute Of Technology Nucleic acid amplification using microfluidic devices
US7816079B2 (en) 2001-06-29 2010-10-19 Agilent Technologies, Inc. Method of DNA sequencing using cleavable tags
EP1407046A4 (fr) * 2001-06-29 2004-12-29 Agilent Technologies Inc Procede de sequen age d'adn au moyen d'etiquettes clivables
EP1407046A1 (fr) * 2001-06-29 2004-04-14 Agilent Technologies Inc. Procede de sequen age d'adn au moyen d'etiquettes clivables
US7427673B2 (en) 2001-12-04 2008-09-23 Illumina Cambridge Limited Labelled nucleotides
US9410200B2 (en) 2001-12-04 2016-08-09 Illumina Cambridge Limited Labelled nucleotides
US10480025B2 (en) 2001-12-04 2019-11-19 Illumina Cambridge Limited Labelled nucleotides
US7772384B2 (en) 2001-12-04 2010-08-10 Illumina Cambridge Limited Labelled nucleotides
EP1451351B1 (fr) 2001-12-04 2017-02-01 Illumina Cambridge Limited Nucleotides marques
US7785796B2 (en) 2001-12-04 2010-08-31 Illumina Cambridge Limited Labelled nucleotides
US9388463B2 (en) 2001-12-04 2016-07-12 Illumina Cambridge Limited Labelled nucleotides
EP2338893B1 (fr) 2001-12-04 2017-06-28 Illumina Cambridge Limited Nucléotides étiquetés
US8148064B2 (en) 2001-12-04 2012-04-03 Illumina Cambridge Limited Labelled nucleotides
US9605310B2 (en) 2001-12-04 2017-03-28 Illumina Cambridge Limited Labelled nucleotides
US10519496B2 (en) 2001-12-04 2019-12-31 Illumina Cambridge Limited Labelled nucleotides
US8158346B2 (en) 2001-12-04 2012-04-17 Illumina Cambridge Limited Labelled nucleotides
US9121062B2 (en) 2001-12-04 2015-09-01 Illumina Cambridge Limited Labelled nucleotides
US7566537B2 (en) 2001-12-04 2009-07-28 Illumina Cambridge Limited Labelled nucleotides
US8658418B2 (en) 2002-04-01 2014-02-25 Fluidigm Corporation Microfluidic particle-analysis systems
US8883092B2 (en) 2002-06-24 2014-11-11 Fluidigm Corporation Recirculating fluidic network and methods for using the same
US9506914B2 (en) 2002-06-24 2016-11-29 Fluidigm Corporation Recirculating fluidic network and methods for using the same
US8168139B2 (en) 2002-06-24 2012-05-01 Fluidigm Corporation Recirculating fluidic network and methods for using the same
US9121060B2 (en) 2002-08-23 2015-09-01 Illumina Cambridge Limited Modified nucleotides
US10513731B2 (en) 2002-08-23 2019-12-24 Illumina Cambridge Limited Modified nucleotides
US9127314B2 (en) 2002-08-23 2015-09-08 Illumina Cambridge Limited Labelled nucleotides
US10487102B2 (en) 2002-08-23 2019-11-26 Illumina Cambridge Limited Labelled nucleotides
US11008359B2 (en) 2002-08-23 2021-05-18 Illumina Cambridge Limited Labelled nucleotides
EP2119722B1 (fr) 2002-08-23 2016-10-26 Illumina Cambridge Limited Nucléotides étiquetées
EP3147292B1 (fr) 2002-08-23 2018-09-26 Illumina Cambridge Limited Nucléotides marqués
US7541444B2 (en) 2002-08-23 2009-06-02 Illumina Cambridge Limited Modified nucleotides
US9410199B2 (en) 2002-08-23 2016-08-09 Illumina Cambridge Limited Labelled nucleotides
US8071739B2 (en) 2002-08-23 2011-12-06 Illumina Cambridge Limited Modified nucleotides
EP3438116B1 (fr) 2002-08-23 2021-02-17 Illumina Cambridge Limited Nucléotides étiquetés
US9388464B2 (en) 2002-08-23 2016-07-12 Illumina Cambridge Limited Modified nucleotides
EP3002289B1 (fr) 2002-08-23 2018-02-28 Illumina Cambridge Limited Nucleotides modifies pour le sequençage de polynucleotide
US10640764B2 (en) 2002-09-12 2020-05-05 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10450560B2 (en) 2002-09-12 2019-10-22 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US10774325B2 (en) 2002-09-12 2020-09-15 Gen9, Inc. Microarray synthesis and assembly of gene-length polynucleotides
US9714443B2 (en) 2002-09-25 2017-07-25 California Institute Of Technology Microfabricated structure having parallel and orthogonal flow channels controlled by row and column multiplexors
US10328428B2 (en) 2002-10-02 2019-06-25 California Institute Of Technology Apparatus for preparing cDNA libraries from single cells
US10940473B2 (en) 2002-10-02 2021-03-09 California Institute Of Technology Microfluidic nucleic acid analysis
US9579650B2 (en) 2002-10-02 2017-02-28 California Institute Of Technology Microfluidic nucleic acid analysis
US8871446B2 (en) 2002-10-02 2014-10-28 California Institute Of Technology Microfluidic nucleic acid analysis
