USRE39793E1 - Compositions for sorting polynucleotides - Google Patents
Compositions for sorting polynucleotides Download PDFInfo
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- USRE39793E1 USRE39793E1 US09/366,081 US36608199A USRE39793E US RE39793 E1 USRE39793 E1 US RE39793E1 US 36608199 A US36608199 A US 36608199A US RE39793 E USRE39793 E US RE39793E
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- polynucleotides
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- WPVQBTNQIXCRIK-UHFFFAOYSA-N CN1=NC=NC(Cl)=N1 Chemical compound CN1=NC=NC(Cl)=N1 WPVQBTNQIXCRIK-UHFFFAOYSA-N 0.000 description 1
- KPSOZQGRMWKZLQ-UHFFFAOYSA-N CNC1=NN(C)=NC=N1 Chemical compound CNC1=NN(C)=NC=N1 KPSOZQGRMWKZLQ-UHFFFAOYSA-N 0.000 description 1
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Definitions
- oligonucleotide tags are used to identify electrophoretically separated bands on a gel that consist of DNA fragments generated in the same sequencing reaction.
- DNA fragments from many sequencing reactions are separated on the same length of a gel which is then blotted with separate solid phase materials on which the fragment bands from the separate sequencing reactions are visualized with oligonucleotide probes that specifically hybridize to complementary tags, Church et al. Science, 240: 185-188 (1988).
- tagging schemes depend in large part on the success in achieving specific hybridization between a tag and its complementary probe. That is, for an oligonucleotide tag to successfully identify a substance, the number of false positive and false negative signals must be minimized. Unfortunately, such spurious signals are not uncommon because base pairing and base stacking free energies vary widely among nucleotides in a duplex or triplex structure. For example, a duplex consisting of a repeated sequence of deoxyadenine (A) and thymidine (T) bound to its complement may have less stability than an equal-length duplex consisting of a repeated sequence of deoxyguanidine (G) and deoxycytidine (C) bound to a partially complementary target containing a mismatch.
- A deoxyadenine
- T thymidine
- C deoxycytidine
- reagents such as tetramethylammonium chloride
- PCR polymerase chain reaction
- Another object of my invention is to provide a method for sorting identical molecules, or subclasses of molecules, especially polynucleotides, onto surfaces of solid phase materials by the specific hybridization of oligonucleotide tags and their complements.
- Another object of my invention is to provide a rapid and reliable method for sequencing target polynucleotides having a length in the range of a few hundred basepairs to several tens of thousands of basepairs.
- An oligonucleotide tag of the invention consists of a plurality of subunits, each subunit consisting of an oligonucleotide of 3 to 6 nucleotides in length. Subunits of an oligonucleotide tag are selected from a minimally cross-hybridizing set. In such a set, a duplex or triplex consisting of a subunit of the set and the complement of any other subunit of the set contains at least two mismatches.
- a subunit of a minimally cross-hybridizing set at best forms a duplex or triplex having two mismatches with the complement of any other subunit of the same set.
- the numbers of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. The number is generally much less than the number of all possible sequences the length of the tag which for a tag nucleotides long would be 4 n . More preferably, subunits are oligonucleotides from 4 to 5 nucleotides in length.
- the population of such beads or regions contains a repertoire of complements with distinct sequences, the size of the repertoire depending on the number of subunits per oligonucleotide tag and the length of the subunits employed.
- the polynucleotides to be sorted each comprises an oligonucleotide tag in the repertoire, such that identical polynucleotides have the same tag and different polynucleotides have different tags.
- the method of my invention comprises the following steps: (a) attaching an oligonucleotide tag from a repertoire of tags to each molecule in a population of molecules (i) such that substantially all the same molecules or same subpopulation of molecules in the population have the same oligonucleotide tag attached and substantially all different molecules or different subpopulations of molecules in the population have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set; and (b) sorting the molecules or subpopulations of molecules of the population by specifically hybridizing the oligonucleotide tags with their respective complements.
- FIGS. 1a-1c illustrates structures of labeled probes employed in a preferred method of “single base” sequencing which may be used with the invention.
- FIG. 5 diagrammatically illustrates an apparatus for carrying out parallel operations, such as polynucleotide sequencing, in accordance with the invention.
- “Complement” or “tag complement” as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex.
- the oligonucleotide tag may be selected to be either double stranded or single stranded.
- the term “couplement” is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.
- oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units.
- ATGCCTG an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5′ ⁇ 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted.
- oligonucleotides of the invention comprise the four natural nucleotides; however, they may also comprise non-natural nucleotide analogs. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.
- “Perfectly matched” in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand.
- the term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed.
- sequences of any two oligonucleotide tags of a repertoire can be even “further” apart, e.g. by designing a minimally cross-hybridizing set such that subunits cannot form a duplex with the complement of another subunit of the same set with less than three mismatched nucleotides, and so on.
- the invention is particularly useful in labeling and sorting polynucleotides for parallel operations, such as sequencing, fingerprinting or other types of analysis.
- nucleotide sequences of the subunits for any minimally cross-hybridizing set are conveniently enumerated by simple computer programs following the general algorithm illustrated in FIG. 3 , and as exemplified by program minhx whose source code is listed in Appendix L minhx computes all minimally cross-hybridizing sets having subunits composed of three kinds of nucleotides and having length of four.
- the algorithm of FIG. 3 is implemented by first defining the characteristic of the subunits of the minimally cross-hybridizing set, i.e. length, number of base differences between members, and composition, e.g. do the consist of two, three, or four kinds of bases.
- minimally cross-hybridizing sets comprise subunits that make approximately equivalent contributions to duplex stability as every other subunit in the set. In this way, the stability of perfectly matched duplexes between every subunit and its complement is approximately equal.
- Guidance for selecting such sets is provided by published techniques for selecting optimal PCR primers and calculating duplex stabilities, e.g. Rychlik et al. Nucleic Acids Research, 17: 8543-8551 (1989) and 18: 6409-6412 (1990); Breslauer et al, Proc. Natl. Acad. Sci., 83: 3746-3750 (1986); Wetmur, Crit. Rev. Biochem. Mol. Biol., 26: 227-259 (1991); and the like.
- tags For shorter tags, e.g. about 30 nucleotides or less, the algorithm described by Rychlik and Wetmur is preferred, and for longer tags, e.g. about 30-35 nucleotides or greater, and algoithm disclosed by Suggs et al, pages 683-693 in Brown, editor, ICN-UCLA Syrup. Dev. Biol., Vol. 23 (Academic Press, New York, 1981) may be conveniently employed.
- minimally cross-hybridizing sets are those whose subunits are made up of three of the four natural nucleotides.
- the absence of one type of nucleotide in the oligonucleotide tags permits target polynucleotides to be loaded onto solid phase supports by use of the 5′ ⁇ 3′ exonuclease activity era DNA polymerase.
- the following is an exemplary minimally cross-hybridizing set of subunits each comprising four nucleotides selected from the group consisting of A, G, and T:
- oligonucleotide tags of the invention and their complements are conveniently synthesized on an automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, using 6 standard chemistries, such as phosphoramidiate chemistry, e.g. disclosed in the following references: Beaucage and Iyer. Tetrahedron, 48: 2223 ⁇ 2311 (1992); Moltco et al, U.S. Pat. No. 4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos.
- coding of tag sequences follows the same principles as for duplex-forming tags; however, there are further constraints on the selection of subunit sequences.
- third strand association via Hoogsteen type of binding is most stable along homopyrimidine-homopurine tracks in a double stranded target.
- base triplets form in T-A*T or C-G*C motifs (where “-” indicates Watson-Crick pairing and “*” indicates Hoogsteen type of binding); however, other motifs are also possible.
- Hoogsteen base pairing permits parallel and antiparallel orientations between the third strand (the Hoogsteen strand) and the purine-rich strand of the duplex to which the third strand binds, depending on conditions and the composition of the strands.
- nucleoside type e.g. whether ribose or deoxyribose nucleosides are employed.
- base modifications e.g. methylated cytosine, and the like in order to maximize, or otherwise regulate, triplex stability as desired in particular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci.
- Oligonudeotide tags of the invention may range in length from 12 to 60 nucleotides or basepairs. Preferably, oligonucleotide tags range in length from 18 to 40 nucleotides or basepairs. More preferably, oligonucleotide tags range in length from 25 to 40 nucleotides or basepairs. Most preferably, oligonucleotide tags are single stranded and specific hybridizing occurs via Watson-Crick pairing with a tag complement.
- sequence, and therefore composition, of such linear polymeric molecules may be encoded within a polynucleotide attached to the tag, as taught by Brenner and Lener (cited above).
- the tag itself or an additional coding segment can be sequenced directly—using a so-called “single base” approach described below—after releasing the molecule of interest, e.g. by restriction digestion of a site engineered into the tag.
- any molecule produced by a sequence of chemical reaction steps compatible with the simultaneous synthesis of the tag moieties can be used in the generation of combinatorial libraries.
- M may be a straight chain, cyclic, or branched organic molecular structure containing from 1 to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur.
- M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms; a heterocycle having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl.
- M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl.
- n may vary significantly depending on the nature of M and L. Usually, n varies from about 3 to about 100. When M is a nucleoside or analog thereof or a nucleoside-sized monomer and L is a phosphorus(V) linkage, then n varies from about 12 to about 100. Preferably, when M is a nucleoside or analog thereof or a nucleoside-sized monomer and L is a phosphorus(V) linkage, then n varies from about 12 to about 40.
- Combinatorial chemical libraries employing tags of the invention are preferably prepared by the method disclosed in Nielson et al (cited above) and illustrated in FIG. 4 for a particular embodiment. Briefly, a solid phase support, such as CPG, is derivatized with a cleavable linker that is compatible with both the chemistry employed to synthesize the tags and the chemistry employed to synthesize the molecule that will undergo some selection process.
- a solid phase support such as CPG
- tags are synthesized using phosphoramidite chemistry as described above and with the modifications recommended by Nielson et al (cited above); that is, DMT-5′-O-protected 3′-phosphoramidite-derivatized subunits having methyl-protected phosphite and phosphate, moieties are added in each synthesis cycle.
- Library compounds are preferably monomers having Fmos—or equivalent—protecting groups masking the functionality to which successive monomer will be coupled.
- a suitable linker for chemistries employing both DMT and Fmoc protecting groups (referred to herein as a sarcosine linker) is disclosed by Brown et al, J. Chem. Soc. Chem. Commun. 1989: 891-893, which reference is incorporated by reference.
- CPG CPG-NHC(O)CN(CH 3 )C(O)CH 2 CH 2 C(O)O(CH 2 ) 6 NHC(O)CH 2 (O-DMT)NC(O)- CH 2 O(CH 2 CH 2 O) 2 CH 2 CH 2 NHC(O)CH 2 O(CH 2 CH 2 O) 2 CH 2 CH 2 NH-Fmoc
- CPG represents a controlled-pore glass support
- DMT represents dimethoxytrityl
- Fmos represents, 9-fluorenylmethoxycarbonyl.
- an oligonucleotide segment 214 is synthesized initially so that in double stranded form a restriction candonuclease site is provided for cleaving the library compound after sorting onto a microparticle, or like substrate. Synthesis proceeds by successive alternative additions of subunits S 1 , S 2 , S 3 , and the like, to form tag 212 , and their corresponding library compound monomers A 1 , A 2 , A 3 , and the like, to form library compound 216 . A “split and mix” technique is employed to generate diversity.
- Solid phase supports for use with the invention may have a wide variety of forms, including microparticles; beads, and membrance, slides, plates micromachined chops, and the like.
- solid phase supports of the invention may comprise a wide variety of compositions, including glass, plastic, silicon, alkanethiolate-dervatized gold, cellulose, low cross-linked and high cross-linked polystyrene, silica gel, polyamide, and the like.
- either a population of discrete particles are employed such that each has a uniform coating, or population, of complementary sequences of the same tag(and no other), of a single or a few supports are employed with spacially discrete regions each containing a uniform coating, or population, or complementary sequences to the same tag (and no other).
- the area of the regions may vary according to particular applications; usually, the regions range in area from several ⁇ m 2 , e.g. 3-5, to several hundred ⁇ m 2 , e.g. 100-500.
- such regions are specifically discrete so that signals generated by events, e.g. fluorescent emissions, at adjacent regions can be resolved by the detection system being employed.
- Tag complements may be used with the solid phase support that they are synthesized on, or they may be separately synthesized and attached to a solid phase support for use, e.g. as disclosed by Lund et al. Nucleic Adds Research, 16: 10861-10880 (1988); Albretsen et al, Anal. Biochem., 189: 40-50 (1990); Wolf et al, Nucleic Acids Research, 15: 2911-2926 (1987); or Ghosh et al, Nucleic Acids Research, 15: 5353-5372 (1987); Preferably, tag complements are synthesized on and used with the same solid phase support; which my comprise a variety of forms and include a variety of linking moieties.
- Such supports may comprise microparticles or arrays, or matrices, of regions where uniform populations of tag complements are synthesized.
- microparticle supports may be used with the invention, including microparticles made of controlled pore glass (CPG), highly cross-linked polstyrene., acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, disclosed in the following exemplary references: Meth. Enzymol, Section A pages 11-147, vol. 44 (Academic Press, New York, 1976); U.S. Pat. No. 4,678,814; 4,413,070; and 4,046;720; and Pon. Chapter 19, in Agrawal, editor, Methods in Molecular Biology, Vol.
- CPG controlled pore glass
- Microparticle supports further include commercially available nucleoside-derivatized CPG and polystyrene beads (e.g. available from Applied Biosystems, Foster City, Calif.); derivatized magnetic beads; polystyrene grafted with polythylene glycol (e.g. TentaGelTM, Rapp Polymere, Tubingen Germany); and the like. Selection of the support characteristics, such as material, porosity, size, shape, and the like, and the type of linking moiety employed depends on the conditions under which the tags are used.
- linking moieties are disclosed in Pon et al, Biotechniques, 6; 768-775 (1988); Webb, U.S. Pat. No. 4,659,774; Barany et al, International patent application PCT/US91/06103; Brown et al, J. Chem. Soc. Commun., 1989: 891-893; Damha et al. Nucleic Acids Research, 18: 3813-3821 (1990); Beattie et al, Clinical Chemistry, 39: 719-722 (1993); Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); and the like.
- tag complements may also be synthesized on a single (or a few) solid phase support to form an array of regions uniformly coated with tag complements. That is, within each region in such an array the same tag complement is synthesized.
- Techniques for synthesizing such arrays are disclosed in McGall et al, International application PCT/US93/03767; Pease et al, Proc. Natl. Acad. Sci., 91: 5022-5026 (1994); Southern and Maskos, International application PCT/GB89/01114; Maskos and Southern (cited above); Southern et al, Genomics, 13: 1008-1017 (1992); and Maskos and Southern, Nucleic Acids Research, 21: 4663-4669 (1993).
- the invention is implemented with microparticles or beads uniformly coated with complements of the same tag sequence.
- Microparticle supports and methods of covalently or noncovalently linking oligonucleotides to their surfaces are well known, as exemplified by the following references: Beaucage and Iyer (cited above); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the references cited above.
- the size and shape of a microparticle is not critical; however, microparticles in the size range of a few, e.g. 1-2, to several hundred, e.g. 200-1000 ⁇ m diameter are preferable, as they facilitate the construction and manipulation of large repertoires of oligonucleotide tags with minimal reagent and sample usage.
- CPG controlled-pore glass
- polystyrene supports are employed as solid phase supports in the invention.
- Such supports come available with base-labile linkers and initial nucleosides attached, e.g. Applied Biosystems (Foster City, Calif.).
- microparticles having pore sizes between 500 and 1000 angstroms are employed.
- An important aspect of the invention is the sorting of populations of identical polynucleotides, e.g. from a cDNA library, and their attachment to microparticles or separate regions of a solid phase support such that each microparticle or region has only a single kind of polynucleotide.
- This latter condition can be essentially met by ligating a repertoire of tags to a population of polynucleotides followed by cloning and sampling of the ligated sequences.
- a repertoire of oligonucleotide tags can be ligated to a population of polynucleotides in a number of ways, such as through direct enzymatic ligation, amplification, e.g.
- the initial ligating step produces a very large populations of tag-polynucleotide conjugates such that a single tag is generally attached to many different polynucleotides.
- the probability of obtaining “doubles,” i.e. the same tag on two different polynucleotide can be made negligible. (Note that it is also possible to obtain different tags with the same polynucleotide in a sample. This case is simply leads to a polynucleotide being processed, e.g. sequenced, twice).
- the probability of obtaining a double in a sample can be estimated by a Poisson distribution since the number of conjugates in a sample will be large, e.g. on the order of thousands or more, and the probability of selecting a particular tag will be small because the tag repertoire is large, e.g. on the order of tens of thousands or more. Generally, the larger the sample the greater the probability of obtaining a double. Thus, a design trade-off exists between selecting a large sample of tag-polynucleotide conjugates—which, for example, ensures adequate coverage of a target polynucleotide in a shotgun sequencing operation, and selecting a small sample which ensures that a minimal number of doubles will be present.
- the presence of double merely adds an additional source of noise or, in the case of sequencing, a minor complication in scanning and signal processing, as microparticles giving multiple fluorescent signals can simply ignored.
- the term “substantially all” in reference to attaching tags to molecules, especially polynucleotides is meant to reflect the statistical nature of the sampling procedure employed to obtain a population of tag-molecule conjugates essentially free of doubles. The meaning of substantially all in terms of actual percentages of tag-molecule conjugates depends on how the tags are being employed. Preferably, for nucleic acid sequencing, substantially all means that at least eighty percent of the tags have unique polynucleotides attached.
- tags More preferably, it means that at least ninety percent of the tags have unique polynucleotides attached. Still more preferably, i. means that at least ninety-five percent of the tags have unique polynucleotides attached. And, more preferably, it means that at least ninety-nine percent of the tags have unique polynucleotides attached.
- oligonucleotides tags are attached by reverse transcribing the mRNA with a set of primers containing complements of tag sequences.
- An exemplary set of such primers could have the following sequence: 5′-mRNA-[A] n -3′ [T] 19 GG[W,W,W,C] 9 AC CAGCTG ATC-5′-biotin where “[W,W,W,C] 9 ” represents the sequence of an oligonucleotide tag of nine subunits of four nucleotides each and “[W,W,W,C]” represents the subunit sequences listed above, i.e. “W” represents T or A.
- the underlined sequences identify an optional restriction endonuclease site that can be used to release the polynucleotide from attachment to a solid phase support via the biotin, if one is employed.
- the complement attached to a microparticle could have the form (SEQ ID NO: 4 ): 5′-[G,W,W,W] 9 TGG-linker-microparticle
- the mRNA is removed, e.g. by RNase H digestion, and the second strand of the cDNA is synthesized using, for example, a primer of the following form (SEQ ID NO:6): 5′-NRRGATGYNN-3′ 5 ′-NRRGATCYNNN- 3 ′ where N is any one of A, T, G, or C; R is a purine-containing nucleotide, and Y is a pyrimidine-containing nucleotide.
- This particular primer creates a Bst Y1 restriction site in the resulting double stranded DNA which, together with the Sal I site, facilitates cloning into a vector with, for example, Bam HI and Xho I sites.
- the exemplary conjugate would have the form (SEQ ID NO: 19 ): 5′-RCGACCA[C,W,W,W,] 9 GG[T] 19 -cDNA-NNR GGT[G,W,W,W] 9 CC[A] 19 -rDNA 0 NNNYCTAG-5′
- the Bst YI and Sal I digested fragments are cloned into a Bam HI-/Xho I-digested vector having the following single-copy restriction sites (SEQ ID NO:1): 5′-GA GGATG CCTTTAT GGATCC A CTCGAG ATCCCAATCCA-3′
- FokI BAmHI XhoI This adds the Fok I site which will allow initiation of the sequencing process discussed more fully below.
- a general method for exposing the single stranded tag after amplification involves digesting a target polynucleotide-containing conjugate with the 5′ ⁇ 3′ 3′ ⁇ 5′ exonuclease activity of T4 DAN polymerase, or a like enzyme.
- T4 DAN polymerase or a like enzyme.
- T4 DAN polymerase When used in the presence of a single nucleoside triphosphate, such a polymerase will cleave nucleotides from 3′ recessed ends present on the non-template strand of a double stranded fragment until a complement of the single nucleoside triphosphate is reached on the template strand.
- the technique may also be used to preferentially methylate interior Fok I sites of a target polynucleotide while leaving a single Folk I site at the terminus of the polynucleotide unmethylated.
- the terminal Folk I site is rendered single stranded using a polymerase with deoxycytidine triphosphate.
- the double stranded portion of the fragment is then methylated, after which the single stranded terminus is filled in with a DNA polymerase in the presence of all four nucleoside triphosphates, thereby regenerating the Folk I site.
- the polynucleotides are mixed with microparticles containing the complementary sequences of the tags under conditions that favor the formation of perfectly matched duplexes between the tags and their complements.
- conditions that favor the formation of perfectly matched duplexes between the tags and their complements.
- the hybridization conditions are sufficiently stringent so that only perfectly matched sequences form stable duplexes. Under such conditions the polynucleotides specifically hybridized through their tags are ligated to the complementary sequences attached to the microparticles. Finally, the microparticles are washed to remove unligated polynucleotides.
- the density of tag complements on the microparticle surface is typically greater than that necessary for some sequencing operations. That is, in sequencing approaches that require successive treatment of the attached polynucleotides with a variety of enzymes, densely spaced polynucleotides may tend to inhibit access of the relatively bulky enzymes to the polynucleotides.
- the polynucleotides are preferably mixed with the microparticles so that tag complements are present in significant excess, e.g. from 10:1 to 100:1, or greater, over the polynucleotides.
- the density of polynucleotides on the microparticle surface will not be so high as to inhibit enzyme access.
- the average interpolynucleotide spacing on the microparticle surface is on the order of 30-100 nm.
- Guidance in selecting ratios for standard CGP supports and Ballotini beads (a type of solid glass support) is found in Maskos and Southern., Nucleic Acids Research, 20: 1679-1684 (1992).
- standard CPG beads of diameter in the range of 20-50 ⁇ m are loaded with about 10 5 polynucleotides.
- the above method may be used to fingerprint mRNA populations when coupled with the parallel sequencing methodology described below. Partial sequence information is obtained simultaneously from a large sample, e.g. ten to a hundred thousand, of cDNAs attched to separate microparticles as described in the above method. The frequency distribution of partial sequences can identify mRNA populations from different cell or tissue types, as well as from diseased tissues, such as cancers. Such mRNA fingerprints are useful in monitoring and diagnosing disease states.
- the present invention can be employed with conventional methods of DNA sequencing, e.g. as disclosed by Hultman et al, Nucleic Acids Research, 17: 4937-4946 (1989).
- a DNA sequencing methodology is preferred that requires neither electrophoretic separation of closely sized DNA fragments nor analysis of cleaved nucleotides by a separate analytical procedure, as in peptide sequencing.
- the methodology permits the stepwise identification of nucleotides, usually one at a time, in a sequence through successive cycles of treatment and detection.
- Such methodologies are referred to herein as “single base” sequencing methods. Single base approaches are disclosed in the following references: Cheeseman, U.S. Pat. No.
- the method comprises the following steps: (a) ligating a probe to an end of the polynucleotide having a protruding strand to form a ligated complex, the probe having a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site; (b) removing unligated probe from the ligated complex; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide by the identity of the ligated probe; (d) cleaving the ligated complex with a nuclease; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.
- identifying the one or more nucleotides can be carried out either before or after cleavage of the ligated complex from the target polynucleotide.
- the method further includes a step of methylating the target polynucleotide at the start of a sequencing operation.
- the probes are double stranded DNA with a protruding strand at one end 10 .
- the probes contain at least one nucleus recognition site 12 and a spacer region 14 between the recognition site and the protruding end 10 .
- probes also include a label 16 , which in this particular embodiment is illustrated at the end opposite of the protruding strand.
- the probes may be labeled by a variety of means and at a variety of locations, the only restriction being that the labeling means selected does not interfere with the ligation step or with the recognition of the probe by the nucleus.
- protruding strand 10 of the probe is a 5′ or 3′ end. However, it is important that the protruding strands of the target polynucleotide and probes be capable of forming perfectly matched duplexes to allow for specific ligation. If the protruding strands of the target polynucleotide and probe are different lengths the resulting gap can be filled in by a polymerase prior to ligation, e.g. as in “gap LCR” disclosed in Backman et al, European patent application 91100959.5.
- the number of nucleotides in the respective protruding strands are the same so that both strands of the probe and target polynucleotide are capable of being ligated without a filling step.
- the protruding strand of the probe is from 2 to 6 nucleotides long. As indicated below, the greater the length of the protruding strand, the greater the complexity of the probe mixture that is applied to the target polynucleotide during each ligation and cleavage cycle.
- the complementary strands of the probes are conveniently synthesized on an automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, using standard chemistries. After synthesis, the complementary strands are combined to form a double stranded probe.
- the protruding strand of a probe is synthesized as a mixture, so that every possible sequence is represented in the protruding portion. For example, if the protruding portion consisted of four nucleotides, in one embodiment four mixtures are prepared as follows:
- X i TTTA Such mixtures are readily synthesized using well known techniques, e.g. as disclosed in Telenius et al (cited above). Generally, these techniques simply call for the application of mixtures of the activated monomers to the growing oligonucleotide during the coupling steps where one desires to introduce the degeneracy. In some embodiments it may be desirable to reduce the degeneracy of the probes. This can be accomplished using degeneracy reducing analogs, such as deoxyinosine, 2-aminopurine, or the like, e.g. as taught in Kong Thoo Lin et al, Nucleic Acids Research, 20: 5149-5152, or by U.S. Pat. No. 5,002,867.
- the duplex forming region of a probe is between about 12 to about 30 basepairs in length; more preferably, its length is between about 15 to about 25 basepairs.
- the 5′ end of the probe may be phosphorylated in some embodiments.
- a 5′ monophosphate can be attached to a second oligonucleotide either chemically or enzymatically with a kinase, e.g. Sambrook et al (cited above). Chemical phosphorylation is described by Horn and Urdea, Tetrahedron Lett, 27: 4705 (1986), and reagents for carrying out the disclosed protocols are commercially available, e.g. 5′ Phosphate-ON(TM) from Clontech Laboratories (Palo Alto, Calif.).
- probes may have the form:
- the above probes can be labeled in a variety of ways, including the direct or indirect attachment of radioactive moieties, fluorescent moieties, colorimetric moieties, chemiluminescene markers, and the like. Many comprehensive reviews of methodologies for labeling DNA and constructing DNA probes provide guidance applicable to constructing probes of the present invention.
- the probes are labeled with one or more fluorescent dyes, e.g. as disclosed by Menchen et al, U.S. Pat. No. 5,188,934; Begot et al International application PCT/US90/05565.
- a probe is ligated to an end of a target polynucleotide to form a ligated complex in each cycle of ligation and cleavage.
- the ligated complex is the double stranded structure formed after the protruding strands of the target polynucleotide and probe anneal and at least one pair of the identically oriented strands of the probe and target are ligated, i.e. are caused to be covalently linked to one another.
- Ligation can be accomplished either enzymatically or chemically. Chemical ligation methods are well known in the art, e.g. Ferris et al, Nucleosides & Nucleotides, 8: 407-414 (1989).
- ligase is carried out enzymatically using a ligase in a standard protocol.
- Many ligases are known and are suitable for use in the invention, e.g. Lehman, Science, 186: 790-797 (1974); Engler et al, DNA Ligases, pages 3-30 in Boyer, editor, The Enzymes, Vol. 15B (Academic Press, New York, 1982); and the like.
- Preferred ligases include T4 DNA ligase, T7 DNA ligase, E.
- ligases require that a 5′ phosphate group be present for ligation to the 3′ hydroxyl of an abutting strand. This is conveniently provided for at least one strand of the target polynucleotide by selecting a nuclease which leaves a 5′ phosphate, e.g. as Fok I.
- An objective of the invention is to sort identical molecules, particularly polynucleotides, onto the surfaces of microparticles by the specific hybridization of tags and their complements. Once such sorting has taken place, the presence of the molecules or operations performed on the can e detected in a number of ways depending on the nature of the tagged molecule, whether microparticles are detected separately or in “batches,” whether repeated measurements are desired, and the like.
