WO2006086209A2 - Analyse genetique par tri specifique de sequences - Google Patents

Analyse genetique par tri specifique de sequences Download PDF

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WO2006086209A2
WO2006086209A2 PCT/US2006/003688 US2006003688W WO2006086209A2 WO 2006086209 A2 WO2006086209 A2 WO 2006086209A2 US 2006003688 W US2006003688 W US 2006003688W WO 2006086209 A2 WO2006086209 A2 WO 2006086209A2
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primer
polynucleotides
different
terminator
population
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WO2006086209A3 (fr
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Sydney Brenner
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Compass Genetics LLC
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Priority claimed from US11/055,187 external-priority patent/US7217522B2/en
Priority claimed from US11/173,465 external-priority patent/US7407757B2/en
Priority claimed from US11/176,927 external-priority patent/US7393665B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the invention relates generally to methods and compositions for analyzing complex populations of polynucleotides, and more particularly, to methods and compositions for partitioning a population of polynucleotides into one or more subpopulations of lesser complexity.
  • a major goal in genetics research is to understand how sequence variations in the genome relate to complex traits, particularly susceptibilities for common diseases such as diabetes, cancer, hypertension, and the like, e.g. Collins et al, Nature, 422: 835-847 (2003).
  • the availability of a sequence of the human genome has provided a highly useful reference for assessing variation, but it is only a first step towards understanding how the estimated 10 million or more common single nucleotide polymorphisms (SNPs), and other polymorphisms, such as inversions, deletions, insertions, and the like, determine or affect states of health and disease.
  • SNPs single nucleotide polymorphisms
  • patent 5,858,656 Unfortunately, most of these techniques rely on some form of subtraction, sequence destruction, or direct or indirect size selection to create subsets, which reduce sensitivity and which are often difficult to implement. Selective analysis of candidate genes thought to be responsible for disease has also been pursued as an alternative to genome-wide analyses, e.g. Cargill et al, Nature Genetics, 22: 231-238 (1999); Cambein et al, Am. J. Hum. Genet., 65: 183-191 (1999); Halushka et al, Nature Genetics, 22: 239-247 (1999). However, such approaches have been limited by the expense of re-sequencing genes from large numbers of individuals.
  • the field of genetic analysis would be advanced by the availability of a method for converting a highly complex population of DNA, such as a mixture of genomes, into subsets having reduced complexity without requiring subtraction, or other sequence destroying, steps.
  • projects for discovering the genetic basis of disease would be advanced by a method that permitted facile identification of sequences that vary from reference sequences, thereby reducing re- sequencing burdens in candidate gene or candidate region approaches to discovering genetic determinant of disease.
  • the invention provides methods and compositions for sorting polynucleotides from a population based on predetennined sequence characteristics.
  • the method of the invention is carried out by the following steps: (i) extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety, (ii) capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety, and (iii) melting the captured polynucleotides from the extended primers to form a subpopulation of polynucleotides having the predetermined sequence characteristics.
  • An exemplary predetermined sequence characteristic is a segment of sequence common to at least one fragment in each of a population of different genomes, such as a segment of an exon, a segment of an intron, a promoter sequence, or the like.
  • the invention includes as method of determining a subset of polynucleotides in a parent population that have sequences that vary from that of a reference sequence.
  • such a method comprises the following steps: (a) annealing a primer to polynucleotides of a parent population to form primer-polynucleotide duplexes; (b) extending the primer in the presence of at least one predetermined terminator having a capture moiety, the predetermined terminator being non-complementary with the reference sequence, so that after extension primer- polynucleotide complexes that contain a polynucleotide having a sequence different from that of the reference sequence have capture moieties; (c) separating the primer-polynucleotide duplexes having an extended primer with a capture moiety from the parent population by specifically binding the capture moiety of the predetermined terminator to a capture agent; (d) melting captured primer- polynucleotide duplexes to form a selected population of polynucleotides having sequences different from that of the reference sequence; (e) shortening polynucleotides of the parent population by from 1 to
  • the population of polynucleotides analyzed by the method of the invention comprises fragments from a population of genomes, wherein the fragments from each genome has the same unique oligonucleotide tag attached.
  • the invention includes a method of determining a frequency of a nucleotide at a predetermined locus in a population of genomes, such method comprising the following steps: (i) separately generating fragments of each genome of the population; (ii) attaching a unique oligonucleotide tag to each genome; (iii) selecting fragments from each genome that contains the predetermined locus; (iv) generating a labeled oligonucleotide tag from each unique oligonucleotide tag, the labeled oligonucleotide tag generating a signal indicative of the nucleotide at the predetermined locus; and (v) determining the frequency of the nucleotide at the predetermined locus by detecting the signals generated by the label
  • the invention provides a method of determining a frequency of a nucleotide at a predetermined locus in a population of genomes, the method comprising the steps of: separately generating fragments of each genome of the population; attaching a unique oligonucleotide tag to each genome; selecting fragments from each genome that contains the predetermined locus; generating a labeled oligonucleotide tag from each unique oligonucleotide tag, the labeled oligonucleotide tag generating a signal indicative of the nucleotide at the predetermined locus; and determining the frequency of the nucleotide at the predetermined locus by detecting the signals generated by the labeled oligonucleotide tags specifically hybridized with their respective tag complements, the respective tag complements being attached in spatially discrete regions on the one or more solid phase supports.
  • Figures IA- IF illustrate the selection of particular fragments by common sequence elements.
  • Figures 2A-2D illustrate the application of the invention for selecting particular haplotypes.
  • Figure 2E illustrates an embodiment of the method of the invention for detecting sequences of a population that vary from a reference sequence.
  • Figures 3A-3D illustrate hybridization tags with "commas” and a hybridization tag with the "comma-less” property.
  • Figure 4 lists melting temperatures of selected tags consisting of four words each having the comma-less property.
  • Figure 5 illustrates tagging of polynucleotides by successive addition of oligonucleotide subunits, or "words.” Definitions
  • Addressable in reference to tag complements means that the nucleotide sequence, or perhaps other physical or chemical characteristics, of a tag complement can be determined from its address, i.e. a one-to-one correspondence between the sequence or other property of the tag complement and a spatial location on, or characteristic of, the solid phase support to which it is attached.
  • an address of a tag complement is a spatial location, e.g. the planar coordinates of a particular region containing copies of the tag complement.
  • tag complements may be addressed in other ways too, e.g. by microparticle size, shape, color, frequency of micro-transponder, or the like, e.g. Chandler et al, PCT publication WO 97/14028.
  • Allele frequency in reference to a genetic locus, a sequence marker, or the site of a nucleotide means the frequency of occurrence of a sequence or nucleotide at such genetic loci or the frequency of occurrence of such sequence marker, with respect to a population of individuals.
  • an allele frequency may also refer to the frequency of sequences not identical to, or exactly complementary to, a reference sequence.
  • Amplicon means the product of an amplification reaction. That is, it is a population of polynucleotides, usually double stranded, that are replicated from one or more starting sequences. The one or more starting sequences may be one or more copies of the same sequence, or it may be a mixture of different sequences. Amplicons may be produced in a polymerase chain reaction (PCR), by replication in a cloning vector, or by linear amplification by an RNA polymerase, such as T7 or SP6, or by like techniques.
  • PCR polymerase chain reaction
  • analyte means any molecule, including organic, inorganic, or biomolecule, whose presence or absence or quantity or concentration in a sample is to be determined in an assay.
  • analytes are oligonucleotides, polynucleotides, genomic fragments, messenger RNAs
  • analytes are genomic fragments, particularly human genomic fragments.
  • “Complement” or “tag complement” as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which an 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 “complement” 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.
  • “Complexity” in reference to a population of double stranded or single stranded polynucleotides means the number of different species of polynucleotide present in the population.
  • the related concept, "kinetic complexity” in reference to genomic DNA means the total number of basepairs present in non-repeating sequences, e.g. Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991); Britten and Davidson, chapter 1 in Hames et al, editors, Nucleic Acid Hybridization: A Practical Approach (IRL Press, Oxford, 1985).