US20160222441A1 (en) * 2002-12-20 2016-08-04 Callida Genomics Materials and Methods Relating to Nano-Tags and Nano-Barcodes
US7771973B2 (en) 2002-12-23 2010-08-10 Illumina Cambridge Limited Modified nucleotides
EP2266699A1 (fr) 2003-04-02 2010-12-29 CLONDIAG GmbH Appareil pour l'amplification et la détection d'acide nucléique
US11028115B2 (en) 2003-08-22 2021-06-08 Illumina Cambridge Limited Labelled nucleotides
US11028116B2 (en) 2003-08-22 2021-06-08 Illumina Cambridge Limited Labelled nucleotides
US10995111B2 (en) 2003-08-22 2021-05-04 Illumina Cambridge Limited Labelled nucleotides
WO2005040425A2 (fr) * 2003-10-20 2005-05-06 Isis Innovation Ltd Methodes de sequençage de polymeres en parallele
EP2270205A2 (fr) 2003-10-20 2011-01-05 Isis Innovation Ltd Procédé de séquençage d'acide nucléique
WO2005040425A3 (fr) * 2003-10-20 2005-07-28 Isis Innovation Methodes de sequençage de polymeres en parallele
EP2270206A2 (fr) 2003-10-20 2011-01-05 Isis Innovation Ltd Procédé de séquençage d'acide nucléique
EP2431481A1 (fr) 2003-10-20 2012-03-21 Kalim Mir Procédés de séquençage de polymère parallèle
US9657344B2 (en) 2003-11-12 2017-05-23 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US20110151449A1 (en) * 2003-11-12 2011-06-23 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
US9012144B2 (en) 2003-11-12 2015-04-21 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
EP2280267A2 (fr) 2004-05-06 2011-02-02 CLONDIAG GmbH Dispositif et procédé pour de detecter des interactions moleculaires
EP2290351A2 (fr) 2004-05-06 2011-03-02 CLONDIAG GmbH Dispositif et procédé pour détecter des interactions moléculaires
EP2256478A2 (fr) 2004-05-06 2010-12-01 CLONDIAG GmbH Dispositif et procede pour de detecter des interactions moleculaires
EP3171155A1 (fr) 2004-05-06 2017-05-24 Clondiag GmbH Dispositif et procede pour de detecter des interactions moleculaires
EP3527287A1 (fr) 2004-05-06 2019-08-21 Alere Technologies GmbH Procéde pour de détecter des interactions moléculaires
EP2299257A2 (fr) 2004-05-06 2011-03-23 CLONDIAG GmbH Dispositif et procédé pour détecter des interactions moléculaires
US7315019B2 (en) 2004-09-17 2008-01-01 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US7313308B2 (en) 2004-09-17 2007-12-25 Pacific Biosciences Of California, Inc. Optical analysis of molecules
US7170050B2 (en) 2004-09-17 2007-01-30 Pacific Biosciences Of California, Inc. Apparatus and methods for optical analysis of molecules
US7302146B2 (en) 2004-09-17 2007-11-27 Pacific Biosciences Of California, Inc. Apparatus and method for analysis of molecules
US7476503B2 (en) 2004-09-17 2009-01-13 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US9909177B2 (en) 2005-06-21 2018-03-06 The Trustees Of Columbia University In The City Of New York Pyrosequencing methods and related compositions
US7805081B2 (en) 2005-08-11 2010-09-28 Pacific Biosciences Of California, Inc. Methods and systems for monitoring multiple optical signals from a single source
US7592435B2 (en) 2005-08-19 2009-09-22 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US7816503B2 (en) 2005-08-19 2010-10-19 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US8212015B2 (en) 2005-08-19 2012-07-03 Illumina Cambridge Limited Modified nucleosides and nucleotides and uses thereof
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US9868978B2 (en) 2005-08-26 2018-01-16 Fluidigm Corporation Single molecule sequencing of captured nucleic acids
US8058031B2 (en) 2005-09-29 2011-11-15 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs and uses therefor
US7777013B2 (en) 2005-09-29 2010-08-17 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs and uses therefor
US7405281B2 (en) 2005-09-29 2008-07-29 Pacific Biosciences Of California, Inc. Fluorescent nucleotide analogs and uses therefor
US7763423B2 (en) 2005-09-30 2010-07-27 Pacific Biosciences Of California, Inc. Substrates having low density reactive groups for monitoring enzyme activity
US7993891B2 (en) 2005-09-30 2011-08-09 Pacific Biosciences Of California, Inc. Method for binding reactive groups in observation area of zero mode waveguide
US8137942B2 (en) 2005-09-30 2012-03-20 Pacific Biosciences Of California, Inc. Method of preparing a modified surface
