WO1997007245A1 - Procedes et dispositifs de sequençage multiplex parallele de polynucleotides - Google Patents

Procedes et dispositifs de sequençage multiplex parallele de polynucleotides Download PDF

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WO1997007245A1
WO1997007245A1 PCT/US1996/013313 US9613313W WO9707245A1 WO 1997007245 A1 WO1997007245 A1 WO 1997007245A1 US 9613313 W US9613313 W US 9613313W WO 9707245 A1 WO9707245 A1 WO 9707245A1
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array
parallel
arrays
separatory
separation
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Ely Michael Rabani
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Ely Michael Rabani
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    • B01J2219/00718—Type of compounds synthesised
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    • B01J2219/00722—Nucleotides
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    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
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    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to the field of molecular biology, and more specifically the determination of polynucleotide sequence composition.
  • each such differently terminated sample aliquot comprises a so-called "nested set" of populations of molecules of different length, up to the length of the input sample molecules, but identically having the specified 3' terminal base composition.
  • the length of each subpopulation within each such sample aliquot corresponds to a position of the base moiety, for which that sable aliquot has been treated, within the sample sequence.
  • the nested sets produced by both of these methods are conventionally separated according to length by electrophoresis through gel matrices, generally of polyacrylamide composition.
  • the sample is labeled with radiolabels or dye moieties such that appropriate detection techniques may localize the position of the bands which are produced by electrophoresis of different length molecules in the gel.
  • this method is implemented by transferring or blotting the gel after electrophoresis onto a nitrocellulose or nylon membrane such that the size-separated polynucleotide molecules are deposited onto said membrane with preservation of spatial configuration of the resultant banding pattern.
  • a further step such as UV illumination of said membrane, is performed to further strengthen the immobilization of said polynucleotide molecules.
  • Probing is generally performed with a radiolabeled, dye labeled or enzyme linked oligonucleotide complementary to the respective tag sequence. After image capture of the banding pattern thus revealed, the oligo probe is denatured from its cognate tag sequence and washed away from the membrane.
  • the membrane is then serially reprobed in the same way until many or all distinctly tagged samples have been detected. Data from each probed sample is used to reconstruct sequence information in the same manner as with Maxam and Gilbert or Sanger sequencing methods. Beyond the reduction of electrophoresis time and effort per sample by thi ⁇ method, distinction with unique tags permits several samples to be pooled together, which enables several samples to be processed in only the four reactions specific to each base, significantly reducing handling steps. A drawback of this method has been the difficulty of automating the serial probing of membranes, though efforts have been made in this regard. 6 ' 7 ' 8
  • Manifold Sample Transfer So-called manifold sequencing, a method developed by A. Lagerkvist et al. ,-*- 1 reduces manual manipulations in the sequencing of many samples by using modified gel combs or similar structures for the transfer of several samples from sample wells of, for example, microtitre plates, wherein reactions such as PCR and enzymatic sequencing are performed, as well as to a slab-gel.
  • Such a method permits handling of more samples than parallel pipettors commercially available with greater simplicity and economy. This is accomplished through the immobilization of sample molecules to the solid phase of the comb teeth, similarly by a method comprising the step of dipping these into wells initially containing sample material. This multiplies the number of samples which may be separately handled in a given manipulation step by the number of comb-teeth used, and has the further advantage of reducing handling errors, an issue critical in clinical applications of sequencing methods.
  • Nanochannel Array Glass R.J. Tonucci et al. 13 have demonstrated a method whereby two or more types of glass, at least one of which is acid-etchable and provided initially in the form of a rod, and one of which is inert, are heated, drawn and fused together repetitively, followed by an annealing step. The number of times such a set of steps is repeated, and the number of contiguous lengths fused together in each step, determine the number of channels ultimately formed, as well as the size of the resulting channels. Channels are formed in a final acid etch step with regularity across the entire medium.
  • A.T. Woolley and R.A. Mathies 14 have recently described microfabricated capillaries integrated on the surface of a solid substrate. These are formed by patterning and then etching plural trenches in a glass substrate according to photolithographic methods, and then sealing these to form a capillary array by fusion of said substrate with a juxtaposed glass plate. Such microfabricated capillary arrays are then filled, by injection, with a separation medium, for which these workers choose hydroxyethyl cellulose. Using devices with 30-120 micron capillary size, these workers demonstrated the separation of the DNA fragments resulting from Haelll restriction digestion of phiX174 replicative form DNA in 120 seconds. Capillaries of similar internal dimension but fabricated by conventional means have been used by these workers to separate sequence reaction products 15 ' 16 rapidly over sufficiently broad size ranges with single base resolution, indicating that sequence determination will be feasible with such microfabricated capillary arrays.
  • Microfabrication with Replication Integrated optical devices and other microscale optical components have been fabricated by the replication of relief patterns into polymeric materials 17 .
  • the optical properties of these materials and the structures resulting from pattern replication determine device function and characteristics.
  • Replication methods used include injection molding and casting into polymeric, elastomeric or metallic molds or mold inserts, and hot embossing with reliefs, for example, metal reliefs. Multiple cycles of replication may be performed to yield a large number of reliefs for the rapid mass production of devices, according to the fidelity of replication and acceptable device tolerances.
  • An original microrelief is formed by the lithographic depth patterning of a relief material, which is then developed to form the desired predetermined structure (first relief) .
  • Electroforming is then performed to yield a metal negative mold insert conforming to the polymer resist structure, and then separated from the original resist.
  • This first generation mold may then be as a mold for the casting or microinjection molding of a second polymeric relief nearly identical to the first relief.
  • the molding or casting process may be repeated with the first generation mold, and the electroforming process may be repeated with second or subsequent generation polymeric relief structures produced with said molds by appropriate methods.
  • M.T. Gale et al., 20 extend methods from the embossed diffractive foil and compact disk (CD) industries by the replication of an original (e.g. microfabricated) microrelief on the surface of a replication shim through electroforming, followed by replication of this first shim by surface passivation followed again by electroforming.
  • CD compact disk
  • These workers find that the shim replication process occurs with only slight ( ⁇ 2nm) increases in surface roughness per generation, thus enabling the reproduction of nanoscale relief features, with corresponding cost advantages.
  • Related injection molding methods are described by A. Neyer et al., 21 and R. Klein and A. Neyer. 22
  • a relief is produced by selectively exposing regions of a photoresist cpated onto a first surface and mechanically transferring either the exposed or non- exposed regions to a polymeric foil second surface by juxtaposing said second surface to the image-exposed polymeric resist and relying on the differential adhesion properties resulting from exposure.
  • a positive image is produced on one surface while the other surface retains the corresponding negative pattern.
  • Such relief patterns may provide differential wetting or liquid-retention properties differing from those of the underlying surfaces, and thus be used as offset lithographic plates.
  • Microcontact Printing with Elastomeric Microreliefs G.M.
  • Whitesides and co-workers have done considerable work developing methods for the use of replicated elastomeric microreliefs for microcontact printing. 24 It is presently important to note that this work demonstrates the conformation of the raised portions of an elastomeric microrelief surface to the shape of another surface to which it is juxtaposed and impressed. These workers have similarly used such reliefs for micromolding, which further indicates that conformed contacts resulting from impression of such microreliefs onto a juxtaposed surface forms a liquid-tight seal.
  • these workers situate azide (alkylazide) functionalities on a substrate surface, and contact a platinum coated AFM tip to these in the presence of dissolved H2, to effect the catalytic hydrogenation of those of said nitriles contacted by said AFM tip under positional control effected by an AFM.
  • the nitrile groups thus contacted are reduced to amines, which these workers detect by reaction to aldehyde modified fluorescently labeled beads, which are observed to bind only to regions which underwent said contact with said platinum.
  • Somorjai 26 have similarly effected positional control of metallic surface catalysis using a platinum-rhodium STM tip in an atmospheric-pressure chemical reactor to catalyze the rehydrogenation of molecules comprising alkyne functionalities on (111) platinum surfaces.
  • Sequencing bv Hybridization Two general variants of sequencing by hybridization methodology have been proposed.
  • a sample under investigation is hybridized to an array of oligonucleotides situated on a surface, fabricated, for example, by the spatially controlled light directed synthesis methods of S.P.A. Fodor et al., 31 , where the composition of an array element is related to the position of said element within said array of oligonucleotides.
  • an array may comprise all possible 10-mer oligos, in which case there are over 10° array elements.