  • the following populations have the indicated sizes and complexities:
  • cccttagctg agggct 8 3 (SEQ ID NO : 8 ) cccttagctg agggct (SEQ ID NO : 8 ) cccttagctg agggct (SEQ ID NO : 8 ) cccttagctg agggctc (SEQ ID NO : 8 )
  • Duplex means at least two oligonucleotides and/or polynucleotides that are fully or partially complementary undergo Watson-Crick type base pairing among all or most of their nucleotides so that a stable complex is formed.
  • annealing and “hybridization” are used interchangeably to mean the formation of a stable duplex.
  • stable duplex means that a duplex structure is not destroyed by a stringent wash, e.g. conditions including tempature of about 5 0 C less that the T n , of a strand of the duplex and low monovalent salt concentration, e.g. less than 0.2 M, or less than 0.1 M.
  • duplex in reference to a duplex means that the poly- or oligonucleotide strands making up the duplex form a double stranded structure with one another such that every nucleotide in each strand undergoes Watson-Crick basepairing with a nucleotide in the other strand.
  • duplex comprehends the pairing of nucleoside analogs, such as deoxyinosine, nucleosides with 2-aminopurine bases, PNAs, and the like, that may be employed.
  • a "mismatch" in a duplex between two oligonucleotides or polynucleotides means that a pair of nucleotides in the duplex fails to undergo Watson-Crick bonding.
  • Melting as used herein means de-annealing. That is, melting means the destabilization of a duplex so that the strands of the duplex separate and become independent single stranded polynucleotides. Melting is usually accomplished by raising the temperature of a reaction mixture to a level above the melting temperature of the duplex. Melting can also be accomplished by adding denaturants, such as urea, sodium hydroxide, or other bases, or organic solvents, such as formamide. "Fragment”, “segment”, or “DNA segment” refers to a portion of a larger DNA polynucleotide or DNA. A polynucleotide, for example, can be broken up, or fragmented into, a plurality of segments.
  • Enzymatic fragmentation may include partial degradation with a DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA-based cleavage methods, such as triplex and hybrid formation methods, that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic acid molecule; or other enzymes or compounds which cleave DNA at known or unknown locations.
  • Physical fragmentation methods may involve subjecting the DNA to a high shear rate.
  • High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing the DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron scale.
  • Other physical methods include sonication and nebulization.
  • Combinations of physical and chemical fragmentation methods may likewise be employed such as fragmentation by heat and ion-mediated hydrolysis. See for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3" 1 Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001) (“Sambrook et al.) which is incorporated herein by reference for all purposes. These methods can be optimized to digest a nucleic acid into fragments of a selected size range.
  • Genetic locus in reference to a genome or target polynucleotide, means a contiguous subregion or segment of the genome or target polynucleotide.
  • genetic locus, or locus may refer to the position of a nucleotide, a gene, or a portion of a gene in a genome, including mitochondrial DNA, or it may refer to any contiguous portion of genomic sequence whether or not it is within, or associated with, a gene.
  • a genetic locus refers to any portion of genomic sequence, including mitochondrial DNA, from a single nucleotide to a segment of few hundred nucleotides, e.g. 100-300, in length.
  • a particular genetic locus may be identified by its nucleotide sequence, or the nucleotide sequence, or sequences, of one or both adjacent or flanking regions.
  • haplotype means a series of alleles found at linked loci on a single chromosome. More particularly, haplotype means a series of single nucleotide polymorphisms at predetermined loci in a genomic DNA fragment.
  • Hybridization refers to the process in which two single-stranded polynucleotides bind non- covalently to form a stable double-stranded polynucleotide.
  • the term “hybridization” may also refer to triple-stranded hybridization.
  • the resulting (usually) double-stranded polynucleotide is a “hybrid” or “duplex.”
  • “Hybridization conditions” will typically include salt concentrations of less than about IM, more usually less than about 500 mM and less than about 200 mM.
  • Hybridization temperatures can be as low as 5° C, but are typically greater than 22° C, more typically greater than about 30° C, and preferably in excess of about 37° C.
  • Hybridizations are usually performed under stringent conditions, i.e. conditions under which a probe will hybridize to its target subsequence.
  • Stringent conditions are sequence-dependent and are different in different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone.
  • stringent conditions are selected to be about 5° C. lower than the T 111 for the specific sequence at s defined ionic strength and pH.
  • Exemplary stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C.
  • conditions of 5 xSSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30° C. are suitable for allele-specific probe hybridizations.
  • stringent conditions see for example, Sambrook, Fritsche and Maniatis. "Molecular Cloning A laboratory Manual" 2 nd Ed.
  • Hybridizing specifically to or “specifically hybridizing to” or like expressions refer to the binding, duplexing, or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Ligation means to form a covalent bond or linkage between the termini of two or more nucleic acids, e.g. oligonucleotides and/or polynucleotides, in a template-driven reaction.
  • the nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically or chemically.
  • ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon of a terminal nucleotide of one oligonucleotide with 3 ' carbon of another oligonucleotide.
  • Microarray refers to a solid phase support, which may be planar or a collection of microparticles, that carries or carry oligo- or polynucleotides fixed or immobilized, usually covalently, at specific addressable locations.
  • a microarray is a solid phase support having a planar surface, which carries an array of nucleic acids, each member of the array comprising identical copies of an oligonucleotide or polynucleotide immobilized to a fixed region, which does not overlap with those of other members of the array.
  • the oligonucleotides or polynucleotides are single stranded and are covalently attached to the solid phase support at known, determinable, or addressable, locations.
  • the density of non-overlapping regions containing nucleic acids in a microarray is typically greater than 100 per cm ⁇ , and more preferably, greater than 1000 per cm ⁇ .
  • Microarray technology is reviewed in the following references: Schena, Editor, Microarrays: A Practical Approach (IRL Press, Oxford, 2000); Southern, Current Opin. Chem. Biol., 2: 404-410 (1998); Nature Genetics Supplement, 21 : 1-60 (1999).
  • Nucleoside as used herein 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 moieties, e.g. described by Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990), or the like, with the proviso that they are capable of specific hybridization.
  • Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, and the like.
  • Polynucleotides comprising analogs with enhanced hybridization or nuclease resistance properties are described in Uhlman and Peyman (cited above); Crooke et al, Exp. Opin. Ther. Patents, 6: 855-870 (1996); Mesmaeker et al, Current Opinion in Structual Biology, 5: 343-355 (1995); and the like.
  • Exemplary types of polynucleotides that are capable of enhancing duplex stability include oligonucleotide N3'— >P5' phosphoramidates (referred to herein as “amidates”), peptide nucleic acids (referred to herein as "PNAs”), oligo-2'-O-alkylribonucleotides, polynucleotides containing C-5 propynylpyrimidines, and like compounds.
  • aminodates oligonucleotide N3'— >P5' phosphoramidates
  • PNAs peptide nucleic acids
  • oligo-2'-O-alkylribonucleotides oligo-2'-O-alkylribonucleotides
  • polynucleotides containing C-5 propynylpyrimidines and like compounds.
  • Such oligonucleotides are either available commercially or may be synthesized using methods described in the literature.
  • Oligonucleotide tag means an oligonucleotide that is attached to a polynucleotide and is used to identify and/or track the polynucleotide in a reaction. Usually, a oligonucleotide tag is attached to the 3 '- or 5'-end of a polynucleotide to fo ⁇ n a linear conjugate, sometime referred to herein as a "tagged polynucleotide.” Oligonucleotide tags may vary widely in size and compositions; the following references provide guidance for selecting sets of oligonucleotide tags appropriate for particular embodiments: Brenner, U.S. patent 5,635,400; Brenner et al, Proc. Natl.
  • oligonucleotide tags each have a length within a range of from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides, respectively.
  • oligonucleotide tags are used in sets, or repertoires, wherein each oligonucleotide tag of the set has a unique nucleotide sequence.
  • each oligonucleotide tag of such a set has a melting temperature that is substantially the same as that of every other member of the same set.
  • the melting temperatures of oligonucleotide tags within a set are within 1O 0 C of one another; in another embodiment, they are within 5 0 C of one another; and in another embodiment, they are within 2 0 C of one another.
  • oligonucleotide tags within a set are maximally orthogonal. That is, they minimally cross-hybridize to their respective complements.