US9297042B2 (en) 2005-10-31 2016-03-29 The Trustees Of Columbia University In The City Of New York Chemically cleavable 3′-O-allyl-dNTP-allyl-fluorophore fluorescent nucleotide analogues and related methods
US9255292B2 (en) 2005-10-31 2016-02-09 The Trustees Of Columbia University In The City Of New York Synthesis of four-color 3′-O-allyl modified photocleavable fluorescent nucleotides and related methods
EP3572789A1 (fr) 2005-11-04 2019-11-27 Alere Technologies GmbH Dispositif et procédé de détection de particules
EP2330215A1 (fr) 2005-11-04 2011-06-08 CLONDIAG GmbH Procédé et dispositif de détection d'interactions moléculaires
US7998717B2 (en) 2005-12-02 2011-08-16 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions
US8415128B2 (en) 2005-12-02 2013-04-09 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions
US7993895B2 (en) 2005-12-02 2011-08-09 Pacific Biosciences Of California, Inc. Mitigation of photodamage in analytical reactions
US8071346B2 (en) 2005-12-02 2011-12-06 Pacific Bioscience Of California, Inc. System for the mitigation of photodamage in analytical reactions
US7995202B2 (en) 2006-02-13 2011-08-09 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US8264687B2 (en) 2006-02-13 2012-09-11 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7715001B2 (en) 2006-02-13 2010-05-11 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7630073B2 (en) 2006-02-13 2009-12-08 Pacific Biosciences Of California Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7626704B2 (en) 2006-02-13 2009-12-01 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US8149399B2 (en) 2006-02-13 2012-04-03 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7961314B2 (en) 2006-02-13 2011-06-14 Pacific Biosciences Of California, Inc. Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US7692783B2 (en) 2006-02-13 2010-04-06 Pacific Biosciences Of California Methods and systems for simultaneous real-time monitoring of optical signals from multiple sources
US8420017B2 (en) 2006-02-28 2013-04-16 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US7815868B1 (en) 2006-02-28 2010-10-19 Fluidigm Corporation Microfluidic reaction apparatus for high throughput screening
US8975216B2 (en) 2006-03-30 2015-03-10 Pacific Biosciences Of California Articles having localized molecules disposed thereon and methods of producing same
US8193123B2 (en) 2006-03-30 2012-06-05 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US8802600B2 (en) 2006-03-30 2014-08-12 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US8772202B2 (en) 2006-03-30 2014-07-08 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US9944980B2 (en) 2006-03-30 2018-04-17 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US11186871B2 (en) 2006-03-30 2021-11-30 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US10655172B2 (en) 2006-03-30 2020-05-19 Pacific Biosciences Of California, Inc. Articles having localized molecules disposed thereon and methods of producing same
US7563574B2 (en) 2006-03-31 2009-07-21 Pacific Biosciences Of California, Inc. Methods, systems and compositions for monitoring enzyme activity and applications thereof
US8053742B2 (en) 2006-09-01 2011-11-08 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8471230B2 (en) 2006-09-01 2013-06-25 Pacific Biosciences Of California, Inc. Waveguide substrates and optical systems and methods of use thereof
US8471219B2 (en) 2006-09-01 2013-06-25 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US7820983B2 (en) 2006-09-01 2010-10-26 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US9587276B2 (en) 2006-09-01 2017-03-07 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US9029802B2 (en) 2006-09-01 2015-05-12 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8618507B1 (en) 2006-09-01 2013-12-31 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8207509B2 (en) 2006-09-01 2012-06-26 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US7838847B2 (en) 2006-09-01 2010-11-23 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US7834329B2 (en) 2006-09-01 2010-11-16 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US9222133B2 (en) 2006-09-01 2015-12-29 Pacific Biosciences Of California, Inc. Substrates, systems and methods for analyzing materials
US8399188B2 (en) 2006-09-28 2013-03-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US9469873B2 (en) 2006-09-28 2016-10-18 Illumina, Inc. Compositions and methods for nucleotide sequencing