  • An unknown sample is denatured and permitted to anneal with said array under stringent conditions. Said array is examined for the presence of bound sample molecules, generally by optical means.
  • the sequence composition of a sufficiently short sample molecule may be reconstructed by using information about which array elements have bound sample molecules and about the compositions of each of these array elements, with algorithms which overlap each of the short sequences (corresponding to the 10-mer oligos in this example) to produce a linear sequence.
  • DNA includes tandem and dispersed repeat elements (including transposons), a particular 10-mer, which is one of the more than IO 6 possible 10-mers may occur many times in naturally occurring sequences much shorter than a megabase. 32 Thus, this method will lead to branch points which ambiguate the sequence data thus obtained.
  • oligo probe libraries or arrays will contain more elements, which thus occur less frequently, but require more surface area and synthesis steps, and will further diminish the ability to discriminate between perfect and mismatched hybridization.
  • hybridization is not perfect for all sequences under any given conditions, as is clear to those familiar with other probe hybridization techniques such as northern blot gel analysis; optimal conditions for a particular probe to form perfect hybrids and not form imperfect or mismatched hybrids vary in a sequence dependent manner, and it is not always simple to predict this optimum even where only a single probe is concerned.
  • optimal conditions for a particular probe to form perfect hybrids and not form imperfect or mismatched hybrids vary in a sequence dependent manner, and it is not always simple to predict this optimum even where only a single probe is concerned.
  • only a subset of elements will display optimal binding discrimination characteristics under any set of particular conditions.
  • the sample sequence (which is generally in the form of a phage or plasmid library) is immobilized, generally in the form of bacteriophage plaque or bacterial colony replicas, on a membrane, which is serially probed with a different labeled oligonucleotide of known sequence and length n.
  • a membrane which is serially probed with a different labeled oligonucleotide of known sequence and length n.
  • conditions may be closely optimized for homoduplex formation in each probing step. Plaques or colonies forming hybrids are noted. Repetition over the full combinatorial library of oligonucleotides of sufficient complexity relative to the complexity of the sample thus provides information about all of the n-mer sequences present in a particular plaque or colony.
  • linear sequence information may be reconstructed as in the first variant of sequencing by hybridization methodology.
  • the two main drawbacks of this method are the requirement for synthesis of large probe libraries, which is much more laborious than the production of surface immobilized arrays by optically patterned deprotection 35 , and that significant linear sequence information may only be reassembled only after many (thousands or more) probing steps.
  • the first variant of sequencing by hybridization is, however, proposed as a confirmatory sequencing method to identify any errors in sequence information obtained by other methods, and the same methodology is proposed as a means of classifying polynucleotides in unknown samples, for example, to detect the presence and type of polynucleotides present in clinical isolates.
  • a characteristic pattern which may serve as a highly specific signature may nonetheless be observed.
  • E.M. Rabani teaches a method for the sequencing of polynucleotides, especially DNA, combining the separation throughput advantages gained in multiplex methods with the detection methods made possible by oligonucleotide arrays.
  • separation is performed so as to effect length dependent fractionation of complex mixtures of tag-primer primed sequencing reaction products, which said fractions are separately scored for the presence of tags, or for labeling moieties in communication with tags, on oligonucleotide arrays which comprise tag-probe sequences (i.e.
  • such arrays may comprise as many distinct tag-probe oligonucleotides as there are distinct elements in such arrays, which may exceed 10 * ⁇ or 10* different oligonucleotide elements per cm**-', depending on the fabrication methods used to produce these.)
  • said tag-primer primed sequencing reaction products are affinity labeled during that reaction with one of four distinct labeling moieties the identity of which corresponds to the identity of the terminal base moiety.
  • the color or identity of the bead bound to a particular array element or tag-probe reveals the identity of the terminal base moiety of the sequencing reaction product fragment thereupon, and hence the identity of the base at the position within the sequence of the tag-primer primed sequencing fragment probed by said tag probe (corresponding to said particular array element) corresponding to the position of said fraction within the series of fractions collected (in a manner precluding the presence of successive base terminated species of the same tag-primer primed sequencing reaction product in the same fraction) and more particularly within the ordered set of labeling data obtained from similar scoring of all successive fractions.
  • fractions may be applied successively to a single array, which may be referred to as serial fraction array tag-probing, or multiple fractions may be applied (on a one to one basis, preserving information regarding ordering) to plural said tag-probe arrays, which may be referred to as parallel fraction array tag- probing.
  • data regarding labeling of array elements may be collected from such tag-probe arrays sequentially by a single suitable photodetection means or in parallel by multiple such photodetection means.
  • CCD based detection is, nonetheless inherently parallel, so this latter distinction refers to hierarchy of detection parallelism.
  • sequence data collection rate with this method remains proportional to the complexity of the probe arrays used (i.e. the number of distinct probes per array) , but the effort or number of automated steps required is primarily determined by the number of fractions thus examined.
  • a third step these samples are each applied to a different separation channel.
  • samples are separated into the constituent molecular species which the nested set comprises according to length or mass separation (e.g. electrophoretic mobility or transport) .
  • the distinct said constituent molecular species in each separation channel are detected (either according to the final position reached in said separation channel at the time said fourth step was concluded by spatial detection method, the coordinate of which corresponds to the order of molecular species along the length of said sequencing channel, or according to the time or time delay at which a fragment is transported passed a detector situated at a fixed position along the length of a separation channel, concurrently with the separation step, in which case the separatory coordinate corresponds to the temporal ordering of transport dependent events; in short, these are spatial and temporal separation coordinate resolution methods, respectively.
  • a sixth step consists in the assembly of sequence information obtained in the preceding steps into longer stretches of sequence data where the sequences of distinct samples presents both sufficient identity and unambiguous or unique match at
  • sample preparation which may include creation of libraries or plasmids, growth, purification and/or amplification steps;
  • base specific fragment generation (sequencing reaction) which may be effected by enzymatic or chemical methods;
  • serial reprobing is made possible by the association of a unique tag sequence, generally as part of the priming molecule, though in the original method part of the sample sequence, by a labeled probe having a complementary sequence to said tag sequence, which such labeled probe will be referred to herein as a tag-probe.
  • a labeled probe having a complementary sequence to said tag sequence, which such labeled probe will be referred to herein as a tag-probe.
  • methods such as the encoding of terminator concentration or relative activity with corresponding effects on band intensity (e.g.
  • dye molecule labels or reporters which may be in communication with primers used (end-labeling), or with deoxynucleotides (length- labeling) permit the separation of the combined mixture of products of four separate sequencing reactions (each corresponding to a different chain terminator moiety and having been labeled with a dye moiety having a distinct absorption and/or emission spectrum) in a single lane, permitting further improvements in this limited sort of multiplexation of separation and detection.
  • the extent of such multiplexation based on distinct labeling is limited by the number of suitable spectrally distinct dyes available.
  • This category of base- moiety multiplexation limited to an individual primer-template sample has, however, been extended, through the use of distinct fluorescent or other optical labels in communication with each of the four chain terminator analog (generally, 2'-3'-dideoxy nucleotide triphosphates) such that the same synthesis event which terminates chain elongation also places a label corresponding to the identity of the incorporated chain terminator in communication with the resulting terminated elongation fragment.
  • This further improvement results in a four-fold reduction of the number of reactions which must be performed to sequence a template sample. Even in this last case, such methods, by themselves, only provide four-fold advantages through the steps of sequencing reactions, separation steps and detection, providing only four-fold advantage at the system throughput level.
  • the essence of the present invention is to combine any of the various multiplexing methods possible at each necessary step of Sanger enzymatic sequencing protocols or Maxam and Gilbert chemical sequencing protocols for use with appropriately prepared complex samples, with the advantageous degrees of parallelism at appropriate steps of sequencing protocols.
  • Prominent multiplex methods among these are multiplexing of samples in reactions directed at generating terminal base specific nested fragment sets or terminal base specific reporter-linked fragment sets, labeling and detection, as well as use of variations on aspects of the multiplex sequencing methods of Church and Gilbert obviating the crosslinking of sample sequencing reaction products to membranes with chemical reagents.
  • Prominent parallelization strategies enabled include parallel separation of sequencing templates or sequencing reaction products from mixtures prior to molecular mass fractionation (e.g.
  • single or plural sample mixtures or sample reaction-product mixtures may be prepared in multiplex, may each be separated in a manner directing them to specific separation channels or other separation means by parallel sample-specific binding means and thereafter separated by plural said specific separation channels or other separation means, combining both multiplexation and parallelism.