  • oligonucleotide tags within a set have at least one mismatch when hybridized to any complement of other members of the same set; in another aspect, oligonucleotide tags within a set have at least two mismatches when hybridized to any complement of other members of the same set; in another aspect, oligonucleotide tags within a set have at least three mismatches when hybridized to any complement of other members of the same set; in another aspect, oligonucleotide tags within a set have at least four mismatches when hybridized to any complement of other members of the same set; in another aspect, oligonucleotide tags within a set have at least five mismatches when hybridized to any complement of other members of the same set.
  • oligonucleotide tags are identified by a hybridization reation, such as by hybridizing a labeled oligonucleotide tag to its complement on a solid phase support.
  • the size of such sets may vary widely.
  • a set oligonucleotide tags may have a size in the range of from several tens to many thousands, or even millions, e.g. 50 to 1.6 x 10 6 . In another embodiment, such a size is in the range of from 200 to 40,000; or from 1000 to 40,000; or from 1000 to 10,000.
  • oligonucletide tags may comprise a concatenation of subunits, such as described by Brenner et al, Proc. Natl. Acad. ScL, 97: 1665-1670 (2000).
  • oligonucleotide subunits or words, can be selected from a set of subunits with the properties of minimal cross-hybridization and substantially equivalent melting temperature. Constructing oligonucleotide tags from a plurality of oligonucleotide subunits permits the convenient and inexpensive formation of very large sets of oligonucleotide tags, e.g. as described by Brenner et al, Proc. Natl. Acad.
  • oligonucleotide tags comprise a plurality of oligonucleotide subunits. Such subunits may vary widely in length.
  • the length of oligonucleotide subunits is in the range of from 2 to 18 nucleotides; in another aspect, the length of oligonucleotide subunits is in the range of from 2 to 8 nucleotides; and in another aspect the length of oligonucleotide subunits is in the range of from 2 to 4 nucleotides.
  • a plurality of oligonucleotide subunits making up an oligonucleotide tag may also vary widely depending on their application. In one aspect, such plurality is a number in the range of 2 to 10; and in another aspect, such plurality is a number in the range of from 2 to 6.
  • the size of a set of oligonucleotide subunits is usually smaller than the size of a set of oligonucleotide tags.
  • a set of oligonucleotide subunits has a size in the range of from 2 to 20; or in another embodiment, from 2 to 10; or in another embodiment, from 4 to 8. It is clear to one of ordinary skill that for subunits only two nucleotides in length that the size of a set of subunits would be smaller than that of subunits having greater lengths.
  • PCR Polymerase chain reaction
  • PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates.
  • the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument.
  • a double stranded target nucleic acid may be denatured at a temperature >90°C, primers annealed at a temperature in the range 50-75 0 C, and primers extended at a temperature in the range 72-78 0 C.
  • PCR encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. Reaction volumes range from a few hundred nanoliters, e.g. 200 nL, to a few hundred ⁇ L, e.g. 200 ⁇ L.
  • Reverse transcription PCR or "RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g. Tecott et al, U.S. patent 5,168,038, which patent is incorporated herein by reference.
  • Real-time PCR means a PCR for which the amount of reaction product, i.e. amplicon, is monitored as the reaction proceeds.
  • Nested PCR means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon.
  • initial primers in reference to a nested amplification reaction mean the primers used to generate a first amplicon
  • secondary primers mean the one or more primers used to generate a second, or nested, amplicon.
  • Multiplexed PCR means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228 (1999)(two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified.
  • Quantitative PCR means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Quantitative measurements are made using one or more reference sequences that may be assayed separately or together with a target sequence.
  • the reference sequence may be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates.
  • Typical endogenous reference sequences include segments of transcripts of the following genes: ⁇ -actin, GAPDH, ⁇ 2 -microglobulin, ribosomal RNA, and the like.
  • Polymorphism or “genetic variant” means a substitution, inversion, insertion, or deletion of one or more nucleotides at a genetic locus, or a translocation of DNA from one genetic locus to another genetic locus.
  • polymorphism means one of multiple alternative nucleotide sequences that may be present at a genetic locus of an individual and that may comprise a nucleotide substitution, insertion, or deletion with respect to other sequences at the same locus in the same individual, or other individuals within a population.
  • An individual may be homozygous or heterozygous at a genetic locus; that is, an individual may have the same nucleotide sequence in both alleles, or have a different nucleotide sequence in each allele, respectively.
  • insertions or deletions at a genetic locus comprises the addition or the absence of from 1 to 10 nucleotides at such locus, in comparison with the same locus in another individual of a population (or another allele in the same individualO.
  • insertions or deletions are with respect to a major allele at a locus within a population, e.g. an allele present in a population at a frequency of fifty percent or greater.
  • Polynucleotide or “oligonucleotide” are used interchangeably and each mean a linear polymer of natural or modified nucleotide monomers.
  • Monomers making up polynucleotides and oligonucleotides include deoxyribonucleotides, ribonucleotides, 2'-deoxy ⁇ 3'-phosphorothioate nucleosides, peptide nucleic acids (PNAs), and the like, that are capable of specifically binding to a natural 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.
  • Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units.
  • oligonucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units.
  • ATGCCTG a sequence of letters (upper or lower case), such as "ATGCCTG”
  • A denotes deoxyadenosine
  • C denotes deoxycytidine
  • G denotes deoxyguanosine
  • T denotes thymidine
  • I denotes deoxyinosine
  • U denotes uridine.
  • polynucleotides comprise the four natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g. including modified bases, sugars, or internucleosidic linkages. 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 polynucleotides consisting solely of natural nucleotides are required.
  • an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g. single stranded DNA, RNA/DNA duplex, or the like
  • selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references.
  • Primer means an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3 ' end along the template so that an extended duplex is formed.
  • the sequence of nucleotides added during the extension process are determined by the sequence of the template polynucleotide.
  • primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 36 nucleotides.
  • Readout means a parameter, or parameters, which are measured and/or detected that can be converted to a number or value.
  • readout may refer to an actual numerical representation of such collected or recorded data.
  • a readout of fluorescent intensity signals from a microarray is the address and fluorescence intensity of a signal being generated at each hybridization site of the microarray; thus, such a readout may be registered or stored in various ways, for example, as an image of the microarray, as a table of numbers, or the like.
  • Sequence determination or "determining a nucleotide sequence” in reference to polynucleotides includes determination of partial as well as full sequence information of the polynucleotide. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, as well as the express identification and ordering of nucleosides, usually each nucleoside, in a target polynucleotide. The term also includes the determination of the identity, ordering, and locations of one, two, or three of the four types of nucleotides within a target polynucleotide.
  • sequence determination may be effected by identifying the ordering and locations of a single type of nucleotide, e.g. cytosines, within the target polynucleotide "CATCGC " so that its sequence is represented as a binary code, e.g. "100101 ... " for "C-(not C)-(not C)-C-(not C)-C ... " and the like.
  • a single type of nucleotide e.g. cytosines
  • “Specific” or “specificity” in reference to the binding of one molecule to another molecule, such as a binding compound, or probe, for a target analyte means the recognition, contact, and formation of a stable complex between the probe and target, together with substantially less recognition, contact, or complex formation of the probe with other molecules.
  • “specific” in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with another molecules in a reaction or sample, it forms the largest number of the complexes with the second molecule. In one aspect, this largest number is at least fifty percent of all such complexes form by the first molecule.
  • molecules involved in a specific binding event have areas on their surfaces or in cavities giving rise to specific recognition between the molecules binding to each other.
  • specific binding include antibody-antigen interactions, enzyme-substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.
  • contact in reference to specificity or specific binding means two molecules are close enough that weak noncovalent chemical interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules.
  • stable complex in reference to two or more molecules means that such molecules form noncovalently linked aggregates, e.g. by specific binding, that under assay conditions are thermodynamically more favorable than a non-aggregated state.
  • “Spectrally resolvable" in reference to a plurality of fluorescent labels means that the fluorescent emission bands of the labels are sufficiently distinct, i.e. sufficiently non-overlapping, that molecular tags to which the respective labels are attached can be distinguished on the basis of the fluorescent signal generated by the respective labels by standard photodetection systems, e.g. employing a system of band pass filters and photomultiplier tubes, or the like, as exemplified by the systems described in U.S. Pat. Nos. 4,230,558; 4,811,218, or the like, or in Wheeless et al, pgs. 21-76, in Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985).