US8808988B2 (en) 2006-09-28 2014-08-19 Illumina, Inc. Compositions and methods for nucleotide sequencing
US9051612B2 (en) 2006-09-28 2015-06-09 Illumina, Inc. Compositions and methods for nucleotide sequencing
EP2548647A1 (fr) 2006-10-20 2013-01-23 CLONDIAG GmbH Dispositifs et méthodes de dosage destinés à la détection de substances á analyser
US9528151B2 (en) 2006-12-01 2016-12-27 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US11098353B2 (en) 2006-12-01 2021-08-24 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US11939631B2 (en) 2006-12-01 2024-03-26 The Trustees Of Columbia University In The City Of New York Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
US10457985B2 (en) 2007-02-02 2019-10-29 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US9512478B2 (en) 2007-02-02 2016-12-06 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US10988806B2 (en) 2007-02-02 2021-04-27 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US11634768B2 (en) 2007-02-02 2023-04-25 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US8053192B2 (en) 2007-02-02 2011-11-08 Illumina Cambridge Ltd. Methods for indexing samples and sequencing multiple polynucleotide templates
US20080199874A1 (en) * 2007-02-16 2008-08-21 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle dna
US8551704B2 (en) 2007-02-16 2013-10-08 Pacific Biosciences Of California, Inc. Controllable strand scission of mini circle DNA
WO2009007294A1 (fr) 2007-07-06 2009-01-15 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Ensemble d'électrodes à membranes
US9732383B2 (en) 2007-07-26 2017-08-15 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US7901889B2 (en) 2007-07-26 2011-03-08 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US8535882B2 (en) 2007-07-26 2013-09-17 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US9051611B2 (en) 2007-07-26 2015-06-09 Pacific Biosciences Of California, Inc. Molecular redundant sequencing
US8304191B2 (en) 2007-09-28 2012-11-06 Pacific Biosciences Of California, Inc. Nucleic acid sequencing methods and systems
US8003330B2 (en) 2007-09-28 2011-08-23 Pacific Biosciences Of California, Inc. Error-free amplification of DNA for clonal sequencing
US7960116B2 (en) 2007-09-28 2011-06-14 Pacific Biosciences Of California, Inc. Nucleic acid sequencing methods and systems
US11242561B2 (en) 2007-10-19 2022-02-08 The Trustees Of Columbia University In The City Of New York DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US9115163B2 (en) 2007-10-19 2015-08-25 The Trustees Of Columbia University In The City Of New York DNA sequence with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US10144961B2 (en) 2007-10-19 2018-12-04 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9175342B2 (en) 2007-10-19 2015-11-03 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US10260094B2 (en) 2007-10-19 2019-04-16 The Trustees Of Columbia University In The City Of New York DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators
US11208691B2 (en) 2007-10-19 2021-12-28 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US9670539B2 (en) 2007-10-19 2017-06-06 The Trustees Of Columbia University In The City Of New York Synthesis of cleavable fluorescent nucleotides as reversible terminators for DNA sequencing by synthesis
US8802424B2 (en) 2008-01-10 2014-08-12 Pacific Biosciences Of California, Inc. Methods and systems for analysis of fluorescent reactions with modulated excitation
US8252911B2 (en) 2008-02-12 2012-08-28 Pacific Biosciences Of California, Inc. Compositions and methods for use in analytical reactions
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US9404146B2 (en) 2008-03-28 2016-08-02 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
US8153375B2 (en) 2008-03-28 2012-04-10 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
US9600626B2 (en) 2008-03-28 2017-03-21 Pacific Biosciences Of California, Inc. Methods and systems for obtaining a single molecule consensus sequence
US8535886B2 (en) 2008-03-28 2013-09-17 Pacific Biosciences Of California, Inc. Methods and compositions for nucleic acid sample preparation
US8309330B2 (en) 2008-03-28 2012-11-13 Pacific Biosciences Of California, Inc. Diagnostic sequencing with small nucleic acid circles