  • separation channels may comprise or suitably articulated with sample recovery means in turn comprising parallel sample separation means, may be detected by parallel detector means (e.g.
  • tag-probe oligonucleotide arrays and photodetector arrays such as CCDs for visible labels
  • signal amplitude encoding schemes where appropriate with detection methods used.
  • Suitable devices and apparata and variations thereupon facilitating such methods are further described.
  • parallelism will generally indicate concurrent performance of similar operations on different populations or sub-populations of molecules or samples across space. Parallelism thus entails the multiplication of devices or articles with the methods of the present invention to gain a corresponding multiplication in throughput.
  • multiplex will generally indicate the concurrent manipulation of different samples (or molecular products yielded by reactions performed collectively upon the ensemble of said different samples) within a continuous region of space, area or fluid volume. Multiplexing thus involves the reduction of handling steps, and the ratio of devices or articles used to accomplish a multiplexed step per sample, by treating different samples identically, which is possible where each of said different samples comprise unique features facilitating subsequent separation and/or discrimination according to the origins of molecules (or of molecular products yielded by reactions performed collectively upon the ensemble of said different samples) of from each of said different samples.
  • multiplex has been applied to arrangements or methods which are more accurately described as parallel. Note that the present usage is consistent with that employed by Church and Gilbert.
  • separation channel is used generically to include such diverse separations means as a mass spectrometer, a slab gel lane, a capillary gel, a capillary or the like.
  • formed channel, formed relief channel and articulated channel all of which may serve as separation channels and may be of varied form and composition, do not apply to structures formed from conventional capillary tubes. Lateral closure of such channels need not be permanent, and thus separated species may be recovered by laterally accessing said thus separated species by reversing said lateral closure of such channels, i.e. separating one or more portions of materials making up said channels so as to expose a substantial proportion of the internal volume of said channels.
  • Figure 1 depicts demultiplexation array means (10) .
  • said demultiplexation means are shown in a sealed channel formed by juxtaposition of trench (20), while in Figure lb, said demultiplexation means are shown each in one among many plural sealed channels formed by juxtaposition of trenches (30) which thus form a separatory channel array.
  • Figure lc shows an isometric cross-section of one of said separatory channels depicted in Figure lb.
  • Figure 2 depicts a bin array (100) comprising a grounded electrode contact (110) across the bottom surface of said bin array (shown with schematic symbol) and shows a portion of a transparent channel array (140) of appropriate pitch offset from said bin array.
  • Figure 3 depicts a superimposed view of and array (two sets of groves shown) comprising fraction transfer grooves and plural surface immobilized tag-probes (330) of a striped-patterned tag-probe array, and electrodes (350a and 350b) for lateral electroelution, positioned facingly over the trenched surface for forming a separatory channel array, with one such trench shown (320) .
  • features 400 and 420 are extensions of the surface comprising said fraction transfer grooves which serve to define two distinct openings to said separation channel trenches when the fraction transfer grove array surface is facingly juxtaposed to the separatory channel trench array surface, and further (420) to prevent cross-transfer of fractions.
  • samples may be prepared (and as necessary, tagged) in multiplex (including multiplex amplification) , subjected to sequencing reactions and/or labeling in multiplex, partially or completely demultiplexed with predetermined transfer to parallel separation channels, separated in multiplex in each of said parallel separation channels or merely in parallel with only one sequence per channel, and detected in parallel (including, as applicable, parallel detection of or by parallel detection of samples separated in multiplex, for each separation channel, favorably performed simultaneously and hence in parallel for all separation channels.)
  • Manifolds may be one dimensional combs as previously described by A. Lagerkvist et al., 36 or may be two dimensional regular arrays of teeth (e.g. rods) projecting from a two dimensional surface, fabricated by suitable means such as molding, including LIGA techniques or variations thereupon, or other suitable means.
  • manifold separation may further provide separation of probe targeted species from complex mixtures.
  • Oligo- probes are situated on manifold teeth and used to fish for probed sequences from a complex pool, e.g. several libraries or complex samples of different origin are individually contacted with one comb tooth, such that a comb may carry several separated sample fragments in a manner ordered according to which tooth was contacted with which sample, which is particularly useful for application to parallel separation channels.
  • This second variation is actually a specific case of demultiplexation, and manifolds with plural distinct oligos situated on said teeth may be seen as a specific case of demultiplexation arrays when used in this way, with multiplexed samples.
  • the present aspect of the present invention involves the use of one- or two- dimensional arrays of sample bins, wells or chambers
  • bins A single sample volume, comprising a number of (individual) molecules equal to the number of wells used, is distributed among these bins.
  • Poisson statistical random sample deposition or simply random isolation.
  • Samples are then (if they had not previously been, or are not otherwise suitable for amplification with standard primers) ligated to PCR primers, and then PCR or other amplification is performed in each chamber, where thermal cycling of all chambers is effected simultaneously, followed by reactions to generate nested set sequencing products with appropriate labels.
  • Other means of effecting parallel amplification are, of course, similarly comprehended by the present aspect of the present invention.
  • a sufficient number of PCR amplification cycles are performed to yield a quantity of DNA that will ensure that sufficient signal will result from the labels in each sequencing reaction fragment for reliable detection.
  • samples comprising distinct sample molecules which each comprise substantially different insert sequences or are each derived from substantially different regions of a genome, are generated in an ensemble of bins or chambers (e.g.
  • Said array of bins is designed so as to conveniently be juxtaposed to a separatory channel array, or alternatively to be used with manifold transfer means for transfer to a separatory channel array; i.e. said array of bins is arranged in either a mirror image of the configuration of entry ports to said separatory channel array with identical array pitch (for transfer by juxtaposition), or arranged identically to the configuration and pitch of said separatory channel array.
  • the approximately two-thirds inefficiency of random bin filling in absence of other measures to fill unfilled bins with single samples, may be greatly offset in various embodiments by the ability to load samples simply and in parallel into a very large number of parallel separation channels.
  • the method and means of the present aspect of the present invention may provide further advantages in cases where a sample is in the form of a complex heterogeneous mixture (e.g. a genomic library or a cDNA library comprising thousands to billions of different chimeric clones) having an estimated number of distinct clones per unit volume.
  • Clones may be isolated by such methods availing statistical separation by first diluting an appropriate volume of said complex heterogeneous mixture such that the desired number of molecules are present in a appropriate volume suitable for application to a bin array according to one of the two variations described above. On average, different clones will appear in a bin array in proportion to their relative copy number or representation within said complex heterogeneous mixture. For example, shotgun libraries may be thus sampled and separated into bins each containing one individual sample molecule, amplified, and directly applied to parallel separation channels or means.
  • transformation steps or phage packing steps may be eliminated in certain cases where a library need not be maintained, by randomly fragmenting genomic material, subjecting to treatment with phosphatase, and Iigating into vectors comprising appropriate amplification primer sequences, sequencing primer sequences, and optionally, tag sequences, and then separating into bin arrays for amplification.
  • genomic material may be shot-gun sequenced directly, without even any transformation steps, providing important rate and cost advantages in certain applications.
  • cDNA libraries may similarly be sequenced without transformation or phage packaging and growth steps.
  • Multiplex PCR products of genomic material may similarly be sequenced directly, for example yielding sequence information regarding several regions of each sample genome.
  • Various assays may be performed on all bins subsequent to sequencing reactions and prior to loading to separation channels to determine whether any polymerization took place within each bin, and further assess the degree of reaction which will depend in part on the number of individual molecules randomly distributed into each of said bins.
  • empty bins may be readily detected, and with sufficiently quantitative photodetection methods, bins containing material amplified from two or more individual polynucleotide molecules may similarly be detected.
  • Such photodetection of polymerization in said bin arrays is favorably accomplished with photon cameras or CCDs or the like interfaced with one or more digital computer.
  • individually controllable electrodes and electrode controlling means or of individually controllable parallel fluid transfer means (e.g. comprising controllable valves)
  • parallel transfer of samples from each bin of said bin array to the corresponding separatory channel of parallel separation means may be individually controlled.
  • the individual transfer controlling means of such an arrangement may be interfaced to one or more digital computer or the like, which may use data gathered as described above or equivalent data pertaining to the occupancy of each bin to control sample transfer of the contents of each bin of said bin array to said parallel separatory channels or separatory channel array such that only the contents of bins containing exactly one or alternatively fewer than two distinct samples are transferred to said separation means, while data concerning which separation channels have not thus received samples are further maintained.