  • T n is used in reference to “melting temperature.”
  • Melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • Tm 81.5 + 0.41 (% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985).
  • Other references e.g., Allawi, H.T.
  • Terminal means a nucleotide that can be incorporated into a primer by a polymerase extension reaction, wherein the nucleotide prevents subsequent incorporation of nucleotides to the primer and thereby halts polymerase-mediated extension.
  • Typical terminators are nucleoside triphosphates that lack a 3'-hydroxyl substituent and include 2',3'-dideoxyribose, 2',3'- didehydroribose, and 2',3'-dideoxy-3'-haloribose, e.g. 3'-deoxy-3'-fluoro-ribose or 2',3'-dideoxy-3'- fluororibose nucleosides, for example.
  • a ribofuranose analog can be used in terminators, such as 2',3'-dideoxy- ⁇ -D-ribofuranosyl, ⁇ -D-arabinofuranosyl, 3'-deoxy- ⁇ -D- arabinofuranosyl, 3'-arnino-2',3'-dideoxy- ⁇ -D-ribofaranosyl, and 2,3'-dideoxy-3'-fluoro- ⁇ -D- ribofuranosyl.
  • terminators such as 2',3'-dideoxy- ⁇ -D-ribofuranosyl, ⁇ -D-arabinofuranosyl, 3'-deoxy- ⁇ -D- arabinofuranosyl, 3'-arnino-2',3'-dideoxy- ⁇ -D-ribofaranosyl, and 2,3'-dideoxy-3'-fluoro- ⁇ -D- ribofuranosyl.
  • nucleotide terminators also include reversible nucleotide terminators, e.g. Metzker et al. Nucleic Acids Res., 22(20):4259 (1994).
  • Terminators of particular interest are terminators having a capture moiety, such as biotin, or a derivative thereof, e.g. Ju, U.S. patent 5,876,936, which is incorporated herein by reference.
  • a "predetermined terminator” is a terminator that basepairs with a desired nucleoside of a template in a reaction extending a primer.
  • a primer extension step is carried out in each of four different reaction vessels, such that each reaction vessel contains a different terminator.
  • the terminator base pairs with a deoxyadenosine i.e., the "desired" nucleoside
  • the terminator base pairs with a deoxycytidine in a first reaction vessel, the terminator base pairs with a deoxycytidine; in a third reaction vessel, the terminator base pairs with a deoxyguanosine; and in a fourth reaction vessel, the terminator base pairs with a deoxythymidine.
  • the invention provides methods for sorting polynucleotides based on predetermined sequence characteristics to form subpopulations of reduced complexity.
  • sorting methods are used to analyze populations of uniquely tagged polynucleotides, such as genome fragments. That is, mixtures may be formed containing fragments of genomic DNA from different individuals such that each individual's DNA is labeled with a unique oligonucleotide tag.
  • the tags may be replicated, labeled and hybridized to a solid phase support, such as a microarray, to provide a simultaneous readout of sequence information related to the genomic DNA.
  • Predetermined sequence characteristics include, but are not limited to, a unique sequence region at a particular locus, or a series of polymorphisms, such as insertions, deletions, or substitutions, at a series of loci, or the like.
  • a predetermined sequence characteristic can be selected from a wide variety of genomic features including, but not limited to, a segment of a selected exon of a selected gene, a selected intron of a selected gene, a transcription regulatory region of a selected gene, such as a promoter, enhancer, repressor, or the like.
  • a predetermined sequence characteristic can also be a segment of a specific binding site of a DNA-binding protein, a CpG island, a locus control region (LCR), a boundary element of a transcriptional unit, a matrix attachment region (MAR), or the like.
  • sorting of uniquely tagged polynucleotides allows massively parallel operations, such as simultaneously sequencing, genotyping, or haplotyping many thousands of genomic DNA fragments from different genomes.
  • sorting of uniquely tagged genomic DNA fragments includes generating a population of fragments enriched for one or more predetermined exons of the same gene from different genomes and comparing such enriched fragments to a reference sequence to determine fragments whose sequences vary from the reference sequence.
  • fragments having sequences that vary from the reference sequence are isolation from the population of all tagged genomic DNA fragments.
  • Exemplary polynucleotides that may be tagged and sorted in accordance with the invention include fragments of genomic DNA, DNA copies of RNAs, such as cDNAs, and the like. Such exemplary polynucleotides also include predetermined sets of genes or exons or other sequence segments of interest, such as promoter regions and the like, that may be selectively amplified from populations of DNA, such as genomic DNA or cDNA libraries. Exemplary techniques for selectively amplifying and/or isolating selected sequences of DNA from complex mixtures are disclosed in the following references: Faham et al, U.S.
  • a plurality of segments of genomic DNA from multiple individuals may be analyzed simultaneously in a single mixture.
  • such a plurality may be in the range of from ten to several hundred, e.g. 500, or from ten to 200, or from ten to 100.
  • such mixture may be formed from multiple individuals in the range of from ten to several thousand, e.g. 3000, or from ten to 1000, or from ten to several hundred, e.g. 200, 300, 400 or 500.
  • One aspect of the complexity-reducing method of the invention is illustrated in Figs. 1 A-IC.
  • Primer binding site (104) has the same, or substantially the same, sequence whenever it is present. That is, there may be differences in the sequences among the primer binding sites (104) in a population, but the primer selected for the site must anneal and be extended by the extension method employed, e.g. DNA polymerase extension.
  • Primer binding site (104) is an example of a predetermined sequence characteristic of polynucleotides in population (100).
  • Parent population (100) also contains polynucleotides that do not contain either a primer binding site (104) or polymorphic region (102).
  • the invention provides a method for isolating sequences from population (100) that have primer binding sites (104) and polymorphic regions (102). This is accomplished by annealing (110) primers (112) to polynucleotides having primer binding sites (104) to form primer-polynucleotide duplexes (113). After primers (112) are annealed, they are extended to incorporate a predetermined terminator having a capture moiety. Extension may be effected by polymerase activity, chemical or enzymatic ligation, or combinations of both.
  • a terminator is incorporated so that successive incorporations (or at least uncontrolled successive incorporations) are prevented.
  • a primer of a primer-polynucleotide duplexes can be fully complementary to its polynucleotide, or it can be complementary only at a portion of its 3 ' end, for example, complementary over a portion of sufficient length so that a DNA polymerase can extend the primer.
  • the 5' end of the primer can contain other sequences that are not complementary to the polynucleotide, e.g. as disclosed in Faham et al, U.S. patent publication 2003/0104459, or the like.
  • template-dependent extension may also be referred to as "template-dependent extension” to mean a process of extending a primer on a template nucleic acid that produces an extension product, i.e. an oligonucleotide that comprises the primer plus one or more nucleotides, that is complementary to the template nucleic acid.
  • template-dependent extension may be carried out several ways, including chemical ligation, enzymatic ligation, enzymatic polymerization, or the like. Enzymatic extensions are preferred because the requirement for enzymatic recognition increases the specificity of the reaction.
  • such extension is carried out using a polymerase in conventional reaction, wherein a DNA polymerase extends primer (112) in the presence of at least one terminator labeled with a capture moiety.
  • a DNA polymerase extends primer (112) in the presence of at least one terminator labeled with a capture moiety.
  • a single capture moiety e.g. biotin
  • extension may take place in four separate reactions, wherein each reaction has a different terminator, e.g. biotinylated dideoxyadenosine triphosphate, biotinylated dideoxycytidine triphosphate, and so on.
  • terminators may be used in a single reaction.
  • the terminators are dideoxynucleoside triphosphates.
  • Such terminators are available with several different capture moieties, e.g. biotin, fluorescein, dinitrophenol, digoxigenin, and the like (Perkin Elmer Lifesciences).
  • the terminators employed are biotinylated dideoxynucleoside triphosphates (biotin-ddNTPs), whose use in sequencing reactions is described by Ju et al, U.S. patent 5,876,936, which is incorporated by reference.
  • each reaction employing only one of the four terminators, biotin-ddATP, biotin-ddCTP, biotin-ddGTP, or biotin- ddTTP.
  • the ddNTPs without capture moieties are also included to minimize misincorporation.
  • the captured polynucleotides contain the primer extended by a single dideoxynucleotide and thereby selects for the templates with the complementary base at the first position.
  • the complexes captured with dideoxythymidine will have all the templates with an initial adenine, which is of lower complexity than the original collection of templates.
  • primer (112) is extended to incorporate a biotinylated dideoxythymidine (118), after which primer-polynucleotide duplexes having the incorporated biotins are captured with a capture agent, which in this illustration is an avidinated (122) (or streptavidinated) solid support, such as a microbead (120).
  • a capture agent which in this illustration is an avidinated (122) (or streptavidinated) solid support, such as a microbead (120).
  • Captured polynucleotides (126) are separated (128) and polynucleotides are melted from the extended primers to form (130) population (132) that has a lower complexity than that of the parent population (100).
  • Other capture agents include antibodies, especially monoclonal antibodies, that form specific and strong complexes with capture moieties.
  • biotin fluorescein, dinitrophenol, digoxigenin, rhodamine, dansyl, and the like
  • Molecular Probes e.g. Molecular Probes, Eugene, OR
  • a biotin is used as a capture moiety and avidin, streptavidin, or like protein, is used as a capture agent.
  • biotins may be used with the method of the invention, including biotin, desthiobiotin, and the like, e.g. Haugland, Handbook of Fluorescent Probes and Research Reagents, Ninth Edition (Molecular Probes, Eugene, OR).
  • the invention also provides a method of carrying out successive selections using a set of overlapping primers of predetermined sequences to isolate a subset of polynucleotides having a common sequence, i.e. a predetermined sequence characteristic.
  • population (140) of Fig. 1 D is formed by digesting a genome or large DNA fragment with one or more restriction endonucleases followed by the ligation of adaptors (142) and (144), e.g. as may be carried out in a conventional AFLP reactions, U.S. patent 6,045,994, which is incorporated herein by reference.
  • Primers (149) are annealed (146) to polynucleotides (151) and extended, for example, by a DNA polymerase to incorporate biotinylated (150) dideoxynucleotide Ni (148). After capture (152) with streptavidinated microbeads (120), selected polynucleotides are separated from primer- polynucleotide duplexes that were not extended (e.g. primer-polynucleotide duplex (147)) and melted to give population (154). Second primers (157) are selected so that when they anneal they basepair with the first nucleotide of the template polynucleotide.
  • second primers (157) are selected so that they anneal to a binding site that is shifted (160) one base into the polynucleotide, or one base downstream, relative to the binding site of the previous primer. That is, in one embodiment, the three-prime most nucleotide of second primers (157) is Ni. In accordance with the invention, primers may be selected that have binding sites that are shifted downstream by more than one base, e.g. two bases. Second primers (157) are extended with a second terminator (158) and are captured by microbeads (163) having an appropriate capture agent to give selected population (164).
  • Successive cycles of annealing primers, extension, capture, and melting may be carried out with a set of primers that permits the isolation of a subpopulation of polynucleotides that all have the same sequence at a region adjacent to a predetermined restriction site.
  • the selected polynucleotides are amplified to increase the quantity of material for subsequent reactions.
  • amplification is carried out by a conventional linear amplification reaction using a primer that binds to one of the flanking adaptors and a high fidelity DNA polymerase.
  • the number of amplification cycles may be in the range of from 1 to 10, and more preferably, in the range of from 4 to 8.
  • the same number of amplification cycles is carried out in each cycle of extension, capturing, and melting.
  • the invention includes a method for selecting a haplotype that comprises a sequence of SNPs adjacent to known sequence regions that are used as primer binding sites.
  • population of polynucleotides (200) may corresponding restriction fragments of different genomes that contain polymorphic loci 1 through 4 adjacent to primer binding sites (201), (203), (205), and (207), respectively.
  • Restriction fragments making up population (200) have adaptors (209) and (213) attached, which may be the same or different, unless oligonucleotide tags are attached to the fragments, as described more fully below.
  • a haplotype is selected from the mixed population by successively selecting polynucleotides in accordance with the invention using primers that specifically anneal to sites (201), (203), (205), and (207).
  • primer (211) anneals (202) to primer binding site (201) and is extended with a biotinylated dideoxycytidine terminator (210) since polynucleotides with a "G" at locus 1 are desired. Consequently, sequences (204) and (208) are selected (214) as described above using a solid phase support (218) having a capture agent.
  • the selected polynucleotides (216) are melted and the next primer (224) is annealed (222) to binding site (203).
  • Primers (224) are extended (226) with a biotinylated dideoxyguanosine terminator (228), because the next SNP of the desired haplotype is deoxycytidine.
  • Solid phase supports (234) having capture agents are added (232) to the reaction so that the extended primers together with their respective polynucleotides are selected. The process is repeated for the remaining loci until the polynucleotides containing the desired haplotype (270) is finally selected.
  • the selection methods described above may be used in another aspect of the invention in which the population of polynucleotides comprises genomes with unique tags. This aspect is illustrated in Fig. IF. Genomes 1 through N are separately fragmented (182), e.g.
  • fragments (180) to which adaptors (181 and 183) of the invention are attached contains a tag synthesized in accordance with the invention, and both adaptors (181 and 183) contain other sequences, such as primer binding sites and restriction sites, necessary to manipulate the fragments, as described above, for example.
  • sequence-specific sorting method of the invention fragments (184) from a predetermined locus from each of the genomes are selected and combined (186) to form a reaction mixture.
  • fragments with adaptors (181 and 183) may be combined prior to selection.
  • the isolated fragments in the reaction mixture may be analyzed (188) by a variety of techniques to identify SNPs or haplotypes, or the like, after which labeled tags are generated (190) to convey information obtained by the analytical reaction to a readout device, which preferably comprises the hybridization (192) of the labeled tags to a microarray (194), or like device.
  • Microarray (194) contains at individual hybridization sites tag complements for every tag used to label the genomic fragments.
  • any population of polynucleotides may be analyzed by the method of the invention, including restriction digests, libraries of genomic fragments, cDNAs, mRNAs, or the like.
  • populations of polynucleotides analyzed by the invention are genomes of organisms whose sequences are known. Such genomes may be from any organism, including plant, animal, bacteria, or the like.
  • genomic DNA is obtained for medical or diagnostic use, it may be obtained from a wide variety of sources, including tissue biopsies, blood samples, amniotic cells, and the like. Genomic DNA is extracted from such tissues by conventional techniques, e.g. as disclosed in Berger and Kimmel, Editors, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, New York, 1987), or the like.
  • selections can be advanced along templates by producing ladders of successively shortened templates so that, either in parallel or serially, each of a plurality of nucleotide positions in a population of templates can be interrogated by selecting only sequences that differ from one or more predetermined references.
  • a population of sequence variants can be obtained for further analysis, while non-variant sequences can be eliminated from further analysis.
  • Sequence selections are made by extending a primer as described above where dNTPs having a capture moiety are only those that different from the reference sequence(s) at the position of incorporation. For example, if a population of templates each derived from the same locus of 100 different individuals were being analyzed and the references had either an A or a C at position 27 of the template, then only dATP and dCTP would have capture moieties, so that only templates having T or G (i.e., not (A or C)) at position 27 would be selected.
  • a method for advancing a template makes use of type Hs restriction endonucleases, e.g. Sfa NI (5'-GCATC(5/9)), and is similar to the process of "double stepping" disclosed in U.S. patent 5,599,675, which is incorporated herein by reference.
  • “Outer cycle” refers to the use of a type Hs restriction enzyme to shorten a template (or population of templates) in order to provide multiple starting points for sequence-based selection, as described above.
  • the above selection methods may be used to isolate fragments from the same locus of multiple genomes, after which multiple outer cycle steps, e.g.
  • K steps are implemented to generated K templates, each one successively shorter (by the "step” size, e.g. 1-20 nucleotides) than the one generated in a previous iteration of the outer cycle.
  • each of these successively shortened templates is in a separate reaction mixture, so that "inner" cycles of primer extensions and sortings can be implemented of the shortened templates separately.
  • an outer cycle is implemented on a mixture of fragments from multiple loci of each of multiple genomes.
  • the primer employed in the extension reaction i.e. the inner cycle
  • starting material has the following form (SEQ ID NO: 23) (where the biotin is optional):
  • biotin-NN ... NNGCATCAAAAGATCNN ... NN ... NNCGTAGTTTTCTAGNN ...
  • biotin-NN ... NNGCATCAAAAG pATCNN ... NN ... NNCGTAGTTTTCTAGNp N ...
  • N dd represents an added dideoxynucleotide.
  • ligated adaptors of the following form (SEQ ID NO: 25):
  • N* represents a nucleotide having a nuclease-resistant linkage, e.g. a phosphorothioate.
  • the specificity of the ligation reaction is not crucial; it is important merely to link the "top” strands together, preserving sequence.
  • N*N*N*NN ... NNNGCATCAAAAATCW N N N N NN ... NNNCGTAGTTTTNNNN dd N The bottom strand is then destroyed by digesting with T7 exonuclease 6, ⁇ exonuclease, or like enzyme. An aliquot of the remaining strand may then be amplified using a first primer of the form:
  • a second primer containing a T7 polymerase recognition site This material can be used to re- enter the outer cycle.
  • Another aliquot is amplified with a non-biotinylated primer (5 '-NN ... GCATCAAAA) and a primer containing a T7 polymerase recognition site eventually to produce an excess of single strands, using conventional methods.
  • These strands may be sorted using the above sequence-specific sorting method where "N" (italicized) above is G, A, T, or C in four separate tubes.
  • the basic outer cycle process may be modified in many details as would be clear to one of ordinary skill in the art.
  • the number of nucleotides removed in an outer cycle may vary widely by selection of different cleaving enzymes and/or by positioning their recognition sites differently in the adaptors.
  • the number of nucleotides removed in one cycle of an outer cycle process is in the range of from 1 to 20; or in another aspect, in the range of from 1 to 12; or in another aspect, in the range of from 1 to 4; or in another aspect, only a single nucleotide is removed in each outer cycle.
  • the number of outer cycles carried out in an analysis may vary widely depending on the length or lengths of nucleic acid segments that are examined. In one aspect, the number of cycles carried out is in the range sufficient for analyzing from 10 to 500 nucleotides, or from 10 to 100 nucleotides, or from 10 to 50 nucleotides.
  • templates that differ from one or more reference sequences, or haplotypes are sorted so that they may be more fully analyzed by other sequencing methods, e.g. conventional Sanger sequencing.
  • reference sequences may correspond to common haplotypes of a locus or loci being examined.
  • actual reagents e.g. primers
  • sequences corresponding to reference sequences need not be generated.
  • extension (or inner) cycle either each added nucleotide has a different capture moiety, or the nucleotides are added in separate reaction vessels for each different nucleotide. In either case, extensions corresponding to the reference sequences and variants are immediately known simply by selecting the appropriate reaction vessel or capture agents.
  • Population of sequences (275) can be restriction fragments (for example), each from the same locus, e.g. an exon of a preselected gene, of a population of individuals.
  • Each sequence has adaptors (279) and (277) (shown in single stranded form) for carrying out operations on the sequences and for identifying the individual from which a sequence came, for example, using the polynucleotide tagging scheme disclosed below.
  • Nucleotides of region (274) of sequences (275) are shown expressly. The nucleotide sequences of the strands are identical in region (274) except at position three (282) and position seven (283).
  • primers are annealed to adaptor strand (279) in four separate reactions and extended preferably with terminators, such as ddNTPs, such that only "not G's,” that is, ddATP's, ddCTP's, and ddTTP's, have capture moieties, such as biotin.
  • variants from the reference sequence are determined by appropriate selection of dsNTPs that have capture moieties. No sequences are selected from the extension at the first position since all sequences have a "C" at that position. Sequences (275) are then combined and shortened (284), after which the cycle is repeated (287) so that position three of the sequences are adjacent to adaptor strand (279). As above, the sequences are divided into four aliquots where primers (290) are separately extended with the four dNTPs wherein only the "not A" ddNTPs are biotinylated.
  • template (285) has an "A,” which is different from the reference sequence; thus, upon extension, the primer incorporates a biotinylated ddTTP (294), by which sequence (285) can be selected (296) using an avidinated or streptavidinated solid support (298). After selection, sequence (285) is replicated for return to the parent population of sequences and/or for analysis if desired.
  • oligonucleotide tags are used to uniquely label members of a population of polynucleotides.
  • a wide variety of oligonucleotide tags may be employed.
  • oligonucleotide tags are selected from the same set of oligonucleotides that have nucleotide sequences that render them mutually discriminable. That is, annealing conditions, or hybridization conditions, are available so that an oligonucleotide tag of a set forms a stable duplex with essentially only its complement and not with the complements of any other oligonucleotide tag of the same set.
  • a set of mutually discriminable oligonucleotide tags may vary widely in sequence, length, and internal structure.
  • each oligonucleotide tag of such a set differs in sequence from every other member of the same set in at least ten percent of its nucleotide positions.
  • each oligonucleotide tag of such a set differs in sequence from every other member of the same set in at least fifteen percent of its nucleotide positions.
  • oligonucleotide tags are selected from a minimally cross-hybridizing set of oligonucleotides, or assembled from oligonucleotide subunits, i.e. "words,” selected from a minimally cross-hybridizing set of oligonucleotides.
  • words selected from a minimally cross-hybridizing set of oligonucleotides.
  • sequences of oligonucleotides of a minimally cross-hybridizing set differ from the sequences of every other member of the same set by at least two nucleotides.
  • each member of such a set cannot form a duplex (or triplex) with the complement of any other member with less than two mismatches.
  • perfectly matched duplexes of tags and tag complements of the same minimally cross-hybridizing set have approximately the same stability, especially as measured by melting temperature and/or dissociation temperature.
  • Complements of hybridization tags, referred to herein as "tag complements” may comprise natural nucleotides or non-natural nucleotide analogs.
  • Hybridization tags when used with their corresponding tag complements provide a means of enhancing the specificity, or discrimination, of hybridization.
  • minimally cross-hybridizing set also includes sets of 2-mers and 3-mers whose members differ from one another by at least a single nucleotide.
  • Minimally cross-hybridizing sets of oligonucleotide tags and tag complements may be synthesized either combinatorially or individually depending on the size of the set desired and the degree to which cross-hybridization is sought to be minimized (or stated another way, the degree to which specificity is sought to be enhanced).
  • a minimally cross-hybridizing set may consist of a set of individually synthesized 10-mer sequences that differ from each other by at least 4 nucleotides, such set having a maximum size of 332, when constructed as disclosed in Brenner et al, International patent application PCT/US96/09513.
  • a minimally cross-hybridizing set of oligonucleotide tags may also be assembled combinatorially from subunits which themselves are selected from a minimally cross-hybridizing set.
  • a set of minimally cross- hybridizing 12-mers differing from one another by at least three nucleotides may be synthesized by assembling 3 subunits selected from a set of minimally cross-hybridizing 4-mers that each differ from one another by three nucleotides.
  • Such an embodiment gives a maximally sized set of 9 , or
  • a hybridization tag When synthesized combinatorially, a hybridization tag preferably consists of a plurality of subunits, each subunit consisting of an oligonucleotide of 2 to 10 nucleotides in length wherein each subunit is selected from the same minimally cross-hybridizing set.
  • the number of hybridization tags available depends on the number of subunits per tag and on the length of the subunits.
  • oligonucleotide tags are hybridized to their complementary sequences, or "anti-tags," which are attached to a solid phase support, such as a microarray.
  • anti-tags which are attached to a solid phase support, such as a microarray.
  • Sets of words with such properties may be constructed in several ways, including by inserting "commas” between words or by using words that inherently possess the above properties, i.e. which result in so-called “comma-less”tags , as discussed below.
  • Tags of word having commas are readily constructed from the minimally cross- hybridizing sets of words disclosed by Brenner in the several references cited above.
  • Either comma-containing or comma-less tags may be used with the invention; however, comma-less tags are preferred, as they generate the maximum degree of instability in a duplex formed after any small (e.g. 1-3 nucleotide) shift of the tag and anti-tag out of perfect alignment, also sometimes referred to herein as a "change of phase.”
  • oligonucleotide tag (300) consisting of words (302) through (312) may align perfectly with its complement (314) to form a perfectly matched duplex.
  • tags in the same repertoire that also form stable duplexes (318), even though the tag (318) is shifted (316), or out of alignment, by one or more bases with complement (314).
  • the stability of such spurious pairings is very close to that of the perfectly aligned pairings, making it difficult to discriminate between correctly hybridized tags and incorrectly hybridized tags.
  • Such spurious hybridizations can be eliminated by designing tags that have large numbers of mismatches whenever the tag and its complement are shifted one or more bases away from the perfectly aligned configuration.
  • such designs can be accomplished by either introducing "commas” between words, or by designing words that inherently have the property that any shift out of perfect alignment introduces large numbers of stability-destroying mismatches.
  • “commas” may be one or more nucleotides (320) introduced between the words (322) of a tag, as illustrated in Fig. 3B.
  • the commas (320) of tag 321 may consist of G's, while the words (322) may consist of only A's, T's, and Cs.
  • tags (332) and its complement (336) are correctly aligned.
  • absence perfect alignment (338) the stability of any duplex (340) that may form will be vastly lower than that of a perfectly aligned tag and its complement.
  • repertoires of tags without commas may be constructed from words that have the same properties as tags with commas.
  • Such tags with the "comma-less" property are illustrated in Fig. 3D. That is, in order to form a perfectly matched duplex between a tag and a complement, the two must be perfectly aligned.
  • Words for a repertoire of comma-less tags may be constructed in a wide variety of lengths, e.g.
  • words may have lengths in the range of from 4 to 10 nucleotides, and may consist of natural or non-natural nucleotides.
  • words are construct from the four natural nucleotides, A, C, G, and T, whenever the resulting tags are operated on by enzymes.
  • words may be constructed from nucleotides selected from the group consisting of A, C, G, T, and I, when the resulting tags (or anti-tags) are not processed by enzymes.
  • Anti-tags synthesized on a solid phase support may typically be constructed from a wider variety of nucleotides than tags that are processed by enzymes.
  • comma-less tags may be constructed from the following words.
  • each set all four differ in both positions from all the other members of the set, but when the four different sets are compared with each other, one base is held in common with one member of the other set.
  • set I eight different words can be created by combining doublets from set I with doublets from set II in the I-II order and the H-I order. Since each of these sets contain doublets that are the reverse complements of the other, the combinations are made such that none of I-II four-base words are the inverse complements of the H-I four-base words.
  • the I-II words are selected as follows: GTCT, TGTC, ACAG, and CAGA
  • the H-I words can be defined only as follows:
  • tags not used in enzymatic processing such as anti-tags synthesized on a solid phase support
  • deoxyinosine may be employed:
  • tag complements 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, or substantially 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 copies of only one type of tag complement having a particular sequence. The population of such beads or regions contains a repertoire of tag complements each with distinct sequences.
  • the term "repertoire" means the total number of different tags or tag complements in a given set or population.
  • Solid phase supports containing tag complements may take a variety of forms, e.g. particulate, single-piece and planar, such as a glass slide, and may be composed of a variety of materials, e.g. glass, plastic, silicon, polystyrene, or the like.
  • Particulate solid phase supports include microspheres, particularly fluorescently labeled microspheres, e.g. Han et al, Nature Biotechnology, 19: 631-635 (2001); Kettman et al, Cytometry, 33: 234-243 (1998); and the like.
  • hybridization tags are detected by hybridizing them to their complementary sequences on a conventional microarray.
  • microarrays may be manufactured by several alternative techniques, such as photo-lithographic optical methods, e.g. Pirrung et al, U.S. patent 5,143,854, Fodor et al, U.S. patents 5,800,992; 5,445,934; and 5,744,305; fluid channel-delivery methods, e.g. Southern et al, Nucleic Acids Research, 20: 1675-1678 and 1679-1684 (1992); Matson et al, U.S. patent 5,429,807, and Coassin et al, U.S. patents 5,583,211 and 5,554,501 ; spotting methods using functionalized oligonucleotides, e.g. Ghosh et al, U.S.
  • Microarrays used with the invention contain from 50 to 500,000 hybridization sites; or from 100 to 250,000 hybridization sites; or from 100 to 40,000 hybridization sites; and preferably, they contain from 100 to 32,000 hybridization sites; or from 100 to 20,000 hybridization sites; or from 100 to 10,000 hybridization sites.
  • application of hybridization tags to a solid phase support includes three steps: treatment with a pre-hybridization buffer, treatment with a hybridization buffer that includes the probes, and washing under stringent conditions.
  • a pre-hybridization step is employed to suppress potential sites for non-specific binding of probe.
  • pre-hybridization and hybridization buffers have a salt concentration of between about 0.8-1.2 M and a pH between about 7.0 and 8.3.
  • a pre-hybridization buffer comprises one or more blocking agents such as Denhardt's solution, heparin, fragmented denature salmon sperm DNA, bovine serum albumin (BSA), SDS or other detergent, and the like.
  • An exemplary pre-hybridization buffer comprises 6X SSC (or 6X SSPE), 5X Denhardt's solution, 0.5% SDS, and 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, or an equivalent defmed-sequence nucleic acid.
  • pre-hybridization buffer comprises 6X-SSPE-T (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA (pH 7.4), 0.005% Triton X- 100) and 0.5 mg/ml BSA.
  • Pre-hybridization and hybridization buffers may also contain organic solvents, such as formamide to control stringency, tetramethylammonium chloride to negate base- specific effects, and the like.
  • An exemplary hybridization buffer is SSPE-T and the desired concentration of isostringency probe. After hybridization, unbound and non-specifically bound isostringency probe is removed by washing the detection support under stringent conditions.
  • stringency of the wash solution is controlled by temperature, organic solvent concentration, or salt concentration. More preferably, the stringency of the wash conditions are determined to be about 2-5 0 C below the melting temperature of the isostringency probes at the salt concentration and pH of the wash solution. Preferably, the salt concentration of the wash solution is between about 0.01 to 0.1 M. Instruments for measuring optical signals, especially fluorescent signals, from labeled tags hybridized to targets on a microarray are described in the following references which are incorporated by reference: Stern et al, PCT publication WO 95/22058; Resnick et al, U.S. patent 4,125,828; Karnaukhov et al, U.S.
  • microbeads made of controlled pore glass (CPG), highly cross-linked polystyrene, 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. patents 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, NJ, 1993).
  • Microbead supports further include commercially available nucleoside-derivatized CPG and polystyrene beads (e.g. available from Applied
  • microbead Generally, the size and shape of a microbead is not critical; however, microbeads 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.
  • glycidal methacrylate (GMA) beads available from Bangs Laboratories (Carmel, IN) are used as microbeads in the invention.
  • GMA glycidal methacrylate
  • Such microbeads are useful in a variety of sizes and are available with a variety of linkage groups for synthesizing tags and/or tag complements.
  • hybridization codes of the invention consist of five bases and are assembled into hybridization tags following a procedure similar to that described in Brenner and Williams (cited above).
  • hybridization tags are constructed that are complements of the anti-tags attached to solid phase supports, such as microarrays.
  • Such tags have the following form (SEQ ID NO: 9):
  • H 1 and H 2 are words of a hybridization tag as described above, for example 4-mer words. Such words may vary in length depending on the embodiment, but generally are in the range of from 2 to 10 nucleotides in length; or they may be in the range of from 3 to 6 nucleotides in length.
  • One factor in selecting word length is whether they are processed by restriction enzymes, such as type Hs restriction enzymes, whose recognition and cleavage characteristics may dictate word length.
  • restriction enzymes such as type Hs restriction enzymes, whose recognition and cleavage characteristics may dictate word length.
  • 64 such di-words are constructed, cloned in conventional vectors, and the DNA can be obtained thereafter by PCR.
  • the cleavage product includes ends complementary to all of the possible ends of the cleavage product of formula (II).
  • an important feature of the invention is attaching oligonucleotide tags to polynucleotides, such as fragments from a genome.
  • fragments from each different genome For simultaneous analysis of fragments from many different genomes, fragments from each different genome have the same oligonucleotide tag attached.
  • the result on a particular fragment, or subset of fragments may be assessed by using their respective oligonucleotide tags, e.g. by labeling, copying, and hybridizing them to a readout platform, such as a microarray.
  • a readout platform such as a microarray.
  • all fragments of each genome of a population of genomes are labeled with one combination of words selected from a set of eight 5-nucleotide words, or subunits.
  • common- sequence fragments e.g. a restriction fragment from a particular locus, can be selected using the method of the invention.
  • the tags may then be used to convey information about the fragments, e.g.
  • each word is cloned in a plasmid with additional elements for aiding in the construction of oligonucleotide tags.
  • 64 di-words are prepared in separate plasmids as described in Brenner and Williams (cited above), which is incorporated by reference.
  • the single word library contains a ten-base sequence [G/T; G/T; A/T] 3 G/T, where "x/T” is an equal mixture of the two bases "x” and “T” at a particular locus.
  • This element is referred to herein as the "Counting Array” or "CAR” element.
  • about 30 copies of each genome are tagged and each is labeled with one unique sequence.
  • any sorted molecule is found to have a unique sequence for this array, it is not a genome difference that should have multiple sequences, and is likely to represent an error in the process which has resulted in an altered molecule. Note that however much any fragment is amplified that it will always possess the original sequences in the counting array, preserving cardinality as distinct from the concentration of DNA.
  • a plasmid having the following characteristics is constructed: (i) no Sapl site, and (ii) a sequence of restriction sites:
  • This embodiment is designed to attach tags to fragments generated by cleaving with the "4OATC" family of restriction endonucleases. These enzymes permit the generation of the fragments of several different lengths:
  • the final construct has the following structure: ... ... Primer X Primer Y Primer Z
  • a library of 64 vectors each containing one of the 64 possible two-word, or "di-word,” concatenations of words from the 8-word library flanked by primer binding sites.
  • This double-word library is then used essentially as described in Brenner and Williams (cited above) to construct oligonucleotide tags.
  • the first flanking primer binding site is that shown above as “Primer X,” and the other contains a recognition site for Fold, 5'-GGATG(9/13), which contains "GG” and therefore cannot cut any of the words described above.
  • word 1 and word 2 refer to both word sequences and their respective complements.
  • word 1 and word 2 refer to both word sequences and their respective complements.
  • a plasmid digested with Apal and BamHI After annealing the above fragments to form a doublestranded element, it is cloned into a plasmid digested with Apal and BamHI. To assure the accuracy of the incorporation, several clones of each "double word" vector are selected and sequenced. Copies of di-words may be conveniently obtained by PCR using a biotinylated X primer and another primer.
  • a procedure for attaching oligonucleotide tags to up to 4096 different genome for simultaneous analysis in accordance with the invention.
  • the procedure is outlined in Fig. 5.
  • Sixty-four groups of 64 mixtures are formed that each contain fragments from a single genome, each group of 64 being represented in the figure by arrays (502), (504), and (506) of 64 dots.
  • This is base tier (500) of submixtures where fragments from each genome may be identified by its position in such a 64-element array, which may correspond to a well in a multi-well plate, a tube in a rack of tubes, or the like.
  • Intermediate tier of submixtures (510) is formed by attaching a different two-word tag to each different genome, as described below.
  • the two-word tag identifies a genome fragment by giving its location within the 64-element array of submixtures.
  • a different tag A through H is attached and combined (514) to form the first mixture (520) in intermediate tier of mixtures (530).
  • the rest of the groups of 64 genomes are treated the same to produce addition mixtures of intermediate tier (530), e.g. mixtures containing gs ⁇ AA to g 57 ( 5 HH; g 577 AA to g 64 oHH; and so on, have words added and are combined (516) to form submixture (522); and so on.
  • Tagged fragments in submixtures (520) to (524) each have a different word attached and are then combined (532) to form mixture (550) of tagged genomes. More specifically, the procedure may be carried out with the following steps.
  • the eight single word libraries, labeled A-H, are amplified and cut with Sapl to generate the following single-word fragment:
  • 64 genomes are tagged in one batch as follows. 64 reaction vessels are arranged in an 8x8 array wherein each row, 1-8, contains 8 vessels labeled A-H. To each vessel a different Bst YI-digested genome is added, after which a different single- word fragment, A-H, is added to vessels 1-8, in each row to give the following array of reaction vessels with the following single- word fragments:
  • the primer is extended using 5-Me-dCTP to give the following (SEQ ID NO: 19 AND SEQ ID NO: 20):
  • the "GATC” overhang is filled in with dGTP and ligated to the following adaptor containing a primer binding site for sequencing (SEQ ID NO: 22): N 20 GC Me ATCAG N 20 CG TAGTCTAGp ⁇
  • the methylated C in the upper strand protects the lefthand site while the right hand portion of the fragments are manipulated.
  • Words are added as follows. First, the Cs of the bottom strand are replaced with 5-methyl-C's. This is accomplished by denaturing the above fragments, priming with a biotinylated Primer X (5'-biotin-GGGCCCN I0 [Sfa Nl site]N 5 ), copying with 5-Me-CTP, and removing the strands with avidinated support. The fragments are released by cleaving with Sfa Nl to give in each of the eight vessels the sequences:
  • AA-HH AA-HH
  • AA-HH means all 64 di-words from AA to HH.
  • the same operation is separately carried out for every one of the sixty-four batches of 64 genomes each, i.e. genomes 65-128, 129-192, ... and 448-512 to give the following 8 mixtures:

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Abstract

L'invention concerne des méthodes servant à trier des polynucléotides issus d'une population en fonction de caractéristiques de séquences prédéterminées. Dans un aspect, la méthode selon l'invention consiste: à étendre une amorce hybridée à des polynucléotides présentant des caractéristiques de séquences prédéterminées afin d'incorporer un terminateur prédéterminé comprenant une fraction de capture; à capturer les polynucléotides comprenant des amorces étendues au moyen d'un agent de capture se liant spécifiquement à la fraction de capture; et à fusionner les polynucléotides capturés à partir des amorces étendues afin de former une sous-population de polynucléotides présentant les caractéristiques de séquences prédéterminées. Dans un autre aspect, la méthode selon l'invention est mise en oeuvre sur une population de polynucléotides marqués, de sorte que, après qu'une sous-population a été sélectionnée, les membres de cette sous-population puissent être analysés simultanément au moyen des marqueurs uniques sur les polynucléotides pour acheminer des informations analytiques vers un réseau d'hybridation pour un affichage.
PCT/US2006/003688 2005-02-10 2006-02-03 Analyse genetique par tri specifique de sequences Ceased WO2006086209A2 (fr)

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US11/055,187 US7217522B2 (en) 2004-02-12 2005-02-10 Genetic analysis by sequence-specific sorting
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US11/173,465 US7407757B2 (en) 2005-02-10 2005-06-30 Genetic analysis by sequence-specific sorting
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EP1502101B1 (fr) * 2001-09-06 2012-11-07 Straus Holdings Inc. Detection rapide et sensible de cellules et de virus
US20040259118A1 (en) * 2003-06-23 2004-12-23 Macevicz Stephen C. Methods and compositions for nucleic acid sequence analysis

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US10202608B2 (en) 2006-08-31 2019-02-12 Gen9, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
EP2395113A1 (fr) * 2007-06-29 2011-12-14 Population Genetics Technologies Ltd. Procédés et compositions d'isolation des variantes de séquence d'acide nucléique
US8241850B2 (en) 2007-06-29 2012-08-14 Population Genetics Technologies Ltd. Methods and compositions for isolating nucleic acid sequence variants
US7635566B2 (en) * 2007-06-29 2009-12-22 Population Genetics Technologies Ltd. Methods and compositions for isolating nucleic acid sequence variants
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US10457935B2 (en) 2010-11-12 2019-10-29 Gen9, Inc. Protein arrays and methods of using and making the same
US11084014B2 (en) 2010-11-12 2021-08-10 Gen9, Inc. Methods and devices for nucleic acids synthesis
US9752176B2 (en) 2011-06-15 2017-09-05 Ginkgo Bioworks, Inc. Methods for preparative in vitro cloning
US11702662B2 (en) 2011-08-26 2023-07-18 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
US10308931B2 (en) 2012-03-21 2019-06-04 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
WO2013163263A3 (fr) * 2012-04-24 2014-03-06 Gen9, Inc. Procédés de tri d'acides nucléiques et de clonage in vitro multiplex préparatoire
US10927369B2 (en) 2012-04-24 2021-02-23 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
US10081807B2 (en) 2012-04-24 2018-09-25 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
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US12241057B2 (en) 2012-06-25 2025-03-04 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing

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