US9910956B2 (en) 2008-03-28 2018-03-06 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US9542527B2 (en) 2008-03-28 2017-01-10 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
US8455193B2 (en) 2008-03-28 2013-06-04 Pacific Biosciences Of California, Inc. Compositions and methods for nucleic acid sequencing
US9556480B2 (en) 2008-03-28 2017-01-31 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US9582640B2 (en) 2008-03-28 2017-02-28 Pacific Biosciences Of California, Inc. Methods for obtaining a single molecule consensus sequence
US9057102B2 (en) 2008-03-28 2015-06-16 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8236499B2 (en) 2008-03-28 2012-08-07 Pacific Biosciences Of California, Inc. Methods and compositions for nucleic acid sample preparation
US9738929B2 (en) 2008-03-28 2017-08-22 Pacific Biosciences Of California, Inc. Nucleic acid sequence analysis
US8834797B2 (en) 2008-04-04 2014-09-16 Life Technologies Corporation Scanning system and method for imaging and sequencing
US11092548B2 (en) 2008-04-04 2021-08-17 Life Technologies Corporation Scanning system and method for imaging and sequencing
US10107758B2 (en) 2008-04-04 2018-10-23 Life Technologies Corporation Scanning system and method for imaging and sequencing
US8835135B2 (en) 2008-08-05 2014-09-16 Pacific Biosciences Of California, Inc. Reaction mixtures for prevention and alleviation of steric hindrance during single molecule synthesis
US8198023B2 (en) 2008-08-05 2012-06-12 Pacific Biosciences Of California, Inc. Prevention and alleviation of steric hindrance during single molecule nucleic acid synthesis by a polymerase
US20100081140A1 (en) * 2008-08-08 2010-04-01 President And Fellows Of Harvard College Chemically cleavable phosphoramidite linkers for sequencing by ligation
US8530156B2 (en) * 2008-08-08 2013-09-10 President And Fellows Of Harvard College Chemically cleavable phosphoramidite linkers for sequencing by ligation
US8795961B2 (en) 2008-09-05 2014-08-05 Pacific Biosciences Of California, Inc. Preparations, compositions, and methods for nucleic acid sequencing
US12043868B2 (en) 2008-09-16 2024-07-23 Pacific Biosciences Of California, Inc. Substrates and optical systems and methods of use thereof
US9222123B2 (en) 2008-09-16 2015-12-29 Pacific Biosciences Of California, Inc. Analytic devices comprising optical waveguides and nanometer-scale apertures and methods of uses thereof
US8274040B2 (en) 2008-09-16 2012-09-25 Pacific Biosciences Of California, Inc. Substrates and optical system having at least one optical waveguide, at least one nanometer-scale aperture and at least one lens array and methods of use thereof
US10697012B2 (en) 2008-09-16 2020-06-30 Pacific Biosciences Of California, Inc. Analytic device comprising a nanohole extending through an opaque mask layer and into a waveguide cladding
US11560591B2 (en) 2008-09-16 2023-01-24 Pacific Biosciences Of California, Inc. Analytic device comprising a substrate, nanometer-scale wells, and shallow waveguide optically coupled to a deep waveguide
US9719138B2 (en) 2008-09-16 2017-08-01 Pacific Biosciences Of California, Inc. Substrates and optical systems and methods of use thereof having a single optically resolvable immobilized reaction component disposed within a nanometer-scale aperture
US10968482B2 (en) 2008-09-16 2021-04-06 Pacific Biosciences Of California, Inc. Substrates and optical systems and methods of use thereof for performing sequencing by synthesis
US10280457B2 (en) 2008-09-16 2019-05-07 Pacific Biosciences Of California, Inc. Substrates and optical systems having a waveguide, nanometer-scale apertures, a lens array, and sensing regions and methods of use thereof
US8921046B2 (en) 2008-09-19 2014-12-30 Pacific Biosciences Of California, Inc. Nucleic acid sequence analysis
US9551028B2 (en) 2008-09-19 2017-01-24 Pacific Biosciences Of California, Inc. Nucleic acid sequence analysis
US8481264B2 (en) 2008-09-19 2013-07-09 Pacific Biosciences Of California, Inc. Immobilized nucleic acid complexes for sequence analysis