- the sorted molecules are exposed to ligands for binding, e.g. in drug development, or are subjected chemical of enzymatic processes, e.g. in polynucleotide sequencing. In both of these uses it is often desirable to simultaneously observe signals corresponding to such events or processes on large numbers of microparticles.
- Microparticles carrying sorted molecules lend themselves to such large scale parallel operations, e.g. as demonstrated by Lam et al (cited above).
- Such scanning systems may be constructed from commercially available components, e.g. x-y translation table controlled by a digital computer used with a detection system comprising one or more photomultiplier tubes, or alternatively, a CCD array, and appropriate optics, e.g. for exciting, collecting, and sorting fluorescent signals. In some embodiments a confocil optical system may be desirable.
- An exemplary scanning system suitable for use in four-color sequencing is illustrated diagrammatically in FIG. 5 .
- Substrate 300 e.g. a microscope slide with fixed microparticles, is placed on x-y translation table 302 , which is connected to and controlled by an appropriately programmed digital computer 304 which may be any of a variety of commercially available personal computers, e.g. 486-based machines or PowerPC model 7100 or 8100 available from Apple Computer (Cupertino, Calif.).
- Computer software for table translation and data collection functions can be provided by commercially available laboratory software, such as Lab Windows, available from National Instruments.
- Substrate 300 and table 302 are operationally associate with microscope 306 having one or more objective lenses 308 which are capable of collecting and delivering light to microparticles fixed to substrate 300 .
- Excitation beam 310 from light source 312 which is preferably a laser, is directed to beam splitter 314 , e.g. a dichoric mirror, which re-directs the beam through microscope 306 and objective lens 308 which, in turn, focuses the beam onto substrate 300 .
- Lens 308 collects fluorescence 316 emitted from the microparticles and directs it through beam splitter 314 to signal distribution optics 318 which, in turn, directs fluorescence to one or more suitable opto-electronic devices for converting some fluorescence characteristic, e.g.
- the stability and reproducibility of the positional location in scanning will determine, to a large extent, the resolution for separating closely spaced microparticles.
- the scanning systems should be capable of resolving closely spaced microparticles, e.g. seperated by a particle diameter.
- the scanning system should at least have the capability of resolving objects on the order of 10-100 ⁇ m. Even higher resolution may be desirable in some embodiments, but with increase resolution, the time required to fully scan a substrate will increase; thus, in some embodiments a compromise may have to be made between speed and resolution.
- microparticle size and scanning system resolution are selected to permit resolution of fluorescently labeled microparticles randomly disposed on a plane at a density between about ten thousand to one hundred thousand microparticles per cm 2 .
- loaded microparticles can be fixed to the surface of a substrate in variety of ways.
- the fixation should be strong enough to allow the microparticles to undergo successive cycles of reagent exposure and washing without significant loss.
- the substrate is glass, its surface may be derivatized with an alkylamino linker using commercially available reagents, e.g. Pierce Chemical, which in turn may be cross-linked to avidin, again using conventional chemistries, to form an avidinated surface, Biotin moieties can be introduced to the loaded microparticles in a number of ways. For example, a fraction, e.g.
- the cloning vectors used to attach tags to polynucleotides are engineered to contain a unique restriction site (providing sticky ends on digestion) immediately adjacent to the polynucleotide insert at an end of the polynucleotide opposite of the tag.
- the site is excised with the polynucleotide and tag for loading onto microparticles. After loading, about 10-15 percent of the loaded polynucleotides will possess the unique restriction site distal from the microparticle surface.
- an appropriate double stranded adapter containing a biotin moiety is ligated to the sticky end. The resulting microparticles are then spread on the avidinated glass surface where they become fixed via the biotin-avidin linkages.
- a mixture of probes is applied to the loaded microparticle: a fraction of the probes contain a type IIs restriction recognition site, as required by the sequencing method, and a fraction of the probes have no such recognition site, but instead contain a biotin moiety at its non-ligating end.
- the mixture comprises about 10-15 percent of the biotylated probe.
- the tagging system of the invention can be used with single base sequencing methods to sequence polynucleotides up to several kilobases in length.
- the tagging system permits many thousands of fragments of a target polynucleotide to be sorted onto one or more solid phase supports and sequenced simultaneously.
- a portion of each sorted fragment is sequenced in a stepwise fashion on each of the many thousands of loaded microparticles which are fixed to a common substrate-such as a microscope slide-associated with a scanning system, such as that described above.
- the size of the portion of the fragments sequenced depends of several factors, such as the number of fragments generated and sorted, the length of the target polynucleotide, the speed and accuracy of the single base method employed, the number of microparticles and/or discrete regions that may be monitored simultaneously; and the like.
- the sequence of the target polynucleotide is determined by collating the 12-50 base fragments via their overlapping regions, e.g. as described in U.S. Pat. No. 5,002,867.
- the length of the target polynucleotide is between 1 kilobase and 50 kilobases. More preferably, the length is between 10 kilobases and 40 kilobases.
- Fragments may be generated from a target polynucleotide in a variety of ways, including so-called “directed” approaches where one attempts to generate sets of fragments covering the target polynucleotide with minimal overlap, and so-called “shotgun” approaches where randomly overlapping fragments are generated.
- “shotgun” approaches to fragment generation are employed because of their simplicity and inherent redundancy.
- randomly overlapping fragments that cover a target polynucleotide are generated in the following conventional “shotgun” sequencing protocol, e.g. as disclosed in Sambrook et al (cited above).
- “cover” in this context means that every portion of the target polynucleotide sequence is represented in each size range, e.g.
- the vector is expanded, purified and digested with the appropriate restriction enzymes to yield about 10-15 ⁇ g of purified insert.
- the protocol results in about 500-1000 subclones per microgram of starting DNA.
- the insert is seperated from the vector fragments by preparative gel electrophoresis, removed from the gel by conventional methods, and resuspended in a standard buffer, such as TE (Tris-EDTA).
- fragments in the 300-500 basepair range are selected and eluted from the gel by conventional means, and ligated into a tag-carrying vector as described above to form a library of tag-fragment conjugates.
- a sample containing several thousand tag-fragment conjugates are taken from the library and expanded after which the tag-fragment inserts are excised from the vector and prepared for specific hybridization to the tag complements on microparticles, as described above.
- multiple samples may be taken from the tag-fragment library and separately expanded, loaded onto microparticles and sequenced. The number of doubles selected will depend on the fraction of the tag repertoire represented in a sample. (The probability of obtaining triples-three different polynucleotides with the same tag-or above can safely be ignored).
- Table IV lists probabilities of obtaining doubles in a sample for giving tag size, sample size, and repertoire diversity
- nucleotides of each of the random fragments are simultaneously sequenced with a single base method.
- sequence of the target polynucleotide is then reconstructed by collating the partial sequences of the random fragments by way of their overlapping portions, using algorithms similar to those used for assembling contigs, or as developed for sequencing by hybridization, disclosed in the above references.
- kits of the invention include kits for carrying out the various embodiments of the invention.
- kits of the invention include a repertoire of tag complements attached to a solid phase support.
- kits of the invention may include the corresponding repertoire of tags, e.g. as primers for amplifying polynucleotides to be sorted or as elements of cloning vectors which can also be used to amplify the polynucleotides to be sorted.
- the repertoire of tag complements are attached to microparticles. Kits may also contain appropriate buffers for enzymatic processing, detector chemistries, e.g.
- sequencing kits may also include substrates, such as a avidinated microscope slides, for fixing loaded nicroparticles for processing.
- a mixture of three target polynucleotide-tag conjugates are obtained as follows: First, the following six oligonucleotides are synthesized and combined pairwise to form tag 1, tag 2, and tag 3 (SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:17) :
- ApUC19 is digested with Sal I and Hind III, the large fragment is purified, and separately ligated with tags 1, 2, and 3, to form pUC19-1, pUC19-2, and pUC19-3, respectively.
- the three recombinants are separately amplified and isolated, after which pUC19-1 is digested with Hind HI and Aat I, pUC19-2 is digested with Hind III and Ssp I, and pUC19-3 is digested with Hind III and Xmn I.
- the small fragments are isolated using conventional protocols to give three double stranded fragments about 250, 375, and 575 basepairs in length, respectively, and each having a recessed 3′ strand adjacent to the tag and a blunt or 3′ protruding strand at the opposite end.
- Approximately 12 nmoles of each fragment are mixed with 5 units T4 DNA polymerase in the manufacturer's recommended reaction buffer containing 33 ⁇ M deoxycytosine triphosphate.
- the reaction mixture is allowed to incubate at 37° C. for 30 minutes, after which the reaction is stopped by placing on ice.
- the fragments are then purified by conventional means.
- CPG microparticles (37-74 mm, particle size, 500 angstrom pore size, Pierce Chemical) are derivatized with the linker disclosed by Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992). After separating into three aliquots, the complements of tags 1, 2, and 3 are synthesized on the microparticles using a conventional automated DNA synthesizer, e.g. a model 392 DNA synthesizer (Applied Biosystems, Foster City, Calif.). Approximately 1 mg of each of the differently derivatized microparticles are placed in separate vessels.
- a conventional automated DNA synthesizer e.g. a model 392 DNA synthesizer (Applied Biosystems, Foster City, Calif.). Approximately 1 mg of each of the differently derivatized microparticles are placed in separate vessels.
- the T4 DNA polymerase-treated fragments excised from pUC19-1, -2, and -3 are resuspended in 50 ⁇ L of the manufacturer's recommended buffer for Taq DNA ligase (New England Biolabs)
- the mixture is then equally divided among the three vessels containing the 1 mg each of derivatized CPG microparticles. 5 units of Taq DNA ligase is added to each vessel, after which they are incubated at 55° C. for 15 minutes. The reaction is stopped by placing on ice and the microparticles are washed several times by repeated centrifugation and resuspension in TE.
- microparticles are resuspended in Nde I reaction buffer (New England Biolabs) where the attached polynucleotides are digested.
- Nde I reaction buffer New England Biolabs
- the polynucleotide fragments released by Nde I digestion are fluorescently labeled by incubating with Sequenase DNA polymerase and fluorescent labeled thymidine triphosphate (Applied Biosystems, Foster City, Calif.).
- Sequenase DNA polymerase and fluorescent labeled thymidine triphosphate (Applied Biosystems, Foster City, Calif.).
- the fragments are thin separately analyzed on a nondenaturing polyacrylamide gel using an Applied Biosystems model 373 DNA sequencer.
- a repertoire of 36-mer tags consisting of nine 4-nucleotide subunits selected from Table I is prepared by separately synthesizing tags and tag complements by a split and mix approach, as described above.
- the repertoire is synthesized so as to permit ligation into a Sma I/Hind III digested M13mp19.
- one set of oligonucleotides begins with the addition of A followed by nine rounds of split and mix synthesis wherein the oligonucleotide is extended subunit-wise by 3′-phosphoramidite derivatized 4-mers corresponding to the subunits of Table I.
- the other set of oligonucleotides begins with the addition of three C's (portion of the Sma I recognition site) and two G's, followed by nine rounds of split and mix synthesis wherein the oligonucleotide is extended by 3′-phosphoramidite derivatized 4-mers corresponding to the complements of the subunits of Table I.
- Synthesis is completed by the nucleotide-by-nucleotide addition of the Hind III recognition site and a 5′-monophosphate.
- the oligonucleotides are mixed under conditions that permit formation of the following duplexes (SEQ ID NO: 18 ): 5′-pGGGCC(w i )(w i )(w i )(w i )(w i )(w i )(w i )(w i )A CCCGG(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′
- the mixture of duplexes is then ligated into a Sma I/Hind III-digested M13mp19.
- a repertoire of tag complements are synthesized on CPG microparticles as described above.
- SV40 DNA is fragmented by sonication following the protocol set forth in Sambrook et al (cited above). The resulting fragments are repaired using standard protocols and separated by size. Fragments in the range of 300-500 basepairs are selected and ligated into the Sma I digested M13 described above to form a library of fragment-tag conjugates, which is then amplified. A sample containing several thousand different fragment-tag conjugates is taken from the library, further amplified, and the fragment-tag inserts are excised by digesting with Eco RI and Hind III. The excised fragment-tag conjugates are treated with T4 DNA polymerase in the presence of deoxycytidine triphosphate, as described in Example I, to expose the oligonucleotide tags for specific hybridization to the CPG microparticles.
- the loaded microparticles are treated with Fok I to produce a 4-nucleotide protruding strand of a predetermined sequence.
- a 10:1 mixture (probe 1:probe 2) of the following probes (SEQ ID NO:3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 ) are ligated to the polynucleotides on microparticles.
- Probe 1 FAM- ATCGGATGAC TAGCCTACTGAGCT Probe 2 biotin- ATCGGATGAC TAGCCTACTGAGCT FAM represents a fluorescein dye attached to the 5′-hydroxyl of the top strand of Probe I through an aminophosphate linker available from Applied Biosystems (Aminolinker). The biotin may also be attached through an Aminolinker moiety and optionally may be further extended via polyethylene oxide linkers, e.g. Jaschke et al (cited above).
- the loaded microparticles are then deposited on the surface of an avidinated glass slide to which and from which reagents and wash solutions can be delivered and removed.
- the avidinated slide with the attached microparticles is examined with a scanning fluorescent microscope (e.g. Zeiss Axiskop equipped with a Newport Model PM500-C motion controller, a Spectra-Physics Model 2020 argon ion laster producing a 488 nm excitation beam, and a 520 nm long-pass emission filter, or like apparatus).
- the excitation beam and fluorescent emissions are delivered and collected, respectively, through the same objective lens.
- the excitation beam and collected fluorescence are separated by a dichroic mirror which directs the collected fluorescence through a series of bandpass filters and to photon-counting devices corresponding to the fluorophors being monitored, e.g. comprising Hamamatsu model 9403-02 photomutlipliers, a Stanford Research Systems model SR445 amplifier and model SR430 multichannel scaler, and digital computer, e.g. a 486-based computer.
- the computer generates a two dimensional map of the slide which registers the positions of the microparticles.
- the polynucleotides on the attached microparticles undergo 20 cycles of probe ligation, washing detection, cleavage, and washing, in accordance with the preferred single base sequencing methodology described below.
- the scanning system records the fluorescent emission corresponding to the base identified at each microparticle. Reactions and washes below are generally carded out with manufacturer's (New England Biolabs') recommended buffers for the enzymes employed, unless otherwise indicated. Standard buffers are also described in Sambrook et al (cited above).
- the bold faced nucleotides are the recognition site for Fok I enidonuclease, and “N” represents any one of the four nucleotides, A, C, G, T.
- TAMRA tetramethylrhodamine
- FAM fluorescein
- ROX rhodamine X
- JOE 2′,7′-dimethoxy-4′,5′-dichlorofluorescein
- the above probes are incubated in approximately 5 molar excess of the target polynucleotide ends as follows: the probes are incubated for 60 minutes at 16° C. with 200 traits of T4 DNA ligase and the anchored target polynculeotide in T4 DNA ligase buffer, after washing, the target polynucleotide is then incubated with 100 units T4 polynucleotide kinase in the manufacturer's, recommended buffer for 30 minutes at 37° C., washed, and again incubated for 30 minutes at 16° C. with 200 units of T4 DNA ligase and the anchored target polynucleotide in T4 DNA ligase buffer.
- Washing is accomplished by successively flowing volumes of wash buffer over the slide, e.g. TE, disclosed in Sambrook et al (cited above). After the cycle of ligation-phosphorylation-ligation and a final washing, the attached microparticles are scanned for the presence of fluorescent label, the positions and characteristics of which are recorded by the scanning system.
- the labeled target polynucleotide i.e. The ligated complex, is then incubated with 10 units of Fok I in the manufacturer's recommended buffer for 30 minutes at 37° C., followed by washing in TE.
- Fok I 10 units of Fok I
- the target polynucleotide is shortened by one nucleotide on each strand and is ready for the next cycle of ligation and cleavage. The process is continued until twenty nucleotides are identified.
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Abstract
The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention each consist of a plurality of subunits 3 to 6 nucleotides in length selected from a minimally cross-hybridizing set. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.
Description
This is a continuation of U.S. patent application Ser. No. 08/358,810 filed 19 Dec. 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/322,348 filed 13 Oct. 1994, now abandoned, which application is incorporated by reference.
The invention relates generally to methods for identifying, sorting, and/or tracking molecules, especially polynucleotides, with oligonucleotide labels, and more particularly, to a method of sorting polynucleotides by specific hybridization to oligonucleotide tags.
Specific hybridization of oligonucleotides and their analogs is a fundamental process that is employed in a wide variety of research, medical, and industrial applications, including the identification of disease-related polynucleotides in diagnostic assays, screening for clones of novel target polynucleotides, identification of specific polynucleotides in blots of mixtures of polynucleotides, amplification of specific target polynucleotides, therapeutic blocking of inappropriately expressed genes. DNA sequencing, and the like, e.g. Sambrook et at, Molecular Cloning: A Laboratory Manual 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Milligan et al. J. Med. Chem., 36: 1923-1937 (1993); Drmanac et al, Science, 260: 1649-1652 (1993); Bains, J. DNA Sequencing and Mapping, 4: 143-150 (1993).
Specific hybridization has also been proposed as a method of tracking, retrieving, and identifying compounds labeled with oligonucleotide tags. For example, in multiplex DNA sequencing oligonucleotide tags are used to identify electrophoretically separated bands on a gel that consist of DNA fragments generated in the same sequencing reaction. In this way, DNA fragments from many sequencing reactions are separated on the same length of a gel which is then blotted with separate solid phase materials on which the fragment bands from the separate sequencing reactions are visualized with oligonucleotide probes that specifically hybridize to complementary tags, Church et al. Science, 240: 185-188 (1988). Similar uses of oligonucleotide tags have also been proposed for identifying explosive, potentially pollutants, such as crude oil, and currency for prevention and detection of counterfeiting, e.g. reviewed by Dollinger, pages 265-274 in Mullis et al, editors. The Polymerase Chain Reaction (Birkhauser, Boston, 1994). More recently, systems employing oligonucleotide tags have also been proposed as a means of manipulating and identifying individual molecules in complex combinatorial chemical libraries, for example, as an aid to screening such libraries for drug candidates, Brenner and Lerner, Proc. Natl. Acad. Sci. 89: 5381-5383 (1992); Alper, Science, 264: 1399-1401 (1994); and Needels et al, Proc. Natl. Acad. Sci., 90: 10700-10704 (1993)
The successful implementation of such tagging schemes depends in large part on the success in achieving specific hybridization between a tag and its complementary probe. That is, for an oligonucleotide tag to successfully identify a substance, the number of false positive and false negative signals must be minimized. Unfortunately, such spurious signals are not uncommon because base pairing and base stacking free energies vary widely among nucleotides in a duplex or triplex structure. For example, a duplex consisting of a repeated sequence of deoxyadenine (A) and thymidine (T) bound to its complement may have less stability than an equal-length duplex consisting of a repeated sequence of deoxyguanidine (G) and deoxycytidine (C) bound to a partially complementary target containing a mismatch. Thus, if a desired compound from a large combinatorial chemical library were tagged with the former oligonucleotide, a significant possibility would exist that, under hybridization conditions designed to detect perfectly matched AT-rich duplexed, undesired compounds labeled with the GC-rich oligonucleotide—even in a mismatched duplex—would be detected along with the perfectly matched duplexes consisting of the AT-rich tag. In the molecular tagging system proposed by Brenner et al (cited above), the related problem of mis-hybridizations of closely related tags was addressed by employing a so-called “commaless” code, which ensures that a probe out of register (or frame shifted) with respect to its complementary tag would result in a duplex with one or more mismatches for each of its five or more three-base words, or “codons.”
Even though reagents, such as tetramethylammonium chloride, are available to negate base-specific stability differences of oligonucleotide duplexes, the effect of such reagents is often limited and their presence can be incompatible with, or render more difficult, further manipulations of the selected compounds, e.g. amplification by polymerase chain reaction (PCR) or the like.
Such problems have made the simultaneous use of multiple hybridization probes in the analysis of multiple or complex genetic loci e.g. via multiplex PCR, reverse dot blotting, or the like, very difficult. As a result, direct sequencing of certain loci, e.g. HLA genes, has been promoted as a reliable alternative to indirected methods employing specific hybridization for the identification of genotypes, e.g. Gyllensten et al, Proc. Nat. Acad. Sci., 85: 7652-7656 (1988).
The ability to sort cloned and identically tagged DNA fragments onto distinct solid phase supports would facilitate such sequencing, particularly when coupled with a non gel-based sequencing methodology simultaneously applicable to many samples in parallel.
In view of the above, it would be useful if there were available an oligonucleotide-based tagging system which provided a large repertoire of tags, but which also minimized the occurrence of false positive and false negative signals without the need to employ special reagents for altering natural base pairing and base stacking free energy differences. Such a tagging system would find applications in many areas, including construction and use of combinatorial chemical libraries, large-scale mapping and sequencing of DNA, genetic identification, medical diagnosis, and the like.
An object of my invention is to provide a molecular tagging system for tracking, retrieving, and identifying compounds.
Another object of my invention is to provide a method for sorting identical molecules, or subclasses of molecules, especially polynucleotides, onto surfaces of solid phase materials by the specific hybridization of oligonucleotide tags and their complements.
A further object of my invention is to provide a combinatorial chemical library whose member compounds are identified by the specific hybridization of oligonucleotide tags and their complements.
A still further object of my invention is to provide a system for tagging and sorting many thousands of fragments, especially randomly overlapping fragments, of a target polynucleotide for simultaneous analysis and/or sequencing.
Another object of my invention is to provide a rapid and reliable method for sequencing target polynucleotides having a length in the range of a few hundred basepairs to several tens of thousands of basepairs.
My invention achieve these and other objects by providing a method and materials for tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. An oligonucleotide tag of the invention consists of a plurality of subunits, each subunit consisting of an oligonucleotide of 3 to 6 nucleotides in length. Subunits of an oligonucleotide tag are selected from a minimally cross-hybridizing set. In such a set, a duplex or triplex consisting of a subunit of the set and the complement of any other subunit of the set contains at least two mismatches. In other words, a subunit of a minimally cross-hybridizing set at best forms a duplex or triplex having two mismatches with the complement of any other subunit of the same set. The numbers of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. The number is generally much less than the number of all possible sequences the length of the tag which for a tag nucleotides long would be 4n. More preferably, subunits are oligonucleotides from 4 to 5 nucleotides in length.
In one aspect of my invention, complements of oligonucleotide tags attached to a solid phase support are used to sort polynucleotides from a mixture of polynucleotides each containing a tag. In this embodiment, complements of the oligonucleotide tags are synthesized on the surface of a solid phase support, such as a microscopic bead or a specific location on an array of synthesis locations on a single support such that populations of identical sequences are produced in specific regions. That is, the surface of each support in the case of a bead, or of each region, in the case of an array, is derivatized by only one type of complement which has a particular sequence. The population of such beads or regions contains a repertoire of complements with distinct sequences, the size of the repertoire depending on the number of subunits per oligonucleotide tag and the length of the subunits employed. Similarly, the polynucleotides to be sorted each comprises an oligonucleotide tag in the repertoire, such that identical polynucleotides have the same tag and different polynucleotides have different tags. Thus, when the populations of supports and polynucleotides are mixed under conditions which permit specific hybridization of the oligonucleotide tags with their respective complements, subpopulations of identical polynucleotides are sorted onto particular beads or regions. The subpopulations of polynucleotides can then be manipulated on the solid phase support by micro-biochemical techniques.
Generally, the method of my invention comprises the following steps: (a) attaching an oligonucleotide tag from a repertoire of tags to each molecule in a population of molecules (i) such that substantially all the same molecules or same subpopulation of molecules in the population have the same oligonucleotide tag attached and substantially all different molecules or different subpopulations of molecules in the population have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set; and (b) sorting the molecules or subpopulations of molecules of the population by specifically hybridizing the oligonucleotide tags with their respective complements.
An important aspect of my invention is the use of the oligonucleotide tags to sort polynucleotides for parallel sequence determination. Preferably, such sequencing is carried out by the following steps: (a) generating from the target polynucleotide a plurality of fragments that cover the target polynucleotide; (b) attaching an oligonucleotide tag from a repertoire of tags to each fragment of the plurality (i) such that substantially all the same fragments have the same oligonucleotide tag attached and substantially all different fragments have different oligonucleotide tags attached and (ii) such that each oligonucleotide tag from the repertoire comprises a plurality of subunits and each subunit of the plurality consists of an oligonucleotide having a length from three to six nucleotides or from three to six basepairs, the subunits being selected from a minimally cross-hybridizing set;
-
- sorting the fragments by specifically hybridizing the oligonucleotide tags with their respective complements; (c) determining the nucleotide sequence of a portion of each of the fragments of the plurality, preferably by a single-base sequencing methodology as described below; and (d) determining the nucleotide sequence of he target polynucleotide by collating the sequences of the fragments.
My invention overcomes a key deficiency of current methods of tagging or labeling molecules with oligonucleotides: By coding the sequences of the tags in accordance with the invention, the stability of any mismatched duplex or triplex between a tag and complement to another tag is far lower than that of any preferably matched duplex between the tag and its own complement. Thus, the problem of incorrect sorting because of mismatch duplexes of GC-rich tags being more stable than perfectly matched AT-rich tags is eliminated.
When used in combination with solid phase supports, such as microscopic beads, my invention provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced and/or analyzed simultaneously.
“Complement” or “tag complement” as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which a oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double stranded or single stranded. Thus, where triplexes are formed, the term “couplement” is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.
The term “oligonucleotide” as used herein includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribunucleosides, α-anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking. Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoramilidate, phosphoramidiate, and the like. Usually oligonucleotides of the invention comprise the four natural nucleotides; however, they may also comprise non-natural nucleotide analogs. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be employed, e.g. where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.
“Perfectly matched” in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one other such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand. The term also comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, and the like, that may be employed. In reference to a triplex, the term means that the triplex consists of a perfectly matched duplex and a third strand in which every nucleotide undergoes Hoogsteen or reverse Hoogsteen association with a basepair of the perfectly matched duplex. Conversely, a “mismatch” in a duplex between a tag and an oligonucleotide means that a pair or triplet of nucleotides in the duplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/or reverse Hoogsteen bonding.
As used herein, “nucleoside” includes the natural nucleosides, including 2′-deoxy and 2′-hydroxyl forms, e.g. as described in Kornberg and Baker, DNA, Replication, 2nd Ed. (Freeman, San Francisco, 1992), “Analogs” in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar modified, e.g. described by Scheti, Nucleotide Analogs (John Wiley, New York, 1980) Uhlman and Peyman Chemical Review, 90: 543-584 (1990), or the like, with the only proviso that they are capable of specific hybridization. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
The invention provides a method of labeling and sorting molecules, particularly polynucleotides, by the use of oligonucleotide tags. The oligonucleotide tags of the invention comprise a plurality of “words” or subunits, selected from minimally cross-hybridizing sets of subunits. Subunits of such sets cannot form a duplex or triplex with the complement of another subunit of the same set with less than two mismatched nucleotides. Thus, the sequences of any two oligonucleotide tags of a repertoire that form duplexes will never be “closer” than differing by two nucleotides. In particular embodiments sequences of any two oligonucleotide tags of a repertoire can be even “further” apart, e.g. by designing a minimally cross-hybridizing set such that subunits cannot form a duplex with the complement of another subunit of the same set with less than three mismatched nucleotides, and so on. The invention is particularly useful in labeling and sorting polynucleotides for parallel operations, such as sequencing, fingerprinting or other types of analysis.
The nucleotide sequences of the subunits for any minimally cross-hybridizing set are conveniently enumerated by simple computer programs following the general algorithm illustrated in FIG. 3 , and as exemplified by program minhx whose source code is listed in Appendix L minhx computes all minimally cross-hybridizing sets having subunits composed of three kinds of nucleotides and having length of four.
The algorithm of FIG. 3 is implemented by first defining the characteristic of the subunits of the minimally cross-hybridizing set, i.e. length, number of base differences between members, and composition, e.g. do the consist of two, three, or four kinds of bases. A table Mn, n=1, is generated (100) that consists of all possible sequences of a given length and composition. An initial subunit S1 is selected and compared (120) with successive subunits S2 for i=n+1 to the end of the cable. Whenever a successive subunit has the required number of mismatches to be a member of the minimally cross-hybridizing set, it is saved in a new table Mn+1 (125), that also contains subunits previously selected in prior passes through step 120. For example, in the first set of comparisons, M2 will contain S1; in the second set of comparisons, M3 will contain S1 and S2; in the third set of comparisons, M4 will contain S1, S2, and S3; and so on. Similarly, comparisons in table Mj will be between Sj and all successive subunits in Mj. Note that each successive table Mn+1 is smaller than its predecessors as subunits are eliminated in successive passes through step 130. After every subunit of table Mn has been compared (140) the old table is replaced by the new table Mn+1, and the next round of comparisons are begun. The process stops (160) when a table Mn is reached that contains no successive subunits to compare to the selected subunit Si, i.e. Mn=Mn+1.
Preferably, minimally cross-hybridizing sets comprise subunits that make approximately equivalent contributions to duplex stability as every other subunit in the set. In this way, the stability of perfectly matched duplexes between every subunit and its complement is approximately equal. Guidance for selecting such sets is provided by published techniques for selecting optimal PCR primers and calculating duplex stabilities, e.g. Rychlik et al. Nucleic Acids Research, 17: 8543-8551 (1989) and 18: 6409-6412 (1990); Breslauer et al, Proc. Natl. Acad. Sci., 83: 3746-3750 (1986); Wetmur, Crit. Rev. Biochem. Mol. Biol., 26: 227-259 (1991); and the like. For shorter tags, e.g. about 30 nucleotides or less, the algorithm described by Rychlik and Wetmur is preferred, and for longer tags, e.g. about 30-35 nucleotides or greater, and algoithm disclosed by Suggs et al, pages 683-693 in Brown, editor, ICN-UCLA Syrup. Dev. Biol., Vol. 23 (Academic Press, New York, 1981) may be conveniently employed.
A preferred embodiment of minimally cross-hybridizing sets are those whose subunits are made up of three of the four natural nucleotides. As will be discussed more fully below, the absence of one type of nucleotide in the oligonucleotide tags permits target polynucleotides to be loaded onto solid phase supports by use of the 5′→3′ exonuclease activity era DNA polymerase. The following is an exemplary minimally cross-hybridizing set of subunits each comprising four nucleotides selected from the group consisting of A, G, and T:
TABLE I | ||||
Word: | W1 | W2 | W3 | W4 |
Sequence: | GATT | TGAT | TAGA | TTTG |
Word: | W5 | W6 | W7 | W8 |
Sequence: | GTAA | AGTA | ATGT | AAAG |
In this set, each member would form a duplex having three mismatched bases with the component of every other member.
Further exemplary minimally cross-hybridizing sets are listed below in Table I. Clearly, additional sets can be generated by substituting different groups of nucleotides, or by using subsets of known minimally cross-hybridizing sets.
TABLE II |
Exemplary Minimally Cross-Hybridizing Sets of 4-mer Subunits |
CATT | ACCC | AAAC | AAAG | AACA | AACG |
CTAA | AGGG | ACCA | ACCA | ACAC | ACAA |
TCAT | CACG | AGGG | AGGC | AGGG | AGGC |
ACTA | CCGA | CACG | CACC | CAAG | CAAC |
TACA | CGAC | CCGC | CCGG | CCGC | CCGG |
TTTC | GAGC | CGAA | CGAA | CGCA | CGCA |
ATCT | GCAG | GAGA | GAGA | GAGA | GAGA |
AAAC | GGCA | GCAG | GCAC | GCCG | GCCC |
AAAA | GGCC | GGCG | GGAC | GGAG | |
AAGA | AAGC | AAGG | ACAG | ACCG | ACGA |
ACAC | ACAA | ACAA | AACA | AAAA | AAAC |
AGCG | AGCG | AGCC | AGGC | AGGC | AGCG |
CAAG | CAAG | CAAC | CAAC | CACC | CACA |
CCCA | CCCC | CCCG | CCGA | CCGA | CACA |
CGGC | CGGA | CGGA | CGCG | CGAG | CGGC |
GACC | GACA | GACA | GAGG | GAGG | GAGG |
GCGG | GCGG | GCGC | GCCC | GCAC | GCCC |
GGAA | GGAC | GGAG | GGAA | GGCA | GGAA |
The oligonucleotide tags of the invention and their complements are conveniently synthesized on an automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, using 6 standard chemistries, such as phosphoramidiate chemistry, e.g. disclosed in the following references: Beaucage and Iyer. Tetrahedron, 48: 2223∝2311 (1992); Moltco et al, U.S. Pat. No. 4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S. Pat. Nos. 4,415,732; 4,458,066; and 4,973,679; and the like. Alternative chemistries, e.g. resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, may also be employed provided that the resulting oligonucleotides are capable of specific hybridization. In some embodiments, tags may comprise naturally occurring nucleotides that permit processing or manipulation by enzymes, while the corresponding tag complements may comprise non-natural nucleotide analogs, such as peptide nucleic acids, or like compounds, that promote the formation of more stable duplexes during sorting.
When microparticles are used as supports, repertoires of oligonucleotide tags and tag complements are preferably generated by subunit-wise synthesis via “split and mix” techniques e.g. as disclosed in Shortle et al, International patent application PCT/US93/03418. Briefly, the basic unit of the synthesis is a subunit of the oligonucleotide tag. Preferably, phosphoramidiate chemistry is used and 3′ phosphoramidiate oligonucleotides are prepared for each subunit in a minimally cross-hybridizing set, e.g. for the set first listed above, there would be eight 4-mer 3′-phosphoramidites. Synthesis proceeds as disclosed by Shortle et al of in direct analogy with the techniques employed to generate diverse oligonucleotide libraries using nucleosidic monomers, e.g. as disclosed in Telenius et al, Genomics, 13: 718-725 (1992); Welsh et al, Nucleic Acids Research, 19: 5275-5279 (1991); Grothues et al, Nucleic Acids Research, 21: 1321-1322 (1993); Hartley, European patent application 90304496.4; Lam et al. Nature; 354: 82-84 (1991); Zuckerman et al, Int. J. Pept. Protein Research, 40: 498-507 (1992) and the like. Generally, these techniques simply call for fine application of mixtures of the activated monomers to the growing oligonucleotide during the coupling steps.
Double standard forms of tags are made by separately synthesized the complementary strands followed by mixing under conditions that permit duplex formation. Such duplex tags may then be inserted into cloning vectors along with target polynucleotides for sorting and manipulation of the target polynucleotide in accordance with the invention.
In embodiments where specific hybridization occurs via triplex formation, coding of tag sequences follows the same principles as for duplex-forming tags; however, there are further constraints on the selection of subunit sequences. Generally, third strand association via Hoogsteen type of binding is most stable along homopyrimidine-homopurine tracks in a double stranded target. Usually, base triplets form in T-A*T or C-G*C motifs (where “-” indicates Watson-Crick pairing and “*” indicates Hoogsteen type of binding); however, other motifs are also possible. For example, Hoogsteen base pairing permits parallel and antiparallel orientations between the third strand (the Hoogsteen strand) and the purine-rich strand of the duplex to which the third strand binds, depending on conditions and the composition of the strands. There is extensive guidance in the literature for selecting appropriate sequences, orientation, conditions, nucleoside type (e.g. whether ribose or deoxyribose nucleosides are employed). base modifications (e.g. methylated cytosine, and the like) in order to maximize, or otherwise regulate, triplex stability as desired in particular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci. 88: 9397-9401 (1991); Roberts et al, Science, 258: 1463-1466 (1992); Distefano et al, Proc. Natl. Acad. Sci. 90: 1179-1183 (1993); Mergny et al, Biochemistry, 30: 9791-9798 (1991); Cheng et al, J. Am. Chem. Soc., 114: 4465-4474 (1992); Beal and Dervan, Nucleic Acids Research, 20: 2773-2776 (1992); Beal and Dervan, J. Am. Chem. Soc, 114: 4976-4982 (1992); Giovannangeli et al, Proc. Natl. Acad. Sci. 89: 8631-8635 (1992); Moser and Dervan, Science, 238: 645-650 (1987); McShan et al, J. Biol. Chem., 267: 5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci., 89: 3840-3844 (1992); Blume et al, Nucleic Acids Research, 20: 1777-1784 (1992); Thuong and Helene, Angew. Chem. Int. Ed. Engl. 32: 666-690 (1993); and the like. Conditions for annealing single-stranded or duplex tags to their single-stranded or duplex complements are well known, e.g. Ji et al, Anal. Chem. 65: 1323-1328 (1993).
Oligonudeotide tags of the invention may range in length from 12 to 60 nucleotides or basepairs. Preferably, oligonucleotide tags range in length from 18 to 40 nucleotides or basepairs. More preferably, oligonucleotide tags range in length from 25 to 40 nucleotides or basepairs. Most preferably, oligonucleotide tags are single stranded and specific hybridizing occurs via Watson-Crick pairing with a tag complement.
Oligonucleotide tags may be attached to many different classes of molecules by a variety of reactive functionalities well known in the art; e.g. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc. Eugene, 1992); Khanna et al, U.S. Pat. No. 4,318,846; or the like. Table III provides exemplary functionalities and counterpart reactive groups that may reside on oligonucleotide tags or the molecules of interest. When the functionalities and counterpart reactants are reacted together, after activation in some cases, a linking group is formed. Moreover, as described more fully below, tags may be synthesized simultaneously with the molecules undergoing selection to form combinatorial chemical libraries.
TABLE III |
Reactive Functionalities and Their Counterpart Reactants |
and Resulting Linking Groups |
Reactive | Counterpart | Linking |
Functionality | Functionality | Group |
—NH2 | —COOH | —CO—NH— |
—NH2 | —NCO | —NHCONH— |
—NH2 | —NCS | —NHCSNH— |
—NH2 |
|
|
—SH | —C═C—CO— | —S—C—C—CO— |
—NH2 | —CHO | —CH2NH— |
—NH2 | —SO2Cl | —SO2NH— |
—OH | —OP(NCH(CH3)2)2 | —OP(═O)(O)O— |
—OP(═O)(O)S | —NHC(═O)CH2Br | —NHC(═O)CH2SP(═O)(O)O— |
A class of molecules particularly convenient for the generation of combinatorial chemical libraries includes linear polymeric molecules of the form:
—(M—L)n—
wherein L is a linker moiety and M is a monomer that may selected from a wide range of chemical structures to provide a range of functions from serving as an inert non-sterically hindering spacer moiety to providing a reactive functionality which can serve as a branching point to attach other components, a site for attaching labels; a site for attaching oligonucleotides or other binding polymers for hybridizing or binding to a therapeutic target; or as a site for attaching other groups for affecting solubility, promotion of duplex and/or triplex formation, such as intercalators, alkylating agents, and the like. The sequence, and therefore composition, of such linear polymeric molecules may be encoded within a polynucleotide attached to the tag, as taught by Brenner and Lener (cited above). However, after a selection event, instead of amplifying then sequencing the tag of the selected molecule, the tag itself or an additional coding segment can be sequenced directly—using a so-called “single base” approach described below—after releasing the molecule of interest, e.g. by restriction digestion of a site engineered into the tag. Clearly, any molecule produced by a sequence of chemical reaction steps compatible with the simultaneous synthesis of the tag moieties can be used in the generation of combinatorial libraries.
—(M—L)n—
wherein L is a linker moiety and M is a monomer that may selected from a wide range of chemical structures to provide a range of functions from serving as an inert non-sterically hindering spacer moiety to providing a reactive functionality which can serve as a branching point to attach other components, a site for attaching labels; a site for attaching oligonucleotides or other binding polymers for hybridizing or binding to a therapeutic target; or as a site for attaching other groups for affecting solubility, promotion of duplex and/or triplex formation, such as intercalators, alkylating agents, and the like. The sequence, and therefore composition, of such linear polymeric molecules may be encoded within a polynucleotide attached to the tag, as taught by Brenner and Lener (cited above). However, after a selection event, instead of amplifying then sequencing the tag of the selected molecule, the tag itself or an additional coding segment can be sequenced directly—using a so-called “single base” approach described below—after releasing the molecule of interest, e.g. by restriction digestion of a site engineered into the tag. Clearly, any molecule produced by a sequence of chemical reaction steps compatible with the simultaneous synthesis of the tag moieties can be used in the generation of combinatorial libraries.
Conveniently there is a wide diversity of phosphate-linked monomers available for generating combinatorial libraries. The following references disclose several phosphoramidite and/or hydrogen phosphonate monomers suitable for use in the present invention and provide guidance for their synthesis and inclusion into oligonucleotides: Newton et al, Nucleic Acids Research, 21: 1155-1162 (1993); Griffin et al, J. Am. Chem. Soc, 114: 7976-7982 (1992); Jaschke et al, Tetrahedron Letters, 34: 301-304 (1992); Ma et al, International application PCT/CA92/00423; Zon et al, International application PCT/US90/06630; Durand et al, Nucleic Acids Research, 18: 6353-6359 (1990); Salunkhe et al, J. Am. Chem. Soc., 114: 8768-8772 (1992); Urdea et al, U.S. Pat. No. 5,093,232; Ruth, U.S. Pat. No. 4,948,882; Cruickshank, U.S. Pat. No. 5,091,519; Haralambidis et al, Nucleic Acids Research, 15: 4857-4876 (1987); and the like. More particularly, M may be a straight chain, cyclic, or branched organic molecular structure containing from 1 to 20 carbon atoms and from 0 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur. Preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 16 carbon atoms; a heterocycle having from 3 to 8 carbon atoms and from 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur; glycosyl; or nucleosidyl. More preferably, M is alkyl, alkoxy, alkenyl, or aryl containing from 1 to 8 carbon atoms; glycosyl; or nucleosidyl.
Preferably, L is a phosphorus (V) linking group which may be phosphodiester, phosphotriester, methyl or ethyl phosphonate, phosphorothioate, phophorodithioate, phosphoramidate, of the like. Generally, linkages derived from phosphoramidite or hydrogen phosphonate precursors are preferred so that the linear polymeric units of the invention can be conveniently synthesized with commercial automated DNA synthesizers, e.g. Applied Biosystems, Inc. (Foster City, Calif.) model 394, or the like.
n may vary significantly depending on the nature of M and L. Usually, n varies from about 3 to about 100. When M is a nucleoside or analog thereof or a nucleoside-sized monomer and L is a phosphorus(V) linkage, then n varies from about 12 to about 100. Preferably, when M is a nucleoside or analog thereof or a nucleoside-sized monomer and L is a phosphorus(V) linkage, then n varies from about 12 to about 40.
Peptides are another preferred class of molecules to which tags of the invention are attached. Synthesis of peptide, oligonucleotide conjugates which may be used in the invention is taught in Nielsen et al, J. Am. Chem. Soc., 115: 9812-9813 (1993); Haralambidis et al (cited above) and International patent application PCT/AU88/004417; Truffert et al, Tetrahedron Letters, 35:2353-2356 (1994); de la Torre et al, Tetrahedron Letters, 35: 2733-2736 (1994); and like references. Preferably, peptide-oligonucleotide conjugates are synthesized as described below. Peptides synthesized in accordance with the invention may consist of the natural amino acid monomers or non-natural monomers, including the D isomers of the natural amino acids and the like.
Combinatorial chemical libraries employing tags of the invention are preferably prepared by the method disclosed in Nielson et al (cited above) and illustrated in FIG. 4 for a particular embodiment. Briefly, a solid phase support, such as CPG, is derivatized with a cleavable linker that is compatible with both the chemistry employed to synthesize the tags and the chemistry employed to synthesize the molecule that will undergo some selection process. Preferably, tags are synthesized using phosphoramidite chemistry as described above and with the modifications recommended by Nielson et al (cited above); that is, DMT-5′-O-protected 3′-phosphoramidite-derivatized subunits having methyl-protected phosphite and phosphate, moieties are added in each synthesis cycle. Library compounds are preferably monomers having Fmos—or equivalent—protecting groups masking the functionality to which successive monomer will be coupled. A suitable linker for chemistries employing both DMT and Fmoc protecting groups (referred to herein as a sarcosine linker) is disclosed by Brown et al, J. Chem. Soc. Chem. Commun. 1989: 891-893, which reference is incorporated by reference.
(CPG)-NHC(O)CN(CH3)C(O)CH2CH2C(O)O(CH2)6NHC(O)CH2(O-DMT)NC(O)- |
CH2O(CH2CH2O)2CH2CH2NHC(O)CH2O(CH2CH2O)2CH2CH2NH-Fmoc |
Here “CPG” represents a controlled-pore glass support, “DMT” represents dimethoxytrityl, and “Fmos” represents, 9-fluorenylmethoxycarbonyl.
In a preferred embodiment, an oligonucleotide segment 214 is synthesized initially so that in double stranded form a restriction candonuclease site is provided for cleaving the library compound after sorting onto a microparticle, or like substrate. Synthesis proceeds by successive alternative additions of subunits S1, S2, S3, and the like, to form tag 212, and their corresponding library compound monomers A1, A2, A3, and the like, to form library compound 216. A “split and mix” technique is employed to generate diversity.
The subunits in a minimally cross-hybridizing set code for the monomer added in the library compound. Thus, a nine word set can unambiguously encode library compounds constructed from nine monomers. If some ambiguity is acceptable, then a single subunit may encode more than one monomer.
After synthesis is completed, the product is cleaved and deprotected (220) to form tagged library compound 225, which then undergoes selection 230, e.g. binding to a predetermined target 235, such as a protein. The subset of library compounds recovered from selection process 230 is then sorted (24) onto a solid phase support 245 via their tag moieties (there complementary subunits and nucleotides are shown in italics). After ligating oligonucleotide splint 242 to tag complement 250 to form restriction site 225, the conjugate is digested with the corresponding restriction endonuclease to cleave the library compound, a peptide in the example of FIG. 4 , from the oligonucleotide moiety. The sequence of the tag, and hence the identity of the library compound, is then determined by the preferred single base sequencing technique of the invention, described below.
Solid phase supports for use with the invention may have a wide variety of forms, including microparticles; beads, and membrance, slides, plates micromachined chops, and the like. Likewise, solid phase supports of the invention may comprise a wide variety of compositions, including glass, plastic, silicon, alkanethiolate-dervatized gold, cellulose, low cross-linked and high cross-linked polystyrene, silica gel, polyamide, and the like. Preferably, either a population of discrete particles are employed such that each has a uniform coating, or population, of complementary sequences of the same tag(and no other), of a single or a few supports are employed with spacially discrete regions each containing a uniform coating, or population, or complementary sequences to the same tag (and no other). In the latter embodiment, the area of the regions may vary according to particular applications; usually, the regions range in area from several μm2, e.g. 3-5, to several hundred μm2, e.g. 100-500. Preferably, such regions are specifically discrete so that signals generated by events, e.g. fluorescent emissions, at adjacent regions can be resolved by the detection system being employed. In some applications, it may be desirable to have regions with uniform coatings of more than one tag complement, e.g. for simultaneous sequence analysis, or for bringing separately tagged molecules into close proximity.
Tag complements may be used with the solid phase support that they are synthesized on, or they may be separately synthesized and attached to a solid phase support for use, e.g. as disclosed by Lund et al. Nucleic Adds Research, 16: 10861-10880 (1988); Albretsen et al, Anal. Biochem., 189: 40-50 (1990); Wolf et al, Nucleic Acids Research, 15: 2911-2926 (1987); or Ghosh et al, Nucleic Acids Research, 15: 5353-5372 (1987); Preferably, tag complements are synthesized on and used with the same solid phase support; which my comprise a variety of forms and include a variety of linking moieties. Such supports may comprise microparticles or arrays, or matrices, of regions where uniform populations of tag complements are synthesized. A wide variety of microparticle supports may be used with the invention, including microparticles made of controlled pore glass (CPG), highly cross-linked polstyrene., acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, disclosed in the following exemplary references: Meth. Enzymol, Section A pages 11-147, vol. 44 (Academic Press, New York, 1976); U.S. Pat. No. 4,678,814; 4,413,070; and 4,046;720; and Pon. Chapter 19, in Agrawal, editor, Methods in Molecular Biology, Vol. 20, (Humana Press, Totowa, N.J., 1993). Microparticle supports further include commercially available nucleoside-derivatized CPG and polystyrene beads (e.g. available from Applied Biosystems, Foster City, Calif.); derivatized magnetic beads; polystyrene grafted with polythylene glycol (e.g. TentaGel™, Rapp Polymere, Tubingen Germany); and the like. Selection of the support characteristics, such as material, porosity, size, shape, and the like, and the type of linking moiety employed depends on the conditions under which the tags are used. For example, in applications involving successive processing with enzymes, supports and linkers that minimize steric hinderance of the enzymes and that facilitate access to substrate are preferred. Exemplary linking moieties are disclosed in Pon et al, Biotechniques, 6; 768-775 (1988); Webb, U.S. Pat. No. 4,659,774; Barany et al, International patent application PCT/US91/06103; Brown et al, J. Chem. Soc. Commun., 1989: 891-893; Damha et al. Nucleic Acids Research, 18: 3813-3821 (1990); Beattie et al, Clinical Chemistry, 39: 719-722 (1993); Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); and the like.
As mentioned above, tag complements may also be synthesized on a single (or a few) solid phase support to form an array of regions uniformly coated with tag complements. That is, within each region in such an array the same tag complement is synthesized. Techniques for synthesizing such arrays are disclosed in McGall et al, International application PCT/US93/03767; Pease et al, Proc. Natl. Acad. Sci., 91: 5022-5026 (1994); Southern and Maskos, International application PCT/GB89/01114; Maskos and Southern (cited above); Southern et al, Genomics, 13: 1008-1017 (1992); and Maskos and Southern, Nucleic Acids Research, 21: 4663-4669 (1993).
Preferably, the invention is implemented with microparticles or beads uniformly coated with complements of the same tag sequence. Microparticle supports and methods of covalently or noncovalently linking oligonucleotides to their surfaces are well known, as exemplified by the following references: Beaucage and Iyer (cited above); Gait, editor, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the references cited above. Generally, the size and shape of a microparticle is not critical; however, microparticles in the size range of a few, e.g. 1-2, to several hundred, e.g. 200-1000 μm diameter are preferable, as they facilitate the construction and manipulation of large repertoires of oligonucleotide tags with minimal reagent and sample usage.
Preferably, commercially available controlled-pore glass (CPG) or polystyrene supports are employed as solid phase supports in the invention. Such supports come available with base-labile linkers and initial nucleosides attached, e.g. Applied Biosystems (Foster City, Calif.). Preferably, microparticles having pore sizes between 500 and 1000 angstroms are employed.
An important aspect of the invention is the sorting of populations of identical polynucleotides, e.g. from a cDNA library, and their attachment to microparticles or separate regions of a solid phase support such that each microparticle or region has only a single kind of polynucleotide. This latter condition can be essentially met by ligating a repertoire of tags to a population of polynucleotides followed by cloning and sampling of the ligated sequences. A repertoire of oligonucleotide tags can be ligated to a population of polynucleotides in a number of ways, such as through direct enzymatic ligation, amplification, e.g. via PCR, using primers containing the tag sequences, and the like. The initial ligating step produces a very large populations of tag-polynucleotide conjugates such that a single tag is generally attached to many different polynucleotides. However, by taking a sufficiently small sample of the conjugates, the probability of obtaining “doubles,” i.e. the same tag on two different polynucleotide, can be made negligible. (Note that it is also possible to obtain different tags with the same polynucleotide in a sample. This case is simply leads to a polynucleotide being processed, e.g. sequenced, twice). As explain more fully below, the probability of obtaining a double in a sample can be estimated by a Poisson distribution since the number of conjugates in a sample will be large, e.g. on the order of thousands or more, and the probability of selecting a particular tag will be small because the tag repertoire is large, e.g. on the order of tens of thousands or more. Generally, the larger the sample the greater the probability of obtaining a double. Thus, a design trade-off exists between selecting a large sample of tag-polynucleotide conjugates—which, for example, ensures adequate coverage of a target polynucleotide in a shotgun sequencing operation, and selecting a small sample which ensures that a minimal number of doubles will be present. In most embodiments, the presence of double merely adds an additional source of noise or, in the case of sequencing, a minor complication in scanning and signal processing, as microparticles giving multiple fluorescent signals can simply ignored. As used herein, the term “substantially all” in reference to attaching tags to molecules, especially polynucleotides, is meant to reflect the statistical nature of the sampling procedure employed to obtain a population of tag-molecule conjugates essentially free of doubles. The meaning of substantially all in terms of actual percentages of tag-molecule conjugates depends on how the tags are being employed. Preferably, for nucleic acid sequencing, substantially all means that at least eighty percent of the tags have unique polynucleotides attached. More preferably, it means that at least ninety percent of the tags have unique polynucleotides attached. Still more preferably, i. means that at least ninety-five percent of the tags have unique polynucleotides attached. And, more preferably, it means that at least ninety-nine percent of the tags have unique polynucleotides attached.
Preferably, when the population of polynucleotides is messenger RNA (mRNA), oligonucleotides tags are attached by reverse transcribing the mRNA with a set of primers containing complements of tag sequences. An exemplary set of such primers could have the following sequence:
5′-mRNA-[A]n-3′
[T]19GG[W,W,W,C]9ACCAGCTGATC-5′-biotin
where “[W,W,W,C]9” represents the sequence of an oligonucleotide tag of nine subunits of four nucleotides each and “[W,W,W,C]” represents the subunit sequences listed above, i.e. “W” represents T or A. The underlined sequences identify an optional restriction endonuclease site that can be used to release the polynucleotide from attachment to a solid phase support via the biotin, if one is employed. For the above primer, the complement attached to a microparticle could have the form (SEQ ID NO:4):
5′-[G,W,W,W]9TGG-linker-microparticle
5′-mRNA-[A]n-3′
[T]19GG[W,W,W,C]9ACCAGCTGATC-5′-biotin
where “[W,W,W,C]9” represents the sequence of an oligonucleotide tag of nine subunits of four nucleotides each and “[W,W,W,C]” represents the subunit sequences listed above, i.e. “W” represents T or A. The underlined sequences identify an optional restriction endonuclease site that can be used to release the polynucleotide from attachment to a solid phase support via the biotin, if one is employed. For the above primer, the complement attached to a microparticle could have the form (SEQ ID NO:4):
5′-[G,W,W,W]9TGG-linker-microparticle
After reverse transcription, the mRNA is removed, e.g. by RNase H digestion, and the second strand of the cDNA is synthesized using, for example, a primer of the following form (SEQ ID NO:6):
5′-NRRGATGYNN-3′
5′-NRRGATCYNNN-3′
where N is any one of A, T, G, or C; R is a purine-containing nucleotide, and Y is a pyrimidine-containing nucleotide. This particular primer creates a Bst Y1 restriction site in the resulting double stranded DNA which, together with the Sal I site, facilitates cloning into a vector with, for example, Bam HI and Xho I sites. After Bst Y1 and Sal I digestion, the exemplary conjugate would have the form (SEQ ID NO:19):
5′-RCGACCA[C,W,W,W,]9GG[T]19-cDNA-NNR
GGT[G,W,W,W]9CC[A]19-rDNA0NNNYCTAG-5′
Preferably, when the ligated-based method of sequencing is employed, the Bst YI and Sal I digested fragments are cloned into a Bam HI-/Xho I-digested vector having the following single-copy restriction sites (SEQ ID NO:1):
5′-GAGGATGCCTTTATGGATCCACTCGAGATCCCAATCCA-3′
FokI BAmHI XhoI
This adds the Fok I site which will allow initiation of the sequencing process discussed more fully below.
5′-NRRGATGYNN-3′
5′-NRRGATCYNNN-3′
where N is any one of A, T, G, or C; R is a purine-containing nucleotide, and Y is a pyrimidine-containing nucleotide. This particular primer creates a Bst Y1 restriction site in the resulting double stranded DNA which, together with the Sal I site, facilitates cloning into a vector with, for example, Bam HI and Xho I sites. After Bst Y1 and Sal I digestion, the exemplary conjugate would have the form (SEQ ID NO:19):
5′-RCGACCA[C,W,W,W,]9GG[T]19-cDNA-NNR
GGT[G,W,W,W]9CC[A]19-rDNA0NNNYCTAG-5′
Preferably, when the ligated-based method of sequencing is employed, the Bst YI and Sal I digested fragments are cloned into a Bam HI-/Xho I-digested vector having the following single-copy restriction sites (SEQ ID NO:1):
5′-GAGGATGCCTTTATGGATCCACTCGAGATCCCAATCCA-3′
FokI BAmHI XhoI
This adds the Fok I site which will allow initiation of the sequencing process discussed more fully below.
A general method for exposing the single stranded tag after amplification involves digesting a target polynucleotide-containing conjugate with the 5′→3′ 3′→5′ exonuclease activity of T4 DAN polymerase, or a like enzyme. When used in the presence of a single nucleoside triphosphate, such a polymerase will cleave nucleotides from 3′ recessed ends present on the non-template strand of a double stranded fragment until a complement of the single nucleoside triphosphate is reached on the template strand. When such a nucleotide is reached the 5′→3′ 3′→5′ digestion effectively ceases, as the polymerase's extension activity adds nucleotides at a higher rate than the excision activity removes nucleotides. Consequently, tags constructed with three nucleotides are readily prepared for loading onto solid phase supports.
The technique may also be used to preferentially methylate interior Fok I sites of a target polynucleotide while leaving a single Folk I site at the terminus of the polynucleotide unmethylated. First, the terminal Folk I site is rendered single stranded using a polymerase with deoxycytidine triphosphate. The double stranded portion of the fragment is then methylated, after which the single stranded terminus is filled in with a DNA polymerase in the presence of all four nucleoside triphosphates, thereby regenerating the Folk I site.
After the oligonucleotide tags are prepared for specific hybridization, e.g. by rendering them single stranded as described above, the polynucleotides are mixed with microparticles containing the complementary sequences of the tags under conditions that favor the formation of perfectly matched duplexes between the tags and their complements. There is extensive guidance in the literature for creating these conditions. Exemplary references providing such guidance include Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 277-259 (1991); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory, New York, 1989); and the like. Preferably, the hybridization conditions are sufficiently stringent so that only perfectly matched sequences form stable duplexes. Under such conditions the polynucleotides specifically hybridized through their tags are ligated to the complementary sequences attached to the microparticles. Finally, the microparticles are washed to remove unligated polynucleotides.
When CPG microparticles conventionally employed as synthesis supports are used, the density of tag complements on the microparticle surface is typically greater than that necessary for some sequencing operations. That is, in sequencing approaches that require successive treatment of the attached polynucleotides with a variety of enzymes, densely spaced polynucleotides may tend to inhibit access of the relatively bulky enzymes to the polynucleotides. In such cases, the polynucleotides are preferably mixed with the microparticles so that tag complements are present in significant excess, e.g. from 10:1 to 100:1, or greater, over the polynucleotides. This ensumes that the density of polynucleotides on the microparticle surface will not be so high as to inhibit enzyme access. Preferably, the average interpolynucleotide spacing on the microparticle surface is on the order of 30-100 nm. Guidance in selecting ratios for standard CGP supports and Ballotini beads (a type of solid glass support) is found in Maskos and Southern., Nucleic Acids Research, 20: 1679-1684 (1992). Preferably, for sequencing applications, standard CPG beads of diameter in the range of 20-50 μm are loaded with about 105 polynucleotides.
The above method may be used to fingerprint mRNA populations when coupled with the parallel sequencing methodology described below. Partial sequence information is obtained simultaneously from a large sample, e.g. ten to a hundred thousand, of cDNAs attched to separate microparticles as described in the above method. The frequency distribution of partial sequences can identify mRNA populations from different cell or tissue types, as well as from diseased tissues, such as cancers. Such mRNA fingerprints are useful in monitoring and diagnosing disease states.
The present invention can be employed with conventional methods of DNA sequencing, e.g. as disclosed by Hultman et al, Nucleic Acids Research, 17: 4937-4946 (1989). However, for parallel, or simultaneous, sequencing of multiple polynucleotides, a DNA sequencing methodology is preferred that requires neither electrophoretic separation of closely sized DNA fragments nor analysis of cleaved nucleotides by a separate analytical procedure, as in peptide sequencing. Preferably, the methodology permits the stepwise identification of nucleotides, usually one at a time, in a sequence through successive cycles of treatment and detection. Such methodologies are referred to herein as “single base” sequencing methods. Single base approaches are disclosed in the following references: Cheeseman, U.S. Pat. No. 5,302,509; Tsien et al, International application WO 91/06678; Rosenthal et al, International application WO 93/21340; Canard et al, Gene, 148: 1-6 (1994); and Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994).
A “single base” method of DNA sequencing which is suitable for use with the present invention and which requires no electrophoretic separation of DNA fragments is described in co-pending U.S. patent application Ser. No. 08/280,441 filed 25 Jul. 1994, which application is incorporated by reference. The method comprises the following steps: (a) ligating a probe to an end of the polynucleotide having a protruding strand to form a ligated complex, the probe having a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site; (b) removing unligated probe from the ligated complex; (c) identifying one or more nucleotides in the protruding strand of the polynucleotide by the identity of the ligated probe; (d) cleaving the ligated complex with a nuclease; and (e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined. As is described more fully below, identifying the one or more nucleotides can be carried out either before or after cleavage of the ligated complex from the target polynucleotide. Preferably, whenever natural protein endonuclease are employed, the method further includes a step of methylating the target polynucleotide at the start of a sequencing operation.
An important feature of the method is the probe ligated to the target polynucleotide. A preferred form of the probes is illustrated in FIG. 1a. Generally, the probes are double stranded DNA with a protruding strand at one end 10. The probes contain at least one nucleus recognition site 12 and a spacer region 14 between the recognition site and the protruding end 10. Preferably, probes also include a label 16, which in this particular embodiment is illustrated at the end opposite of the protruding strand. The probes may be labeled by a variety of means and at a variety of locations, the only restriction being that the labeling means selected does not interfere with the ligation step or with the recognition of the probe by the nucleus.
It is not critical whether protruding strand 10 of the probe is a 5′ or 3′ end. However, it is important that the protruding strands of the target polynucleotide and probes be capable of forming perfectly matched duplexes to allow for specific ligation. If the protruding strands of the target polynucleotide and probe are different lengths the resulting gap can be filled in by a polymerase prior to ligation, e.g. as in “gap LCR” disclosed in Backman et al, European patent application 91100959.5. Preferably, the number of nucleotides in the respective protruding strands are the same so that both strands of the probe and target polynucleotide are capable of being ligated without a filling step. Preferably, the protruding strand of the probe is from 2 to 6 nucleotides long. As indicated below, the greater the length of the protruding strand, the greater the complexity of the probe mixture that is applied to the target polynucleotide during each ligation and cleavage cycle.
The complementary strands of the probes are conveniently synthesized on an automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer, using standard chemistries. After synthesis, the complementary strands are combined to form a double stranded probe. Generally, the protruding strand of a probe is synthesized as a mixture, so that every possible sequence is represented in the protruding portion. For example, if the protruding portion consisted of four nucleotides, in one embodiment four mixtures are prepared as follows:
-
- X1X2 . . . XlNNNA,
- X1X2 . . . XiNNNC,
- X1X2 . . . XrNNNG, and
- X1X2 . . . XtNNNT
where the “NNNs” represent every possible 3-mer and the “Xs” represent the duplex forming portion of the strand. Thus, each of the four probes listed above contains 43 or 64 distinct sequences; or, in other words, each of the four probes has a degeneracy of 64. For example, X1X2 . . . XiNNNA contains the following sequences:
X1X2 | . . . | XiAAAA | ||
X1X2 | . . . | XiAACA | ||
X1X2 | . . . | XiAAGA | ||
X1X2 | . . . | XiAATA | ||
X1X2 | . . . | XiACAA | ||
. | ||||
. | ||||
. | ||||
X1X2 | . . . | XiTGTA | ||
X1X2 | . . . | XiTTAA | ||
X1X2 | . . . | XiTTCA | ||
X1X2 | . . . | XiTTGA | ||
X1X2 | . . . | XiTTTA | ||
Such mixtures are readily synthesized using well known techniques, e.g. as disclosed in Telenius et al (cited above). Generally, these techniques simply call for the application of mixtures of the activated monomers to the growing oligonucleotide during the coupling steps where one desires to introduce the degeneracy. In some embodiments it may be desirable to reduce the degeneracy of the probes. This can be accomplished using degeneracy reducing analogs, such as deoxyinosine, 2-aminopurine, or the like, e.g. as taught in Kong Thoo Lin et al, Nucleic Acids Research, 20: 5149-5152, or by U.S. Pat. No. 5,002,867.
Preferably, for oligonucleotides with phosphodiester linkages, the duplex forming region of a probe is between about 12 to about 30 basepairs in length; more preferably, its length is between about 15 to about 25 basepairs.
When conventional ligases are employed in the invention, as described more fully below, the 5′ end of the probe may be phosphorylated in some embodiments. A 5′ monophosphate can be attached to a second oligonucleotide either chemically or enzymatically with a kinase, e.g. Sambrook et al (cited above). Chemical phosphorylation is described by Horn and Urdea, Tetrahedron Lett, 27: 4705 (1986), and reagents for carrying out the disclosed protocols are commercially available, e.g. 5′ Phosphate-ON(TM) from Clontech Laboratories (Palo Alto, Calif.). Thus, in some embodiments, probes may have the form:
5′-X1X2 | . . . | XiTTGA |
Y1Y2 | . . . | YiP |
where the Y's are the complementary nucleotides of the X's and “p” is a monophosphate group.
The above probes can be labeled in a variety of ways, including the direct or indirect attachment of radioactive moieties, fluorescent moieties, colorimetric moieties, chemiluminescene markers, and the like. Many comprehensive reviews of methodologies for labeling DNA and constructing DNA probes provide guidance applicable to constructing probes of the present invention. Such reviews include Kricka, editor, Nonisotopic DNA Probe Techniques (Academic Press, San Diego, 1992); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, 1992); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); and Eckstein, editor, Oligonucleotides and Analogues; A Practical Approach IRL Press, Oxford, 1991,(Kessler, editor, Nonradioactive Labeling and Detection of Biomolecules (Springer-Verlag, Berlin, 1992); Wetmur (cited above); and the like.
Preferably, the probes are labeled with one or more fluorescent dyes, e.g. as disclosed by Menchen et al, U.S. Pat. No. 5,188,934; Begot et al International application PCT/US90/05565.
In accordance with the method, a probe is ligated to an end of a target polynucleotide to form a ligated complex in each cycle of ligation and cleavage. The ligated complex is the double stranded structure formed after the protruding strands of the target polynucleotide and probe anneal and at least one pair of the identically oriented strands of the probe and target are ligated, i.e. are caused to be covalently linked to one another. Ligation can be accomplished either enzymatically or chemically. Chemical ligation methods are well known in the art, e.g. Ferris et al, Nucleosides & Nucleotides, 8: 407-414 (1989). Shabarova et al, Nucleis Acids Research, 19: 4247-4251 (1991); and the like. Preferably, however, ligation is carried out enzymatically using a ligase in a standard protocol. Many ligases are known and are suitable for use in the invention, e.g. Lehman, Science, 186: 790-797 (1974); Engler et al, DNA Ligases, pages 3-30 in Boyer, editor, The Enzymes, Vol. 15B (Academic Press, New York, 1982); and the like. Preferred ligases include T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taq ligase, Pfu ligase, and Tth ligase. Protocols for their use are well know, e.g. Sambrook et al (cited above); Barneym PCR Methods and Applications, 1: 5-16 (1991); Marsh et al, Strategies, 5: 73-76 (1992); and the like. Generally, ligases require that a 5′ phosphate group be present for ligation to the 3′ hydroxyl of an abutting strand. This is conveniently provided for at least one strand of the target polynucleotide by selecting a nuclease which leaves a 5′ phosphate, e.g. as Fok I.
In an embodiment of the sequencing method employing unphosphorylated probes, the step of ligating includes (i) ligating the probe to the target polynucleotide with ligase so that a ligated complex is formed having a nick on one strand, (ii) phosphorylating the 5′ hydroxyl at the nick with a kinase using conventional protocols, e.g. Sambrook et al (cited above), and (iii) ligating again to covalently join the strands at the nick, i.e. to remove the nick.
An objective of the invention is to sort identical molecules, particularly polynucleotides, onto the surfaces of microparticles by the specific hybridization of tags and their complements. Once such sorting has taken place, the presence of the molecules or operations performed on the can e detected in a number of ways depending on the nature of the tagged molecule, whether microparticles are detected separately or in “batches,” whether repeated measurements are desired, and the like. Typically, the sorted molecules are exposed to ligands for binding, e.g. in drug development, or are subjected chemical of enzymatic processes, e.g. in polynucleotide sequencing. In both of these uses it is often desirable to simultaneously observe signals corresponding to such events or processes on large numbers of microparticles. Microparticles carrying sorted molecules (referred to herein as “loaded” microparticles) lend themselves to such large scale parallel operations, e.g. as demonstrated by Lam et al (cited above).
Preferably, whenever light-generating signals, e.g. chemiluminescent, fluorescent, or the like, are employed to detect events or processes, loaded microparticles are spread on a planar substrate, e.g. a glass slide, for examination with a scanning system, such as described in International patent applications PCT/US91/09217 and PCT/NL90/00081. The scanning system should be able to reproducibly scan the substrate and to define the positions of each microparticle in a predetermined region by way of a coordinate system. In polynucleotide sequencing applications, it is important that the positional identification of microparticles be repeatable in successive scan step.
Such scanning systems may be constructed from commercially available components, e.g. x-y translation table controlled by a digital computer used with a detection system comprising one or more photomultiplier tubes, or alternatively, a CCD array, and appropriate optics, e.g. for exciting, collecting, and sorting fluorescent signals. In some embodiments a confocil optical system may be desirable. An exemplary scanning system suitable for use in four-color sequencing is illustrated diagrammatically in FIG. 5. Substrate 300, e.g. a microscope slide with fixed microparticles, is placed on x-y translation table 302, which is connected to and controlled by an appropriately programmed digital computer 304 which may be any of a variety of commercially available personal computers, e.g. 486-based machines or PowerPC model 7100 or 8100 available from Apple Computer (Cupertino, Calif.). Computer software for table translation and data collection functions can be provided by commercially available laboratory software, such as Lab Windows, available from National Instruments.
The stability and reproducibility of the positional location in scanning will determine, to a large extent, the resolution for separating closely spaced microparticles. Preferably, the scanning systems should be capable of resolving closely spaced microparticles, e.g. seperated by a particle diameter. Thus, for most applications, e.g. using CPG microparticles, the scanning system should at least have the capability of resolving objects on the order of 10-100 μm. Even higher resolution may be desirable in some embodiments, but with increase resolution, the time required to fully scan a substrate will increase; thus, in some embodiments a compromise may have to be made between speed and resolution. Increases in scanning time can be achieved by a system which only scans positions where microparticles are known to be located, e.g. from an initial full scan. Preferably, microparticle size and scanning system resolution are selected to permit resolution of fluorescently labeled microparticles randomly disposed on a plane at a density between about ten thousand to one hundred thousand microparticles per cm2.
In sequencing applications, loaded microparticles can be fixed to the surface of a substrate in variety of ways. The fixation should be strong enough to allow the microparticles to undergo successive cycles of reagent exposure and washing without significant loss. When the substrate is glass, its surface may be derivatized with an alkylamino linker using commercially available reagents, e.g. Pierce Chemical, which in turn may be cross-linked to avidin, again using conventional chemistries, to form an avidinated surface, Biotin moieties can be introduced to the loaded microparticles in a number of ways. For example, a fraction, e.g. 10-15 percent, of the cloning vectors used to attach tags to polynucleotides are engineered to contain a unique restriction site (providing sticky ends on digestion) immediately adjacent to the polynucleotide insert at an end of the polynucleotide opposite of the tag. The site is excised with the polynucleotide and tag for loading onto microparticles. After loading, about 10-15 percent of the loaded polynucleotides will possess the unique restriction site distal from the microparticle surface. After digestion with the associated restriction endonuclease, an appropriate double stranded adapter containing a biotin moiety is ligated to the sticky end. The resulting microparticles are then spread on the avidinated glass surface where they become fixed via the biotin-avidin linkages.
Alternatively and preferably when sequencing by ligation is employed, in the initial ligation step a mixture of probes is applied to the loaded microparticle: a fraction of the probes contain a type IIs restriction recognition site, as required by the sequencing method, and a fraction of the probes have no such recognition site, but instead contain a biotin moiety at its non-ligating end. Preferably, the mixture comprises about 10-15 percent of the biotylated probe.
The tagging system of the invention can be used with single base sequencing methods to sequence polynucleotides up to several kilobases in length. The tagging system permits many thousands of fragments of a target polynucleotide to be sorted onto one or more solid phase supports and sequenced simultaneously. In accordance with a preferred implementation of tha method, a portion of each sorted fragment is sequenced in a stepwise fashion on each of the many thousands of loaded microparticles which are fixed to a common substrate-such as a microscope slide-associated with a scanning system, such as that described above. The size of the portion of the fragments sequenced depends of several factors, such as the number of fragments generated and sorted, the length of the target polynucleotide, the speed and accuracy of the single base method employed, the number of microparticles and/or discrete regions that may be monitored simultaneously; and the like. Preferably, from 12-50 bases are identified at each microparticle or region; and more preferably, 18-30 bases are identified at each microparticle of region. With this information, the sequence of the target polynucleotide is determined by collating the 12-50 base fragments via their overlapping regions, e.g. as described in U.S. Pat. No. 5,002,867. The following references provide additional guidance in determining the portion of the fragments that must be sequenced for successful reconstruction of a target polynucleotide of a given length: Drmanac et al, Genomics, 4: 114-128 (1989); Bains, DNA Sequencing and Mapping, 4: 143-150 (1993); Bains, Genomics, 11: 294-301 (1991); Drmanac et al, J. Biomolecular Structure and Dynamics, 8: 1085-1102 (1991); and Pevzner, J. Biomolecular Structure and Dynamics, 7: 63-73 (1989). Preferably, the length of the target polynucleotide is between 1 kilobase and 50 kilobases. More preferably, the length is between 10 kilobases and 40 kilobases.
Fragments may be generated from a target polynucleotide in a variety of ways, including so-called “directed” approaches where one attempts to generate sets of fragments covering the target polynucleotide with minimal overlap, and so-called “shotgun” approaches where randomly overlapping fragments are generated. Preferably, “shotgun” approaches to fragment generation are employed because of their simplicity and inherent redundancy. For example, randomly overlapping fragments that cover a target polynucleotide are generated in the following conventional “shotgun” sequencing protocol, e.g. as disclosed in Sambrook et al (cited above). As used herein, “cover” in this context means that every portion of the target polynucleotide sequence is represented in each size range, e.g. all fragments between 100 and 200 basepairs in length, of the generated fragments. Briefly, starting with a target polynucleotide as an insert in a n appropriate cloning vector, e.g. A phage, the vector is expanded, purified and digested with the appropriate restriction enzymes to yield about 10-15 μg of purified insert. Typically, the protocol results in about 500-1000 subclones per microgram of starting DNA. The insert is seperated from the vector fragments by preparative gel electrophoresis, removed from the gel by conventional methods, and resuspended in a standard buffer, such as TE (Tris-EDTA). The restriction enzymes selected to excise the insert from the vector preferably leave compatible sticky ends on the insert, so that the insert can be self-ligated in preparation for generating randomly overlapping fragments, As explained in Sanbrook et al (cited above), the circularized DNA yields a better random distribution of fragments than linear DNA in the fragmentation methods employed below. After self-ligating the inset, e.g. with T4 ligase using conventional protocols, the purified ligated insert is fragmented by a standard protocol, e.g. sonication or DNAase I digestion in the presence of Mn++. After fragmentation the ends of the fragments are repair, e.g. as described in sambrook et al (cited above), and the repaired fragments are seperated by size using gel electrophoresis. Fragments in the 300-500 basepair range are selected and eluted from the gel by conventional means, and ligated into a tag-carrying vector as described above to form a library of tag-fragment conjugates.
As described below, a sample containing several thousand tag-fragment conjugates are taken from the library and expanded after which the tag-fragment inserts are excised from the vector and prepared for specific hybridization to the tag complements on microparticles, as described above. Depending of the size of the target polynucleotide, multiple samples may be taken from the tag-fragment library and separately expanded, loaded onto microparticles and sequenced. The number of doubles selected will depend on the fraction of the tag repertoire represented in a sample. (The probability of obtaining triples-three different polynucleotides with the same tag-or above can safely be ignored). As mentioned above, the probability of doubles in a sample can be estimated from the Poisson distribution p(double)=m2e−m/2, where m is the fraction of the tag repertoire in the sample. Table IV below lists probabilities of obtaining doubles in a sample for giving tag size, sample size, and repertoire diversity
TABLE IV | ||||
Number of | ||||
words in | Fraction of | |||
tag from 8 | Size of tag | repertoire | Probability of | |
word set | repertoire | Size of sample | sampled | double |
7 | 2.1 × 106 | 3000 | 1.43 × 10−3 | 10−6 |
8 | 1.68 × 107 | 3 × 104 | 1.78 × 10−3 | 1.6 × 10−6 |
3000 | 1.78 × 10−4 | 1.6 × 10−8 | ||
9 | 1.34 × 108 | 3 × 105 | 2.24 × 10−3 | 2.5 × 10−6 |
3 × 104 | 2.24 × 10−4 | 2.5 × 10−8 | ||
10 | 1.07 × 109 | 3 × 106 | 2.8 × 10−3 | 3.9 × 10−6 |
3 × 105 | 2.8 × 10−4 | 3.9 × 10−8 | ||
In any case, the loaded microparticles are then dispersed and fixed onto a glass microscope slide, preferably via an avidin-biotin coupling. Preferably, at least 15-20 nucleotides of each of the random fragments are simultaneously sequenced with a single base method. The sequence of the target polynucleotide is then reconstructed by collating the partial sequences of the random fragments by way of their overlapping portions, using algorithms similar to those used for assembling contigs, or as developed for sequencing by hybridization, disclosed in the above references.
The invention includes kits for carrying out the various embodiments of the invention. Preferably, kits of the invention include a repertoire of tag complements attached to a solid phase support. Additionally, kits of the invention may include the corresponding repertoire of tags, e.g. as primers for amplifying polynucleotides to be sorted or as elements of cloning vectors which can also be used to amplify the polynucleotides to be sorted. Preferably, the repertoire of tag complements are attached to microparticles. Kits may also contain appropriate buffers for enzymatic processing, detector chemistries, e.g. fluorescent or chemiluscent tags, and the like, instructions for use, processing enzymes, such al ligases, polymerases, transferases, and so on. In an important embodiment for sequencing kits may also include substrates, such as a avidinated microscope slides, for fixing loaded nicroparticles for processing.
A mixture of three target polynucleotide-tag conjugates are obtained as follows: First, the following six oligonucleotides are synthesized and combined pairwise to form tag 1, tag 2, and tag 3 (SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:17):
5′-pTCGACC(w1)(w2)(w3)(w4)(w5)(w6)(w7)(w8)(w1)A | |
GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5 | |
Tag | |
1 | |
5′-pTCGACC(w6)(w7)(w8)(w1)(w2)(w6)(w4)(w2)(w1)A | |
GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′ | |
Tag 2 | |
5′-pTCGACC(w3)(w2)(w1)(w1)(w5)(w8)(w8)(w4)(w4)A | |
GG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5 | |
Tag | |
3 |
where “p” indicates a monophosphate, the wi's represent the subunits define in Table I, and the terms “(**)” represent their respective complements. ApUC19 is digested with Sal I and Hind III, the large fragment is purified, and separately ligated with
CPG microparticles (37-74 mm, particle size, 500 angstrom pore size, Pierce Chemical) are derivatized with the linker disclosed by Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992). After separating into three aliquots, the complements of tags 1, 2, and 3 are synthesized on the microparticles using a conventional automated DNA synthesizer, e.g. a model 392 DNA synthesizer (Applied Biosystems, Foster City, Calif.). Approximately 1 mg of each of the differently derivatized microparticles are placed in separate vessels.
The T4 DNA polymerase-treated fragments excised from pUC19-1, -2, and -3 are resuspended in 50 μL of the manufacturer's recommended buffer for Taq DNA ligase (New England Biolabs) The mixture is then equally divided among the three vessels containing the 1 mg each of derivatized CPG microparticles. 5 units of Taq DNA ligase is added to each vessel, after which they are incubated at 55° C. for 15 minutes. The reaction is stopped by placing on ice and the microparticles are washed several times by repeated centrifugation and resuspension in TE. Finally, the microparticles are resuspended in Nde I reaction buffer (New England Biolabs) where the attached polynucleotides are digested. After separation from the microparticles the polynucleotide fragments released by Nde I digestion are fluorescently labeled by incubating with Sequenase DNA polymerase and fluorescent labeled thymidine triphosphate (Applied Biosystems, Foster City, Calif.). The fragments are thin separately analyzed on a nondenaturing polyacrylamide gel using an Applied Biosystems model 373 DNA sequencer.
A repertoire of 36-mer tags consisting of nine 4-nucleotide subunits selected from Table I is prepared by separately synthesizing tags and tag complements by a split and mix approach, as described above. The repertoire is synthesized so as to permit ligation into a Sma I/Hind III digested M13mp19. Thus, as in Example I, one set of oligonucleotides begins with the addition of A followed by nine rounds of split and mix synthesis wherein the oligonucleotide is extended subunit-wise by 3′-phosphoramidite derivatized 4-mers corresponding to the subunits of Table I. The synthesis of then completed with the nucleotide-by-nucleotide addition of one half of the Sma I recognition site (GGG), two C's, and a 5′-monophosphate, e.g. via the Phosphate-ON reagent available from Clontech Laboratories (Palo Alto, Calif.). The other set of oligonucleotides begins with the addition of three C's (portion of the Sma I recognition site) and two G's, followed by nine rounds of split and mix synthesis wherein the oligonucleotide is extended by 3′-phosphoramidite derivatized 4-mers corresponding to the complements of the subunits of Table I. Synthesis is completed by the nucleotide-by-nucleotide addition of the Hind III recognition site and a 5′-monophosphate. After separation from the synthesis supports the oligonucleotides are mixed under conditions that permit formation of the following duplexes (SEQ ID NO:18):
5′-pGGGCC(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)A
CCCGG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′
The mixture of duplexes is then ligated into a Sma I/Hind III-digested M13mp19. A repertoire of tag complements are synthesized on CPG microparticles as described above.
5′-pGGGCC(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)(wi)A
CCCGG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5′
The mixture of duplexes is then ligated into a Sma I/Hind III-digested M13mp19. A repertoire of tag complements are synthesized on CPG microparticles as described above.
Next the following adapter (SEQ ID NO:2 and SEQ ID NO:7) is prepared which contains a Fok I site and portion of Eco RI and Sma I sites:
5′-pAATTCGGATGATGCATGCATCGACCC | |
GCCTACTACGTACGTAGCTGGGp-5′ | |
Eco RI Fok I Sma I |
The adapter is ligated into the Eco RI/Sma I digested M13 described above.
Separately, SV40 DNA is fragmented by sonication following the protocol set forth in Sambrook et al (cited above). The resulting fragments are repaired using standard protocols and separated by size. Fragments in the range of 300-500 basepairs are selected and ligated into the Sma I digested M13 described above to form a library of fragment-tag conjugates, which is then amplified. A sample containing several thousand different fragment-tag conjugates is taken from the library, further amplified, and the fragment-tag inserts are excised by digesting with Eco RI and Hind III. The excised fragment-tag conjugates are treated with T4 DNA polymerase in the presence of deoxycytidine triphosphate, as described in Example I, to expose the oligonucleotide tags for specific hybridization to the CPG microparticles.
After hybridization and ligation, as described in Example I, the loaded microparticles are treated with Fok I to produce a 4-nucleotide protruding strand of a predetermined sequence. A 10:1 mixture (probe 1:probe 2) of the following probes (SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 ) are ligated to the polynucleotides on microparticles.
| FAM- | ATCGGATGAC | ||
TAGCCTACTGAGCT | ||||
Probe 2 | biotin- | ATCGGATGAC | ||
TAGCCTACTGAGCT | ||||
FAM represents a fluorescein dye attached to the 5′-hydroxyl of the top strand of Probe I through an aminophosphate linker available from Applied Biosystems (Aminolinker). The biotin may also be attached through an Aminolinker moiety and optionally may be further extended via polyethylene oxide linkers, e.g. Jaschke et al (cited above).
The loaded microparticles are then deposited on the surface of an avidinated glass slide to which and from which reagents and wash solutions can be delivered and removed. The avidinated slide with the attached microparticles is examined with a scanning fluorescent microscope (e.g. Zeiss Axiskop equipped with a Newport Model PM500-C motion controller, a Spectra-Physics Model 2020 argon ion laster producing a 488 nm excitation beam, and a 520 nm long-pass emission filter, or like apparatus). The excitation beam and fluorescent emissions are delivered and collected, respectively, through the same objective lens. The excitation beam and collected fluorescence are separated by a dichroic mirror which directs the collected fluorescence through a series of bandpass filters and to photon-counting devices corresponding to the fluorophors being monitored, e.g. comprising Hamamatsu model 9403-02 photomutlipliers, a Stanford Research Systems model SR445 amplifier and model SR430 multichannel scaler, and digital computer, e.g. a 486-based computer. The computer generates a two dimensional map of the slide which registers the positions of the microparticles.
After cleavage with Fok I to remove the initial probe, the polynucleotides on the attached microparticles undergo 20 cycles of probe ligation, washing detection, cleavage, and washing, in accordance with the preferred single base sequencing methodology described below. Within each detection step, the scanning system records the fluorescent emission corresponding to the base identified at each microparticle. Reactions and washes below are generally carded out with manufacturer's (New England Biolabs') recommended buffers for the enzymes employed, unless otherwise indicated. Standard buffers are also described in Sambrook et al (cited above).
The following four sets of mixed probes (SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15) are provided for addition to the target polynucleotides:
TAMRA- | ATCGGATGACATCAAC | ||
TAGCCTACTGTAGTTGANNN | |||
FAM- | ATCGGATGACATCAAC | ||
TAGCCTACTGTAGTTGCNNN | |||
ROX- | ATCGGATGACATCAAC | ||
TACCCTACTGTAGTTGGNNN | |||
JOE- | ATCGGATGACATCAAC | ||
TACCCTACTGTAGTTGTNNN | |||
where TAMRA, FAM, ROX and JOE are spectrally resolvable fluorescent lables attached by way of Aminolinker II (all being available from Applied Biosystems, Inc., Foster City, Calif.); the bold faced nucleotides are the recognition site for Fok I enidonuclease, and “N” represents any one of the four nucleotides, A, C, G, T. TAMRA (tetramethylrhodamine), FAM (fluorescein), ROX (rhodamine X), and JOE (2′,7′-dimethoxy-4′,5′-dichlorofluorescein) and their attachment to oligonucleotides is also described in Fung et al, U.S. Pat. No. 4;855,225.
The above probes are incubated in approximately 5 molar excess of the target polynucleotide ends as follows: the probes are incubated for 60 minutes at 16° C. with 200 traits of T4 DNA ligase and the anchored target polynculeotide in T4 DNA ligase buffer, after washing, the target polynucleotide is then incubated with 100 units T4 polynucleotide kinase in the manufacturer's, recommended buffer for 30 minutes at 37° C., washed, and again incubated for 30 minutes at 16° C. with 200 units of T4 DNA ligase and the anchored target polynucleotide in T4 DNA ligase buffer. Washing is accomplished by successively flowing volumes of wash buffer over the slide, e.g. TE, disclosed in Sambrook et al (cited above). After the cycle of ligation-phosphorylation-ligation and a final washing, the attached microparticles are scanned for the presence of fluorescent label, the positions and characteristics of which are recorded by the scanning system. The labeled target polynucleotide, i.e. The ligated complex, is then incubated with 10 units of Fok I in the manufacturer's recommended buffer for 30 minutes at 37° C., followed by washing in TE. As a result the target polynucleotide is shortened by one nucleotide on each strand and is ready for the next cycle of ligation and cleavage. The process is continued until twenty nucleotides are identified.
APPENDIX I |
Exemplary computer program for generating |
minimally cross hybridizing sets |
Program minxh |
c |
c |
c |
integer*2 sub1 (6) ,mset1(1000,6) ,mset2(1000,6) | |
dimension nbase(6) | |
c | |
c | |
write(*,*)‘ENTER SUBUNIT LENGTH’ | |
read(*,100)nsub | |
100 | format(i1) |
open(1,file=‘sub4.dat’,form=‘formatted’,status=‘new’) | |
c | |
c | |
nset=0 | |
do 7000 m1=1,3 |
do 7000 m2=1,3 |
do 7000 m3=1,3 |
do 7000 m4=1,3 |
sub1(1)=m1 | |
sub1(2)=m2 | |
sub1(3)=m3 | |
sub1(4)=m4 |
c | |
c | |
ndiff=3 | |
c | |
c |
c | Generate set of subunits differing from |
c | sub1 by at least ndiff nucleotides. |
c | Save in mset1. |
c | |
c |
jj=1 | |
do 900 J=1,nsub |
900 | mset1(1,j)=sub1(j) |
c | |
c |
do 1000 k1=1,3 |
do 1000 k2=1,3 |
do 1000 k3=1,3 |
do 1000 k4=1,3 |
c | |
c | |
nbase(1)=k1 | |
nbase(2)=k2 | |
nbase(3)=k3 | |
nbase(4)=k4 | |
c |
n=0 |
do 1200 j=1,nsub |
if(sub1(j) .eq.1 .and. nbase(j) .ne.1 .or. |
1 | sub1(j) .eq.2 .and. nbase(j) .ne.2 .or. | |
3 | sub1(j) .eq.3 .and. nbase(j) .ne.3) then | |
n=n+1 | ||
endif |
1200 | continue |
c | |
c |
if(n.ge.ndiff) then |
c | |
c | |
c | If number of mismatches |
c | is greater than or equal |
c | to ndiff then record |
c | subunit in matrix mset |
c |
jj=jj+1 | |
do 1100 i=1,nsub |
1100 | mset1(jj,i)=nbase(i) |
endif |
c | |
c | |
1000 | continue |
c | |
c |
do 1325 j2=1,nsub | |
mset2(1,j2)=mset1(1,j2) | |
1325 | mset2(2,j2)=mset1(2,j2) |
c | |
c |
c | Compare subunit 2 from |
c | mset1 with each successive |
c | subunit in mset1, i.e. 3, |
c | 4,5, . . . etc. Save those |
c | with mismatches .ge. ndiff |
c | in matrix mset2 starting at |
c | position 2. |
c | Next transfer contents |
c | of mset2 into mset1 and |
c | start |
c | comparisons again this time |
c | starting with |
c | Continue until all subunits |
c | undergo the comparisons. |
c | |
c |
npass=0 | |
c | |
c | |
1700 | continue |
kk=npass+2 | |
npass=npass+1 | |
c | |
c |
do 1500 m=npass+2,jj |
n=0 | |
do 1600 j=1,nsub |
if(mset1(npass+1,j) .eq.1.and.mset1(m,j) .ne.1.or. |
2 | mset1(npass+1,j) .eq.2.and.mset1(m,j) .ne.2.or. | |
2 | mset1(npass+1,j) .eq.3.and.mset1(m,j) .ne.3) then | |
n=n+1 | ||
endif |
1600 | continue |
if(n.ge.ndiff) then |
kk=kk+1 | |
do 1625 i=1,nsub |
1625 | nset2(kk,i)=mset1(m,i) |
endif | |
1500 | continue |
c | |
c |
c | kk is the number of subunits |
c | stored in mset2 |
c | |
c | |
c | Transfer contents of mset2 |
c | into mset1 for next pass. |
c | |
c |
do 2000 k=1,kk |
do 2000 m=1,nsub |
2000 | mset1(k, m)=mset2 (k, m) |
if(kk.1t.jj) then |
jj=kk | |
goto 1700 | |
endif | |
c | |
c | |
nset=nset+1 |
write (1,7009) |
7009 | format(/) |
do 7008 k=1,kk |
7008 | write(1,7010)(mset1(k,m),m=1,nsub) |
7010 | format(4i1) |
write(*,*) | |
write(*,120) kk, |
|
120 | format(1x,‘Subunits in set=’,i5,2x,‘Set No=’,i5) |
7000 | continue |
close(1) | |
c | |
c | |
end | |
Claims (7)
1. A composition of matter comprising:
a solid phase support having one or more spacially discrete regions; and
a uniform population of substantially identical oligonucleotide tag complements covalently attached to the solid phase support in at least one of the one or more spacially discrete regions, the oligonucleotide tag complements comprising a plurality of subunits, each subunit consisting of an oligonucleotide having a length from three to six nucleotides and each subunit being selected from a minimally cross-hybridizing set, wherein a subunit of the set and a component of any other subunit of the set would have at least two mismatches.
2. The composition of matter of claim 1 wherein said plurality of said subunits is in the range of from 4 to 10.
3. The composition of matter of claim 2 wherein said solid phase support is a microparticle having a single spacially discrete region.
4. The composition of matter of claim 3 wherein said microparticles is selected from the group consisting of glass microparticles, magnetic beads, and polystyrene microparticles.
5. A composition of matter comprising a plurality of from ten thousand to a hundred thousand different polynucleotides, selected from cDNA molecules or fragments of a target polynucleotide to be analyzed or sequenced, said composition including a mixture of microparticles,
wherein each microparticle has identical polynucleotides of the plurality attached thereto,
and wherein substantially all different polynucleotides in the plurality are attached to different microparticles.
6. The composition of claim 5 wherein each microparticle has about 10 5 identical polynucleotides attached thereto.
7. A composition of matter comprising a plurality of different polynucleotides, selected from cDNA molecules or fragments of a target polynucleotide to be analyzed or sequenced, said composition including a mixture of microparticles,
wherein tag complements are attached to each said microparticle,
and wherein each said cDNA molecule or fragment has an oligonucleotide tag attached, such that substantially all the same molecules have the same oligonucleotide tag attached and substantially all different molecules have different oligonucleotide tags attached,
such that perfectly matched duplexes are formed between the tag complements of said microparticles and the oligonucleotide tags of said cDNA molecules or fragments,
whereby, each microparticle has identical polynucleotides of the plurality attached thereto, and substantially all different polynucleotides in the plurality are attached to different microparticles.
Priority Applications (1)
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US09/366,081 USRE39793E1 (en) | 1994-10-13 | 1999-08-02 | Compositions for sorting polynucleotides |
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US32234894A | 1994-10-13 | 1994-10-13 | |
US08/358,810 US5604097A (en) | 1994-10-13 | 1994-12-19 | Methods for sorting polynucleotides using oligonucleotide tags |
US08/484,712 US5654413A (en) | 1994-10-13 | 1995-06-07 | Compositions for sorting polynucleotides |
US09/366,081 USRE39793E1 (en) | 1994-10-13 | 1999-08-02 | Compositions for sorting polynucleotides |
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US08/484,712 Reissue US5654413A (en) | 1994-10-13 | 1995-06-07 | Compositions for sorting polynucleotides |
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US08/478,238 Expired - Lifetime US5635400A (en) | 1994-10-13 | 1995-06-07 | Minimally cross-hybridizing sets of oligonucleotide tags |
US08/484,712 Ceased US5654413A (en) | 1994-10-13 | 1995-06-07 | Compositions for sorting polynucleotides |
US09/366,081 Expired - Lifetime USRE39793E1 (en) | 1994-10-13 | 1999-08-02 | Compositions for sorting polynucleotides |
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US08/478,238 Expired - Lifetime US5635400A (en) | 1994-10-13 | 1995-06-07 | Minimally cross-hybridizing sets of oligonucleotide tags |
US08/484,712 Ceased US5654413A (en) | 1994-10-13 | 1995-06-07 | Compositions for sorting polynucleotides |
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WO2012045012A2 (en) | 2010-09-30 | 2012-04-05 | Raindance Technologies, Inc. | Sandwich assays in droplets |
WO2013126741A1 (en) | 2012-02-24 | 2013-08-29 | Raindance Technologies, Inc. | Labeling and sample preparation for sequencing |
WO2013165748A1 (en) | 2012-04-30 | 2013-11-07 | Raindance Technologies, Inc | Digital analyte analysis |
US8738300B2 (en) | 2012-04-04 | 2014-05-27 | Good Start Genetics, Inc. | Sequence assembly |
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US9359601B2 (en) | 2009-02-13 | 2016-06-07 | X-Chem, Inc. | Methods of creating and screening DNA-encoded libraries |
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US9562896B2 (en) | 2010-04-21 | 2017-02-07 | Dnae Group Holdings Limited | Extracting low concentrations of bacteria from a sample |
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US11667951B2 (en) | 2016-10-24 | 2023-06-06 | Geneinfosec, Inc. | Concealing information present within nucleic acids |
US11674135B2 (en) | 2012-07-13 | 2023-06-13 | X-Chem, Inc. | DNA-encoded libraries having encoding oligonucleotide linkages not readable by polymerases |
US11840730B1 (en) | 2009-04-30 | 2023-12-12 | Molecular Loop Biosciences, Inc. | Methods and compositions for evaluating genetic markers |
US12098419B2 (en) | 2018-08-23 | 2024-09-24 | Ncan Genomics, Inc. | Linked target capture and ligation |
Families Citing this family (462)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5800992A (en) | 1989-06-07 | 1998-09-01 | Fodor; Stephen P.A. | Method of detecting nucleic acids |
US5547839A (en) * | 1989-06-07 | 1996-08-20 | Affymax Technologies N.V. | Sequencing of surface immobilized polymers utilizing microflourescence detection |
DE69132905T2 (en) | 1990-12-06 | 2002-08-01 | Affymetrix, Inc. (N.D.Ges.D.Staates Delaware) | Detection of nucleic acid sequences |
US6156501A (en) * | 1993-10-26 | 2000-12-05 | Affymetrix, Inc. | Arrays of modified nucleic acid probes and methods of use |
US7375198B2 (en) * | 1993-10-26 | 2008-05-20 | Affymetrix, Inc. | Modified nucleic acid probes |
US6654505B2 (en) | 1994-10-13 | 2003-11-25 | Lynx Therapeutics, Inc. | System and apparatus for sequential processing of analytes |
US6280935B1 (en) | 1994-10-13 | 2001-08-28 | Lynx Therapeutics, Inc. | Method of detecting the presence or absence of a plurality of target sequences using oligonucleotide tags |
US5846719A (en) | 1994-10-13 | 1998-12-08 | Lynx Therapeutics, Inc. | Oligonucleotide tags for sorting and identification |
US6013445A (en) * | 1996-06-06 | 2000-01-11 | Lynx Therapeutics, Inc. | Massively parallel signature sequencing by ligation of encoded adaptors |
USRE43097E1 (en) | 1994-10-13 | 2012-01-10 | Illumina, Inc. | Massively parallel signature sequencing by ligation of encoded adaptors |
US8236493B2 (en) * | 1994-10-21 | 2012-08-07 | Affymetrix, Inc. | Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA |
US6974666B1 (en) * | 1994-10-21 | 2005-12-13 | Appymetric, Inc. | Methods of enzymatic discrimination enhancement and surface-bound double-stranded DNA |
EP0832287B1 (en) * | 1995-06-07 | 2007-10-10 | Solexa, Inc | Oligonucleotide tags for sorting and identification |
AU718357B2 (en) * | 1995-06-07 | 2000-04-13 | Lynx Therapeutics, Inc. | Oligonucleotide tags for sorting and identification |
US5780231A (en) * | 1995-11-17 | 1998-07-14 | Lynx Therapeutics, Inc. | DNA extension and analysis with rolling primers |
US6312893B1 (en) * | 1996-01-23 | 2001-11-06 | Qiagen Genomics, Inc. | Methods and compositions for determining the sequence of nucleic acid molecules |
US6613508B1 (en) * | 1996-01-23 | 2003-09-02 | Qiagen Genomics, Inc. | Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques |
EP0880598A4 (en) * | 1996-01-23 | 2005-02-23 | Affymetrix Inc | Nucleic acid analysis techniques |
US6027890A (en) * | 1996-01-23 | 2000-02-22 | Rapigene, Inc. | Methods and compositions for enhancing sensitivity in the analysis of biological-based assays |
US6852487B1 (en) | 1996-02-09 | 2005-02-08 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays |
US6458530B1 (en) * | 1996-04-04 | 2002-10-01 | Affymetrix Inc. | Selecting tag nucleic acids |
US5948615A (en) * | 1996-04-16 | 1999-09-07 | Hitachi, Ltd. | Method for analysis of nucleic acid and DNA primer sets for use therein |
US7144119B2 (en) * | 1996-04-25 | 2006-12-05 | Bioarray Solutions Ltd. | System and method for programmable illumination pattern generation |
EP0907889B1 (en) | 1996-04-25 | 2007-07-04 | BioArray Solutions Ltd. | Light-controlled electrokinetic assembly of particles near surfaces |
US6387707B1 (en) * | 1996-04-25 | 2002-05-14 | Bioarray Solutions | Array Cytometry |
US7041510B2 (en) | 1996-04-25 | 2006-05-09 | Bioarray Solutions Ltd. | System and method for programmable illumination pattern generation |
US6958245B2 (en) | 1996-04-25 | 2005-10-25 | Bioarray Solutions Ltd. | Array cytometry |
US6048691A (en) * | 1996-05-13 | 2000-04-11 | Motorola, Inc. | Method and system for performing a binding assay |
CA2255774C (en) | 1996-05-29 | 2008-03-18 | Cornell Research Foundation, Inc. | Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions |
PL331513A1 (en) * | 1996-06-06 | 1999-07-19 | Lynx Therapeutics | Method of sequencing, by a ligand effect, specific encoded adapters and composition containing double-string oligonucleotidic adapters |
CA2301230A1 (en) | 1996-09-20 | 1998-03-26 | Digital Drives, Inc. | Spatially addressable combinatorial chemical arrays in cd-rom format |
GB9620769D0 (en) * | 1996-10-04 | 1996-11-20 | Brax Genomics Ltd | Nucleic acid sequencing |
US5858671A (en) | 1996-11-01 | 1999-01-12 | The University Of Iowa Research Foundation | Iterative and regenerative DNA sequencing method |
US6258533B1 (en) * | 1996-11-01 | 2001-07-10 | The University Of Iowa Research Foundation | Iterative and regenerative DNA sequencing method |
US6133436A (en) | 1996-11-06 | 2000-10-17 | Sequenom, Inc. | Beads bound to a solid support and to nucleic acids |
US6060240A (en) * | 1996-12-13 | 2000-05-09 | Arcaris, Inc. | Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom |
US20030027126A1 (en) | 1997-03-14 | 2003-02-06 | Walt David R. | Methods for detecting target analytes and enzymatic reactions |
US7622294B2 (en) * | 1997-03-14 | 2009-11-24 | Trustees Of Tufts College | Methods for detecting target analytes and enzymatic reactions |
GB9708606D0 (en) * | 1997-04-28 | 1997-06-18 | Webster Andrew | Sequencing |
US6007990A (en) * | 1997-04-29 | 1999-12-28 | Levine; Robert A. | Detection and quantification of one or more nucleotide sequence target analytes in a sample using spatially localized target analyte replication |
JP2002502237A (en) * | 1997-05-12 | 2002-01-22 | ライフ テクノロジーズ,インコーポレイテッド | Methods for generation and purification of nucleic acid molecules |
US6969488B2 (en) * | 1998-05-22 | 2005-11-29 | Solexa, Inc. | System and apparatus for sequential processing of analytes |
AU736321B2 (en) | 1997-05-23 | 2001-07-26 | Lynx Therapeutics, Inc. | System and apparatus for sequential processing of analytes |
WO1998055657A1 (en) * | 1997-06-05 | 1998-12-10 | Cellstore | Methods and reagents for indexing and encoding nucleic acids |
US6096496A (en) * | 1997-06-19 | 2000-08-01 | Frankel; Robert D. | Supports incorporating vertical cavity emitting lasers and tracking apparatus for use in combinatorial synthesis |
AU739963B2 (en) * | 1997-06-27 | 2001-10-25 | Lynx Therapeutics, Inc. | Method of mapping restriction sites in polynucleotides |
WO1999004042A1 (en) * | 1997-07-16 | 1999-01-28 | Burden David W | Synthetic oligonucleotide-peptide conjugates and methods of use |
US6607878B2 (en) * | 1997-10-06 | 2003-08-19 | Stratagene | Collections of uniquely tagged molecules |
US6265163B1 (en) | 1998-01-09 | 2001-07-24 | Lynx Therapeutics, Inc. | Solid phase selection of differentially expressed genes |
US6054276A (en) * | 1998-02-23 | 2000-04-25 | Macevicz; Stephen C. | DNA restriction site mapping |
US6136537A (en) * | 1998-02-23 | 2000-10-24 | Macevicz; Stephen C. | Gene expression analysis |
US6019896A (en) * | 1998-03-06 | 2000-02-01 | Molecular Dynamics, Inc. | Method for using a quality metric to assess the quality of biochemical separations |
AU758565B2 (en) * | 1998-03-18 | 2003-03-27 | Board Of Trustees Of The University Of Illinois, The | Selection subtraction approach to gene identification |
WO1999055886A1 (en) * | 1998-04-24 | 1999-11-04 | Genova Pharmaceuticals Corporation | Function-based gene discovery |
US6780591B2 (en) | 1998-05-01 | 2004-08-24 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
US7875440B2 (en) | 1998-05-01 | 2011-01-25 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
NZ508548A (en) * | 1998-05-13 | 2003-06-30 | Spectra Science Corp | Micro-lasing beads and structures, and associated methods |
CA2335951C (en) * | 1998-06-24 | 2013-07-30 | Mark S. Chee | Decoding of array sensors with microspheres |
US7399844B2 (en) * | 1998-07-09 | 2008-07-15 | Agilent Technologies, Inc. | Method and reagents for analyzing the nucleotide sequence of nucleic acids |
US20100130368A1 (en) * | 1998-07-30 | 2010-05-27 | Shankar Balasubramanian | Method and system for sequencing polynucleotides |
US20040106110A1 (en) * | 1998-07-30 | 2004-06-03 | Solexa, Ltd. | Preparation of polynucleotide arrays |
US6703228B1 (en) | 1998-09-25 | 2004-03-09 | Massachusetts Institute Of Technology | Methods and products related to genotyping and DNA analysis |
EP1001037A3 (en) * | 1998-09-28 | 2003-10-01 | Whitehead Institute For Biomedical Research | Pre-selection and isolation of single nucleotide polymorphisms |
WO2000023458A1 (en) * | 1998-10-19 | 2000-04-27 | The Board Of Trustees Of The Leland Stanford Junior University | Dna-templated combinatorial library chemistry |
US6480791B1 (en) * | 1998-10-28 | 2002-11-12 | Michael P. Strathmann | Parallel methods for genomic analysis |
CA2349836A1 (en) * | 1998-11-02 | 2000-05-11 | Lynx Therapeutics, Inc. | Method for making complementary oligonucleotide tag sets |
ATE250784T1 (en) * | 1998-11-27 | 2003-10-15 | Synaptics Uk Ltd | POSITION SENSOR |
AU2206800A (en) | 1998-12-11 | 2000-06-26 | Regents Of The University Of California, The | Targeted molecular bar codes and methods for using the same |
NO986133D0 (en) * | 1998-12-23 | 1998-12-23 | Preben Lexow | Method of DNA Sequencing |
EP1157131A2 (en) * | 1999-02-22 | 2001-11-28 | Lynx Therapeutics, Inc. | Polymorphic dna fragments and uses thereof |
GB9905807D0 (en) * | 1999-03-12 | 1999-05-05 | Amersham Pharm Biotech Uk Ltd | Analysis of differential gene expression |
AU3519900A (en) * | 1999-03-19 | 2000-10-09 | Aclara Biosciences, Inc. | Methods for single nucleotide polymorphism detection |
JP2002539849A (en) * | 1999-03-26 | 2002-11-26 | ホワイトヘッド インスチチュート フォアー バイオメディカル リサーチ | Universal array |
EP1190092A2 (en) | 1999-04-06 | 2002-03-27 | Yale University | Fixed address analysis of sequence tags |
US20060275782A1 (en) * | 1999-04-20 | 2006-12-07 | Illumina, Inc. | Detection of nucleic acid reactions on bead arrays |
EP1190100B1 (en) * | 1999-05-20 | 2012-07-25 | Illumina, Inc. | Combinatorial decoding of random nucleic acid arrays |
US6544732B1 (en) * | 1999-05-20 | 2003-04-08 | Illumina, Inc. | Encoding and decoding of array sensors utilizing nanocrystals |
US8481268B2 (en) | 1999-05-21 | 2013-07-09 | Illumina, Inc. | Use of microfluidic systems in the detection of target analytes using microsphere arrays |
US8080380B2 (en) * | 1999-05-21 | 2011-12-20 | Illumina, Inc. | Use of microfluidic systems in the detection of target analytes using microsphere arrays |
US20020042081A1 (en) * | 2000-10-10 | 2002-04-11 | Eric Henderson | Evaluating binding affinities by force stratification and force panning |
US20030186311A1 (en) * | 1999-05-21 | 2003-10-02 | Bioforce Nanosciences, Inc. | Parallel analysis of molecular interactions |
US20030073250A1 (en) * | 1999-05-21 | 2003-04-17 | Eric Henderson | Method and apparatus for solid state molecular analysis |
US6573369B2 (en) * | 1999-05-21 | 2003-06-03 | Bioforce Nanosciences, Inc. | Method and apparatus for solid state molecular analysis |
US8137906B2 (en) | 1999-06-07 | 2012-03-20 | Sloning Biotechnology Gmbh | Method for the synthesis of DNA fragments |
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 |
US6465183B2 (en) * | 1999-07-01 | 2002-10-15 | Agilent Technologies, Inc. | Multidentate arrays |
US6403319B1 (en) * | 1999-08-13 | 2002-06-11 | Yale University | Analysis of sequence tags with hairpin primers |
WO2001012862A2 (en) | 1999-08-18 | 2001-02-22 | Illumina, Inc. | Compositions and methods for preparing oligonucleotide solutions |
WO2001014589A2 (en) * | 1999-08-20 | 2001-03-01 | Luminex Corporation | Liquid array technology |
US7244559B2 (en) * | 1999-09-16 | 2007-07-17 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
US7211390B2 (en) * | 1999-09-16 | 2007-05-01 | 454 Life Sciences Corporation | Method of sequencing a nucleic acid |
US6274320B1 (en) | 1999-09-16 | 2001-08-14 | Curagen Corporation | Method of sequencing a nucleic acid |
WO2001020043A1 (en) * | 1999-09-17 | 2001-03-22 | Affymetrix, Inc. | Method of cluster analysis of gene expression profiles |
US6428957B1 (en) | 1999-11-08 | 2002-08-06 | Agilent Technologies, Inc. | Systems tools and methods of assaying biological materials using spatially-addressable arrays |
US7809382B2 (en) * | 2000-04-11 | 2010-10-05 | Telecommunication Systems, Inc. | Short message distribution center |
US6235483B1 (en) * | 2000-01-31 | 2001-05-22 | Agilent Technologies, Inc. | Methods and kits for indirect labeling of nucleic acids |
GB0002389D0 (en) | 2000-02-02 | 2000-03-22 | Solexa Ltd | Molecular arrays |
CA2401962A1 (en) | 2000-02-07 | 2001-08-09 | Illumina, Inc. | Nucleic acid detection methods using universal priming |
US7361488B2 (en) * | 2000-02-07 | 2008-04-22 | Illumina, Inc. | Nucleic acid detection methods using universal priming |
US7611869B2 (en) * | 2000-02-07 | 2009-11-03 | Illumina, Inc. | Multiplexed methylation detection methods |
DE60136166D1 (en) | 2000-02-07 | 2008-11-27 | Illumina Inc | NUCLEIC ACID PROOF METHOD WITH UNIVERSAL PRIMING |
US7582420B2 (en) | 2001-07-12 | 2009-09-01 | Illumina, Inc. | Multiplex nucleic acid reactions |
US7955794B2 (en) | 2000-09-21 | 2011-06-07 | Illumina, Inc. | Multiplex nucleic acid reactions |
US8076063B2 (en) * | 2000-02-07 | 2011-12-13 | Illumina, Inc. | Multiplexed methylation detection methods |
AU3838901A (en) | 2000-02-16 | 2001-08-27 | Illumina Inc | Parallel genotyping of multiple patient samples |
US6749756B1 (en) * | 2000-02-18 | 2004-06-15 | University Of Pittsburgh | Reaction and separation methods |
US6783985B1 (en) | 2000-02-18 | 2004-08-31 | Elitra Pharmaceuticals Inc. | Gene disruption methodologies for drug target discovery |
US6897015B2 (en) * | 2000-03-07 | 2005-05-24 | Bioforce Nanosciences, Inc. | Device and method of use for detection and characterization of pathogens and biological materials |
US7157564B1 (en) | 2000-04-06 | 2007-01-02 | Affymetrix, Inc. | Tag nucleic acids and probe arrays |
US6368801B1 (en) | 2000-04-12 | 2002-04-09 | Molecular Staging, Inc. | Detection and amplification of RNA using target-mediated ligation of DNA by RNA ligase |
CN1592792B (en) * | 2000-05-19 | 2010-12-01 | 伊拉根生物科学公司 | Materials and methods for detection of nucleic acids |
WO2001094625A2 (en) * | 2000-06-06 | 2001-12-13 | Tm Bioscience Corporation | Capture moieties for nucleic acids and uses thereof |
ES2259666T3 (en) * | 2000-06-21 | 2006-10-16 | Bioarray Solutions Ltd | MOLECULAR ANALYSIS OF MULTIPLE ANALYTICS USING SERIES OF RANDOM PARTICLES WITH APPLICATION SPECIFICITY. |
US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
AU2001271722B2 (en) | 2000-06-30 | 2006-04-13 | Qiagen, Gmbh | Signal amplification with lollipop probes |
EP1350106A2 (en) * | 2000-08-11 | 2003-10-08 | Agilix Corporation | Ultra-sensitive detection systems |
US6911204B2 (en) | 2000-08-11 | 2005-06-28 | Favrille, Inc. | Method and composition for altering a B cell mediated pathology |
AU2001283272A1 (en) * | 2000-08-11 | 2002-02-25 | Favrille, Inc. | A molecular vector identification system |
EP1348113B1 (en) * | 2000-08-15 | 2008-07-30 | Bioforce Nanosciences, Inc. | Nanoscale molecular arrayer |
GB0021367D0 (en) * | 2000-09-01 | 2000-10-18 | Sec Dep Of The Home Department | Improvements in and relating to marking |
US7057704B2 (en) * | 2000-09-17 | 2006-06-06 | Bioarray Solutions Ltd. | System and method for programmable illumination pattern generation |
AU1317502A (en) | 2000-10-14 | 2002-04-29 | Eragen Biosciences Inc | Solid support assay systems and methods utilizing non-standard bases |
US20030045005A1 (en) * | 2000-10-17 | 2003-03-06 | Michael Seul | Light-controlled electrokinetic assembly of particles near surfaces |
ATE380883T1 (en) | 2000-10-24 | 2007-12-15 | Univ Leland Stanford Junior | DIRECT MULTIPLEX CHARACTERIZATION OF GENOMIC DNA |
US20040018491A1 (en) * | 2000-10-26 | 2004-01-29 | Kevin Gunderson | Detection of nucleic acid reactions on bead arrays |
US6861222B2 (en) * | 2000-11-09 | 2005-03-01 | Yale University | Nucleic acid detection using structured probes |
WO2002061129A2 (en) * | 2000-11-15 | 2002-08-08 | Minerva Biotechnologies Corporation | Oligonucleotide identifiers |
US20030180953A1 (en) * | 2000-12-29 | 2003-09-25 | Elitra Pharmaceuticals, Inc. | Gene disruption methodologies for drug target discovery |
US20030049616A1 (en) * | 2001-01-08 | 2003-03-13 | Sydney Brenner | Enzymatic synthesis of oligonucleotide tags |
US6958217B2 (en) * | 2001-01-24 | 2005-10-25 | Genomic Expression Aps | Single-stranded polynucleotide tags |
US7226737B2 (en) * | 2001-01-25 | 2007-06-05 | Luminex Molecular Diagnostics, Inc. | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
WO2002059354A2 (en) | 2001-01-25 | 2002-08-01 | Tm Bioscience Corporation | Polynucleotides for use as tags and tag complements, manufacture and use thereof |
AU2002231933A1 (en) * | 2001-01-30 | 2002-08-12 | Solexa Ltd. | The preparation of polynucleotide arrays |
EP1390468A4 (en) * | 2001-04-23 | 2004-09-22 | Elitra Pharmaceuticals Inc | Identification of essential genes of aspegillus fumigatus and methods of use |
EP1262563A3 (en) * | 2001-05-31 | 2003-02-05 | Takara Bio Inc. | Method for immobilizing DNA on a surface |
US20070184436A1 (en) * | 2001-06-07 | 2007-08-09 | Joel Myerson | Generic capture probe arrays |
US20060234231A1 (en) * | 2001-06-20 | 2006-10-19 | Nuevolution A/S | Microarrays displaying encoded molecules |
US7727713B2 (en) | 2001-06-20 | 2010-06-01 | Nuevolution A/S | Templated molecules and methods for using such molecules |
US7262063B2 (en) | 2001-06-21 | 2007-08-28 | Bio Array Solutions, Ltd. | Directed assembly of functional heterostructures |
AU2002329606A1 (en) * | 2001-07-17 | 2003-03-03 | Bioforce Nanosciences, Inc. | Combined molecular blinding detection through force microscopy and mass spectrometry |
US6872529B2 (en) * | 2001-07-25 | 2005-03-29 | Affymetrix, Inc. | Complexity management of genomic DNA |
US7297778B2 (en) * | 2001-07-25 | 2007-11-20 | Affymetrix, Inc. | Complexity management of genomic DNA |
US7026123B1 (en) * | 2001-08-29 | 2006-04-11 | Pioneer Hi-Bred International, Inc. | UTR tag assay for gene function discovery |
US7042488B2 (en) | 2001-09-27 | 2006-05-09 | Fujinon Corporation | Electronic endoscope for highlighting blood vessel |
AU2002360272A1 (en) * | 2001-10-10 | 2003-04-22 | Superarray Bioscience Corporation | Detecting targets by unique identifier nucleotide tags |
US6767733B1 (en) | 2001-10-10 | 2004-07-27 | Pritest, Inc. | Portable biosensor apparatus with controlled flow |
US20040002073A1 (en) | 2001-10-15 | 2004-01-01 | Li Alice Xiang | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
US7132236B2 (en) * | 2001-10-25 | 2006-11-07 | Agilent Technologies, Inc. | Composition and method for optimized hybridization using modified solutions |
JPWO2003038091A1 (en) * | 2001-10-29 | 2005-04-07 | 独立行政法人科学技術振興機構 | Oligonucleotide sequence capable of avoiding mishybridization and its design method |
US20030124595A1 (en) * | 2001-11-06 | 2003-07-03 | Lizardi Paul M. | Sensitive coded detection systems |
DK1314783T3 (en) * | 2001-11-22 | 2009-03-16 | Sloning Biotechnology Gmbh | Nucleic acid linkers and their use in gene synthesis |
GB0129012D0 (en) | 2001-12-04 | 2002-01-23 | Solexa Ltd | Labelled nucleotides |
US20040020993A1 (en) * | 2001-12-28 | 2004-02-05 | Green Larry R. | Method for luminescent identification and calibration |
AU2003214031A1 (en) * | 2002-03-15 | 2003-09-29 | Nuevolution A/S | An improved method for synthesising templated molecules |
EP1497465B1 (en) * | 2002-04-26 | 2009-02-18 | Solexa, Inc | Constant length signatures for parallel sequencing of polynucleotides |
US20040060987A1 (en) * | 2002-05-07 | 2004-04-01 | Green Larry R. | Digital image analysis method for enhanced and optimized signals in fluorophore detection |
US20050239193A1 (en) * | 2002-05-30 | 2005-10-27 | Bioforce Nanosciences, Inc. | Device and method of use for detection and characterization of microorganisms and microparticles |
AU2003243720A1 (en) * | 2002-06-21 | 2004-01-06 | Bayer Bioscience N.V. | Method for detecting foreign dna in a host genome |
US20050186573A1 (en) * | 2002-07-24 | 2005-08-25 | Janeczko Richard A. | Polynucleotides for use as tags and tag complements in the detection of nucleic acid sequences |
AU2003247266A1 (en) * | 2002-08-01 | 2004-02-23 | Nuevolution A/S | Multi-step synthesis of templated molecules |
US7541444B2 (en) | 2002-08-23 | 2009-06-02 | Illumina Cambridge Limited | Modified nucleotides |
US7414116B2 (en) | 2002-08-23 | 2008-08-19 | Illumina Cambridge Limited | Labelled nucleotides |
US11008359B2 (en) | 2002-08-23 | 2021-05-18 | Illumina Cambridge Limited | Labelled nucleotides |
US20040043384A1 (en) * | 2002-08-28 | 2004-03-04 | Oleinikov Andrew V. | In vitro protein translation microarray device |
US7157228B2 (en) * | 2002-09-09 | 2007-01-02 | Bioarray Solutions Ltd. | Genetic analysis and authentication |
EP1545762A2 (en) * | 2002-09-27 | 2005-06-29 | Carlsberg A/S | Spatially encoded polymer matrix |
US20040259105A1 (en) * | 2002-10-03 | 2004-12-23 | Jian-Bing Fan | Multiplex nucleic acid analysis using archived or fixed samples |
NZ538993A (en) | 2002-10-30 | 2008-04-30 | Nuevolution As | Method for the synthesis of a bifunctional complex |
US20040086892A1 (en) * | 2002-11-06 | 2004-05-06 | Crothers Donald M. | Universal tag assay |
WO2004047007A1 (en) | 2002-11-15 | 2004-06-03 | Bioarray Solutions, Ltd. | Analysis, secure access to, and transmission of array images |
ATE450609T1 (en) | 2002-12-19 | 2009-12-15 | Nuevolution As | SYNTHESIS METHOD GUIDED BY QUASI-RANDOM STRUCTURES AND FUNCTIONS |
US9487823B2 (en) | 2002-12-20 | 2016-11-08 | Qiagen Gmbh | Nucleic acid amplification |
AU2003300156A1 (en) * | 2003-01-02 | 2004-07-29 | Bioforce Nanoscience, Inc. | Method and apparatus for molecular analysis in small sample volumes |
ATE417129T1 (en) * | 2003-01-17 | 2008-12-15 | Eragen Biosciences Inc | NUCLEIC ACID AMPLIFICATION WITH NON-STANDARD BASES |
CA2513985C (en) * | 2003-01-21 | 2012-05-29 | Illumina Inc. | Chemical reaction monitor |
AU2004209426B2 (en) * | 2003-01-29 | 2008-04-17 | 454 Life Sciences Corporation | Method for preparing single-stranded DNA libraries |
US7575865B2 (en) * | 2003-01-29 | 2009-08-18 | 454 Life Sciences Corporation | Methods of amplifying and sequencing nucleic acids |
US20060234238A1 (en) * | 2003-02-06 | 2006-10-19 | Salerno John C | Polymerase-based protocols for generating chimeric oligonucleotides |
US20060228786A1 (en) * | 2003-02-06 | 2006-10-12 | Salerno John C | Polymerase-based protocols for the introduction of deletions and insertions |
US20060257876A1 (en) * | 2003-02-06 | 2006-11-16 | Rensselaer Polytechnis Institute | Polymerase-based protocols for generating chimeric and combinatorial... |
US20060134624A1 (en) * | 2003-02-06 | 2006-06-22 | Salerno John C | Polymerase-based protocols for the introductions of deletions and insertions |
US20070026397A1 (en) | 2003-02-21 | 2007-02-01 | Nuevolution A/S | Method for producing second-generation library |
WO2004074501A2 (en) * | 2003-02-21 | 2004-09-02 | Nuevolution A/S | A method for obtaining structural information about an encoded molecule |
US20100022414A1 (en) | 2008-07-18 | 2010-01-28 | Raindance Technologies, Inc. | Droplet Libraries |
EP1608748B1 (en) | 2003-03-20 | 2009-03-04 | Nuevolution A/S | Ligational encoding of small molecules |
US8043834B2 (en) | 2003-03-31 | 2011-10-25 | Qiagen Gmbh | Universal reagents for rolling circle amplification and methods of use |
GB0307428D0 (en) * | 2003-03-31 | 2003-05-07 | Medical Res Council | Compartmentalised combinatorial chemistry |
US20060078893A1 (en) | 2004-10-12 | 2006-04-13 | Medical Research Council | Compartmentalised combinatorial chemistry by microfluidic control |
GB0307403D0 (en) * | 2003-03-31 | 2003-05-07 | Medical Res Council | Selection by compartmentalised screening |
WO2004090153A2 (en) | 2003-04-01 | 2004-10-21 | Eragen Biosciences,Inc. | Polymerase inhibitor and method of using same |
US7195875B2 (en) * | 2003-04-18 | 2007-03-27 | Beckman Coulter, Inc. | Oligonucleotide pairs for multiplexed binding assays |
JP2004355294A (en) * | 2003-05-29 | 2004-12-16 | National Institute Of Advanced Industrial & Technology | Designing method of dna code as information carrier |
US20040259118A1 (en) * | 2003-06-23 | 2004-12-23 | Macevicz Stephen C. | Methods and compositions for nucleic acid sequence analysis |
WO2005010494A2 (en) * | 2003-07-21 | 2005-02-03 | Jie Wu | Multiplexed analyte detection |
WO2005026686A2 (en) * | 2003-09-09 | 2005-03-24 | Compass Genetics, Llc | Multiplexed analytical platform |
WO2005029705A2 (en) * | 2003-09-18 | 2005-03-31 | Bioarray Solutions, Ltd. | Number coding for identification of subtypes of coded types of solid phase carriers |
EP1685380A2 (en) * | 2003-09-18 | 2006-08-02 | Parallele Bioscience, Inc. | System and methods for enhancing signal-to-noise ratios of microarray-based measurements |
ATE447626T1 (en) | 2003-09-18 | 2009-11-15 | Nuevolution As | METHOD FOR OBTAINING STRUCTURAL INFORMATION FROM ENCODED MOLECULES AND FOR SELECTING COMPOUNDS |
JP4564959B2 (en) * | 2003-09-22 | 2010-10-20 | バイオアレイ ソリューションズ リミテッド | Surface-immobilized polyelectrolyte with multiple functional groups that can be covalently bonded to biomolecules |
NZ547492A (en) | 2003-10-28 | 2009-12-24 | Bioarray Solutions Ltd | Optimization of gene expression analysis using immobilized capture probes of different lengths and densities |
US20050089916A1 (en) * | 2003-10-28 | 2005-04-28 | Xiongwu Xia | Allele assignment and probe selection in multiplexed assays of polymorphic targets |
PT1694859E (en) | 2003-10-29 | 2015-04-13 | Bioarray Solutions Ltd | Multiplexed nucleic acid analysis by fragmentation of double-stranded dna |
US20050094807A1 (en) * | 2003-11-04 | 2005-05-05 | John Silzel | Accuracy array assay system and method |
EP1687406B1 (en) * | 2003-11-10 | 2010-01-27 | Geneohm Sciences, Inc. | Nucleic acid detection method having increased sensitivity |
US7169560B2 (en) | 2003-11-12 | 2007-01-30 | Helicos Biosciences Corporation | Short cycle methods for sequencing polynucleotides |
WO2005065814A1 (en) | 2004-01-07 | 2005-07-21 | Solexa Limited | Modified molecular arrays |
CA2552858A1 (en) * | 2004-01-23 | 2005-08-04 | Lingvitae As | Improving polynucleotide ligation reactions |
EP1557464B1 (en) | 2004-01-23 | 2010-09-29 | Sloning BioTechnology GmbH | De novo enzymatic production of nucleic acid molecules |
WO2005081776A2 (en) * | 2004-01-30 | 2005-09-09 | Eragen Biosciences, Inc. | Materials and methods for the detection of sars |
GB0402895D0 (en) * | 2004-02-10 | 2004-03-17 | Solexa Ltd | Arrayed polynucleotides |
US20080108515A1 (en) * | 2004-02-10 | 2008-05-08 | Gormley Niall A | Arrayed polynucleotides |
CA2555377A1 (en) * | 2004-02-12 | 2005-09-01 | Compass Genetics, Llc | Genetic analysis by sequence-specific sorting |
WO2005078122A2 (en) * | 2004-02-17 | 2005-08-25 | Nuevolution A/S | Method for enrichment involving elimination by mismatch hybridisation |
US7981604B2 (en) | 2004-02-19 | 2011-07-19 | California Institute Of Technology | Methods and kits for analyzing polynucleotide sequences |
WO2005085477A1 (en) * | 2004-03-02 | 2005-09-15 | Orion Genomics Llc | Differential enzymatic fragmentation by whole genome amplification |
DK1730277T3 (en) | 2004-03-22 | 2010-03-01 | Nuevolution As | Coding by ligation using building block oligonucleotides |
US20050221339A1 (en) * | 2004-03-31 | 2005-10-06 | Medical Research Council Harvard University | Compartmentalised screening by microfluidic control |
EP1582599A1 (en) * | 2004-03-31 | 2005-10-05 | Takara Bio Inc. | Method for purifying microbeads |
US20050266447A1 (en) * | 2004-04-19 | 2005-12-01 | Pioneer Hi-Bred International, Inc. | Method for identifying activators of gene transcription |
WO2005111242A2 (en) * | 2004-05-10 | 2005-11-24 | Parallele Bioscience, Inc. | Digital profiling of polynucleotide populations |
EP2290069A3 (en) | 2004-05-28 | 2011-08-10 | Asuragen, Inc. | Methods and compositions involving microRNA |
US7363170B2 (en) * | 2004-07-09 | 2008-04-22 | Bio Array Solutions Ltd. | Transfusion registry network providing real-time interaction between users and providers of genetically characterized blood products |
WO2006014869A1 (en) * | 2004-07-26 | 2006-02-09 | Parallele Bioscience, Inc. | Simultaneous analysis of multiple genomes |
US7848889B2 (en) | 2004-08-02 | 2010-12-07 | Bioarray Solutions, Ltd. | Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification |
EP1623996A1 (en) * | 2004-08-06 | 2006-02-08 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Improved method of selecting a desired protein from a library |
DE602005025782D1 (en) | 2004-09-21 | 2011-02-17 | Life Technologies Corp | TWO-TONE REAL-TIME / END POINT QUANTIFICATION OF MICRO-RNAS (MIRNAS) |
US7968287B2 (en) | 2004-10-08 | 2011-06-28 | Medical Research Council Harvard University | In vitro evolution in microfluidic systems |
WO2006047791A2 (en) * | 2004-10-27 | 2006-05-04 | The Board Of Trustees Of The Leland Stanford Junior University | Dna-templated combinatorial library device and method for use |
ES2503742T3 (en) | 2004-11-12 | 2014-10-07 | Asuragen, Inc. | Procedures and compositions involving miRNA and miRNA inhibitor molecules |
DE502005003509D1 (en) * | 2004-11-19 | 2008-05-08 | Ebm Papst St Georgen Gmbh & Co | ARRANGEMENT WITH ONE FAN AND ONE PUMP |
EP2236628A3 (en) | 2005-02-01 | 2010-10-13 | AB Advanced Genetic Analysis Corporation | Reagents, methods and libraries for bead-based sequencing |
EP2230316A1 (en) * | 2005-02-01 | 2010-09-22 | AB Advanced Genetic Analysis Corporation | Nucleic acid sequencing by performing successive cycles of duplex extension |
US20070207555A1 (en) * | 2005-02-03 | 2007-09-06 | Cesar Guerra | Ultra-sensitive detection systems using multidimension signals |
US7393665B2 (en) | 2005-02-10 | 2008-07-01 | Population Genetics Technologies Ltd | Methods and compositions for tagging and identifying polynucleotides |
US7407757B2 (en) * | 2005-02-10 | 2008-08-05 | Population Genetics Technologies | Genetic analysis by sequence-specific sorting |
US20060204971A1 (en) * | 2005-03-11 | 2006-09-14 | Varde Shobha A | Oligonucleotides for multiplexed binding assays |
US20060211030A1 (en) * | 2005-03-16 | 2006-09-21 | Sydney Brenner | Methods and compositions for assay readouts on multiple analytical platforms |
US7498136B2 (en) * | 2005-03-18 | 2009-03-03 | Eragen Biosciences, Inc. | Methods for detecting multiple species and subspecies of Neisseria |
US8309303B2 (en) | 2005-04-01 | 2012-11-13 | Qiagen Gmbh | Reverse transcription and amplification of RNA with simultaneous degradation of DNA |
US8486629B2 (en) | 2005-06-01 | 2013-07-16 | Bioarray Solutions, Ltd. | Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation |
CA2610863C (en) * | 2005-06-07 | 2017-01-17 | Eragen Biosciences, Inc. | Methods for detection and typing of nucleic acids |
AU2006259565B2 (en) | 2005-06-15 | 2011-01-06 | Complete Genomics, Inc. | Single molecule arrays for genetic and chemical analysis |
WO2007025594A1 (en) * | 2005-07-07 | 2007-03-08 | Pamgene Bv | Method for detection and quantification of target nucleic acids in a sample |
WO2007021971A2 (en) * | 2005-08-12 | 2007-02-22 | Pharmorx Inc. | Labeling compositions and methods of use for deterrent trackability |
US7666593B2 (en) | 2005-08-26 | 2010-02-23 | Helicos Biosciences Corporation | Single molecule sequencing of captured nucleic acids |
EP1762627A1 (en) | 2005-09-09 | 2007-03-14 | Qiagen GmbH | Method for the activation of a nucleic acid for performing a polymerase reaction |
DK3018206T3 (en) * | 2005-12-01 | 2021-11-15 | Nuevolution As | ENZYMATIC ENCODING METHODS FOR EFFICIENT SYNTHESIS OF LARGE LIBRARIES |
WO2007081387A1 (en) | 2006-01-11 | 2007-07-19 | Raindance Technologies, Inc. | Microfluidic devices, methods of use, and kits for performing diagnostics |
WO2007087310A2 (en) * | 2006-01-23 | 2007-08-02 | Population Genetics Technologies Ltd. | Nucleic acid analysis using sequence tokens |
WO2007087312A2 (en) * | 2006-01-23 | 2007-08-02 | Population Genetics Technologies Ltd. | Molecular counting |
US20070264694A1 (en) * | 2006-04-07 | 2007-11-15 | Eragen Biosciences, Inc. | Use of non-standard bases and proximity effects for gene assembly and conversion of non-standard bases to standard bases during dna synthesis |
AU2007237909A1 (en) * | 2006-04-19 | 2007-10-25 | Applied Biosystems, Llc. | Reagents, methods, and libraries for gel-free bead-based sequencing |
ATE540750T1 (en) | 2006-05-11 | 2012-01-15 | Raindance Technologies Inc | MICROFLUIDIC DEVICE AND METHOD |
US9562837B2 (en) | 2006-05-11 | 2017-02-07 | Raindance Technologies, Inc. | Systems for handling microfludic droplets |
US7799542B2 (en) * | 2006-06-29 | 2010-09-21 | The Invention Science Fund I, Llc | Apparatus for arbitrary peptide synthesis |
US7910695B2 (en) * | 2006-06-29 | 2011-03-22 | The Invention Science Fund I, Llc | Methods for arbitrary peptide synthesis |
US7777002B2 (en) * | 2006-06-29 | 2010-08-17 | The Invention Science Fund I, Llc | Methods for arbitrary peptide synthesis |
US7879975B2 (en) * | 2006-06-29 | 2011-02-01 | The Invention Science Fund I, Llc | Methods for arbitrary peptide synthesis |
US7923533B2 (en) * | 2006-06-29 | 2011-04-12 | The Invention Science Fund I, Llc | Methods for arbitrary peptide synthesis |
US9012390B2 (en) | 2006-08-07 | 2015-04-21 | Raindance Technologies, Inc. | Fluorocarbon emulsion stabilizing surfactants |
US20090002703A1 (en) * | 2006-08-16 | 2009-01-01 | Craig Edward Parman | Methods and systems for quantifying isobaric labels and peptides |
WO2008027558A2 (en) | 2006-08-31 | 2008-03-06 | Codon Devices, Inc. | Iterative nucleic acid assembly using activation of vector-encoded traits |
US20090131348A1 (en) | 2006-09-19 | 2009-05-21 | Emmanuel Labourier | Micrornas differentially expressed in pancreatic diseases and uses thereof |
CA2663962A1 (en) | 2006-09-19 | 2008-03-27 | Asuragen, Inc. | Mir-15, mir-26, mir-31,mir-145, mir-147, mir-188, mir-215, mir-216, mir-331, mmu-mir-292-3p regulated genes and pathways as targets for therapeutic intervention |
US7955802B2 (en) | 2006-12-13 | 2011-06-07 | Luminex Corporation | Systems and methods for multiplex analysis of PCR in real time |
US8772046B2 (en) | 2007-02-06 | 2014-07-08 | Brandeis University | Manipulation of fluids and reactions in microfluidic systems |
US8592221B2 (en) | 2007-04-19 | 2013-11-26 | Brandeis University | Manipulation of fluids, fluid components and reactions in microfluidic systems |
AR066922A1 (en) | 2007-06-08 | 2009-09-23 | Monsanto Technology Llc | METHODS OF MOLECULAR IMPROVEMENT OF THE GERMOPLASMA OF A PLANT BY DIRECTED SEQUENCING |
US7635566B2 (en) * | 2007-06-29 | 2009-12-22 | Population Genetics Technologies Ltd. | Methods and compositions for isolating nucleic acid sequence variants |
WO2009040682A2 (en) * | 2007-09-26 | 2009-04-02 | Population Genetics Technologies Ltd. | Methods and compositions for reducing the complexity of a nucleic acid sample |
WO2009059199A2 (en) * | 2007-11-02 | 2009-05-07 | Hunch Inc. | Interactive machine learning advice facility |
US20090148849A1 (en) * | 2007-11-02 | 2009-06-11 | Barbara Galvan-Goldman | One-step target detection assay |
US8815576B2 (en) * | 2007-12-27 | 2014-08-26 | Lawrence Livermore National Security, Llc. | Chip-based sequencing nucleic acids |
US9115352B2 (en) | 2008-03-31 | 2015-08-25 | Sloning Biotechnology Gmbh | Method for the preparation of a nucleic acid library |
US9249455B2 (en) * | 2008-04-18 | 2016-02-02 | Luminex Corporation | Methods for detection and quantification of small RNA |
JP2009268665A (en) * | 2008-05-07 | 2009-11-19 | Canon Inc | Inhalation device |
EP2990487A1 (en) | 2008-05-08 | 2016-03-02 | Asuragen, INC. | Compositions and methods related to mirna modulation of neovascularization or angiogenesis |
US8198028B2 (en) | 2008-07-02 | 2012-06-12 | Illumina Cambridge Limited | Using populations of beads for the fabrication of arrays on surfaces |
US12038438B2 (en) | 2008-07-18 | 2024-07-16 | Bio-Rad Laboratories, Inc. | Enzyme quantification |
EP3216874A1 (en) | 2008-09-05 | 2017-09-13 | TOMA Biosciences, Inc. | Methods for stratifying and annotating cancer drug treatment options |
US9528160B2 (en) | 2008-11-07 | 2016-12-27 | Adaptive Biotechnolgies Corp. | Rare clonotypes and uses thereof |
US9506119B2 (en) | 2008-11-07 | 2016-11-29 | Adaptive Biotechnologies Corp. | Method of sequence determination using sequence tags |
US8748103B2 (en) | 2008-11-07 | 2014-06-10 | Sequenta, Inc. | Monitoring health and disease status using clonotype profiles |
US9394567B2 (en) | 2008-11-07 | 2016-07-19 | Adaptive Biotechnologies Corporation | Detection and quantification of sample contamination in immune repertoire analysis |
US9365901B2 (en) | 2008-11-07 | 2016-06-14 | Adaptive Biotechnologies Corp. | Monitoring immunoglobulin heavy chain evolution in B-cell acute lymphoblastic leukemia |
EP3699296A1 (en) | 2008-11-07 | 2020-08-26 | Adaptive Biotechnologies Corporation | Methods of monitoring conditions by sequence analysis |
US8628927B2 (en) | 2008-11-07 | 2014-01-14 | Sequenta, Inc. | Monitoring health and disease status using clonotype profiles |
WO2010083456A1 (en) | 2009-01-15 | 2010-07-22 | Imdaptive Inc. | Adaptive immunity profiling and methods for generation of monoclonal antibodies |
US8528589B2 (en) | 2009-03-23 | 2013-09-10 | Raindance Technologies, Inc. | Manipulation of microfluidic droplets |
WO2010115100A1 (en) | 2009-04-03 | 2010-10-07 | L&C Diagment, Inc. | Multiplex nucleic acid detection methods and systems |
EP2248914A1 (en) | 2009-05-05 | 2010-11-10 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | The use of class IIB restriction endonucleases in 2nd generation sequencing applications |
AU2010263172B2 (en) | 2009-06-25 | 2016-03-31 | Fred Hutchinson Cancer Research Center | Method of measuring adaptive immunity |
CN102686728B (en) * | 2009-06-29 | 2018-09-18 | 卢米耐克斯公司 | Chimeric primers with hairpin conformation and its application method |
US20110059453A1 (en) * | 2009-08-23 | 2011-03-10 | Affymetrix, Inc. | Poly(A) Tail Length Measurement by PCR |
WO2011037990A1 (en) | 2009-09-22 | 2011-03-31 | President And Fellows Of Harvard College | Entangled mate sequencing |
EP2486409A1 (en) | 2009-10-09 | 2012-08-15 | Universite De Strasbourg | Labelled silica-based nanomaterial with enhanced properties and uses thereof |
JP5938348B2 (en) * | 2009-10-23 | 2016-06-22 | ルミネックス コーポレーション | Amplification primers containing non-standard bases for increased reaction specificity |
US9315857B2 (en) | 2009-12-15 | 2016-04-19 | Cellular Research, Inc. | Digital counting of individual molecules by stochastic attachment of diverse label-tags |
US8835358B2 (en) | 2009-12-15 | 2014-09-16 | Cellular Research, Inc. | Digital counting of individual molecules by stochastic attachment of diverse labels |
EP2517025B1 (en) | 2009-12-23 | 2019-11-27 | Bio-Rad Laboratories, Inc. | Methods for reducing the exchange of molecules between droplets |
US9399797B2 (en) | 2010-02-12 | 2016-07-26 | Raindance Technologies, Inc. | Digital analyte analysis |
US10351905B2 (en) | 2010-02-12 | 2019-07-16 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
EP4435111A1 (en) | 2010-02-12 | 2024-09-25 | Bio-Rad Laboratories, Inc. | Digital analyte analysis |
US9366632B2 (en) | 2010-02-12 | 2016-06-14 | Raindance Technologies, Inc. | Digital analyte analysis |
ES2631458T3 (en) | 2010-03-04 | 2017-08-31 | Interna Technologies B.V. | MRNA molecule defined by its source and its therapeutic uses in cancer associated with EMT |
ES2713873T3 (en) | 2010-04-16 | 2019-05-24 | Nuevolution As | Bifunctional complexes and methods for making and using such complexes |
DK2567226T3 (en) | 2010-05-06 | 2016-10-10 | Adaptive Biotechnologies Corp | Monitoring the health and disease status using klonotypeprofiler |
US8828688B2 (en) | 2010-05-27 | 2014-09-09 | Affymetrix, Inc. | Multiplex amplification methods |
NZ719520A (en) | 2010-07-06 | 2017-07-28 | Int Tech Bv | Mirna and its diagnostic and therapeutic uses in diseases or conditions associated with melanoma, or in diseases or conditions associated with activated braf pathway |
DK2623613T3 (en) | 2010-09-21 | 2016-10-03 | Population Genetics Tech Ltd | Increasing the reliability of the allele-indications by molecular counting |
IN2013MN00522A (en) | 2010-09-24 | 2015-05-29 | Univ Leland Stanford Junior | |
US8759038B2 (en) | 2010-09-29 | 2014-06-24 | Illumina Cambridge Limited | Compositions and methods for sequencing nucleic acids |
US8753816B2 (en) | 2010-10-26 | 2014-06-17 | Illumina, Inc. | Sequencing methods |
EP2637780B1 (en) | 2010-11-12 | 2022-02-09 | Gen9, Inc. | Protein arrays and methods of using and making the same |
JP6118725B2 (en) | 2010-11-12 | 2017-04-19 | ジェン9・インコーポレイテッドGen9,INC. | Methods and devices for nucleic acid synthesis |
AU2011329772B2 (en) | 2010-11-17 | 2017-05-04 | Interpace Diagnostics, Llc | miRNAs as biomarkers for distinguishing benign from malignant thyroid neoplasms |
EP2474617A1 (en) | 2011-01-11 | 2012-07-11 | InteRNA Technologies BV | Mir for treating neo-angiogenesis |
US9364803B2 (en) | 2011-02-11 | 2016-06-14 | Raindance Technologies, Inc. | Methods for forming mixed droplets |
MX2013009863A (en) | 2011-02-28 | 2013-12-06 | Univ Iowa Res Found | ANTI-MÃœLLERIAN HORMONE CHANGES IN PREGNANCY AND PREDICTION OFADVERSE PREGNANCY OUTCOMES AND GENDER. |
US10501779B2 (en) | 2011-05-12 | 2019-12-10 | President And Fellows Of Harvard College | Oligonucleotide trapping |
EP3709018A1 (en) | 2011-06-02 | 2020-09-16 | Bio-Rad Laboratories, Inc. | Microfluidic apparatus for identifying components of a chemical reaction |
US8841071B2 (en) | 2011-06-02 | 2014-09-23 | Raindance Technologies, Inc. | Sample multiplexing |
US9752176B2 (en) | 2011-06-15 | 2017-09-05 | Ginkgo Bioworks, Inc. | Methods for preparative in vitro cloning |
AU2012271487B2 (en) * | 2011-06-15 | 2017-05-25 | Gen9, Inc. | Methods for preparative in vitro cloning |
US8658430B2 (en) | 2011-07-20 | 2014-02-25 | Raindance Technologies, Inc. | Manipulating droplet size |
EP2748318B1 (en) | 2011-08-26 | 2015-11-04 | Gen9, Inc. | Compositions and methods for high fidelity assembly of nucleic acids |
US10385475B2 (en) | 2011-09-12 | 2019-08-20 | Adaptive Biotechnologies Corp. | Random array sequencing of low-complexity libraries |
WO2013040251A2 (en) | 2011-09-13 | 2013-03-21 | Asurgen, Inc. | Methods and compositions involving mir-135b for distinguishing pancreatic cancer from benign pancreatic disease |
CN108192952A (en) | 2011-09-29 | 2018-06-22 | 露美内克丝公司 | Hydrolysis probes |
EP2768982A4 (en) | 2011-10-21 | 2015-06-03 | Adaptive Biotechnologies Corp | Quantification of adaptive immune cell genomes in a complex mixture of cells |
US20130157884A1 (en) | 2011-10-26 | 2013-06-20 | Asuragen, Inc. | Methods and compositions involving mirna expression levels for distinguishing pancreatic cysts |
EP2771487A1 (en) | 2011-10-27 | 2014-09-03 | Asuragen, INC. | Mirnas as diagnostic biomarkers to distinguish benign from malignant thyroid tumors |
US9499865B2 (en) | 2011-12-13 | 2016-11-22 | Adaptive Biotechnologies Corp. | Detection and measurement of tissue-infiltrating lymphocytes |
EP2794927B1 (en) | 2011-12-22 | 2017-04-12 | Ibis Biosciences, Inc. | Amplification primers and methods |
US11177020B2 (en) | 2012-02-27 | 2021-11-16 | The University Of North Carolina At Chapel Hill | Methods and uses for molecular tags |
ES2663234T3 (en) | 2012-02-27 | 2018-04-11 | Cellular Research, Inc | Compositions and kits for molecular counting |
WO2013128281A1 (en) | 2012-02-28 | 2013-09-06 | Population Genetics Technologies Ltd | Method for attaching a counter sequence to a nucleic acid sample |
US10077478B2 (en) | 2012-03-05 | 2018-09-18 | Adaptive Biotechnologies Corp. | Determining paired immune receptor chains from frequency matched subunits |
US9150853B2 (en) | 2012-03-21 | 2015-10-06 | Gen9, Inc. | Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis |
EP3543350B1 (en) | 2012-04-24 | 2021-11-10 | Gen9, Inc. | Methods for sorting nucleic acids and multiplexed preparative in vitro cloning |
EP2831276B1 (en) | 2012-05-08 | 2016-04-20 | Adaptive Biotechnologies Corporation | Compositions and method for measuring and calibrating amplification bias in multiplexed pcr reactions |
US9012022B2 (en) | 2012-06-08 | 2015-04-21 | Illumina, Inc. | Polymer coatings |
WO2013188756A1 (en) | 2012-06-15 | 2013-12-19 | The University Of Chicago | Oligonucleotide-mediated quantitative multiplexed immunoassays |
CA2877823A1 (en) | 2012-06-25 | 2014-01-03 | Gen9, Inc. | Methods for nucleic acid assembly and high throughput sequencing |
US20150152499A1 (en) | 2012-07-03 | 2015-06-04 | Interna Technologies B.V. | Diagnostic portfolio and its uses |
US11913065B2 (en) | 2012-09-04 | 2024-02-27 | Guardent Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
US20160040229A1 (en) | 2013-08-16 | 2016-02-11 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
PL3591073T3 (en) | 2012-09-04 | 2022-03-28 | Guardant Health, Inc. | Methods to detect rare mutations and copy number variation |
US10876152B2 (en) | 2012-09-04 | 2020-12-29 | Guardant Health, Inc. | Systems and methods to detect rare mutations and copy number variation |
ES2660027T3 (en) | 2012-10-01 | 2018-03-20 | Adaptive Biotechnologies Corporation | Evaluation of immunocompetence by the diversity of adaptive immunity receptors and clonal characterization |
US20140100124A1 (en) | 2012-10-04 | 2014-04-10 | Asuragen, Inc. | Diagnostic mirnas for differential diagnosis of incidental pancreatic cystic lesions |
US9476089B2 (en) | 2012-10-18 | 2016-10-25 | President And Fellows Of Harvard College | Methods of making oligonucleotide probes |
US9365896B2 (en) | 2012-10-19 | 2016-06-14 | Agilent Technologies, Inc. | Addition of an adaptor by invasive cleavage |
US10597650B2 (en) | 2012-12-21 | 2020-03-24 | New England Biolabs, Inc. | Ligase activity |
US10597710B2 (en) | 2012-12-21 | 2020-03-24 | New England Biolabs, Inc. | Ligase activity |
US9683230B2 (en) | 2013-01-09 | 2017-06-20 | Illumina Cambridge Limited | Sample preparation on a solid support |
US9868992B2 (en) | 2013-03-15 | 2018-01-16 | Baylor Research Institute | Tissue and blood-based miRNA biomarkers for the diagnosis, prognosis and metastasis-predictive potential in colorectal cancer |
US9540685B2 (en) | 2013-03-15 | 2017-01-10 | President And Fellows Of Harvard College | Methods of identifying homologous genes using FISH |
ES2935257T3 (en) | 2013-03-15 | 2023-03-03 | Univ Chicago | Methods and Compositions Related to T Cell Activity |
EP3366785A3 (en) | 2013-03-15 | 2018-09-19 | Baylor Research Institute | Ulcerative colitis (uc)-associated colorectal neoplasia markers |
US9593373B2 (en) | 2013-03-15 | 2017-03-14 | Illumina Cambridge Limited | Modified nucleosides or nucleotides |
EP2981622A1 (en) | 2013-04-04 | 2016-02-10 | Georgia State University Research Foundation, Inc. | Rna microchip detection using nanoparticle-assisted signal amplification |
CA2909972A1 (en) | 2013-05-23 | 2014-11-27 | The Board Of Trustees Of The Leland Stanford Junior University | Transposition into native chromatin for personal epigenomics |
EP3011056B1 (en) | 2013-06-19 | 2019-03-06 | Luminex Corporation | Real-time multiplexed hydrolysis probe assay |
US9708657B2 (en) | 2013-07-01 | 2017-07-18 | Adaptive Biotechnologies Corp. | Method for generating clonotype profiles using sequence tags |
CN105555971B (en) | 2013-08-09 | 2017-08-29 | 卢米耐克斯公司 | Probe for improving molten chain resolution and multiplicity in nucleic acid determination |
KR102402446B1 (en) | 2013-08-28 | 2022-05-30 | 벡톤 디킨슨 앤드 컴퍼니 | Massively parallel single cell analysis |
US11901041B2 (en) | 2013-10-04 | 2024-02-13 | Bio-Rad Laboratories, Inc. | Digital analysis of nucleic acid modification |
JP2017504307A (en) | 2013-10-07 | 2017-02-09 | セルラー リサーチ, インコーポレイテッド | Method and system for digitally counting features on an array |
EP3061831A4 (en) | 2013-10-21 | 2017-04-19 | KIM, Sung-Chun | Method and apparatus for analyzing biomolecules by using oligonucleotide |
CA2935138C (en) | 2013-10-22 | 2021-04-20 | Sung-Chun Kim | Marker for generating binding information on biomolecules and nucleic acids, preparation method therefor, and method and apparatus for analyzing biomolecule by using same |
US9944977B2 (en) | 2013-12-12 | 2018-04-17 | Raindance Technologies, Inc. | Distinguishing rare variations in a nucleic acid sequence from a sample |
ES2784450T3 (en) | 2013-12-28 | 2020-09-25 | Guardant Health Inc | Methods and systems to detect genetic variants |
WO2015105179A1 (en) | 2014-01-10 | 2015-07-16 | 国立大学法人京都大学 | Rna micro-array for detecting interaction between protein and conformation-containing rna |
US20170292149A1 (en) | 2014-03-05 | 2017-10-12 | Adaptive Biotechnologies Corporation | Methods using randomer-containing synthetic molecules |
US11390921B2 (en) | 2014-04-01 | 2022-07-19 | Adaptive Biotechnologies Corporation | Determining WT-1 specific T cells and WT-1 specific T cell receptors (TCRs) |
US10066265B2 (en) | 2014-04-01 | 2018-09-04 | Adaptive Biotechnologies Corp. | Determining antigen-specific t-cells |
ES2777529T3 (en) | 2014-04-17 | 2020-08-05 | Adaptive Biotechnologies Corp | Quantification of adaptive immune cell genomes in a complex mixture of cells |
JP6664381B2 (en) | 2014-08-11 | 2020-03-13 | ルミネックス コーポレーション | Probes for improved melting discrimination and multiplexing in nucleic acid assays |
US10093967B2 (en) | 2014-08-12 | 2018-10-09 | The Regents Of The University Of Michigan | Detection of nucleic acids |
US10174383B2 (en) | 2014-08-13 | 2019-01-08 | Vanadis Diagnostics | Method of estimating the amount of a methylated locus in a sample |
EP3183358B1 (en) | 2014-08-19 | 2020-10-07 | President and Fellows of Harvard College | Rna-guided systems for probing and mapping of nucleic acids |
EP3189157A1 (en) * | 2014-09-05 | 2017-07-12 | Qiagen GmbH | Preparation of adapter-ligated amplicons |
US10040048B1 (en) | 2014-09-25 | 2018-08-07 | Synthego Corporation | Automated modular system and method for production of biopolymers |
EP3715455A1 (en) | 2014-10-29 | 2020-09-30 | Adaptive Biotechnologies Corp. | Highly-multiplexed simultaneous detection of nucleic acids encoding paired adaptive immune receptor heterodimers from many samples |
US10246701B2 (en) | 2014-11-14 | 2019-04-02 | Adaptive Biotechnologies Corp. | Multiplexed digital quantitation of rearranged lymphoid receptors in a complex mixture |
WO2016086029A1 (en) | 2014-11-25 | 2016-06-02 | Adaptive Biotechnologies Corporation | Characterization of adaptive immune response to vaccination or infection using immune repertoire sequencing |
EP3766988B1 (en) | 2015-02-19 | 2024-02-14 | Becton, Dickinson and Company | High-throughput single-cell analysis combining proteomic and genomic information |
US10246702B1 (en) | 2015-02-23 | 2019-04-02 | New England Biolabs, Inc. | Compositions and methods for labeling target nucleic acid molecules |
WO2016138122A1 (en) | 2015-02-24 | 2016-09-01 | Adaptive Biotechnologies Corp. | Methods for diagnosing infectious disease and determining hla status using immune repertoire sequencing |
US9727810B2 (en) | 2015-02-27 | 2017-08-08 | Cellular Research, Inc. | Spatially addressable molecular barcoding |
JP7508191B2 (en) | 2015-03-30 | 2024-07-01 | ベクトン・ディキンソン・アンド・カンパニー | Methods and compositions for combinatorial barcoding |
EP3277294B1 (en) | 2015-04-01 | 2024-05-15 | Adaptive Biotechnologies Corp. | Method of identifying human compatible t cell receptors specific for an antigenic target |
CN107580632B (en) | 2015-04-23 | 2021-12-28 | 贝克顿迪金森公司 | Methods and compositions for whole transcriptome amplification |
WO2016196229A1 (en) | 2015-06-01 | 2016-12-08 | Cellular Research, Inc. | Methods for rna quantification |
EP3307908B1 (en) | 2015-06-09 | 2019-09-11 | Life Technologies Corporation | Methods for molecular tagging |
US10647981B1 (en) | 2015-09-08 | 2020-05-12 | Bio-Rad Laboratories, Inc. | Nucleic acid library generation methods and compositions |
JP6940484B2 (en) | 2015-09-11 | 2021-09-29 | セルラー リサーチ, インコーポレイテッド | Methods and compositions for library normalization |
WO2017046775A1 (en) | 2015-09-18 | 2017-03-23 | Vanadis Diagnostics | Probe set for analyzing a dna sample and method for using the same |
US10465232B1 (en) | 2015-10-08 | 2019-11-05 | Trace Genomics, Inc. | Methods for quantifying efficiency of nucleic acid extraction and detection |
CN108603228B (en) | 2015-12-17 | 2023-09-01 | 夸登特健康公司 | Method for determining tumor gene copy number by analyzing cell-free DNA |
US9931638B2 (en) | 2016-02-24 | 2018-04-03 | Latonya Stewart | Garbage disposal cleaning system |
ES2956757T3 (en) | 2016-05-02 | 2023-12-27 | Becton Dickinson Co | Accurate molecular barcode coding |
US10301677B2 (en) | 2016-05-25 | 2019-05-28 | Cellular Research, Inc. | Normalization of nucleic acid libraries |
CN109074430B (en) | 2016-05-26 | 2022-03-29 | 贝克顿迪金森公司 | Molecular marker counting adjustment method |
US10240196B2 (en) | 2016-05-27 | 2019-03-26 | Agilent Technologies, Inc. | Transposase-random priming DNA sample preparation |
US10640763B2 (en) | 2016-05-31 | 2020-05-05 | Cellular Research, Inc. | Molecular indexing of internal sequences |
US10202641B2 (en) | 2016-05-31 | 2019-02-12 | Cellular Research, Inc. | Error correction in amplification of samples |
EP3500669A4 (en) | 2016-08-16 | 2020-01-22 | The Regents of the University of California | Method for finding low abundance sequences by hybridization (flash) |
EP4219746A3 (en) | 2016-09-02 | 2023-10-18 | New England Biolabs, Inc. | Analysis of chromatin using a nicking enzyme |
US10428325B1 (en) | 2016-09-21 | 2019-10-01 | Adaptive Biotechnologies Corporation | Identification of antigen-specific B cell receptors |
WO2018057928A1 (en) | 2016-09-23 | 2018-03-29 | Grail, Inc. | Methods of preparing and analyzing cell-free nucleic acid sequencing libraries |
KR102363716B1 (en) | 2016-09-26 | 2022-02-18 | 셀룰러 리서치, 인크. | Determination of protein expression using reagents having barcoded oligonucleotide sequences |
WO2018081604A1 (en) | 2016-10-28 | 2018-05-03 | Grail, Inc. | Methods for single-stranded nucleic acid library preparation |
EP3538672A1 (en) | 2016-11-08 | 2019-09-18 | Cellular Research, Inc. | Methods for cell label classification |
EP3539035B1 (en) | 2016-11-08 | 2024-04-17 | Becton, Dickinson and Company | Methods for expression profile classification |
US20180163201A1 (en) | 2016-12-12 | 2018-06-14 | Grail, Inc. | Methods for tagging and amplifying rna template molecules for preparing sequencing libraries |
EP3559255A1 (en) | 2016-12-23 | 2019-10-30 | Grail, Inc. | Methods for high efficiency library preparation using double-stranded adapters |
EP3568234B1 (en) | 2017-01-13 | 2023-09-06 | Cellular Research, Inc. | Hydrophilic coating of fluidic channels |
WO2018144240A1 (en) | 2017-02-01 | 2018-08-09 | Cellular Research, Inc. | Selective amplification using blocking oligonucleotides |
US11274344B2 (en) | 2017-03-30 | 2022-03-15 | Grail, Inc. | Enhanced ligation in sequencing library preparation |
WO2018183942A1 (en) | 2017-03-31 | 2018-10-04 | Grail, Inc. | Improved library preparation and use thereof for sequencing-based error correction and/or variant identification |
US11118222B2 (en) | 2017-03-31 | 2021-09-14 | Grail, Inc. | Higher target capture efficiency using probe extension |
US10914729B2 (en) | 2017-05-22 | 2021-02-09 | The Trustees Of Princeton University | Methods for detecting protein binding sequences and tagging nucleic acids |
AU2018272848B2 (en) * | 2017-05-23 | 2022-03-31 | Rutgers, The State University Of New Jersey | Target mediated |
US10676779B2 (en) | 2017-06-05 | 2020-06-09 | Becton, Dickinson And Company | Sample indexing for single cells |
WO2018229547A1 (en) | 2017-06-15 | 2018-12-20 | Genome Research Limited | Duplex sequencing using direct repeat molecules |
US11180804B2 (en) | 2017-07-25 | 2021-11-23 | Massachusetts Institute Of Technology | In situ ATAC sequencing |
EP3704250A1 (en) | 2017-11-03 | 2020-09-09 | InteRNA Technologies B.V. | Mirna molecule, equivalent, antagomir, or source thereof for treating and/or diagnosing a condition and/or a disease associated with neuronal deficiency or for neuronal (re)generation |
US11254980B1 (en) | 2017-11-29 | 2022-02-22 | Adaptive Biotechnologies Corporation | Methods of profiling targeted polynucleotides while mitigating sequencing depth requirements |
WO2019118925A1 (en) | 2017-12-15 | 2019-06-20 | Grail, Inc. | Methods for enriching for duplex reads in sequencing and error correction |
EP3728636B1 (en) | 2017-12-19 | 2024-09-11 | Becton, Dickinson and Company | Particles associated with oligonucleotides |
WO2019126803A1 (en) | 2017-12-22 | 2019-06-27 | Grail, Inc. | Error removal using improved library preparation methods |
AU2018353924A1 (en) | 2017-12-29 | 2019-07-18 | Clear Labs, Inc. | Automated priming and library loading device |
EP3553182A1 (en) | 2018-04-11 | 2019-10-16 | Université de Bourgogne | Detection method of somatic genetic anomalies, combination of capture probes and kit of detection |
CN112272710A (en) | 2018-05-03 | 2021-01-26 | 贝克顿迪金森公司 | High throughput omics sample analysis |
EP3788170A1 (en) | 2018-05-03 | 2021-03-10 | Becton, Dickinson and Company | Molecular barcoding on opposite transcript ends |
KR102209178B1 (en) * | 2018-07-17 | 2021-01-29 | 이윤경 | Method for preserving and utilizing genome and genome information |
US11639517B2 (en) | 2018-10-01 | 2023-05-02 | Becton, Dickinson And Company | Determining 5′ transcript sequences |
EP3877520A1 (en) | 2018-11-08 | 2021-09-15 | Becton Dickinson and Company | Whole transcriptome analysis of single cells using random priming |
CN113195717A (en) | 2018-12-13 | 2021-07-30 | 贝克顿迪金森公司 | Selective extension in single cell whole transcriptome analysis |
AU2019398307A1 (en) | 2018-12-13 | 2021-06-10 | Dna Script | Direct oligonucleotide synthesis on cells and biomolecules |
US20220356510A1 (en) | 2019-01-03 | 2022-11-10 | Dna Script | One Pot Synthesis of Sets of Oligonucleotides |
US11371076B2 (en) | 2019-01-16 | 2022-06-28 | Becton, Dickinson And Company | Polymerase chain reaction normalization through primer titration |
WO2020154247A1 (en) | 2019-01-23 | 2020-07-30 | Cellular Research, Inc. | Oligonucleotides associated with antibodies |
CN113454234A (en) | 2019-02-14 | 2021-09-28 | 贝克顿迪金森公司 | Hybrid targeting and whole transcriptome amplification |
EP3938541B9 (en) | 2019-03-14 | 2023-10-04 | Genome Research Limited | Method for sequencing a direct repeat |
US20220162198A1 (en) | 2019-04-12 | 2022-05-26 | The Regents Of The University Of California | Compositions and methods for increasing muscle mass and oxidative metabolism |
WO2020214642A1 (en) | 2019-04-19 | 2020-10-22 | Becton, Dickinson And Company | Methods of associating phenotypical data and single cell sequencing data |
EP4004231A1 (en) | 2019-07-22 | 2022-06-01 | Becton, Dickinson and Company | Single cell chromatin immunoprecipitation sequencing assay |
WO2021092386A1 (en) | 2019-11-08 | 2021-05-14 | Becton Dickinson And Company | Using random priming to obtain full-length v(d)j information for immune repertoire sequencing |
US11649497B2 (en) | 2020-01-13 | 2023-05-16 | Becton, Dickinson And Company | Methods and compositions for quantitation of proteins and RNA |
EP4150118A1 (en) | 2020-05-14 | 2023-03-22 | Becton Dickinson and Company | Primers for immune repertoire profiling |
US11932901B2 (en) | 2020-07-13 | 2024-03-19 | Becton, Dickinson And Company | Target enrichment using nucleic acid probes for scRNAseq |
EP4189112A1 (en) | 2020-07-31 | 2023-06-07 | Becton Dickinson and Company | Single cell assay for transposase-accessible chromatin |
WO2023012521A1 (en) | 2021-08-05 | 2023-02-09 | Inivata Limited | Highly sensitive method for detecting cancer dna in a sample |
WO2022029688A1 (en) | 2020-08-05 | 2022-02-10 | Inivata Ltd. | Highly sensitive method for detecting cancer dna in a sample |
WO2022109343A1 (en) | 2020-11-20 | 2022-05-27 | Becton, Dickinson And Company | Profiling of highly expressed and lowly expressed proteins |
EP4413582A1 (en) | 2021-10-04 | 2024-08-14 | F. Hoffmann-La Roche AG | Online base call compression |
WO2024003332A1 (en) | 2022-06-30 | 2024-01-04 | F. Hoffmann-La Roche Ag | Controlling for tagmentation sequencing library insert size using archaeal histone-like proteins |
WO2024028794A1 (en) | 2022-08-02 | 2024-02-08 | Temple Therapeutics BV | Methods for treating endometrial and ovarian hyperproliferative disorders |
Citations (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0303459A2 (en) | 1987-08-11 | 1989-02-15 | The President And Fellows Of Harvard College | Multiplex sequencing |
EP0304845A2 (en) * | 1987-08-28 | 1989-03-01 | Profile Diagnostic Sciences Inc. | Method and kit for assaying gene expressions |
WO1990003382A1 (en) | 1988-09-21 | 1990-04-05 | Isis Innovation Limited | Support-bound oligonucleotides |
EP0392546A2 (en) | 1989-04-14 | 1990-10-17 | Ro Institut Za Molekularnu Genetiku I Geneticko Inzenjerstvo | Process for determination of a complete or a partial contents of very short sequences in the samples of nucleic acids connected to the discrete particles of microscopic size by hybridization with oligonucleotide probes |
US5028545A (en) | 1987-06-16 | 1991-07-02 | Wallac Oy | Biospecific multianalyte assay method |
CA2036946A1 (en) | 1990-04-06 | 1991-10-07 | Kenneth V. Deugau | Indexing linkers |
WO1992000091A1 (en) | 1990-07-02 | 1992-01-09 | Bioligand, Inc. | Random bio-oligomer library, a method of synthesis thereof, and a method of use thereof |
US5104791A (en) | 1988-02-09 | 1992-04-14 | E. I. Du Pont De Nemours And Company | Particle counting nucleic acid hybridization assays |
WO1992010588A1 (en) | 1990-12-06 | 1992-06-25 | Affymax Technologies N.V. | Sequencing by hybridization of a target nucleic acid to a matrix of defined oligonucleotides |
WO1992010587A1 (en) | 1990-12-06 | 1992-06-25 | Affymax Technologies N.V. | Sequencing of surface immobilized polymers utilizing microfluorescence detection |
WO1993006121A1 (en) | 1991-09-18 | 1993-04-01 | Affymax Technologies N.V. | Method of synthesizing diverse collections of oligomers |
US5206143A (en) | 1985-11-01 | 1993-04-27 | Smithkline Beecham Corporation | Method and reagents for performing subset analysis using quantitative differences in fluorescence intensity |
WO1993017126A1 (en) | 1992-02-19 | 1993-09-02 | The Public Health Research Institute Of The City Of New York, Inc. | Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids |
WO1993021203A1 (en) | 1992-04-15 | 1993-10-28 | The Johns Hopkins University | Synthesis of diverse and useful collections of oligonucleotides |
WO1993022684A1 (en) | 1992-04-29 | 1993-11-11 | Affymax Technologies N.V. | Factorial chemical libraries |
WO1993022680A1 (en) | 1992-04-24 | 1993-11-11 | Affymax Technologies N.V. | Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces |
US5302509A (en) | 1989-08-14 | 1994-04-12 | Beckman Instruments, Inc. | Method for sequencing polynucleotides |
WO1994008051A1 (en) | 1992-10-01 | 1994-04-14 | The Trustees Of Columbia University In The City Of New York | Complex combinatorial chemical libraries encoded with tags |
US5405746A (en) | 1988-03-23 | 1995-04-11 | Cemu Bioteknik Ab | Method of sequencing DNA |
WO1995020053A1 (en) | 1994-01-21 | 1995-07-27 | Medical Research Council | Sequencing of nucleic acids |
US5482836A (en) | 1993-01-14 | 1996-01-09 | The Regents Of The University Of California | DNA purification by triplex-affinity capture and affinity capture electrophoresis |
US5512439A (en) | 1988-11-21 | 1996-04-30 | Dynal As | Oligonucleotide-linked magnetic particles and uses thereof |
US5518883A (en) | 1992-07-02 | 1996-05-21 | Soini; Erkki J. | Biospecific multiparameter assay method |
US5567627A (en) | 1991-07-16 | 1996-10-22 | Trans-Med Biotech, Incorporated | Method and composition for the simultaneous and discrete analysis of multiple analytes |
Family Cites Families (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4046720A (en) | 1974-01-17 | 1977-09-06 | California Institute Of Technology | Crosslinked, porous, polyacrylate beads |
US4318846A (en) | 1979-09-07 | 1982-03-09 | Syva Company | Novel ether substituted fluorescein polyamino acid compounds as fluorescers and quenchers |
US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4973679A (en) | 1981-03-27 | 1990-11-27 | University Patents, Inc. | Process for oligonucleo tide synthesis using phosphormidite intermediates |
US4415732A (en) | 1981-03-27 | 1983-11-15 | University Patents, Inc. | Phosphoramidite compounds and processes |
US4413070A (en) | 1981-03-30 | 1983-11-01 | California Institute Of Technology | Polyacrolein microspheres |
US4678814A (en) | 1981-03-30 | 1987-07-07 | California Institute Of Technology | Polyacrolein microspheres |
US4948882A (en) | 1983-02-22 | 1990-08-14 | Syngene, Inc. | Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis |
DE3329892A1 (en) | 1983-08-18 | 1985-03-07 | Köster, Hubert, Prof. Dr., 2000 Hamburg | METHOD FOR PRODUCING OLIGONUCLEOTIDES |
US4659774A (en) | 1985-11-01 | 1987-04-21 | American Hoechst Corporation | Support for solid-phase oligonucleotide synthesis |
US5093232A (en) | 1985-12-11 | 1992-03-03 | Chiron Corporation | Nucleic acid probes |
US4855225A (en) | 1986-02-07 | 1989-08-08 | Applied Biosystems, Inc. | Method of detecting electrophoretically separated oligonucleotides |
FR2596761B1 (en) | 1986-04-08 | 1988-05-20 | Commissariat Energie Atomique | NUCLEOSIDE DERIVATIVES AND THEIR USE FOR SYNTHESIS OF OLIGONUCLEOTIDES |
US5091519A (en) | 1986-05-01 | 1992-02-25 | Amoco Corporation | Nucleotide compositions with linking groups |
ZA877772B (en) * | 1986-10-23 | 1988-04-20 | Amoco Corporation | Target and background capture methods and apparatus for affinity assays |
EP0972779A3 (en) | 1987-10-28 | 2004-10-20 | Howard Florey Institute Of Experimental Physiology And Medicine | Oligonucleotide-polyamide conjugates |
US5002867A (en) | 1988-04-25 | 1991-03-26 | Macevicz Stephen C | Nucleic acid sequence determination by multiple mixed oligonucleotide probes |
US5043272A (en) | 1989-04-27 | 1991-08-27 | Life Technologies, Incorporated | Amplification of nucleic acid sequences using oligonucleotides of random sequence as primers |
US5143854A (en) | 1989-06-07 | 1992-09-01 | Affymax Technologies N.V. | Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof |
US5366860A (en) | 1989-09-29 | 1994-11-22 | Applied Biosystems, Inc. | Spectrally resolvable rhodamine dyes for nucleic acid sequence determination |
CA2044616A1 (en) | 1989-10-26 | 1991-04-27 | Roger Y. Tsien | Dna sequencing |
US5188934A (en) | 1989-11-14 | 1993-02-23 | Applied Biosystems, Inc. | 4,7-dichlorofluorescein dyes as molecular probes |
WO1991007092A1 (en) | 1989-11-17 | 1991-05-30 | Applied Biosystems, Inc. | Poly(alkyl and alkenyl phosphate)s and their thiophosphate and selenophosphate derivatives as antiviral agents |
EP0439182B1 (en) | 1990-01-26 | 1996-04-24 | Abbott Laboratories | Improved method of amplifying target nucleic acids applicable to both polymerase and ligase chain reactions |
WO1992003461A1 (en) * | 1990-08-24 | 1992-03-05 | Ixsys, Inc. | Methods of synthesizing oligonucleotides with random codons |
DE69130333T2 (en) | 1990-08-31 | 1999-03-04 | Regents Of The University Of Minnesota, Minneapolis, Minn. | Resins for solid peptide synthesis |
JPH057490A (en) * | 1991-05-25 | 1993-01-19 | Sumitomo Electric Ind Ltd | Method for separating and collecting dna fragment |
AU2674092A (en) * | 1991-09-09 | 1993-04-05 | Baylor College Of Medicine | Method and device for rapid dna or rna sequencing determination by a base addition sequencing scheme |
AU2657392A (en) | 1991-09-27 | 1993-04-27 | Allelix Biopharmaceuticals Inc. | Duplex-forming, polynucleotide conjugates |
DE69231003T2 (en) * | 1991-12-24 | 2000-10-19 | Tepnel Medical Ltd., Manchester | MANIPULATION OF NUCLEIC ACID SEQUENCES |
GB9208733D0 (en) | 1992-04-22 | 1992-06-10 | Medical Res Council | Dna sequencing method |
GB9214873D0 (en) * | 1992-07-13 | 1992-08-26 | Medical Res Council | Process for categorising nucleotide sequence populations |
AU4779493A (en) * | 1992-07-21 | 1994-02-14 | Bunsen Rush Laboratories Inc. | Oligomer library formats and methods relating thereto |
GB9315847D0 (en) * | 1993-07-30 | 1993-09-15 | Isis Innovation | Tag reagent and assay method |
US5631734A (en) | 1994-02-10 | 1997-05-20 | Affymetrix, Inc. | Method and apparatus for detection of fluorescently labeled materials |
DE69535428T2 (en) | 1994-02-14 | 2007-12-06 | Smithkline Beecham Corp. | Method for finding differentially expressed genes |
US5552278A (en) | 1994-04-04 | 1996-09-03 | Spectragen, Inc. | DNA sequencing by stepwise ligation and cleavage |
-
1994
- 1994-12-19 US US08/358,810 patent/US5604097A/en not_active Expired - Lifetime
-
1995
- 1995-06-07 US US08/478,238 patent/US5635400A/en not_active Expired - Lifetime
- 1995-06-07 US US08/484,712 patent/US5654413A/en not_active Ceased
- 1995-10-12 HU HU9801187A patent/HUT77916A/en unknown
- 1995-10-12 AU AU42778/96A patent/AU712929B2/en not_active Ceased
- 1995-10-12 EP EP06007602A patent/EP1724348A3/en not_active Withdrawn
- 1995-10-12 AT AT95941325T patent/ATE323159T1/en not_active IP Right Cessation
- 1995-10-12 EP EP95941325A patent/EP0793718B1/en not_active Expired - Lifetime
- 1995-10-12 KR KR1019970702433A patent/KR970707279A/en not_active Application Discontinuation
- 1995-10-12 WO PCT/US1995/012791 patent/WO1996012014A1/en active IP Right Grant
- 1995-10-12 DE DE69534930T patent/DE69534930T2/en not_active Expired - Lifetime
- 1995-10-12 CZ CZ97866A patent/CZ86697A3/en unknown
- 1995-10-12 CA CA002202167A patent/CA2202167C/en not_active Expired - Fee Related
- 1995-10-12 EP EP09008365A patent/EP2110445A3/en not_active Withdrawn
- 1995-10-12 JP JP51329896A patent/JP4206130B2/en not_active Expired - Lifetime
-
1996
- 1996-06-06 KR KR1019970709024A patent/KR19990022543A/en active IP Right Grant
-
1997
- 1997-04-09 FI FI971473A patent/FI971473A/en not_active IP Right Cessation
- 1997-04-10 NO NO971644A patent/NO971644L/en not_active Application Discontinuation
-
1999
- 1999-08-02 US US09/366,081 patent/USRE39793E1/en not_active Expired - Lifetime
- 1999-10-04 AU AU52663/99A patent/AU784741B2/en not_active Ceased
-
2003
- 2003-10-08 JP JP2003350142A patent/JP4480380B2/en not_active Expired - Lifetime
-
2004
- 2004-09-08 JP JP2004261709A patent/JP2005052146A/en not_active Withdrawn
- 2004-11-01 JP JP2004318624A patent/JP4480544B2/en not_active Expired - Lifetime
-
2006
- 2006-05-01 JP JP2006128002A patent/JP2006320321A/en active Pending
-
2008
- 2008-03-03 JP JP2008052711A patent/JP4712822B2/en not_active Expired - Lifetime
Patent Citations (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5206143A (en) | 1985-11-01 | 1993-04-27 | Smithkline Beecham Corporation | Method and reagents for performing subset analysis using quantitative differences in fluorescence intensity |
US5028545A (en) | 1987-06-16 | 1991-07-02 | Wallac Oy | Biospecific multianalyte assay method |
EP0303459A2 (en) | 1987-08-11 | 1989-02-15 | The President And Fellows Of Harvard College | Multiplex sequencing |
EP0304845A2 (en) * | 1987-08-28 | 1989-03-01 | Profile Diagnostic Sciences Inc. | Method and kit for assaying gene expressions |
US5104791A (en) | 1988-02-09 | 1992-04-14 | E. I. Du Pont De Nemours And Company | Particle counting nucleic acid hybridization assays |
US5405746A (en) | 1988-03-23 | 1995-04-11 | Cemu Bioteknik Ab | Method of sequencing DNA |
WO1990003382A1 (en) | 1988-09-21 | 1990-04-05 | Isis Innovation Limited | Support-bound oligonucleotides |
US5512439A (en) | 1988-11-21 | 1996-04-30 | Dynal As | Oligonucleotide-linked magnetic particles and uses thereof |
EP0392546A2 (en) | 1989-04-14 | 1990-10-17 | Ro Institut Za Molekularnu Genetiku I Geneticko Inzenjerstvo | Process for determination of a complete or a partial contents of very short sequences in the samples of nucleic acids connected to the discrete particles of microscopic size by hybridization with oligonucleotide probes |
US5302509A (en) | 1989-08-14 | 1994-04-12 | Beckman Instruments, Inc. | Method for sequencing polynucleotides |
CA2036946A1 (en) | 1990-04-06 | 1991-10-07 | Kenneth V. Deugau | Indexing linkers |
WO1992000091A1 (en) | 1990-07-02 | 1992-01-09 | Bioligand, Inc. | Random bio-oligomer library, a method of synthesis thereof, and a method of use thereof |
WO1992010587A1 (en) | 1990-12-06 | 1992-06-25 | Affymax Technologies N.V. | Sequencing of surface immobilized polymers utilizing microfluorescence detection |
WO1992010588A1 (en) | 1990-12-06 | 1992-06-25 | Affymax Technologies N.V. | Sequencing by hybridization of a target nucleic acid to a matrix of defined oligonucleotides |
US5567627A (en) | 1991-07-16 | 1996-10-22 | Trans-Med Biotech, Incorporated | Method and composition for the simultaneous and discrete analysis of multiple analytes |
WO1993006121A1 (en) | 1991-09-18 | 1993-04-01 | Affymax Technologies N.V. | Method of synthesizing diverse collections of oligomers |
WO1993017126A1 (en) | 1992-02-19 | 1993-09-02 | The Public Health Research Institute Of The City Of New York, Inc. | Novel oligonucleotide arrays and their use for sorting, isolating, sequencing, and manipulating nucleic acids |
WO1993021203A1 (en) | 1992-04-15 | 1993-10-28 | The Johns Hopkins University | Synthesis of diverse and useful collections of oligonucleotides |
WO1993022680A1 (en) | 1992-04-24 | 1993-11-11 | Affymax Technologies N.V. | Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces |
WO1993022684A1 (en) | 1992-04-29 | 1993-11-11 | Affymax Technologies N.V. | Factorial chemical libraries |
US5518883A (en) | 1992-07-02 | 1996-05-21 | Soini; Erkki J. | Biospecific multiparameter assay method |
WO1994008051A1 (en) | 1992-10-01 | 1994-04-14 | The Trustees Of Columbia University In The City Of New York | Complex combinatorial chemical libraries encoded with tags |
US5482836A (en) | 1993-01-14 | 1996-01-09 | The Regents Of The University Of California | DNA purification by triplex-affinity capture and affinity capture electrophoresis |
WO1995020053A1 (en) | 1994-01-21 | 1995-07-27 | Medical Research Council | Sequencing of nucleic acids |
Non-Patent Citations (34)
Cited By (91)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7622281B2 (en) | 2004-05-20 | 2009-11-24 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for clonal amplification of nucleic acid |
US11168321B2 (en) | 2009-02-13 | 2021-11-09 | X-Chem, Inc. | Methods of creating and screening DNA-encoded libraries |
US9359601B2 (en) | 2009-02-13 | 2016-06-07 | X-Chem, Inc. | Methods of creating and screening DNA-encoded libraries |
US11840730B1 (en) | 2009-04-30 | 2023-12-12 | Molecular Loop Biosciences, Inc. | Methods and compositions for evaluating genetic markers |
US9476812B2 (en) | 2010-04-21 | 2016-10-25 | Dna Electronics, Inc. | Methods for isolating a target analyte from a heterogeneous sample |
US11073513B2 (en) | 2010-04-21 | 2021-07-27 | Dnae Group Holdings Limited | Separating target analytes using alternating magnetic fields |
US11448646B2 (en) | 2010-04-21 | 2022-09-20 | Dnae Group Holdings Limited | Isolating a target analyte from a body fluid |
US9970931B2 (en) | 2010-04-21 | 2018-05-15 | Dnae Group Holdings Limited | Methods for isolating a target analyte from a heterogenous sample |
US10677789B2 (en) | 2010-04-21 | 2020-06-09 | Dnae Group Holdings Limited | Analyzing bacteria without culturing |
US9671395B2 (en) | 2010-04-21 | 2017-06-06 | Dnae Group Holdings Limited | Analyzing bacteria without culturing |
US9562896B2 (en) | 2010-04-21 | 2017-02-07 | Dnae Group Holdings Limited | Extracting low concentrations of bacteria from a sample |
US9696302B2 (en) | 2010-04-21 | 2017-07-04 | Dnae Group Holdings Limited | Methods for isolating a target analyte from a heterogeneous sample |
US9869671B2 (en) | 2010-04-21 | 2018-01-16 | Dnae Group Holdings Limited | Analyzing bacteria without culturing |
US8278049B2 (en) * | 2010-04-26 | 2012-10-02 | Ann & Robert H. Lurie Children's Hospital of Chicago | Selective enrichment of CpG islands |
US20110263434A1 (en) * | 2010-04-26 | 2011-10-27 | Hehuang Xie | Selective enrichment of cpg islands |
EP3447155A1 (en) | 2010-09-30 | 2019-02-27 | Raindance Technologies, Inc. | Sandwich assays in droplets |
WO2012045012A2 (en) | 2010-09-30 | 2012-04-05 | Raindance Technologies, Inc. | Sandwich assays in droplets |
US11041851B2 (en) | 2010-12-23 | 2021-06-22 | Molecular Loop Biosciences, Inc. | Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction |
US11041852B2 (en) | 2010-12-23 | 2021-06-22 | Molecular Loop Biosciences, Inc. | Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction |
US11768200B2 (en) | 2010-12-23 | 2023-09-26 | Molecular Loop Biosciences, Inc. | Methods for maintaining the integrity and identification of a nucleic acid template in a multiplex sequencing reaction |
EP3736281A1 (en) | 2011-02-18 | 2020-11-11 | Bio-Rad Laboratories, Inc. | Compositions and methods for molecular labeling |
US10865409B2 (en) | 2011-09-07 | 2020-12-15 | X-Chem, Inc. | Methods for tagging DNA-encoded libraries |
US9228233B2 (en) | 2011-10-17 | 2016-01-05 | Good Start Genetics, Inc. | Analysis methods |
US10370710B2 (en) | 2011-10-17 | 2019-08-06 | Good Start Genetics, Inc. | Analysis methods |
US9822409B2 (en) | 2011-10-17 | 2017-11-21 | Good Start Genetics, Inc. | Analysis methods |
WO2013126741A1 (en) | 2012-02-24 | 2013-08-29 | Raindance Technologies, Inc. | Labeling and sample preparation for sequencing |
EP3309262A1 (en) | 2012-02-24 | 2018-04-18 | Raindance Technologies, Inc. | Labeling and sample preparation for sequencing |
US10604799B2 (en) | 2012-04-04 | 2020-03-31 | Molecular Loop Biosolutions, Llc | Sequence assembly |
US11149308B2 (en) | 2012-04-04 | 2021-10-19 | Invitae Corporation | Sequence assembly |
US11155863B2 (en) | 2012-04-04 | 2021-10-26 | Invitae Corporation | Sequence assembly |
US8738300B2 (en) | 2012-04-04 | 2014-05-27 | Good Start Genetics, Inc. | Sequence assembly |
US11667965B2 (en) | 2012-04-04 | 2023-06-06 | Invitae Corporation | Sequence assembly |
US9298804B2 (en) | 2012-04-09 | 2016-03-29 | Good Start Genetics, Inc. | Variant database |
US8812422B2 (en) | 2012-04-09 | 2014-08-19 | Good Start Genetics, Inc. | Variant database |
US10227635B2 (en) | 2012-04-16 | 2019-03-12 | Molecular Loop Biosolutions, Llc | Capture reactions |
US12110537B2 (en) | 2012-04-16 | 2024-10-08 | Molecular Loop Biosciences, Inc. | Capture reactions |
EP3524693A1 (en) | 2012-04-30 | 2019-08-14 | Raindance Technologies, Inc. | Digital analyte analysis |
WO2013165748A1 (en) | 2012-04-30 | 2013-11-07 | Raindance Technologies, Inc | Digital analyte analysis |
US11674135B2 (en) | 2012-07-13 | 2023-06-13 | X-Chem, Inc. | DNA-encoded libraries having encoding oligonucleotide linkages not readable by polymerases |
US9902949B2 (en) | 2012-12-19 | 2018-02-27 | Dnae Group Holdings Limited | Methods for universal target capture |
US9599610B2 (en) | 2012-12-19 | 2017-03-21 | Dnae Group Holdings Limited | Target capture system |
US10000557B2 (en) | 2012-12-19 | 2018-06-19 | Dnae Group Holdings Limited | Methods for raising antibodies |
US9995742B2 (en) | 2012-12-19 | 2018-06-12 | Dnae Group Holdings Limited | Sample entry |
US11603400B2 (en) | 2012-12-19 | 2023-03-14 | Dnae Group Holdings Limited | Methods for raising antibodies |
US10584329B2 (en) | 2012-12-19 | 2020-03-10 | Dnae Group Holdings Limited | Methods for universal target capture |
US11016086B2 (en) | 2012-12-19 | 2021-05-25 | Dnae Group Holdings Limited | Sample entry |
US9804069B2 (en) | 2012-12-19 | 2017-10-31 | Dnae Group Holdings Limited | Methods for degrading nucleic acid |
US9551704B2 (en) | 2012-12-19 | 2017-01-24 | Dna Electronics, Inc. | Target detection |
US10379113B2 (en) | 2012-12-19 | 2019-08-13 | Dnae Group Holdings Limited | Target detection |
US10745763B2 (en) | 2012-12-19 | 2020-08-18 | Dnae Group Holdings Limited | Target capture system |
US9115387B2 (en) | 2013-03-14 | 2015-08-25 | Good Start Genetics, Inc. | Methods for analyzing nucleic acids |
US9677124B2 (en) | 2013-03-14 | 2017-06-13 | Good Start Genetics, Inc. | Methods for analyzing nucleic acids |
US10202637B2 (en) | 2013-03-14 | 2019-02-12 | Molecular Loop Biosolutions, Llc | Methods for analyzing nucleic acid |
WO2014172288A2 (en) | 2013-04-19 | 2014-10-23 | Raindance Technologies, Inc. | Digital analyte analysis |
US10706017B2 (en) | 2013-06-03 | 2020-07-07 | Good Start Genetics, Inc. | Methods and systems for storing sequence read data |
US9535920B2 (en) | 2013-06-03 | 2017-01-03 | Good Start Genetics, Inc. | Methods and systems for storing sequence read data |
US10577646B2 (en) | 2013-08-19 | 2020-03-03 | Abbott Molecular Inc. | Nucleotide analogs |
US10036013B2 (en) | 2013-08-19 | 2018-07-31 | Abbott Molecular Inc. | Next-generation sequencing libraries |
WO2015026845A2 (en) | 2013-08-19 | 2015-02-26 | Abbott Molecular Inc. | Nucleotide analogs |
US10995363B2 (en) | 2013-08-19 | 2021-05-04 | Abbott Molecular Inc. | Nucleotide analogs |
US10865410B2 (en) | 2013-08-19 | 2020-12-15 | Abbott Molecular Inc. | Next-generation sequencing libraries |
US9932623B2 (en) | 2013-08-19 | 2018-04-03 | Abbott Molecular Inc. | Nucleotide analogs |
EP3626866A1 (en) | 2013-08-19 | 2020-03-25 | Abbott Molecular Inc. | Next-generation sequencing libraries |
EP3879012A1 (en) | 2013-08-19 | 2021-09-15 | Abbott Molecular Inc. | Next-generation sequencing libraries |
US11041203B2 (en) | 2013-10-18 | 2021-06-22 | Molecular Loop Biosolutions, Inc. | Methods for assessing a genomic region of a subject |
US10851414B2 (en) | 2013-10-18 | 2020-12-01 | Good Start Genetics, Inc. | Methods for determining carrier status |
US12077822B2 (en) | 2013-10-18 | 2024-09-03 | Molecular Loop Biosciences, Inc. | Methods for determining carrier status |
WO2015103367A1 (en) | 2013-12-31 | 2015-07-09 | Raindance Technologies, Inc. | System and method for detection of rna species |
US11053548B2 (en) | 2014-05-12 | 2021-07-06 | Good Start Genetics, Inc. | Methods for detecting aneuploidy |
US11408024B2 (en) | 2014-09-10 | 2022-08-09 | Molecular Loop Biosciences, Inc. | Methods for selectively suppressing non-target sequences |
US10429399B2 (en) | 2014-09-24 | 2019-10-01 | Good Start Genetics, Inc. | Process control for increased robustness of genetic assays |
EP3835429A1 (en) | 2014-10-17 | 2021-06-16 | Good Start Genetics, Inc. | Pre-implantation genetic screening and aneuploidy detection |
US10829813B2 (en) | 2014-11-04 | 2020-11-10 | Boreal Genomics, Inc. | Methods of sequencing with linked fragments |
US11827930B2 (en) | 2014-11-04 | 2023-11-28 | Ncan Genomics, Inc. | Methods of sequencing with linked fragments |
US10000799B2 (en) | 2014-11-04 | 2018-06-19 | Boreal Genomics, Inc. | Methods of sequencing with linked fragments |
US11680284B2 (en) | 2015-01-06 | 2023-06-20 | Moledular Loop Biosciences, Inc. | Screening for structural variants |
US10066259B2 (en) | 2015-01-06 | 2018-09-04 | Good Start Genetics, Inc. | Screening for structural variants |
US10801059B2 (en) | 2016-03-28 | 2020-10-13 | Boreal Genomics, Inc. | Droplet-based linked-fragment sequencing |
EP4282974A2 (en) | 2016-03-28 | 2023-11-29 | Ncan Genomics, Inc. | Linked duplex target capture |
US10961568B2 (en) | 2016-03-28 | 2021-03-30 | Boreal Genomics, Inc. | Linked target capture |
US11021742B2 (en) | 2016-03-28 | 2021-06-01 | Boreal Genomics, Inc. | Linked-fragment sequencing |
US10961573B2 (en) | 2016-03-28 | 2021-03-30 | Boreal Genomics, Inc. | Linked duplex target capture |
WO2017168332A1 (en) | 2016-03-28 | 2017-10-05 | Boreal Genomics, Inc. | Linked duplex target capture |
US11905556B2 (en) | 2016-03-28 | 2024-02-20 | Ncan Genomics, Inc. | Linked target capture |
US11667951B2 (en) | 2016-10-24 | 2023-06-06 | Geneinfosec, Inc. | Concealing information present within nucleic acids |
US11879151B2 (en) | 2016-12-09 | 2024-01-23 | Ncan Genomics, Inc. | Linked ligation |
US11268137B2 (en) | 2016-12-09 | 2022-03-08 | Boreal Genomics, Inc. | Linked ligation |
US11332736B2 (en) | 2017-12-07 | 2022-05-17 | The Broad Institute, Inc. | Methods and compositions for multiplexing single cell and single nuclei sequencing |
US12098419B2 (en) | 2018-08-23 | 2024-09-24 | Ncan Genomics, Inc. | Linked target capture and ligation |
US11473136B2 (en) | 2019-01-03 | 2022-10-18 | Ncan Genomics, Inc. | Linked target capture |
WO2020141464A1 (en) | 2019-01-03 | 2020-07-09 | Boreal Genomics, Inc. | Linked target capture |
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