  • Such a procedure is repeated with additional bin arrays, such that separatory channels not previously receiving samples will receive samples from any of said additional bin array if and only if the juxtaposed or otherwise corresponding bin contains either exactly one or alternatively fewer than two distinct samples.
  • the probability that any given separatory channel has not received an individual sample decreases by a factor of approximately one-third.
  • the probability that any separatory channel of said parallel separation means or separatory channel array has received sample material amplified from exactly one molecules is given by l-(2/3) n , for n>0, which indicates that after 4 transfer cycles over 80% of said separatory channels will have received sample material amplified from exactly one molecules, which percentage increases to over 91% after 6 transfer cycles.
  • bin-array transfer cycles may not represent significant costs of time or effort which maximizing the quantity of useful information obtained per unit time from a parallel separatory channel array of a given degree of parallelism.
  • the present aspect of the present invention uses one or more immobilized probes or tag-probes (which are generally oligonucleotides, but may, for example be sequence specific binding proteins, etc.) to selectively cause only polynucleotide sample molecules comprising complementary sequences to bind to a selected surface region or spatial region, to facilitate the transfer of only bound polynucleotide molecules to a particular separation channel.
  • immobilized probes or tag-probes which are generally oligonucleotides, but may, for example be sequence specific binding proteins, etc.
  • the ensemble of distinct said immobilized probes or tag-probes, each of which is chosen and/or synthesized to be substantially distinct from any other probes or probe-tags of the ensemble, are referred to as a demultiplexation array, which term also refers to the surface and object on which they are situated when used most generally.
  • said probes or tag-probes are provided as a linear array and are situated with a pitch equal to that of the entry ports of a separatory channel array such each element of said demultiplexation array is in, or may be placed in, convenient proximity to exactly one of said entry ports, and such that upon release of said sample molecules from said probes or tag-probes, said sample molecules are either situated within or are readily transported in a targeted manner to the corresponding said entry port.
  • release of bound sample molecules may, for example, be effected by increasing temperature sufficiently, or exposure to a sufficient concentration of denaturing reagents (e.g. 7M urea) .
  • a complex sample (termed a multiplexed sample) is contacted as a solution with said demultiplexing array such that the sample molecules therein may bind to complementary probes and such that most or all of the probe elements of said demultiplexation array will bind sample molecules of substantially identical origin.
  • a flat surface with a linear trench may be juxtaposed with said trench oriented lengthwise juxtaposedly along said demultiplexation array such that said trench passes over all array elements and forms a channel.
  • a sample mixture containing sequencing reaction product fragments for example, which are derived from plural distinct sequence regions (i.e.
  • the products of a multiplex sequencing reaction in this example is flowed through the channel thus formed such that it passes over each of the elements of said demultiplexation array, and non-denaturing conditions of sufficient stringency are maintained. Then a rinse solution is passed through said channel thus formed to carry away and unbound molecules, as non ⁇ denaturing conditions of sufficient stringency are maintained.
  • Said array elements of said demultiplexation arrays are designed and produced so as to comprise a sufficient number of probe molecules to capture a sufficient number of sequencing reaction product fragments for reliable detection of each species corresponding to termination at a particular base with the detection method used.
  • Said sample mixture is prepared so as to either not have any two identical tag sequences in communication with different sample sequences to eliminate probing ambiguity, or is produced such that probing ambiguity will be improbable.
  • said probe sequences or other binding means are chosen so as to target only one sequence in said multiplex sample based either on knowledge about the sample or statistical predictions concerning probe sequence occurrence.
  • Said flat surface with a linear trench is then removed from said demultiplexation array, which is then positioned juxtaposedly to the array of entry ports of a separatory channel array, if not part of the same structure.
  • said demultiplexation array is situated on the same surface of an article comprising separatory channels as the input ports of said channels, then a surface conforming to said demultiplexation array and input port array surface but comprising trenches for juxtaposedly forming channels permitting the transport of molecules from each of said demultiplexation array elements to the corresponding separatory channel input port is then appropriately juxtaposed, and either temperature is briefly increased or denaturants are introduced into said trenches, in either case as the electrophoresis field is applied to initiate separation of the thus demultiplexed sequencing reaction products in parallel.
  • Distinct tag-probe oligos or internal sequence probes or other such probes of different composition are situated at the entry port to each of two or more, and preferably several, and most preferably thousands or more, separation channels which may be capillary tubes or may be a channel array glass (or microchannel or nanochannel array glass) or other types of separation channels.
  • probe-tag arrays which may favorably be of triangular or hexagonal geometry, are either fabricated on the surface of said channel array glass, which surface comprises said entry ports or openings, or are most favorably situated on a surface which is juxtaposed to the surface comprising openings of said channel array glass in a loading step.
  • probe tag arrays or demultiplexation arrays Complex mixtures of different sample molecules are first bound to said probe tag arrays or demultiplexation arrays, and unbound species are removed by one or more wash steps.
  • sample molecules are bound to said probe tag array which is then washed before juxtaposition or the contacting of said probe-tag array with said channel array glass.
  • An electric field directed along the axis of said separatory channels and oriented with an increasingly positive electrical potential from said probe-array to the distal opening (exit port) of said separatory channels is established, and conditions (e.g.
  • sample molecules enter particular separatory channels according to the array element to which said sample molecules first bound, and according to which separatory channel said array element was juxtaposed.
  • means, including automated instrumentation, embodying this aspect of the present invention should preferably juxtapose such arrays in a repeatable manner to said surface such that the sample molecules bound to a known array element will be separated by a particular, thus predetermined, separatory channel.
  • a variation comprises the transfer of sample molecules from a probe-tag array to a surface of a manifold, with binding of sample molecules to said surface of a manifold, after which said manifold is used to carry sample molecules into arrays of vessels, if any, to perform reactions, if any, and comprises transfer of sample molecules or reaction products therefrom to separatory channel arrays, channel glass arrays or capillary arrays.
  • said channel array glass must be transparent at least to the excitation frequencies used, and where emitted photons traveling in any direction other than that of the channel axis direction, further transparent to the wavelength of emission of the dyes used.
  • Fractionated species may be detected by scanning a laser beam of appropriate frequency through said arrays, and detecting fluorescence of fluorescent labels as a function of beam position and linear position along said beam, as proposed by S. Esener et al. 37 for three dimensional optical memories with one-beam addressing.
  • Such a one- beam approach may comprise the scanning of said one beam across planes comprising the axis of each of many channels or across a plurality of channels at a fixed position along the length of all such channels as separation proceeds, and in either case recording the dye emission signals thus associated with particular positions or times with each channel of said array.
  • Planar beams also used by Esener et al. for two dimensional readout, may similarly be used with two-dimensional photodetection means (e.g.
  • CCD array camera to either collect spatial separation data along the lengths of those channels whose axes substantially occur in the plane of said planar beams, or to collect signals regarding the passage of species through said plane (i.e. according to a temporal separation coordinate), here oriented perpendicularly to the axis of each channel
  • the useful minimal dimensions of glass channel arrays will be determined by the resolution limit, i.e. related to the wavelengths of the light used.
  • the second detection scheme used with channel array glass parallel separation means molecular species are detected as they exit separation channels.
  • the output port comprising surface of said channel array glass may be briefly contacted with a receiving surface, such as an ion exchange paper surface or nylon membrane, onto which sample molecules are transported by the action of applied electrical field (or fluid flow in the case of ion-exchange border matrix capillary modifications described herein) and are bound to said receiving surface (e.g. by crosslinking with UV radiation in the case of nylon membranes.)
  • Said contacting is performed such that sample molecule species bind to the portion of said sample receiving surface immediately juxtaposed to the channel from which each such molecule exited (i.e. such that sample molecules of an individual molecular species do not diffuse to regions juxtaposed to channels from which they did not respectively exit) .
  • This may be facilitated by performing said contacting with said receiving surface under high pressure normal to the juxtaposed surfaces, and may further be facilitated by the use of elastomeric surfaces or underlayers to cause said receiving surface to conform to said channel array glass surface comprising exit ports.
  • Such brief contacting steps are repeated with fresh receiving surfaces as separation proceeds, preserving information as to the order in which receiving surfaces or receiving surface regions were contacted.
  • Said receiving surfaces are then examined for the spatial location or pattern of labels, from which information about the sample sequences separated in each respective channel is reconstructed.
  • Said pattern may favorably be captured by imaging visual labels (e.g. fluorescent labels, affinity labels bound by receptor-comprising beads, etc.,) with photodetector arrays such as CCD cameras, and analyzed by digital computer for information content, which is then reordered and further analyzed to glean sequence information.
  • imaging visual labels e.g. fluorescent labels, affinity labels bound by receptor-comprising beads, etc.
  • a second category of treatments depend on the chemical modification or derivatization of said internal surfaces.
  • organosilane compounds and derivatives thereof e.g. RSiX ⁇ , R2SiX2 or R3Si where R groups may be identical or distinct
  • surface hydroxyl groups e.g. formed on surfaces with exposed Si ⁇ 2 including native silicon oxides
  • Other chemistries may similarly be used to derivatize such surfaces. This fact has been exploited in capillary zonal electrophoresis 40 .
  • Derivatives conventionally include polyacrylamide derivatives, though these have generally been used for the reduction of protein adsorption to such surfaces.
  • the embodiments of the present invention availing capillary tubes and channel array glass may further avail similar treatments.
  • such treatments may provide for a channel comprising a surface localized separation matrix (border matrix) which may occupy a substantial portion of the inner diameter of such channels.
  • border matrix may comprise a predetermined polymer chain length or length distribution or composition distribution.
  • ionizable chemical functional groups situated on such border matrices may be used to modulate the degree of interaction of polynucleotides with such matrices, in analogy to ion-exchange resins.
  • pH of a running buffer (which occupies such channels and may flow through these independently of any border matrix) may be chosen to effect the desired degree of border matrix ionization, up to the maximum determined by the.surface density of ionizable groups.
  • Other conditions, such as temperature, affecting the ionization of chemical groups may similarly be exploited.
  • Polynucleotide fragments comprise negatively charged phosphate linkages in direct proportion to length; for separation of polynucleotides, said ionizable groups are chosen to yield cations upon ionization, in order to bind said negatively charged phosphate linkages. Degree of border matrix ionization will affect the probability that each negatively charged phosphate group associates with an ionized group of said border matrix. The different number of anionic groups corresponding to the different polynucleotide molecules will result in a difference degree of association of different length polynucleotides to such surfaces.
  • Conditions and border matrix composition are favorably chosen such that each polynucleotide fragment in the size range to be separated has an appreciable characteristic mobility, fractional association time or holdup, arising from differences in the mean number of associations with said border matrix per unit length.
  • Optimal conditions and border matrix compositions will vary according to border matrix composition and size fractionation range, and the many parameters which may be varied to effect a desired result provide numerous possible combinations for many desirable fractionation properties.
  • the mechanisms involved in these types of size fractionation may, for many cases, be envisaged as the association at a few points of polynucleotide backbones with a border matrix, where a characteristic proportion of said associations spontaneously dissociate per unit time, and said polynucleotide molecules thus locally freed (at one or more previously bound points along the length of said polynucleotide molecules) translate, to the extent possible under the constraint of other points of association persisting at that moment, in the direction of an applied electrical field or fluid flow.
  • a polynucleotide advances in the direction of said applied field or fluid flow, at a rate which is negatively correlated with length (or more specifically, for polyions or polyanions in general, with number of cationic and/or anionic groups.
  • mobility will depend on the ratio of time which any such ion comprising species subjected to such separation spends in association with a border matrix ion to the time which it spends unassociated, and further depend on the mobility of said ion comprising species when unbound and in either the border matrix and overlying fluid phase (and the fraction of time spent in each of these, and mobilities in each of these.)
  • ion-exchange resolved electrophoresis or ion-exchange zonal electrophoresis
  • fluid flow such methods may be termed
  • Such methods bear some similarity to thin-layer chromatography, but are crucially distinguished by the presence of a free-fluid or mobile phase constrained to a limited proximity to said border matrix by channel dimensions.
  • binary masking methods used with physical masks may be used to pattern surface density gradients or other distribution patterns of, for example, molecules comprising ionic or ionizable groups for separation methods involving ion exchange, where a surface used for forming a formed channel is exposed.
  • Such a surface is a flat surface comprising linear trenches.
  • Another such surface is -a substantially flat surface which may be juxtaposed to said flat surface comprising linear trenches.
  • a large number of surface modification compositions and combinations thereof may be tested by such binary masking means for any desired sample type. This provide ⁇ a method for the rapid optimization of surface modifying resolution media.
  • This aspect of the present invention combines partial demultiplexation, parallel multiplex separation and conventional serial reprobing of sequences separated in multiplex and transferred to membranes or the like.
  • Parallel formed separatory channels capable of being opened length-wise may be used to enable transfer of separated species to membranes for probing.
  • transfer to a moving membrane from the outlet port of capillaries or separatory channels simultaneous with separation may accomplish transfer of the fractionated sample conserving a separation coordinate (i.e. the time of exit coordinate is thus transformed to a positional membrane transfer coordinate on said membrane, related by the velocity of said membrane relative to said capillaries or separatory channel) .
  • each serial reprobing cycle yields data up to a maximum equal to read length times the number of separation channels used. For example, for a read length of 500 bases with 1000 separatory channels and a serial reprobing time (probing, washing, probe detection and probe removal) of one hour, 12 million bases of sequence information may be collected per membrane per day. Of course, reprobing of multiple membranes (i.e. patterns transferred from multiple sequencing runs) will increase this rate correspondingly.
  • a preferred probe oligo labeling method in this aspect of the present invention is involves labeling, (e.g. with biotin) and detection with dye labeled or fluorescent labeled affinity modified beads (e.g.
  • intensity encoding with four monochromatic levels in one lane (referring to a region of the membrane) or channel is the preferred means for reducing the number of channels required.
  • Data capture is again performed with a CCD and digital computer.
  • This aspect of the present invention gains maximum multiplexation of sample preparation and of sequencing reaction multiplexation, exploits parallel separation and further exploits parallel detection of multiplexed species in each separatory channel, simultaneously (i.e. a further level of parallelism) for different separatory channels.
  • the maximum number of sequence reads which may thus be obtained is equal to the degree of parallelism of partial demultiplexation (which equals the number of separatory channels used) times the number of parallel detection elements for each fraction. Variations on and methods and means for this aspect of the present invention are described below.
  • multiplex loading and parallel separation methods and means of the present invention are combined with the parallel multiplex methods described by E.M. Rabani, comprising the simultaneous detection of plural different species within the same separation fraction.
  • Such methods and means permit the sequence determination rate possible with a given separation rate (r) and a given separatory channel parallelism (s) to be multiplied by the parallelism of detection (d) , yielding a maximal rate of (r) (s) (d) determined bases per unit time.
  • lateral transfer which is to say transfer of all fractions in one step or substantially simultaneously to detection arrays in a manner such that each fraction or each portion of a fraction will be transferred to a distinct detection array or distinct detection array region.
  • separatory channels which may be laterally opened or have many lateral ports, such that transfer may occur laterally as in the case of transfer from slab gels to laterally juxtaposed or opposingly placed membranes in, for example. Southern blots.
  • lateral transfer from one or more separatory channel array facilitates the use of parallel detection arrays, preferably comprising stripes of plural distinct tag-probe oligonucleotides or other probing compositions, with an array repetition of a similar pitch to that of said separatory channel array, such that a said stripe of each of said distinct tag- probe oligonucleotides or other probing compositions occurs for each separatory channel of said separatory channel array.
  • Said detection array comprising stripes are aligned parallel to said separatory channels during transfer, such that the contents of any fraction in each of said separatory channels will be exposed, upon transfer to at least one, and preferably exactly one, repetition of said stripes of said detection array.
  • said array may comprise stripes of fine pitch which will be oriented perpendicularly to said separatory channels, provided that the pitch with which a full repetition of said array is less than half the length occupied by a species of a particular length in said separatory channel, such that the molecules in each fraction (many of which may be identical to each other but different from most molecules in the multiplex fraction) will be juxtaposed to all detection array elements.
  • separatory channels are preferably formed from molded or cast microreliefs or reliefs (first reliefs), preferably of elastomeric composition, comprising narrow parallel trenches, which define the ultimate separatory channels, and with surfaces composed or treated so as to have good affinity to any resolution media used but no undesirable affinity to sample molecules (in cases of separation by a gel or low viscosity medium, as opposed to separation on border matrices, in which case the surfaces of said reliefs are treated accordingly.) Said reliefs are juxtaposed to either a substantially flat surface or a second relief surface comprising arrays (fraction transfer arrays) of fraction transfer grooves and electrodes for lateral electroelution, and preferably also integrated tag-probe arrays; such juxtaposition is followed by the application of sufficient normal pressure to compress regions of greatest extremity on said relief or reliefs (or in the case of non- elastomeric relief surface, of an elastomeric film or an elastomeric second relief surface comprising arrays of fraction transfer groves
  • Fluid separatory media such as low viscosity media including polyethylene-oxide or polyacrylamide may then be injected into the resulting channels.
  • a liquid resolution media from which bubbles have been removed, e.g. by degassing
  • said flat surface which is in this case preferably flexible, which is applied with a uniform unrolling motion or a squeegeeing motion such that only one meniscus forms and is evenly translated so as to prevent the formation of bubbles.
  • Transfer may be favorably accomplished as follows. Subsequent to separation of plural different multiplex samples on said formed separatory channel array, a surface of said formed separatory channel array comprising lengthwise portions of the internal surface area of each of said separatory channels is removed, and later replaced with a transfer surface, which is thus juxtaposed to the surface comprising the remainder of said formed separatory channel array.
  • lateral opening of said channels is performed by removing, for example, said flat surface from said relief surface, the aspect ratio or recession of the trenches therein favoring retention of any resolution media.
  • This removal step is performed, preferably with a lateral sliding motion to prevent adhesion of resolution media, such that resolution media and sample material substantially remains in the remainder of said formed separatory channel array, principally or exclusively within the trenches defining formed channels.
  • said relief surface comprising trenches Said transfer surface is substantially free of separation media before juxtaposition to said surface comprising the remainder of said formed separatory channel array.
  • separatory channels may be performed by juxtaposition, as in the other case, but further with relative positioning such that said trenches of said first relief are immediately opposite a flat portion of said second relief surface.
  • Said second relief is preferably coated with a degassed liquid layer to facilitate electroelution before said juxtaposition is performed, and the resulting meniscus is caused to translate evenly to avoid the formation of bubbles.
  • said second relief surface is translated by a lateral sliding motion along the surface of said first relief such that two openings of each of said fraction transfer grooves (which preferably run perpendicularly to said formed separatory channel trenches) are opposite each trench on said first relief which defines said separatory channel.
  • a voltage is applied to the electrodes on of said second relief so as to electroelute fractions from said separatory channels and cause molecules in said fractions to pass into a restricted volume contacting said detection array.
  • said second relief may be translated so as to minimize the opportunity for denaturants to seep from said separation medium in said separatory channels into said restricted volume.
  • Conditions are then changed to establish the desired binding stringency and thus permit selective binding of tag sequences with the tag-probes of said detection array.
  • appropriate means are used for signal amplification, if any, and detection.
  • detection methods and means may include contacting said parallel detection array to receptor or antibody labeled colored or fluorescent beads targeted to particular affinity labels used to label sequencing reaction products, e.g. according to terminator moiety.
  • suitable photodetection means for example comprising a CCD array or photon camera and appropriate filters, and recorded by a digital computer interfaced to said photodetection means.
  • Said photodetection means and said detection array are preferably mounted on a system or translation table which moves one or both of these relative to each other such that data may conveniently be collected across the entire array by means of such relative motion or scanning. After data collection, said detection array is subjected to denaturing conditions and repetitive rinses to remove labels and sample molecules and thus prepare said detection array for reuse, care as always being taken not to contaminate said detection array.
  • a pool of oligonucleotides of a particular nucleotide length n is randomly synthesized from all for nucleotide monomer reactants by a suitable oligonucleotide synthesis chemistry (e.g. phosphoramidite) .
  • Length n must be chosen such that number of different sequences in said pool, 4 n , exceeds the size N (number of bases) of the sample by some factor greater than, for example, two.
  • a sample of size N can comprise no more than N distinct n-mer sequences (N»n) .
  • said pool comprises a large number of n-mers not present in said sample. The non-randomness and repetitivity of evolved DNA sequences increases this number.
  • Such a pool is produced in quantity sufficient to ensure that on average at least, for example, ten thousand copies of each possible sequence occur. At least half of this quantity is left on the solid phase support used in synthesis but is nonetheless subjected to removal of protecting groups.
  • a quantity equivalent to 100 copies of each sequence in the pool is fractionated against genomic DNA.
  • This quantity of such an oligonucleotide pool is mixed with a tenfold excess of genomic DNA (which may have previously been filtered to eliminate small fragments from the sample) .
  • the resulting mixture is subjected to denaturing conditions and then hybridized under reduced stringency hybridization conditions.
  • Reduced stringency conditions are favorable because of the variation optimal hybridization conditions for highest stringency with (oligo) sequence; reduced stringency conditions will eliminate sequences which, although they do not occur in said genome, nonetheless may bind to genomic sequences under the set of conditions used, while minimizing the likelihood that any oligos with sequences occurring in said genome will fail to bind.
  • Unbound oligonucleotides are recovered after hybridization, for example by ultrafiltration (care being taken to avoid excessive fragmentation by shearing, etc.,) .
  • the unbound fraction may be subjected to repeated rounds of depletion by repetition of the foregoing mixing, denaturing, hybridization and filtration steps, against the same quantity of genomic DNA used initially, to greatly enrich for sequences not occurring in said genomic DNA and greatly reduce the presence of genomic sequences in said unbound oligonucleotide fractions. Different conditions may be used in different depletion rounds. Following depletion against genomic DNA, the fraction unbound after the final depletion cycle may optionally then be subjected to selection for binding of the undepleted pool, under more stringent conditions.
  • This is again performed by mixing the undepleted, solid phase bound pool in, for example, fifty-fold excess, denaturing and hybridizing under more stringent conditions. Washing or filtering is then performed to eliminate the unbound fraction. The bound fraction may then be recovered by denaturation and washing, and will appear in the eluted fraction. Such a selection will enrich for sequences which bind their complementary sequences under more stringent conditions. Such enrichment will reduce the number of oligo sequences not bound to genomic DNA but exhibiting unacceptable non- specificity of binding against the lager complexity random pool, due to the increased stringency used. This treatment does not, however, formally preclude the possibility of recovery of oligos which bound to less than fully complementary targets because no discrimination of such events is possible, and these are only reduced with increasing stringency.
  • binding of oligos in this fraction to more than one tag-probe cannot be excluded, but will be improbable under sufficiently stringent conditions.
  • Longer random tag sequences (relative to a fixed sample genome complexity) prepared as above and used under correspondingly higher stringency binding conditions in sequencing protocols and other related protocols may address this issue by rendering the likelihood of one probe-tag binding multiple tags or one tag binding to other tags correspondingly less likely in finite sample sizes. Further, to the extent that such events occur, these will only reduce overall efficiency of parallel detection or demultiplexing across a large sample diversity (e.g. samples comprising many different distinctly tagged sequence inserts); these inefficiencies are readily offset by the parallelism and multiplexing possible with the methods of the present invention.
  • the fraction thus recovered comprises sequences favorably used as tag-probes, sequences complementary to these may favorably be used as tags for library clone tagging.
  • Sequence determination of the oligos obtained by the above methods may be performed as follows. Two 5' phosphorylated oligos, complementary to each other over most of their length but having, for example, a random three-base 3' overhang at one end (but blunt at the other) are annealed to an excess of depleted tag-probes, and then ligated to these, such that a ligation event occurs at the 3' end of oligos from the pool of said depleted tag-probes.
  • the resulting products are then subjected to polymerization (primer extension) to fill in the overhangs thus formed comprising 13 of the 16 bases of said depleted tag-probes.
  • the resulting mixture is subjected to blunt-end ligation (e.g. with T-4 DNA Ligase) to form large multimers, which are then ligated into a sequencing vector, or a vector comprising priming sites suitable for PCR amplification of the insert material.
  • These vectors are then transformed into hosts, or the inserted material is amplified by PCR after dilution and distribution into wells (e.g. of a microtitre dish suitable for PCR cycling) at a concentration yielding on average one amplifiable molecule per well.
  • Said second tag sequence comprising a limited number of variable bases at predetermined positions, e.g. 6 bases, not necessarily all contiguous, such that probe arrays complementary to all of the possible second tags may be formed and used for one or more of the demultiplexing or parallel detection methods of the present invention.
  • a third tag sequence of predetermined complexity may similarly be included, for example, adjacent to said second tag sequence, and may comprise a similar complexity but a distinct consensus sequence, and be used in a step other than where said second tags were used for demultiplexing or parallel detection, said third tags used for parallel detection or demultiplexing.
  • a set of tags from the depleted pool may be sequenced, and the methods of the present invention may be used to rapidly determine the sequences thus recovered, where said second and said third tags permit, for example, demultiplexing (here, partial demultiplexation) for separation of a complex pool in parallel separatory channels by binding to arrays comprising probes for said second tags, and parallel detection is accomplished with arrays of probes complementary to said third tags.
  • Sequence information gained about sequences not occurring with such a genome is favorably then used to generate probe tag arrays for used with said genome.
  • the cloned or amplified depleted tag material may be constructed such that individual tags are liberated by digestion with a predetermined restriction enzyme, and these may then be used in the generation of a pool of tagged derivatives of a library vector construct.
  • sequence information regarding said depleted tag material may be used to score consensus sequences and m-mers (m ⁇ n) which may be used as elements in the synthesis of pools of oligos comprising sequences of highly unlikely occurrence in said genome.
  • tag sequences are preferably placed within a library vector immediately downstream from a sequencing primer region, and immediately upstream from a cloning site.
  • tag sequences will be copied (or otherwise included) during the generation of sequencing reaction products, and all but the shortest sequencing products will comprise the tag sequence.
  • probe-tag array oligonucleotide ensemble composition must comprise oligonucleotides having sequence composition which will generally, and preferably uniformly, each probe a distinct tag of such vectors under sufficiently stringent binding conditions.
  • one aspect of the present invention is the demultiplexation based separation of libraries or other pools of different sequences constructed from the vectors of G.M. Church and S. Kieffer-Higgins.
  • Lithographic plates such as those taught by T. Suzuki and F. Shinozaki in U.S. Patent Number 5,380,620, or those reviewed by B.
  • Copolymer types may include peptides and oligo- and poly-nucleotides, as well as other copolymers which may be synthesized by deprotection step-controlled chemistry on solid phase supports. This method is in analogy to that of S.P.A. Fodor et al., but substitutes spatial control of reagent placement according to the wettability of an juxtaposed surface contacted with an array substrate for spatial control of removal of photolabile protecting groups.
  • lithographic plates are used to contact regions of said array, corresponding to those regions desired to undergo deprotection of the copolymers or initiators thereupon, with deprotection reagents included in said liquids ("inks") on said plates.
  • the entire surface of said array is then exposed to the reactant (comonomer) to be added only to the copolymers or initiators deprotected by the preceding treatment.
  • Such steps are repeated, using different lithographic plates aligned relative to the position of precedingly used lithographic plates, in an overall procedure otherwise resembling the light controlled method of S.P.A. Fodor et al., to produce the desired pattern of copolymers with predetermined sequence composition and situated in a predetermined manner which corresponds to said sequence composition on said array substrate surface.
  • This first variation may be termed lithographic plate patterned deprotection.
  • a second, contrasting, variation may be termed lithographic plate patterned comonomer contacting and addition.
  • the totality of one or more such array is contacted with deprotection reagents to remove any protecting groups from any preceding steps, and then contacted with a lithographic plate "inked" with a liquid comprising a single comonomer reactant type.
  • Comonomers are thus added only to those copolymers or initiators in those array regions contacted with said liquid, i.e. contacted with said reactant.
  • a wash step is favorably performed using reagents that will inactivate unadded comonomer reactants, such that the reactivity of these is neutralized before they are swept into possible contact with regions comprising copolymers or initiators with which said reactants are not desired to react.
  • the advantage of this variation lies in the reduction of steps from the identity of patterning and reactant contacting steps.
  • the final pattern is determined by the lithographic plate pattern used, the order in which these are used, and the alignment and precision of alignment with which they are repeatably contacted with said array substrate.
  • the third variation combines the patterning methods of the previous variations, and may be termed lithographically patterned deprotection with redundantly patterned comonomer contacting and addition.
  • lithographic-plate control is exerted, as in the first variation above, to effect the deprotection of initiators or copolymers situated in spatially predetermined array regions in a first cycle step, and in a second cycle step either the same lithographic plate or another plate with an identical pattern is used, as in the second variation above, to contact reactants with only the desired predetermined regions substantially identical in location and extent to those deprotected in said first cycle step, after which a wash step is performed as in the second variation above.
  • Such a cycle is repeated with different lithographic plates to produce the desired final copolymer array pattern.
  • Patent Number 5,384,261 comprising steps of flowing reagents through groves on the surface of a channel block juxtaposed to a substrate, or those taught by R.E. Southern and co-workers in U.S. Patent Number 5,324,663, may be employed to produce the linear probe-tag arrays disclosed herein as useful for parallel multiplex sequencing with direct spatial transfer including parallel multiplex sequencing with parallel separation and direct spatial transfer.
  • lithographic plates used in this aspect of the present invention may have patterns comprising isolated inked or isolated wettable regions, as retention of applied solutions (rather than transport through channels to desired surface areas, as in some prior art methods) does not require transport through contiguous regions.
  • lithographic plates of this kind are favorably produced by the microfabrication methods described and referenced above, and thus generally comprise films of polymeric resist-type coatings which are usually of 50 nm or greater depth from the surface, being deposited by such methods as spin-coating.
  • this method comprises the steps of: (a) producing lithographic plates that specify the desired spatial patterns of chemical reactions, in particular, the desired spatial patterns of addition of reagents or reactants; (b) applying said reactants or reagents to one of said lithographic plates for use in a reaction cycle according to the final pattern desired, with the result that solutions are only retained in selected predetermined regions according to the pattern of the respective lithographic plate; (c) juxtaposing the lithographic plate of step (b) to the target surface on which copolymer initiators or intermediate products are situated such that reagents or reactants are contacted and thus permitted to react with only the desired regions specified by the pattern of said lithographic plate, where said juxtaposing constitutes opposing said lithographic plate to said target surface in correct alignment, register and relative position (e.g.
  • Probe-tag arrays arrays comprising oligonucleotides which need not be probe-tags, and most generally copolymer arrays may be produced by patterned synthesis comprising one or more steps of spatially controlled catalysis, particularly spatially controlled deprotection or activation.
  • One aspect of this method comprising the catalytic reduction of nitrile groups in the presence of dissolved H2 to yield amine groups will be presented here for illustration. It will be noted, however, that the essence of this method concerns the exertion of spatially predetermined step control in the synthesis of copolymers situated on surfaces, and may be adapted to other chemistries and corresponding reactions catalyzed by metal surfaces.
  • nucleotide synthesis reactant monomers comprising 5' nitrile groups, and not comprising phosphoester protecting groups which include nitriles (such as is the case for cyanoethyl groups used with phosphoramidite oligonucleotide synthesis chemistries) or any unsubstituted amides are used.
  • the active acceptor group in preferred reactant monomers is a 3' -(OPOCH3CI) activated phosphate.
  • This group is chosen to be reactive with amines (by attack of amine nitrogen lone pair at the partially positive phosphate, with liberation of the chlorine as a stable anionic leaving group) , and to not comprise any nitrile or unsubstituted amide functionalities.
  • Initiators used which are preferably of substantially linear structure, comprise a surface binding group (e.g. an organosilane for glass substrate surfaces), a linker of sufficient but not excessive length (e.g. having linkers 10-1000 atoms or bonds in length), and a terminal nitrile group. Longer initiator linkers will impose limitations on minimum array element size and spacing.
  • nitrile groups may be reduced by platinum in the presence of dissolved H2. 45
  • Patterned platinum catalyst structures or microstructures which may, for example, be patterned and fabricated by LIGA methods, comprising substantially flat surfaces from which a pattern is recessed or omitted (in the form of voids), provides for the spatially predetermined patterned activation of nitrile groups situated on a surface.
  • the patterned surface of said patterned platinum catalyst structure is contacted, in the presence of dissolved H2, with said substrate surface on which said nitriles are situated; said nitriles experiencing sufficient contact with the surface of said platinum are thus transformed to amine groups.
  • Said contacting is performed in a manner and with means such that a particular platinum catalyst structure may be repeatably contacted with the same surface with high spatial accuracy, or with high accuracy stepping (as in semiconductor microlithography) .
  • nucleotide reactant monomers such as those described above comprising the base moiety (in protected form) desired to be added to the amines formed in the preceding step; nitriles are substantially inert to methoxyphosphorochloride ester addition, as evidenced by their use as backbone protecting groups in other oligonucleotide synthesis chemistries employing PCI3 derivatives.
  • a catalytic patterning step followed by a monomer addition step comprise a patterned addition cycle. Other steps necessary are identical to those used in standard phosphoramidite chemistries; these include capping and oxidation steps.
  • Patterned addition cycles are repeated with different patterned platinum catalyst structures or microstructures, or the same structures in different registry or juxtaposed orientation, and different reactant monomers such as those described above comprising different base moieties each of which said moieties is desired to be added to the amines formed in the preceding catalytic patterned activation step.
  • the oligonucleotide synthesized by nitrile/amine step control methods such as these will comprise backbone linkages replacing 5'CH2 ⁇ (NH)-P02-0-3'C for the usual 5'CH2*-0-P ⁇ 2-0-3'C. Under conditions where this backbone nitrogen is not further protonated (i.e.
  • hybridization properties of oligonucleotides with such modifications are expected to be similar to those of unmodified oligonucleotides of identical seguence.
  • the logic and considerations pertaining to the spatial patterns chosen and order of comonomer addition to yield an array with a desired oligonucleotide array sequence compositional configuration are substantially identical to those pertaining to the methods of S.P.A. Fodor et al., 46 or other similar methods.
  • biomimetic copolymers which may be prepared from monomer precursors comprising nitrile groups may likewise be thus prepared.
  • Copolymers of particular interest which may be produced by this method are backbone-modified-DNA, backbone-modified-RNA, peptide- nucleic acids, peptides and the various peptide analogues or mimics, and obvious variations upon all of these.
  • Patterned Activation with Metal Catalvst AFM Tip Surface The same synthesis methods used above with patterned metal catalyst surface controlled activation patterning may be used to synthesize copolymer arrays with nanometer resolution by the use of scanning probe positioning means (e.g. a scanning probe microscope) to position a catalyst metal coated probe (e.g. an AFM tip), as was done by W.T. Muller, P.G. Schultz, et al. 47 but otherwise according to the above oligonucleotide synthetic methods.
  • scanning probe positioning means e.g. a scanning probe microscope
  • a catalyst metal coated probe e.g. an AFM tip
  • activation of initiator nitriles or nucleotide or oligonucleotide nitriles, to form amines which will react as donors with phosphate derivative groups on nucleotide monomer reactants is performed here by the positioning and contacting of the platinum on an AFM or other similar tip with said substrate surface, but synthesis is otherwise conducted as above.
  • Arrays used with the present invention may be used to bind polynucleotide species labeled with one among several different affinity labels, which affinity labels may be detected with beads comprising receptors or antibodies or other binding means specific for each of said different affinity labels.
  • Each of said receptors or antibodies or other binding means is placed in communication with a perceptibly distinct bead (e.g. streptavidin with red beads and antidigoxigenin with yellow beads) .
  • tag-probe arrays may be synthesized on high dielectric, low dielectric or conducting materials with appropriate initiators (e.g. on Au(lll) with linkers comprising terminal -SH groups) or on membranes of polymeric composition (e.g.
  • Probe-tag arrays situated on surfaces of such compositions favorable for blotting transfer may be produced by the methods and means disclosed herein, by those of known art including those photolithography based methods of S.P.A. Fodor et al. or any other equivalent means compatible with such surface ⁇ , appropriate initiators and linkers being chosen.

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Abstract

Ces procédés permettent de multiplexer des réactions de séquençage, tel que le procédé Sanger, lorsqu'on les utilise avec des échantillons complexes correctement préparés. L'avantage de tels procédés est de permettre le séquençage simultané de nombreux polynucléotides. Ils reposent essentiellement sur les réactions de séquençage multiplex de Church et Gilbert car les polynucléotides à séquencer sont recombinés de façon à contenir un marquage spécifique des bases terminales. De plus, ces procédés évitent de devoir réticuler des produits de réaction sur des membranes avec des réactifs chimiques. Un tel procédé consiste à séparer en parallèle des matrices de séquençage, ou des produits de réaction de séquençage, d'un mélange avant une électrophorèse sur gel, puis à détecter en parallèle différent échantillons ou produits de réaction. Pour obtenir cette séparation, on applique plusieurs échantillons à des canaux spécifiques de séparation en les y liant par un moyen spécifique aux échantillons parallèles, ce qui permet de combiner un multiplexage et le parallélisme. La détection s'effectue grâce à un dispositif de détection comprenant des arrangements étiquettes-sondes. On décrit aussi des dispositifs permettant de mettre en oeuvre un tel procédé.
PCT/US1996/013313 1995-08-14 1996-08-14 Procedes et dispositifs de sequençage multiplex parallele de polynucleotides WO1997007245A1 (fr)

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US6514699B1 (en) 1996-10-04 2003-02-04 Pe Corporation (Ny) Multiplex polynucleotide capture methods and compositions
US6124092A (en) * 1996-10-04 2000-09-26 The Perkin-Elmer Corporation Multiplex polynucleotide capture methods and compositions
US7413852B2 (en) 1996-12-31 2008-08-19 High Throughput Genomics Multiplexed diagnostic and therapeutics
US6238869B1 (en) 1997-12-19 2001-05-29 High Throughput Genomics, Inc. High throughput assay system
US6458533B1 (en) 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs
US20030096232A1 (en) 1997-12-19 2003-05-22 Kris Richard M. High throughput assay system
US6232066B1 (en) 1997-12-19 2001-05-15 Neogen, Inc. High throughput assay system
US7659063B2 (en) 1998-07-02 2010-02-09 High Throughput Genomics, Inc. High throughput assay system
WO2001016598A2 (fr) * 1999-09-01 2001-03-08 Medalys Corporation Evaluation a fort rendement du profil d'un produit chimique
WO2001016284A1 (fr) * 1999-09-01 2001-03-08 Medalys Corporation Systeme automatise etablissant des profils d'agents d'essai
WO2001016598A3 (fr) * 1999-09-01 2001-11-22 Medalys Corp Evaluation a fort rendement du profil d'un produit chimique
WO2001025479A3 (fr) * 1999-10-07 2002-06-20 Europ Lab Molekularbiolog Sequençage multiplex a fluorescence
WO2001025479A2 (fr) * 1999-10-07 2001-04-12 Europäisches Laboratorium für Molekularbiologie (EMBL) Sequençage multiplex a fluorescence
EP1186886A3 (fr) * 2000-09-04 2004-01-07 Fuji Photo Film Co., Ltd. Méthode d'analyse biochimique et appareil pour utiliser cette méthode
EP1186886A2 (fr) * 2000-09-04 2002-03-13 Fuji Photo Film Co., Ltd. Méthode d'analyse biochimique et appareil pour utiliser cette méthode
US9243284B2 (en) 2000-12-01 2016-01-26 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9845500B2 (en) 2000-12-01 2017-12-19 Life Technologies Corporation Enzymatic nucleic acid synthesis: compositions and methods for inhibiting pyrophosphorolysis
US9920380B2 (en) 2001-07-03 2018-03-20 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
US9890419B2 (en) 2005-12-23 2018-02-13 Nanostring Technologies, Inc. Nanoreporters and methods of manufacturing and use thereof
US10077466B2 (en) 2008-08-14 2018-09-18 Nanostring Technologies, Inc. Stable nanoreporters
US9856519B2 (en) 2008-08-14 2018-01-02 Nanostring Technologies, Inc. Stable nanoreporters
US9714937B2 (en) 2009-10-13 2017-07-25 Nanostring Technologies, Inc. Protein detection via nanoreporters
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US9714446B2 (en) 2010-02-11 2017-07-25 Nanostring Technologies, Inc. Compositions and methods for the detection of small RNAs
US9034173B2 (en) 2010-03-18 2015-05-19 Robert Bosch Gmbh Microfluidic dielectrophoresis system
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DE102010003001B4 (de) 2010-03-18 2024-02-08 Robert Bosch Gmbh Mikrofluidisches Dielektrophorese-System
US9758834B2 (en) 2011-03-28 2017-09-12 Nanostring Technologies, Inc. Compositions and methods for diagnosing cancer
US10501777B2 (en) 2015-07-17 2019-12-10 Nanostring Technologies, Inc. Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue
US10640816B2 (en) 2015-07-17 2020-05-05 Nanostring Technologies, Inc. Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
US11708602B2 (en) 2015-07-17 2023-07-25 Nanostring Technologies, Inc. Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
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