US8383369B2 (en) 2008-09-24 2013-02-26 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8628940B2 (en) 2008-09-24 2014-01-14 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US11214830B2 (en) 2008-09-24 2022-01-04 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US10563255B2 (en) 2008-09-24 2020-02-18 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US8143030B2 (en) 2008-09-24 2012-03-27 Pacific Biosciences Of California, Inc. Intermittent detection during analytical reactions
US11866780B2 (en) 2008-10-02 2024-01-09 Illumina Cambridge Limited Nucleic acid sample enrichment for sequencing applications
US8728764B2 (en) 2008-10-02 2014-05-20 Illumina Cambridge Limited Nucleic acid sample enrichment for sequencing applications
US9702002B2 (en) 2008-10-02 2017-07-11 Illumina, Inc. Nucleic acid sample enrichment for sequencing applications
US10745750B2 (en) 2008-11-19 2020-08-18 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US10161002B2 (en) 2008-11-19 2018-12-25 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US9551031B2 (en) 2008-11-19 2017-01-24 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US9879319B2 (en) 2008-11-19 2018-01-30 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US8252910B2 (en) 2008-11-19 2012-08-28 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US8846881B2 (en) 2008-11-19 2014-09-30 Pacific Biosciences Of California, Inc. Modular nucleotide compositions and uses therefor
US8370079B2 (en) 2008-11-20 2013-02-05 Pacific Biosciences Of California, Inc. Algorithms for sequence determination
US8993230B2 (en) 2008-12-04 2015-03-31 Pacific Biosciences of Californ, Inc. Asynchronous sequencing of biological polymers
US9175341B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
US11844666B2 (en) 2008-12-11 2023-12-19 Pacific Biosciences Of California, Inc. Classification of nucleic acid templates
US10793903B2 (en) 2008-12-11 2020-10-06 Pacific Biosciences Of California, Inc. Identifying organisms in a sample using sequencing kinetic signatures
US9175338B2 (en) 2008-12-11 2015-11-03 Pacific Biosciences Of California, Inc. Methods for identifying nucleic acid modifications
US9951383B2 (en) 2008-12-11 2018-04-24 Pacific Biosciences Of California, Inc. Methods of sequencing and identifying the position of a modified base in a nucleic acid
US10294523B2 (en) 2008-12-11 2019-05-21 Pacific Biosciences Of California, Inc. Identification of nucleic acid template-linked barcodes comprising nucleic acid modifications
US9200320B2 (en) 2009-04-27 2015-12-01 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8940507B2 (en) 2009-04-27 2015-01-27 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8501405B2 (en) 2009-04-27 2013-08-06 Pacific Biosciences Of California, Inc. Real-time sequencing methods and systems
US8501406B1 (en) 2009-07-14 2013-08-06 Pacific Biosciences Of California, Inc. Selectively functionalized arrays
US8541172B2 (en) 2009-09-15 2013-09-24 Illumina Cambridge Limited Method for sequencing a polynucelotide template
US9758825B2 (en) 2009-09-15 2017-09-12 Illumina Cambridge Limited Centroid markers for image analysis of high density clusters in complex polynucleotide sequencing
US8182994B2 (en) 2009-09-15 2012-05-22 Illumina Cambridge Limited Centroid markers for image analysis of high denisty clusters in complex polynucleotide sequencing
US8795971B2 (en) 2009-09-15 2014-08-05 Illumina Cambridge Limited Centroid markers for image analysis of high density clusters in complex polynucleotide sequencing
US8518643B2 (en) 2010-02-04 2013-08-27 Pacific Biosciences Of California, Inc. Method to improve single molecule analyses
US8603741B2 (en) 2010-02-18 2013-12-10 Pacific Biosciences Of California, Inc. Single molecule sequencing with two distinct chemistry steps
US9587275B2 (en) 2010-02-18 2017-03-07 Pacific Biosciences Of California, Inc. Single molecule sequencing with two distinct chemistry steps
US9447464B2 (en) 2010-02-18 2016-09-20 Pacific Biosciences Of California, Inc. Single molecule sequencing with two distinct chemistry steps
US9169515B2 (en) 2010-02-19 2015-10-27 Life Technologies Corporation Methods and systems for nucleic acid sequencing validation, calibration and normalization
US10337058B2 (en) 2010-02-19 2019-07-02 Life Tech Nologies Corporation Methods and systems for nucleic acid sequencing validation, calibration and normalization
US10337057B2 (en) 2010-02-19 2019-07-02 Life Technologies Corporation Methods and systems for nucleic acid sequencing validation, calibration and normalization
US8318094B1 (en) 2010-06-18 2012-11-27 Pacific Biosciences Of California, Inc. Substrate analysis systems
US9732382B2 (en) 2010-08-12 2017-08-15 Pacific Biosciences Of California, Inc. Photodamage mitigation compounds and systems
US8834847B2 (en) 2010-08-12 2014-09-16 Pacific Biosciences Of California, Inc. Photodamage mitigation compounds and systems
US8465922B2 (en) 2010-08-26 2013-06-18 Pacific Biosciences Of California, Inc. Methods and systems for monitoring reactions
US10934585B2 (en) 2011-03-23 2021-03-02 Pacific Biosciences Of California, Inc. Loading extended polymerase-nucleic acid complexes
US8658364B2 (en) 2011-03-23 2014-02-25 Pacific Biosciences Of California, Inc. Isolation of polymerase-nucleic acid complexes
US9381517B2 (en) 2011-03-23 2016-07-05 Pacific Biosciences Of California, Inc. Apparatus for loading molecules onto substrates
US10000805B2 (en) 2011-03-23 2018-06-19 Pacific Biosciences Of California, Inc. Isolation of polymerase-nucleic acid complexes
US9475054B2 (en) 2011-03-23 2016-10-25 Pacific Biosciences Of California, Inc. Isolation of polymerase-nucleic acid complexes
US8715930B2 (en) 2011-03-23 2014-05-06 Pacific Biosciences Of California, Inc. Loading molecules onto substrates
US11827934B2 (en) 2011-03-23 2023-11-28 Pacific Biosciences Of California, Inc. Methods for isolating nucleic acids
US9611510B2 (en) 2011-04-06 2017-04-04 The University Of Chicago Composition and methods related to modification of 5-methylcytosine (5-mC)
US9624539B2 (en) 2011-05-23 2017-04-18 The Trustees Of Columbia University In The City Of New York DNA sequencing by synthesis using Raman and infrared spectroscopy detection
US10273537B2 (en) 2011-10-14 2019-04-30 Pacific Biosciences Of California, Inc. Real-time redox sequencing methods
US10941443B2 (en) 2011-10-14 2021-03-09 Pacific Biosciences Of California, Inc. Real-time redox sequencing chips
US11014958B2 (en) 2012-02-15 2021-05-25 Pacific Biosciences Of California, Inc. Fluorescent polymerase enzyme substrates having protein shields
US10023605B2 (en) 2012-02-15 2018-07-17 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs having protein shields
US9062091B2 (en) 2012-02-15 2015-06-23 Pacific Biosciences Of California, Inc. Polymerase enzyme substrates with protein shield
US11718639B2 (en) 2012-02-15 2023-08-08 Pacific Biosciences Of California, Inc. Fluorescent polymerase enzyme substrates having protein shields
US9238836B2 (en) 2012-03-30 2016-01-19 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing modified nucleic acids
US10590484B2 (en) 2012-03-30 2020-03-17 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing modified nucleic acids
US9175348B2 (en) 2012-04-24 2015-11-03 Pacific Biosciences Of California, Inc. Identification of 5-methyl-C in nucleic acid templates
US10982277B2 (en) 2013-03-15 2021-04-20 Illumina Cambridge Limited Modified nucleosides or nucleotides
US10407721B2 (en) 2013-03-15 2019-09-10 Illumina Cambridge Limited Modified nucleosides or nucleotides
US20170166961A1 (en) 2013-03-15 2017-06-15 Illumina Cambridge Limited Modified nucleosides or nucleotides
US10648026B2 (en) 2013-03-15 2020-05-12 The Trustees Of Columbia University In The City Of New York Raman cluster tagged molecules for biological imaging
US9593373B2 (en) 2013-03-15 2017-03-14 Illumina Cambridge Limited Modified nucleosides or nucleotides
US12060383B2 (en) 2013-08-05 2024-08-13 Pacific Biosciences Of California, Inc. Protected fluorescent reagent compounds
US11578093B2 (en) 2013-08-05 2023-02-14 Pacific Biosciences Of California, Inc. Protected fluorescent reagent compounds
US9957291B2 (en) 2013-08-05 2018-05-01 Pacific Biosciences Of California, Inc. Protected fluorescent reagent compounds
US10800805B2 (en) 2013-08-05 2020-10-13 Pacific Biosciences Of California, Inc. Protected fluorescent reagent compounds
US9212394B2 (en) 2013-08-19 2015-12-15 Singular Bio, Inc. Assays for single molecule detection and use thereof
US11326204B2 (en) 2013-08-19 2022-05-10 Invitae Corporation Assays for single molecule detection and use thereof
US10626450B2 (en) 2013-08-19 2020-04-21 Singular Bio, Inc. Assays for single molecule detection and use thereof
US9758814B2 (en) 2013-08-19 2017-09-12 Singular Bio, Inc. Assays for single molecule detection and use thereof
US9416414B2 (en) 2013-10-24 2016-08-16 Pacific Biosciences Of California, Inc. Delaying real-time sequencing
US10081836B2 (en) 2013-10-24 2018-09-25 Pacific Biosciences Of California, Inc. Delaying real-time sequencing
US11629376B2 (en) 2013-10-24 2023-04-18 Pacific Biosciences Of California, Inc. Delaying real-time sequencing
US11269199B2 (en) 2015-01-23 2022-03-08 Pacific Biosciences Of California, Inc. Producing bragg gratings in optical waveguides
US10302972B2 (en) 2015-01-23 2019-05-28 Pacific Biosciences Of California, Inc. Waveguide transmission
US10787573B2 (en) 2015-02-04 2020-09-29 Pacific Biosciences Of California, Inc. Multimeric protected fluorescent reagents
US10150872B2 (en) 2015-02-04 2018-12-11 Pacific Biosciences Of California, Inc. Multimeric protected fluorescent reagents
US11739371B2 (en) 2015-02-18 2023-08-29 Invitae Corporation Arrays for single molecule detection and use thereof
WO2017087823A1 (fr) 2015-11-18 2017-05-26 Mir Kalim U Séquençage super-résolution
US10781483B2 (en) 2015-11-20 2020-09-22 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
US11203689B2 (en) 2015-11-20 2021-12-21 Pacific Biosciences Of California, Inc. Protected dye-labeled reagents
US11359235B2 (en) 2015-11-20 2022-06-14 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
US11466319B2 (en) 2015-11-20 2022-10-11 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
US11884826B2 (en) 2015-11-20 2024-01-30 Pacific Biosciences Of California, Inc. Protected dye-labeled reagents
US12000000B2 (en) 2015-11-20 2024-06-04 Pacific Biosciences Of California, Inc. Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
US12037641B2 (en) 2015-11-20 2024-07-16 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
US10669299B2 (en) 2015-11-20 2020-06-02 Pacific Biosciences Of California, Inc. Protected dye-labeled reagents
US10676788B2 (en) 2015-11-20 2020-06-09 Pacific Biosciences Of California, Inc. Modified nucleotide reagents
US11827931B2 (en) 2018-12-26 2023-11-28 Illumina Cambridge Limited Methods of preparing growing polynucleotides using nucleotides with 3′ AOM blocking group
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
US11787831B2 (en) 2020-06-22 2023-10-17 Illumina Cambridge Limited Nucleosides and nucleotides with 3′ acetal blocking group

Also Published As

Publication number Publication date
AU5171696A (en) 1996-09-18

Similar Documents

Publication Publication Date Title
WO1996027025A1 (fr) Dispositif, composes, algorithmes, et procedes de caracterisation et manipulation avec parallelisme moleculaire
JP4294740B2 (ja) 分析物の系列的プロセシングのためのシステムおよび装置
JP4741079B2 (ja) 拡大タグを使用したシークエンシング方法
US7282370B2 (en) System and apparatus for sequential processing of analytes
US20030022207A1 (en) Arrayed polynucleotides and their use in genome analysis
US20020137052A1 (en) System and apparatus for sequential processing of analytes
US20220228201A1 (en) Molecular arrays and methods for generating and using the arrays
US20060040314A1 (en) Methods for screening polypeptides
JP2001513109A (ja) 核酸含有重合可能錯体
WO1997013877A1 (fr) Mesure de profils d'expression genique pour evaluer la toxicite
CA2402263A1 (fr) Surfaces revetues de polymere pour micro-echantillons
JP2007521833A (ja) 配列特異的分類による遺伝的分析
CA2339121A1 (fr) Biomolecules en rangees et leur utilisation dans une procedure de sequencage
WO2004050915A1 (fr) Determination de la methylation de sequences d'acide nucleique
EP1597397A1 (fr) Analyse de sequence d'adn
AU2098999A (en) Solid-phase tips and uses relating thereto
WO2005021786A1 (fr) Methode de sequençage d'acides nucleiques par ligature d'oligonucleotiques marques
US6451525B1 (en) Parallel sequencing method
JP2002531106A (ja) 不連続プライマ−伸長による核酸反復配列の長さ決定
JP2001508308A (ja) 核酸配列決定
US7297479B2 (en) DNA-based analog neural networks
EP1026258A2 (fr) Génotypage multiplex de populations d'individus
WO2005030987A1 (fr) Oligonucleotides en epingle a cheveu partiellement double brin immobilisables par l'extremite en boucle et comprenant un site de restriction
US20100130368A1 (en) Method and system for sequencing polynucleotides
WO2004050916A1 (fr) Recuperation de modele d'origine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase