EP1268859A2 - Procedes de genotypage par analyse d'hybrydation - Google Patents

Procedes de genotypage par analyse d'hybrydation

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Publication number
EP1268859A2
EP1268859A2 EP01934221A EP01934221A EP1268859A2 EP 1268859 A2 EP1268859 A2 EP 1268859A2 EP 01934221 A EP01934221 A EP 01934221A EP 01934221 A EP01934221 A EP 01934221A EP 1268859 A2 EP1268859 A2 EP 1268859A2
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EP
European Patent Office
Prior art keywords
diversity
nucleic acid
array
hybridization
organisms
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EP01934221A
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German (de)
English (en)
Inventor
Andrzej Kilian
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CAMBIA
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Center for Application of Molecular Biology to International Agriculture
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Priority claimed from PCT/IB2001/000833 external-priority patent/WO2001073119A2/fr
Publication of EP1268859A2 publication Critical patent/EP1268859A2/fr
Withdrawn legal-status Critical Current

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Definitions

  • This invention relates generally to determining the genotype of organisms by hybridization analysis and, more specifically, to establishing the relatedness of individual organisms within a species.
  • a genotype is the genetic constitution of an individual or group. Variations in genotype are essential for commercial breeding programs, diagnostics, monitoring genetic-based diseases, following spread of pathogens, detemiining parentage, and the like.
  • genomic DNA While determining the nucleic acid sequence of genomic DNA is one way to unambiguously establish a genotype of an individual, it is not currently practicable to accomplish, especially in organisms with complex genomes.
  • Genotypes can be more readily described in terms of genetic markers.
  • a genetic marker identifies a specific region or locus in the genome. Thus, the more genetic markers, the finer defined is the genotype.
  • a genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual.
  • RFLP restriction fragment length polymorphism
  • SSR single-sequence repeats
  • the present invention discloses methods and compositions for performing high throughput genotype determinations by basing analyses on hybridization of unselected nucleic acids to genomic nucleic acids immobilized to solid state materials, and further provides other related advantages.
  • the present invention relates to methods and compositions for determi-ning and relating genotypes of organisms.
  • a nucleic acid molecule that contains a polymorphism is identified. Two organisms are selected, one may be referred to as a reference organism and the other may be referred to as the tester organism. Nucleic acids from each of these organisms are separately amplified. Amplified material from the ' tester organism is cloned or otherwise separated (by e.g., gel electrophoresis, HPLC), and individual clones or separated amplified material is placed into an addressable array. The amplified material from the reference organism, which contains a detectable label is hybridized to the array. Clones on the array that do not evidence detectable hybridization are thus identified as containing a polymorphism.
  • the genotype of an organism is determined.
  • nucleic acids from two or more organisms are pooled and used to generate a first diversity panel.
  • the diversity panel is generated by amplification.
  • the diversity panel is generated by restriction enzyme digestion, a combination of amplification and restriction digestion, or other means that creates a reproducible pattern.
  • the first diversity panel is then separated on the basis of sequence or molecular weight, e.g., by cloning, gel electrophoresis, HPLC, or the like, and individual elements of the diversity panel, e.g., clones, are placed into an addressable array.
  • Nucleic acids from another organism which may be one of the organisms in the initial pool, the selected organism, is used to generate a second diversity panel.
  • the polymorphisms detected are caused by insertion elements, such as transposons.
  • the diversity panels are generated by amplification, and in some embodiments amplification in conjunction with restriction enzyme digestion and ligation of adapters. Amplification is performed with a primer pair in which one of the primers anneals to a sequence found in a family of insertion elements.
  • the first and second diversity panels are generated by the same technique and using the same primers, enzymes, or methods.
  • the techniques differ, and in yet other embodiments, the techniques are the same but the primers or enzymes used to generate the two diversity panels are different.
  • the second diversity panel contains a detectable label, such as a fluorochrome, chemiluminescent molecule, radiolabel, enzyme, ligand, and the like.
  • a detectable label such as a fluorochrome, chemiluminescent molecule, radiolabel, enzyme, ligand, and the like.
  • the array is then hybridized with the second diversity panel. A pattern of hybridization to the array is established.
  • the genotype of the selected organism is thus determined. Briefly, the more elements of the array that hybridize with the diversity panel of the selected organism, the more related the selected organism is to the organisms constituting the array. By generating a diversity panel from each of the organisms in the pool and hybridizing them individually to the array, the genotypes and the relatedness of all the organisms can be determined. .
  • a first diversity panel is generated and placed onto an array as described for the second aspect.
  • the array will thus comprise the genomes of two or more organisms.
  • a second diversity panel is generated from a selected organism, that may or may not be represented in the first diversity panel.
  • the second diversity panel is hybridized to the array, and a pattern of hybridization is detected. The genotype ofthe selected organism is established.
  • a third, fourth, and so on diversity panels are generated from individual organisms and mixed with the second diversity panel.
  • the second, third, and so on diversity panels contain a detectable label, and each diversity panel contains a label distinguishable from the others.
  • the labels are fluorochromes or mass-spectometry tags.
  • the mixture of diversity panels is hybridized to the array, and a pattern of hybridization with each diversity panel is detected. The genotypes of the selected organisms are thus determined from the patterns of hybridization.
  • genomic nucleic acids from two or more organisms are digested with a restriction enzyme.
  • the restriction enzyme may be an enzyme sensitive to methylation.
  • the polymorphisms detected are modifications (methylation) of bases.
  • fragments are selected on the basis of size to comprise a pool of fragments in a desired size range.
  • the digested fragments are cloned into a vector and placed into an addressable array on a solid surface, such as a glass slide.
  • Another organism whose genotype is to be determined (called here organism X), and which may or may not be the same orgamsm as one in the first group, is digested with the same restriction enzyme.
  • adapter sequences are ligated to the fragments and also used as primers for amplification.
  • the amplified fragments are also labeled with one of the labels described below.
  • Labeled fragments are hybridized to the addressable array, nonhybridized fragments are washed off, and the array is then analyzed for the label. In this way a pattern of hybridization is obtained. That pattern is the genotype of the organism X.
  • an element in the array hybridizes, it indicates that the organisms share sequence similarity for that fragment.
  • an element in the array does not hybridize, it indicates a polymorphism.
  • the polymorphism is analogous to a restriction fragment length polymorphism and arises because the restriction fragment in organism X is too long to be amplified or too short to hybridize.
  • kits and arrays are provided that comprise diversity panels for genotyping.
  • Figures 1A, IB, and 1C present a schematic representation of various embodiments of the present invention.
  • A Generation of a diversity panel. Genomic DNAs of various organisms to be studied are pooled together. The DNA is cut with a chosen restriction enzyme and ligated to adapters. The complexity of the sub-genomic profile is reduced in this case by amplification using primers with selective overhangs. The amplified sub-genomic fragments are cloned. Clones are selected and inserts are amplified, purified and arrayed onto the solid support.
  • B Contrasting two samples using diversity array technology. Two genomic samples are converted to diversity panels comprising sub- genomic samples.
  • Each sub-genomic sample is labeled with a green or red fluorescent dye, mixed and hybridized to the diversity array.
  • the ratio of green/red signal intensity is measured at each array element. Significant differences in the signal ratio indicate array elements (and the relevant fragment of the genome) for which the two samples differ.
  • C Genetic fingerprinting.
  • the DNA sample for analysis is converted to a sub-genomic sample and labeled with green fluorescent dye. Fragments of the cloning vector common to all elements of the array are labeled with red fluorescence and hybridized to the diversity panels together with the sub-genomic sample.
  • the ratio of signal intensity is measured at each array feature. The ratios across the diversity array provide genetic fingerprint information for the sample analyzed.
  • Figures 2 A and 2B show differences between fingerprints of two rice cultivars, IR64 and Millin.
  • A Synthetic array image of 96 spots printed 4 times from an EcoRI-generated diversity panel. The rice cultivars IR64 and Millin are labeled with Cy3- green and Cy5-red respectively.
  • B Histogram of green to red normalized signal intensity ratios shows tri-modal distribution. The majority ofthe array features show signal intensity ratios are around 1 indicating equal hybridization intensity for Millin and IR64. The green and red "tails" are seen at signal intensity ratios above 2.9 indicate features ofthe diversity panel that differentiate IR64 and Millin DNA.
  • FIGs 3A and 3B Two clones (F4 and F8), representing two polymorphic features on the EcoRI diversity panel are used as molecular probes.
  • Four varieties of rice are analyzed simultaneously, lane 1, Bala; lane 2, Millin; lane 3, IR64, lane 4, IR20.
  • B Hybridization of labeled F4 and F8 probes to Southern blots of diversity panels of sub-genomic samples generated from genomic DNA samples.
  • Figure 4 shows the result of hybridization of monomorphic clone FI 1 to EcoRI-digested genomic DNA from strains Millin, Bala, IR20, and IR64.
  • Figures 5A and 5B show hybridization of Cy3-labeled IR20 diversity panel and Cy5-labeled Millin diversity panel (Fig 5A) and Cy3-labeled IR64 diversity panel and Cy5-labeled Millin diversity panel (Fig 5B) to duplicate addressable arrays of a mixture of diversity panels.
  • Figures 6A and 6B show hybridization of Cy3-labeled IR20 diversity panel and Cy5-labeled Millin diversity panel (Fig 6A) and Cy3-labeled IR20 diversity panel and Cy5-labeled vector DNA (Fig 6B) to duplicate addressable arrays of a mixture of diversity panels.
  • Figures 7A, 7B, and 7C show cumulative distribution functions for non- polymorphic fragments (A), polymorphic fragments (B), and a reference fragment (C).
  • A Cumulative distribution function of log transformed normalized signal ratios for 4 different non-polymorphic spots across 18 different slides. Classification as non-polymorphic is based on the monomodal distribution of the ratios across the 18 slides.
  • B Cumulative distribution, function of log transformed normalized signal ratios for 4 different polymorphic spots across 18 different slides. Classification as polymorphic is based on a clear bimodal distribution across the 18 slides. The algorithm calculates the best value for separation of the high (value of 1) and low (value of 0) clusters shown as a cross on the curves.
  • Figure 8 presents a histogram of unique and replicate features from the Msp ⁇ diversity panel. Clones are considered to be replicates if they have the same apparent gel mobility and the same polymorphism patterns among the rice cultivars analyzed. A total of 50 polymorphic spots are analyzed here. The red bars indicate the actual numbers of spots found in each category; the blue bars indicate the expected total number, of spots in the diversity panel in each category by extrapolation from 50 to 384 spots in the panel.
  • Figure 9 shows dendrograms generated from Mspl (A) and Pstl (B) diversity panels.
  • Figure 10 presents the results of a reconstruction experiment using mixed (rice and several microorganisms) diversity panels.
  • a Millin diversity panel is labeled with red fluorescent dye and an Enterobacter spiked Millin diversity panel is labeled with green fluorescent dye.
  • the image and histogram are created using the Pathways program.
  • the left half of the array (mostly yellow features) represents rice Mspl diversity array.
  • the right half of the array contains features from Mspl diversity panels from seven bacterial species and one from yeast.
  • the green spots in the right part of the array correspond to the elements ofthe panel developed from the Enterobacter DNA source.
  • B Histogram ofthe signal ratios for the array presented at (A).
  • the Enterobacter spike is detected as the green peak seen at the left edge ofthe distribution.
  • Figure 11 presents the result of a diversity array containing DNA from 3 barley cultivars (Steptoe, Morex, Harrington) and a wild barley Hordeum spont ⁇ neum hybridized with Cy3-labeled Morex diversity panel and Cy5-labeled Steptoe diversity panel.
  • Figure 12 presents the result of a mouse cDNA diversity array hybridized with Cy3-labeled C57B1/6 diversity panel and Cy5 labeled NOD K diversity panel.
  • Figure 13 presents the result of a rice diversity array hybridized with Cy5 labeled callus-diversity panel and a Cy5-labeled seedling root diversity panel (upper array) an a Cy5-labeled callus diversity panel and a Cy3-labeled immature embryo diversity panel:
  • Figure 14 presents the result of a Southern hybridization of various clones identified as differentially methylated in fertilized ovary an stigma to DNA prepared from 12 different diversity panels.
  • an "addressable array” or an “array” means a workspace in which nucleic acid molecules are positioned in discrete locations, which can be either physically or temporally defined, such that each location is uniquely identifiable.
  • the workspace is a solid substrate in which the locations are an identifiable pattern or at regular intervals. Examples of substrates suitable for this invention include, but are not limited to, glass slides, silicon chips, or light fiber optic tubes.
  • a "fingerprint” comprises a distinct pattern of nucleic acid molecules that is a characteristic of the genotype of the organism that the nucleic acids are prepared from.
  • the patterns can be generated by a variety of techniques, such as restriction enzyme, digestion, amplification, a combination of enzyme digestion and amplification, or other method.
  • Fingerprints can reveal sequence differences between nucleic acid samples and can be used to establish a genotype of an organism or groups of organisms. Generally, fingerprints are used to analyze and compare DNA from different species or different individuals of the same species. The differences that are detected are called polymorphisms, if pre-existing in the population, individual, or gene pool, or mutations, if exogenously or spontaneously induced of newly emergent. The precise names given to the differences, however, does not change the outcomes.
  • a “diversity panel” as used herein refers to nucleic acid fragments prepared from organismal nucleic acids (e.g., genomic DNA) by a method that can reveal polymorphisms or mutations (e.g., sequence differences) between samples.
  • a diversity panel is applied to an array, it is called herein a “diversity array.”
  • organism refers to an individual entity or a uniform set of individuals (e.g., species, strain, etc.).
  • polymorphism and “mutation” mean a difference in DNA sequences among individuals. Differences include, without limitation, changes, modifications (e.g., methylation, bromination, animation, and the like), insertions, and deletions or combinations of these differences and may involve one or more bases.
  • changes e.g., changes, modifications (e.g., methylation, bromination, animation, and the like), insertions, and deletions or combinations of these differences and may involve one or more bases.
  • the present invention provides addressable arrays, also referred to herein as arrays, comprising diversity panels of nucleic acid molecules, in which the molecules on the array are addressable or uniquely identifiable in some fashion.
  • these diversity panels are generated from nucleic acid samples isolated from multiple organisms.
  • a diversity panel refers to nucleic acid fragments prepared from organismal nucleic acids by a method that can reveal sequence differences between nucleic acid samples. As taught herein, a variety of methods may be used to generate diversity arrays.
  • the nucleic acid products of the diversity panel are separated for application in a uniquely addressable format, generally onto. a substrate, hereinafter called an array or an addressable array. Separation may be achieved on the basis of physical parameters, e.g., length, molecular weight, or by genetic methods, e.g., cloning.
  • the separated diversity panel is then delivered onto a substrate to create an addressable array.
  • nucleic acid molecules are deposited or synthesized on a glass or silicon wafer in an ordered array.
  • Other types of arrays can also be used, such as those that comprise nucleic acid molecules immobilized on microspheres that are uniquely encoded and randomly deposited in wells of a chemically-etched optical imaging fiber.
  • the codes on the beads or particles permit positional registration of beads of a particular sensor type after assembly.
  • the addressing is accomplished by the unique coding signature of each microsphere.
  • nucleic acids for generating diversity panels are isolated from a variety of organisms.
  • Exemplary organisms include viruses (e.g., HIN and other lentiviruses, papilloma viruses, cytomegalovirus (CMV), retroviruses, hepadnaviruses, etc.); bacteria (e.g., enterobacteria, rhizhobia, Hemophilus, etc.); plants, including commercially important crops and weedy plants; fungi, animals, including parasites (e.g., malaria, Giardia, etc.), food animals, rare or endangered species (e.g., condors, Zealandn devils, spotted owl, etc.); and humans.
  • viruses e.g., HIN and other lentiviruses, papilloma viruses, cytomegalovirus (CMV), retroviruses, hepadnaviruses, etc.
  • bacteria e.g., enterobacteria, r
  • the cellular source of the nucleic acids for generating diversity panels may be genomic DNA, genomic RNA, such as for retroviruses, organelle DNA, such as mitochondrial DNA, mRNA or cDNA, and the like. Methods for isolation and preparation of nucleic acid molecules are well known (see, e.g., Ausebel et al. "Current Protocols in Molecular Biology” Greene Publishing, 2000).
  • the nucleic acid molecules used to generate diversity panels may furthermore be a mixture of two or more of these types of nucleic acids-
  • the source ofthe nucleic acids may be from multiple organisms or specific sub-fractions of an organism.
  • a soil sample may contain a variety of bacterial species, animals, protozoa, plant parts and the like.
  • mRNA or cDNAs
  • the choice of the cellular source depends in part upon the complexity of the organism, for example a multicellular versus unicellular organism, and the intended use ofthe fingerprint analysis.
  • generating a diversity panel entails using a method that can reveal sequence differences between nucleic acid samples. Then by determining and comparing the fingerprints of different DNA samples, the genetic relatedhess of the organisms may be established.
  • At least two diversity panels are generated.
  • one panel is arrayed onto a solid substrate and hybridized to the other panel which is in liquid phase and is the panel being fingerprinted.
  • Either the same method or different methods may be used to generate the two or more diversity panels. While it is not necessary to reduce the complexity of the nucleic acids when generating diversity panels for this invention, at times it may be desirable to do so. Many ofthe methods described herein will result in a diversity panel with reduced complexity compared to the starting nucleic acids.
  • the diversity panel that is being fingerprinted can be a subset or a superset of the diversity panel that is arrayed. In preferred embodiments, the probing diversity panel is a superset ofthe arrayed diversity panel. a. Amplification methods
  • a wide variety of amplification methods may be used to generate diversity panels. Such methods include adapter-mediated amplification (U.S. Patent No. 5,710,000); U.S. Patent No. 5,728,524, AFLP (U.S. Patent No. 6,100,030) and other indexing methods (U.S. Patent No. 5,994,068; U.S. Patent No. 5,858,656; U.S. Patent No. 5,508,169), arbitrarily-primed polymerase chain reaction (U.S. Patent No. 5,487,985; U.S. Patent No. 5,413,909; U.S. Patent No. 5,126,239; U.S. Patent No. 5,861,245; U.S. Patent No.
  • AFLP AFLP
  • complexity is reduced by digesting the DNA with a restriction enzyme, ligating adapters to the fragments, and then amplifying the fragments using a primer that corresponds to the adapter and restriction site sequences and contains one or more bases at the 3' end of the primer. If the primer has one extra base, on average, only 1/16 of the fragments will amplify (only 1 in 4 fragments will have a complement to the extra base at one end of the fragment and 1 in 4 will have a complement at the other end of the fragment).
  • the choice of primers will determine, at least in part, the fraction ofthe genome that is represented in the diversity panel. For example, more extra bases at the 3' end of the primer or primers used for amplification will result in a smaller fraction of the genome that will be amplified.
  • Other parameters that can be altered to control the fraction of the represented genome include the DNA polymerase used, such as whether the enzyme can synthesize long stretches of nucleic acids, amplification reaction conditions, such as cycling times and temperatures, amount or type of cofactor in the reaction and the like. These and other parameters are known to those in the art and are widely used to affect the outcome of amplifications.
  • regions comprising insertion elements are amplified. Insertion elements are common in some organisms, may be mobile or immobilized, and many groups of such elements have been described. For example, transposable elements in plants (e.g., Ac, Ds, miniature inverted-repeat transposable elements (MITE) elements), insects (e.g., Drosophila P, gypsy), fungi (e.g., impala element, Scooter), animals (e.g., Tigger, mariner-like elements, B2 elements, long- interspersed elements (LINE)), bacteria, and the like, are well known and characterized.
  • transposable elements in plants e.g., Ac, Ds, miniature inverted-repeat transposable elements (MITE) elements
  • insects e.g., Drosophila P, gypsy
  • fungi e.g., impala element, Scooter
  • animals e.g., Tigger, mariner-like elements, B2 elements, long- interspersed
  • Amplification of these regions such that polymorphisms are revealed may be achieved with
  • a suitable primer pair comprises a primer that anneals to sequences that are conserved in the chosen family of insertion elements and the second primer anneals to genomic sequences flanking one side of the insertion.
  • the sequence ofthe second primer may be chosen arbitrarily, such as for the arbitrarily-primed PCR methods cited above.
  • the sequence can comprise (ordered from the 3' end) five (or more) arbitrarily chosen bases optionally linked to several or more bases in which all four bases are represented at each position followed by a defined sequence of at least 11 bases (e.g., at least 12 bases, at least 13 bases, at least 14 bases, and so on).
  • the first round of amplification uses this primer pair and to obtain a greater degree of specificity, subsequent rounds of amplification use the first primer and for the second primer use the defined sequence.
  • primer sequences such as incorporating a restriction site and the like, and variation on methodology, such as performing nested PCR, are well known and commonly employed by those skilled in the art.
  • the nucleic acid molecules are digested first with a restriction enzyme, preferably one that does not cut within the insertion element.
  • Adapters are ligated to the fragments, and the fragments are amplified with.
  • a primer pair in which the first primer anneals to sequences that are conserved in the chosen family of insertion elements and the second primer anneals to the adapter sequence.
  • restriction digestion using enzymes that recognize at least a six base sequence containing one or more degenerate bases, enzymes that cut infrequently, enzymes that cut DNA both 5' and 3' of the recognition sequence, enzymes that are sensitive or insensitive to methylation, or the like may be used.
  • Other methods include primer-directed synthesis of DNA and the like.
  • fragments are generated by restriction enzyme digestion and ligated with an adapter sequence. These ligated fragments are then amplified with primers comprising the adapter sequence.
  • Other exemplary embodiments are presented above.
  • the discrete nucleotide sequences ofthe diversity panel are preferably separated prior to applying them to the array.
  • the discrete sequences of the diversity panel that are used to probe the array are preferably not separated. Separation may be achieved by any of a variety of methods. Such methods are known in the art and include, but are not limited to, cloning, gel electrophoresis, chromatography, e.g., HPLC, and dilution.
  • the diversity panel products are cloned into a suitable vector. Techniques for cloning are well known in the art (see e.g., Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing, 1999).
  • the products need to be prepared.
  • the products will either be digested with one or more appropriate restriction enzymes or treated with a DNA polymerase (e.g., E. coli DNA pol I) in the presence of all four dNTPs to produce blunt ends.
  • the diversity panel products are then ligated to the cloning vector.
  • the cloning vector is one that will replicate in bacteria.
  • Many such vectors are commercially available (New England Biolabs, MA USA; Invitrogen, CA, USA; etc.) and include pBluescript, pET series vectors, pUC series vectors, and the like.
  • the recombinants are transformed- into a bacterial host, typically E. coli, and transformed bacteria are selected for or screened for.
  • the diversity panel products may be separated by gel electrophoresis, including capillary electrophoresis.
  • Apparatuses for capillary electrophoresis are commercially available (e.g., Hewlett-Packard; CA USA; SpectruMedix, PA, USA).
  • separation by electrophoresis fractionates the nucleic acids by length, to an approximation.
  • the separated diversity panel products are collected by means known in the art and transferred to the array substrate.
  • chromatography can also be employed. Such technologies include HPLC (high-performance liquid chromatography) and matched ion polynucleotide chromatography (Transgenomic, Inc. USA; U.S. Patent No. 5,986,085; U.S. Patent No. 5,997,742).
  • HPLC high-performance liquid chromatography
  • matched ion polynucleotide chromatography Transgenomic, Inc. USA; U.S. Patent No. 5,986,085; U.S. Patent No. 5,997,742
  • Another technique for separation, although less efficient that the other methods, is dilution of the diversity panel sample to a point where the sample drop to be applied to the array contains a discrete nucleotide molecule.
  • nucleic acid molecule can be directly bound to the solid support or bound through a linker arm, which is typically positioned between the nucleic acid sequence and the solid support.
  • a linker arm that increases the distance between the nucleic acid molecule and the substrate can increase hybridization efficiency. There are a number of ways to position a linker arm.
  • the solid support is coated with a polymeric layer that provides linker arms with a lot of reactive ends/sites.
  • a common example of this type is glass slides coated with polylysine (see, U.S. Patent No. 5667976), which are commercially available *
  • the linker arm may be synthesized as part of or conjugated to the nucleic acid molecule, and then this complex is bonded to the solid support.
  • one approach takes advantage of the extremely high affinity biotin-streptavidin interaction.
  • streptavidin-biotinylated reaction is stable enough to withstand stringent washing conditions and is sufficiently stable that it is not cleaved by laser pulses used in some detection systems, such as matrix- assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Therefore, streptavidin may be covalently attached to a solid support, and the nucleic acid molecule is labeled with a biotin group (or vice versa). The biotinylated nucleic acid molecule effectively sticks wherever it is placed on the streptavidin-covered support surface.
  • MALDI-TOF matrix- assisted laser desorption/ionization time of flight
  • an amino-coated silicon wafer is reacted with the «-hydroxysuccinimido-ester of biotin and complexed with streptavidin.
  • Biotinylated oligonucleotides are bound to the surface at a concentration of about 20 finol DNA per mm 2 .
  • the support is coated with hydrazide groups, then treated with carbodiimide.
  • Carboxy-modified nucleic acid molecules are then coupled to the treated support.
  • Epoxide-based chemistries are also being employed with amine modified oligonucleotides.
  • Other chemistries for coupling nucleic acid molecules to solid substrates are known to those of skill in the art.
  • the nucleic acid molecules must be delivered to the substrate material. Because of the miniaturization of the arrays, delivery techniques must be capable of positioning very small amounts of liquids (e.g., less than 1 nanoliter) in very small regions (e.g., 100 ⁇ m diameter dots), very close to one another (e.g., 250 ⁇ m separation) and amenable to automation. Several techniques and apparatus are available to achieve such delivery. Among these are mechanical mechanisms (e.g., arrayers from GeneticMicroSystems, MA, USA) and ink-jet technology. Very fine pipets may also be used.
  • a 96-well format with fixation of the nucleic acids to a nitrocellulose or nylon membrane may also be employed.
  • the probes After the nucleic acid molecules have been bound to the solid support, it is often essential to block reactive sites on the solid support that are not consumed in binding to the nucleic acid molecule. Otherwise, the probes will, to some extent, bind directly to the solid support itself, giving rise to so-called non-specific binding. Non-specific binding can defeat the ability to detect low levels of specific binding.
  • a variety of effective blocking agents e.g., milk powder, serum albumin or other proteins with free amine groups, polyvinylpyrrolidine
  • the choice depends at least in part upon the binding chemistry.
  • the nucleic acid molecules of the diversity panel that are used to probe the array are preferably directly detectable.
  • a detectable molecule also referred to herein as a label
  • a label will be incorporated or added to the diversity panel nucleic acid sequences.
  • Many types of molecules can be used within the context of this invention. Such molecules include, but are not limited to, fluorochromes, chemiluminescent molecules, chromogenic molecules, radioactive molecules, mass spectometry tags, proteins, and the like.
  • Other labels will be readily apparent to one skilled in the art- Indirect detection can also be used within the context of this' invention. Proteins and other molecules are available that will bind to double-stranded DNA but not to single- stranded DNA. Thus, hybridization can be measured.
  • diversity panels that are used as probes may be mixed prior to hybridization as long as each diversity panel can be distinguished.
  • the products of each diversity panel in the mixture comprises a different detectable molecule.
  • the number of diversity panels that can then be mixed and applied to the array at a single' time is dependent on the number of distinguishable detectable molecules.
  • diversity panel products are labeled with fluorochromes.
  • fluorochromes A plethora of fluorochromes are commercially available or can be chemically synthesized. An extensive list of suitable fluorochromes, procedures for using them and detecting them is available in "Handbook of Fluorescent Probes and Research
  • the nucleic acid molecules are directly or indirectly coupled to an enzyme.
  • a chromogenic substrate is applied and the colored product is detected by a camera, such as a charge-coupled camera.
  • enzymes include alkaline phosphatase, horseradish peroxidase and the like.
  • the invention also provides methods of labeling nucleic acid molecules with cleavable mass spectrometry tags (CMST) (see for example, U.S. Patent No: 60279890). ⁇ After an assay is complete, and the uniquely CMST-labeled probes are distributed across the array, a laser beam is sequentially directed to each member ofthe array.
  • CMST cleavable mass spectrometry tags
  • the light from the laser beam both cleaves the unique tag from the tag-nucleic acid molecule conjugate and volatilizes it.
  • the volatilized tag is directed into a mass spectrometer. Based on the mass spectrum ofthe tag and knowledge of how the tagged nucleotides were prepared, one can unambiguously identify the nucleic acid molecules to which the tag was attached (see, e.g., WO9905319).
  • the nucleic acids can be labeled readily by any of a variety of techniques.
  • the nucleic acids can be labeled during the reaction by incorporation of a labeled dNTP or use of labeled amplification primer.
  • the amplification primers include a promoter for an RNA polymerase, a post- reaction labeling can be achieved by synthesizing RNA in the presence of labeled NTPs.
  • Amplified fragments that were unlabeled during amplification or unamplified nucleic acid molecules can be labeled by one of a number of end labeling techniques or by a transcription method, such as nick-translation, random-primed DNA synthesis.
  • the invention provides hybridization of a diversity panel to a diversity array, which is an addressable array containing products of diversity panels.
  • stringent hybridization and washing conditions are used for nucleic acid molecules over about 500 bp.
  • Stringent hybridization conditions include a solution comprising about 1 M Na+ at 25° to 30°C below the Tm; e.g., 5 x SSPE, 0.5% SDS, at 65°C; see, Ausubel, et al, Current Protocols in Molecular Biology, Greene Publishing, 1995; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989).
  • Tm is dependent on both the G+C content and the concentration of Na+.
  • Tm 40°C below Tm, and are used for short fragments, e.g., less than about.500 bp.
  • the Tm decreases about 1.5°C for every fewer 50 bp than 500.
  • a formula for calculating Tm is 2°C for each AT pair and 4°C for each GC pair. Very high stringency hybridizations are performed at conditions approximately 10°C below Tm.
  • Hybridization conditions are tailored to the length and GC content of the oligonucleotide. Suitable hybridization conditions may be found in Sambrook et al., supra, Ausubel et al., supra, and furthermore hybridization solutions may contain additives such as tetramethylammonium chloride or other chaotropic reagents or hybotropic reagents to increase specificity of hybridization (see for example, PCT/US97/17413).
  • Hybridization may be detected in a variety of ways and with a variety of equipment.
  • the methods may be categorized as those that rely upon detectable molecules incorporated into the diversity panels and those that rely upon measurable properties of double-stranded nucleic acids (i.e., hybridized nucleic acids) that distinguish them from single-stranded nucleic acids (i.e., unhybridized nucleic acids).
  • the latter category of methods includes intercalation of dyes, such as ethidium bromide, into double- stranded nucleic acids, differential absorbance properties of double and single stranded nucleic acids, binding of proteins that preferentially bind double-stranded nucleic acids, and the like.
  • the diversity panels applied to the addressable arrays are labeled with a detectable molecule.
  • labels are discussed above.
  • some means of detecting a successful reaction must be addressed.
  • the means of detection depend on the type of label used. For example, if a radioactive label is used, autoradiography or storage phosphor screens (Phosphorlmager) are common methods of detection.
  • Other systems including chemiluminescent and fluorescent labels in conjunction with autoradiography, charge-coupled cameras or confocal microscopy, are part of an arsenal of detection systems.
  • An alternative detection system that can be used with radioactive, fluorescent or chemiluminescent labels is a CCD integrated silicon wafer.
  • a charge-coupled device designed to detect high energy beta particles or photons, is placed in direct contact with a silicon support for an array.
  • a radioisotope decay product or photon is generated.
  • Electron-hole pairs are generated in the silicon and then electrons are collected by the CCD.
  • An alternative detection system for fluorescent molecules is a lens based camera detecting one or more fluorescent labels. As mentioned above, these cameras include epifluorescent microscopes, confocal microscopes, and charge-coupled cameras.
  • a laser excites a fluorescent label, the emitted light is collected through a bandpass filter, and the signal is detected by a photomultiplier tube that has electronics for counting photons.
  • labels are also amenable to use with either a lens-based camera or a CCD.
  • chemiluminescent labels or chromogenic substrates can be detected with a lens-based charge-coupled camera.
  • the label is a cleavable mass-spectrometry tag.
  • Such labels are then detected using a mass-spectrometer.
  • Many detection systems are commercially available (e.g., Affymetrix, Santa Clara, GA).
  • Affymetrix e.g., Affymetrix, Santa Clara, GA.
  • One skilled in the art is able to choose an appropriate detection means and equipment for the label used. 2. Analysis
  • a genotype of an organism is determined by the pattern of hybridization. Patterns can be expressed as presence or absence of hybridization, the degree of hybridization, or some combination of these. The simplest analysis is performed .by determining the presence or absence of hybridization. When the complexity ofthe genome ' - of the organism to be genotyped is greater than the complexity of the genome(s) . represented on the . array, the absence of hybridization conclusively signifies a polymorphism. When the complexity is less than on the array, the absence of hybridization can signify either a polymorphism or a lack of representation of those sequences in the probing diversity panel. The presence of hybridization, however, does not necessarily signify the absence of a polymorphism under either scenario. As described in more detail below, the pattern of hybridization is informative.
  • each addressable area is queried for hybridization using a method appropriate to the label. For example, when fluorescent labels are used, such as Cy3 and Cy5, both green and red signals are assayed.
  • a method appropriate to the label For example, when fluorescent labels are used, such as Cy3 and Cy5, both green and red signals are assayed.
  • positive and negative controls are included on the array, signals are compared to the controls and each addressable area is assigned a value, e.g., 1 for detectable hybridization and 0 for no detectable hybridization. In general, a value of 1 is assigned for detection over a threshold level and 0 assigned for detection under a threshold level. It will be appreciated by those skilled in the art that detection of polymorphisms is based primarily on finding a binary distribution of signal values for any particular array feature when hybridized with multiple diversity panels..
  • the panels are the same as those used to create the diversity array (see Example 5).
  • a diversity panel is generated from a heterozygote for a polymorphism, one will then detect a trimodal distribution.
  • two threshold values are calculated, the: first threshold separates the "0" cluster (lack of hybridization) from the "0/1" cluster (heterozygote) and the second threshold separates the "0/1” cluster from the "1" cluster (hybridization present).
  • Conventional statistical methods may be used to determine the threshold levels.
  • the genotype of the organism may then be expressed as a value for each addressable area.
  • the addressable array is a 96- spot format (a grid of 8 rows (A-G) x 12 columns (1-12)), and the value for hybridization is
  • visualization of a hypothetical genotype from one such grid may look like:
  • genotyping by hybridization facilitates many different genetic studies, such as breeding of animals or plants, trait selection, introgression of traits, genetic disease diagnosis, forensic analysis, viral family detection, genomic mapping, determining origin of germplasm, establishing relatedness of germplasm, and the like. 1. Genotvping / detection of polymorphisms
  • this invention provides methods and compositions for establishing the genotype of an organism.
  • the genotype is expressed as presence/absence or extent of hybridization to individual nucleic acid molecules from two or more organisms.
  • genotypes have been expressed in such ways as complete nucleotide sequence, explicit restriction fragment or amplified fragment lengths, a collection of genetic traits and the like. The present invention now allows genotypes to be written as it were by hybridization profiles.
  • genotyping is the determination of a number of individuals or strains within a species.
  • samples of a plant species are collected from around the world. Nucleic acids are extracted from these individuals.
  • Genotypes of each individual are determined using the methods taught herein. Comparisons of the genotypes can reveal the relatedness of the individuals. Briefly, the closer the patterns of hybridization, the more related the individuals. In this way, for example gene flow can be documented.
  • the gene flow or relatedness of viruses can be tracked.
  • the genotype of HTV infections is becoming crucial for predicting disease progression, selecting effective therapies, and the like.
  • Other viruses or parasites, such as trypanosomes, that display extensive genotypic variation are useful candidates for the present invention.
  • breeding programs for both plants and animals will benefit from this invention: For example, when there is a small population of rare animals that are being bred, it is believed important to interbreed unrelated individuals. Similarly, for plant breeding, it would be advantageous to characterize at the molecular level the diversity available to the plant breeder, so that he can choose the most appropriate individuals to work on before embarking on an extensive crossing and selection program. Most current means of determining relatedness are cumbersome, laborious and yield limited information. In contrast, the present invention allows high throughput and yields extensive information. The present invention also provides methods for identifying polymorphisms.
  • a polymorphism is identified by amplifying nucleic acids from two different organisms, preparing diversity panels from the two organisms, placing the diversity panel from one into an addressable array and hybridizing the array with the diversity panel of the other organism.
  • a polymorphism is identified by the absence of hybridization.
  • the arrayed diversity panel is cloned first and individual cloned molecules are placed into the array.
  • This approach can be applied to the identification of a polymorphism genetically linked to a phenotypic trait: the strategy commonly known as Bulk Segregant Analysis can benefit from the present invention.
  • Classically a large number of individuals are scored for a particular trait or phenotype and each individual is placed in one of two possible categories. The DNA of individuals in each category is pooled and interrogated to identify markers specifically present in one ofthe two categories.
  • a clear advantage ofthe present invention to perform this analysis is its parallel nature: in a single experiment, a large number of markers will be interrogated simultaneously. The chance of detecting a polymorphic marker distinguishing between the two categories is therefore higher.
  • the nucleic acid molecule comprising a detected polymorphism is isolated using techniques known in the art.
  • the nucleic acid molecule may be cloned in an appropriate vector if not already cloned. In turn, the clone may be mapped on the genome using conventional techniques or mapped to a collection of BAC or YAC clones.
  • the nucleotide sequence may be determined as well.
  • the polymorphic nucleic acid molecule may be used to transform a host cell, either a plant or animal. Methods to make transgenic plants are known in the art. Depending upon the nature of the transgenic sequence it may be desirable to operatively link the sequence to a promoter that are active in plants.
  • Such promoters may be constitutive, such as the 35S CaMN promoter, tissue-dependent, such as those active only in root tissues, stage-dependent, such as those active during embryogenesis, or the like. Examples of promoters are readily found in public databases (e.g., GenBank).
  • Introgression of specific alleles is a goal frequently pursued in plant breeding as well as laboratory animal breeding programs.
  • the end product is a plant or animal nearly identical to the desired parent except for a specific region ofthe genome that is contributed by another individual.
  • the advent of mice strains with identical backgrounds but differing at the Major Histocompatibility Complex locus was instrumental in understanding the effect of MHC differences on organ transplantation.
  • a desirable trait such as disease resistance, may be identified in a plant, but is generally introgressed into elite varieties that are better suited to the local environment, soil and climate or to consumer preferences than the original plant.
  • the introgression is usually performed by repeated backcrosses of the new individual with the elite parent.
  • the introgression of the genes that account for the traits means to follow that trait are necessary.
  • the trait may not be assayable in the field except under defined conditions (e.g., challenge with the pathogen). It is advantageous, however, to have a marker for the gene i.e. a polymorphism genetically linked to the desired trait, which can then be assayed to identify suitable plants for the breeding program.
  • a marker for the gene i.e. a polymorphism genetically linked to the desired trait, which can then be assayed to identify suitable plants for the breeding program.
  • it is also important to monitor that the rest of the genome is as similar as possible to the elite parent.
  • the determination of a genotype encompassing a large number of markers in parallel is a distinct advantage.
  • the present invention provides the means to follow specific markers linked to a desirable traits, as well as genome-wide markers measuring the extent of reconversion of the genome, and allows for high throughput screening.
  • the present invention provides the means to build rapidly a genetic map, even for organism for which little or no molecular data is available. Once the genotype of two individuals is determined according to the invention, the progeny arising from a cross between these individuals can be genotyped in a similar manner. Each individual from the progeny is genotyped. Commonly used softwares such as Mapmaker will then extract from the individual genotypes the co-segregation ratio between markers and calculate a linkage map of the markers.
  • phenotypic data such as qualitative or quantitative traits
  • genetic data such as molecular markers linked to the trait, markers for Quantitative Trait Loci and the like
  • molecular data such as DNA sequence associated with the markers, surrounding the markers, comprised between the markers, and the like.
  • the fingerprint of an individual as determined by the present invention can be used to identify the individual unambiguously. Due to the parallel analysis of a large number of markers provided by the present invention, the identification is highly reliable and the fingerprinting process has a high throughput and a low cost. This reliable and cheap method for identifying plant or animal varieties is useful in a large range of activities: it will facilitate the detection by plant or animal breeders of unlawful copying of their registered varieties and it will facilitate quality control of identity preserved crops. Fingerprinting as provided by the present invention can also be used to identify the genotype of grains delivered by a producer, for example for the purpose of collecting " royalties ,on the production of specific varieties.
  • Representative samples of rice germplasm are identified for genotyping.
  • the samples are chosen solely for demonstration purposes and are chosen on the basis of other knowledge for being a diverse set of genotypes. This is done, mostly through analyzing dendrograms based on sequence and/or molecular marker polymorphism in order to pick up members of separate groupings.
  • representative genotypes can be identified as representatives of separate clusters if the results (like Principal Component Analysis) or clustering algorithms are available.
  • representative genotypes can be identified through single pass sequencing of rapidly evolving segment of the genome followed by similarity/dissimilarity analysis. DNAs from a sampling of genotypes representing genetic diversity of rice species (usually 10-15) are Used to generate DNA diversity panels through a number of techniques, one of which is exemplified below.
  • genomic DNA prepared from 9 rice cultivars: Azucena, IR20, IR64, Italica, Karolina, Labelle, L203, Millin and Nipponbare.
  • Three different restriction endonuclease (Table 1) digestions ofthe DNAs generate fragments, which are ligated with adapters, amplified and cloned.
  • primers for amplification are chosen such that the resulting products comprise a subset of the restriction fragments. With this method, complexity of the genome is reduced by 100 to 1000-fold compared to total genomic samples.
  • Genomic DNA is extracted from young seedlings (Murray and Thompson Nucleic Acid Res. 8: 4321-4326 (1980)). About 5 ng of DNA from each cultivar is digested at 37°C for 1 hour with 2 units of restriction enzyme in a volume of 8 ⁇ l. Following digestion, 2 ⁇ l of ligase mixture is added, and the reaction is incubated at 37°C for 3 hours.
  • Ligase mixture comprises 0.2 ⁇ l T4 ligase (NewEngland Biolabs, MA), 0.2 ⁇ l lOxligase buffer, 0.1 ⁇ l lOOxBSA (NewEngland Biolabs, MA), 0.2 ⁇ l 50 mM ATP, 1.2 ⁇ l MilliQ (MQ) H 2 O and 0.1 ⁇ l of enzyme-specific adapter (Table 1) at 50 pmol/ ⁇ l for Mspl-specific adapter and 5 pmol/ ⁇ l for EcoRI- and P-ftl-specific adapters.
  • the mixture is diluted to 500 ⁇ l with MQ H 2 O and 2 ⁇ l is used as a template in a 50 ⁇ l amplification reaction with 2 units of RedTaqTM polymerase (Sigma Chemicals, St Louis, MO, USA) and one of the primers (1.5 ⁇ l at 50 ng/ ⁇ l) listed in Table 1.
  • RedTaqTM polymerase Sigma Chemicals, St Louis, MO, USA
  • the primers 1.5 ⁇ l at 50 ng/ ⁇ l listed in Table 1.
  • the reactions are cycled 30 times: at 94°C for 30 sec, 60°C for 45 sec and 72°C for 1 rnin.
  • a final extension cycle is performed at 72°C for 8 min.
  • amplified fragments are ligated into PCR2.1-TOPO vector using the TOPOTM cloning kit and transformed into heat-shock competent E. coli strain TOP10F' (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, amplified products may be purified to reduce adapter and primer contamination.
  • the ligation mixture which contains approximately 2 ⁇ l of amplified products, is incubated for 5 min and terminated. About 2 - 2.5 ⁇ l of the ligation reaction is used to transform E. coli.
  • Approximately 20-50 ⁇ l of the transformed E. coli is plated on L plates containing ampicillin for selection and X-gal for blue/white visualization to identify recombinant plasmids. Approximately 1000-2000 recombinants are typically isolated. This number represents a similar complexity as the diversity panels that are used for detecting polymorphisms.
  • the products are precipitated with one vol of isopropanol (100 ⁇ l) at room temperature.
  • the plate is then centrifuged at 3200 rpm for 20 min at 4°C. All the liquid is removed, and the pellet is washed quickly with 100 ⁇ l of 70% EtOH.
  • the plate is then further centrifuged for 10 min at 4°C. The EtOH is removed, and the plate is air dried.
  • the pellet is resuspended at a concentration of about 20 ng/ ⁇ l in MQ water, 3x SSC, or lx SSC, 0.01% sarcosyl.
  • the amplified DNA inserts are transferred into 384-well plates (Genetix) and arrayed using a microarrayer (e.g., 417 microarrayer; Affymetrix, Palo Alto, CA) onto PolysineTM microscope slides (MenzelGlazer, Germany) or in-house polylysine-coated microscope slides. Arrays are made with six replicates per fragment. The average center to center spot spacing is 250 ⁇ m.
  • slides are processed by hydration in IX SSC, quick drying, blocking for 15 min in a solution of NaBrH 4 /PBS (prepared by dissolving 1 g NaBrH 4 in 300 ml PBS, pH 7.0) (see also http://www.microarrays.org/protocols.html, Protocol for Post Processing Microarrays; June 2000, except that the succinate anhydride pyrolidone is replaced with NaBrH4 in PBS as the blocking solution). Slides are then dipped in boiling water for 30 sec to denature the DNA and followed by a 10 sec dip in 100%) EtOH. Slides are dried by centrifugation at 1000 rpm in a slide rack on microtiter plate carriers for 1 minute.
  • a diversity panel of one or more specific genotypes is generated and labeled with a fluorescent dye.
  • a number of genotypes can be compared, the number being equal to number of labels that can be unequivocally detected and resolved.
  • an Affymetrix 418 scanner is equipped with "green” and “red” lasers, allowing for simultaneous analysis of two different samples.
  • Genomic DNA 200 ng-2 ⁇ g/ ⁇ l
  • EcoRI adapters 1.5 ⁇ l of 5 pmoles/ ⁇ l
  • T4 ligase 40 units
  • An amplification reaction contains 2.5 units RedTaqTM (Sigma, St. Louis, MO USA), 1-5 ⁇ l of ligated genomic DNA from above, 10 ⁇ l lOx buffer (lOx buffer contains 500 mM KCl, 1 M Tris-HCl (pH 8.8), 0.1% Triton X-100, 15 mM MgCl 2 ), 10 ⁇ l of 2mM dNTPs, and 1 ⁇ l of 20 pmol/ ⁇ l primers. Because the DNA fragments are ligated with an adapter a single primer identical to one strand of the adapter is used with one or more additional bases added to the 5' end. In some experiments a mix of primers is used that are identical to one strand ofthe adapter but have one or more additional bases at the 3' end of the primer. Such a mix serves to limit the complexity of the resulting fingerprint.
  • Amplification conditions are 1 cycle of 95°C for 2 min, 30 cycles of 94°C for 30 sec, 54°C for 30 sec, 72°C for 1.1 min, 1 cycle of 72°C for 5 min and hold at 4°C.
  • Amplification products are purified using Qiagen Quick PCRTM purification columns to remove the dNTPs, which otherwise will affect the labeling steps.
  • Amplified material is labeled by incorporating dUTP-Cy3 or dUTP-Cy5 using a random priming method. In this method, up to 11 ⁇ l of DNA in MQ water is mixed with 2 ⁇ l of E.
  • Cy5 are pooled and purified together using Qiagen columns according to the manufacturer's recommendations, except that one extra wash using 0.5 ml wash buffer is performed.
  • the labeled nucleic acid molecules are eluted in ⁇ 30 ⁇ l of water.
  • amplified material can be labeled using a Deca-random-prime DNA labeling kit from Fermentas (Vilnius). When this kit is used, minor deviations from the manufacturer's instructions are used, specifically the reaction volume is reduced to 5 ⁇ l, the time increased to 1 hour and 0.4 ⁇ l of 1 mM Cy3-dUTP or Cy5-dUTP is used instead of 32 P-dNTPs. Probes are not purified for hybridization.
  • the labeled material Prior to hybridization, 5 ⁇ l of the labeled material is mixed with 2 ⁇ l of 20 mg/ml herring sperm DNA which is dissolved in Express HybridizationTM buffer (Clontech, Palo Alto, CA, USA), and the mixture is denatured at 96°C for 3 min.
  • the denatured probes are mixed with 10 to 15 ⁇ l of ExpressHyb hybridization solution, pipetted directly onto the microarray surface and covered with a glass cover slip (24 mm x 24 mm Mediglass, Australia). Slides are then quickly placed into a homemade humidification chamber in a 65°C water bath for overnight hybridization.
  • the coverslips are removed, and the slides are rinsed in lxSSC with 0.1% SDS for 5 min; lxSSC for 2 min; 0.2 xSSC for 2 min; and 0.02 x SSC for 20 sec; all solutions are at room temperature. Slides are quickly dried by centrifugation at 1000 rpm in a slide rack on microtiter plate carriers for 1 min.
  • the intensity of fluorescence at each spot is measured by scanning the slide with an array reader (for example Affymetrix 418 microarray scanner). Fluorescence is read using scanner settings appropriate for the fluorescent dyes used in labeling reaction.
  • array reader for example Affymetrix 418 microarray scanner. Fluorescence is read using scanner settings appropriate for the fluorescent dyes used in labeling reaction.
  • Cy3 dye the green laser is set to PMT 60 and laser power at 100%
  • Cy5 the red laser is set to PMT 90 and laser power at 100%. Scanning conditions are adjusted if necessary. Identification of polymorphic clones may be made by visual inspection of a graphic file representing an overlay of scanning results for two genotypes to be compared.
  • An overlay can be a result of single hybridization or, alternatively scans from independent hybridizations can be overlayed.
  • Polymorphic clones may be identified as those hybridizing to only one of the two samples compared. Numerous statistical methods are available to facilitate conversion of signal intensities into binary (presence/absence) characters. Large populations of genotypes can be analyzed in pairs to develop similarity/dissimilarity measures matrix for the whole population.
  • fingerprints of rice cultivars are determined by hybridization of labeled diversity panels to a diversity array comprising a diversity panels generated from a mixture of 9 rice genomes.
  • a schematic of this type of experiment are compared on a single array as exemplified in Fig IB.
  • the diversity panels are generated using 9 cultivars of rice (3 indica and 6 japonica types).
  • Several panels are constructed using the pair-wise combination of restriction enzymes and primers described in Table 1.
  • the resulting fragments in the diversity panel range from 0.3 to 2.4 kb with an average insert size of around 1 kb.
  • an array feature or element is scored when the signal is at least 3 times the level of local background for the vector control (TOPO). At least 90% of array elements are scored for the panels analyzed in these examples. Furthermore, this value is reached without purification of amplification products.
  • Fingerprints for four rice cultivars are determined by hybridization of a diversity panel from each cultivar to the EcoRI-generated diversity panel of the 9 mixed rice genomes. Pairs of the rice cultivars (e.g., Millin and
  • IR64; Bala and IR20 are labeled with two different dyes for ease of detection.
  • a comparison between Millin (sub-genomic sample labeled with Cy5 dye) and IR64 (sub- genomic sample labeled with Cy3 dye) shows a high level of variation in signal intensity (brightness of array features) and Cy3/Cy5 signal ratios among array elements (Fig 2A).
  • a histogram showing green to red channel normalized signal intensity ratios (Fig. 2B) shows a tri-modal distribution. The majority of the array features cluster around a ratio of 1, indicating equal signal intensity for Millin and IR64 samples (monomorphic features). The red and green "tails" represent the groups of "polymorphic" spots.
  • Genomic DNA (2 ⁇ g) is cut with EcoRI, resolved in 0.8% agarose gel and transferred to nylon membranes (Fig 3A).
  • Diversity panels are prepared as described above, resolved using 1.5% agarose gel and transferred to positively charged nylon membrane (Boehringer Mannheim) (Fig 3B).
  • Fig 3 shows the results of hybridization of candidate clone F4, which is polymorphic by fingerprint analysis when a diversity panel of Millin is tested against diversity panels of Bala, IR20 and IR64.
  • F4 hybridizes strongly with Millin diversity panel (lane 2), whereas F4 does not detectably hybridize to Bala (lane 1), IR64 (lane 3) and IR20 (lane 4) diversity panels.
  • a second candidate polymorphic fragment, clone F8, also shows polymorphism on Southern analysis.
  • a smaller EcoRI fragment 1.3 kb
  • both IR20 and IR64 DNA display a 1.5 kb fragment (lanes 3 and 4) (Fig. 3A).
  • the band intensities are similar, in the diversity panel Southern, the hybridization strength to J-R64 and J-R20 are much weaker compared to the Millin and Bala bands.
  • the difference in the abundance of specific amplified material in the diversity panel translates into easily detectable polymorphism in microarray experiment when Millin is contrasted with r-R20 or IR64.
  • an RFLP is converted to a quantitative polymorphism detected by signal intensity differences between Millin and I-R64 sub-genomic samples on the array.
  • Fll One additional clone, Fll, is characterized in this example. Fll scores as monomorphic when analyzed against four rice cultivars, i.e., approximately equal signal intensity is observed for this clone when the array containing it is probed against any ofthe four labeled diversity panels. Fll is also tested as a probe against a Southern blot of diversity panels from these genotypes. Fig. 4 shows clearly that similar size ' (and abundance) products hybridize with the FI 1 probe in all four genotypes.
  • This EcoRI-generated diversity panel is also used to determine the minimal amount of DNA required for generation of reproducible diversity panels.
  • Four different amounts of adapter ligation products, from 0.2 ng to 12.5 ng, are used for amplification of four genotypes (Bala, Millin, IR64 and IR20) and hybridization results are analyzed for polymorphisms. All genotypes are scored reproducibly as either present (1) or absent (0) for 14 elements identified as polymorphic at the four DNA amount levels (data not shown).
  • Typical distributions of normalized ratios of signal intensities (the signal for Mspl sub-genomic sample labeled with Cy3 divided by the signal for Topo vector control labeled with Cy5) for four examples of non-polymorphic (Fig 7A) and polymorphic (Fig 7B) spots are presented.
  • Fig 7A non-polymorphic spots
  • Fig 7B polymorphic spots
  • Fig 7A it is apparent that the range of ratios is larger for spots with an average ratio value below zero (in which the signal from the sub-genomic sample is weaker than the Topo control signal).
  • Distribution of the ratios for all 384 features of the Mspl panel for the same set of 18 slides shows more variation between slides at lower values (especially below -0.2).
  • the presence of a different number of "positive" spots among genotypes tested is likely to be one of the sources of the between slide variation.
  • the proportion of the polymorphic spots is relatively low this result most likely indicates that array features that hybridize weakly to the sub-genomic sample (around 30% ofthe total number) are more influenced by the noise in our system compared to the more strongly hybridizing ones.
  • the number of array features found as polymorphic among nine rice cultivars is 50 (14.5% of scored spots) for the Mspl diversity panel. Apart from providing an estimate of polymorphism level detectable by this system, identification of polymorphic features allows assessment of the level of redundancy among them. DNA fragments representing array elements displaying the same pattern of polymorphism (same binary scoring) among the nine rice cultivars are resolved on an. agarose gel. DNA fragments with the same apparent mobility are scored as repeats (Fig 8). The analysis revealed that 50 polymorphic spots represented 28 unique clones of which most (20) had just one copy in the Mspl panel of almost 400 clones.
  • the binary scoring table for the 28 unique polymorphic features is used to calculate the distances between the cultivars.
  • a distance table is used to produce dendrograms showing the relatedness of the cultivars.
  • Binary scoring tables of 28 unique features from Mspl and 28 from Pstl are clustered by Cluster program (Stanford University) using similarity metric setting of correlation uncentered and presented by treeview . (Stanford University). Differentiation among the cultivars analyzed and separation between japonica and indica types is apparent in both dendrograms.
  • Figure 9 A shows the separation between indica and japonica rice cultivar classes based on fingerprints established from using the Mspl diversity panel. Similar results are found using the Pstl- generated diversity panel (Fig 9B).
  • DH doubled haploid lines developed from the cross between IR64 and Azucena (REF) are used for genetic mapping. All 40 polymorphisms segregating in the DH lines population are successfully mapped on the microsatellite-based framework without any apparent clustering ofthe new markers.
  • EXAMPLE 8 FINGERPRINTING USING A COMPLEX MIXTURE OF DIVERSITY PANELS
  • complex DNA samples are analyzed to demonstrate that minor amounts of a genome are detectable.
  • DNA fragments from diversity panels developed from 8 species are arrayed on the same slide..
  • the mix included rice and 7 species of micro organisms.
  • This composite panel is then used as a target for hybridization with a diversity panel comprising sub-genomic samples from rice with or without a DNA admixture from microorganisms.
  • the diversity panel from rice cultivar Millin which is labeled with Cy5 dye
  • Barley diversity panels are generated using DNA from 3 barley cultivars: Steptoe, Morex, Harrington, and from Hordeum spontaneum (wild barley) accession OSU15. Diversity panels are constructed according to the Examples above, except that the restriction enzyme P-?tI is used to generate panels having complexities 100 to 1000 fold less than total genomic samples (below). Varying complexities of panels are achieved by the choice of primers used in amplification. Fragments from the panels are cloned, and inserts are individually amplified from bacterial colonies before arraying on glass slides.
  • Genomic DNA is extracted from seedlings of various cultivars. Genomic DNA (50 ng) is digested at 37°C for 1 hour with 2 units of Pstl restriction enzyme in a volume of 8 ⁇ l. After digestion, 2 ⁇ l of ligase mixture is added. Ligase mixture consists of 0.2 ⁇ l T4 ligase (New England Biolabs, USA), 0.2 ⁇ l lOx ligase buffer, 0.1 ⁇ l lOOx BSA (New England Biolabs, USA), 0.2 ⁇ l 50mM ATP, 1.2 ⁇ l MilliQ (MQ) H 2 O and 0.1 ⁇ l (5 pmoles) of Pstl adapter:
  • the mixture is diluted to 500 ⁇ l with MQ H 2 O.
  • 2 ⁇ l of the diluted ligated DNA is used as template from amplification in a 50 ⁇ l reaction using 2 units of RedTaqTM polymerase (Sigma, USA).
  • the sequence of the amplification primers are either GATGGATCCAGTGCAG (SEQ ID No: 12) or GATGGATCCAGTGCAG-X (SEQ ID No: 13) where X is A, C, G or T.
  • Single primer for SEQ ID No: 12 or a combination of primers of SEQ ID No: 13 are used in amplification to achieve various levels of complexity reduction.
  • Amplification parameters are 1 cycle at 95 °C for 3 min, 30 cycles at 94°C for 30 sec, 60°C for 45 sec, 72°C for 1 min, followed by 1 cycle at 72°C for 8 min.
  • the amplification products are cloned, amplified and arrayed according to methods in Examples 1 and 2.
  • Diversity panels are prepared as above from cultivars Morex and Steptoe.
  • the amplification primer used has the sequence 5'-GATGGATCCAGTGCAG-3' (SEQ ID No: 14).
  • the amplification products are labeled with fluorescent dyes (Cy3 for the Morex diversity panel and Cy5 for the Steptoe diversity panel) and the hybridized to slides containing the Pstl diversity panels from above. Hybridization, washing, image capture and analysis is done . according to methods described in Examples 3 and 4.
  • Fig 11 shows a fragment of the array with polymorphic array features indicated.
  • the frequency of polymorphic array features detected between Morex and Steptoe varied from 10-15%.
  • diversity arrays prepared from cDNA are used for genotyping analysis.
  • cDNA or EST sequences may be used as a diversity panel that can be arrayed and used to establish genotypes.
  • a cDNA library from multiple mouse strains and tissues is arrayed on glass slides (>5000 independent cDNA clones per slide). Arraying and slide processing is done as in Example 2. Diversity panels for probing the cDNA are prepared according to the methods taught in the examples from two mouse strains, strain C57B1/6 and strain NOD K. Briefly, 0.1 microgram of genomic DNA is digested by Mspl restriction endonuclease, an adapter with an spl-compatible end is ligated to the restriction fragments, and the fragments are amplified using an adapter- specific primer. Amplification products are labeled using fluorescent dyes (Cy3 and Cy5) and hybridized to the cDNA diversity arrays using Quick HybTM buffer (Clonetech). Hybridization, washing, image capture and analysis is carried out as described in Examples 4-6.
  • the diversity panel is generated from genomic DNA by a method that does not utilize amplification. Instead, the DNA is digested with a restriction enzyme and a range of lengths of the restriction fragments are chosen and isolated. The panel is then labeled with fluorescent dye and hybridized along with a similarly prepared diversity panel from a second sample to a diversity array comprising a large collection of cDNAs.
  • mouse cDNA diversity arrays are prepared using 4000 cDNA clones. Diversity panels are created from two mouse inbred strains, NOD K and C57B1/6 by Mspl digestion of 10 ⁇ g of total genomic DNA. Digested DNA is electrophoresed in a 2.0 % agarose gel, and a section of the gel containing fragments from 300 bp to 700 bp is isolated. The DNA is extracted from the agarose and purified using a gel extraction kit (Qiagen). The purified DNA is labeled with Cy3 (strain C57B1 6) or Cy5 dye (strain NOD K), respectively, using a method described in Example 3. Hybridization, washing and image analysis is done using techniques described in Examples 4 and 5. Polymorphic array features are identified as those with Cy3/Cy5 signal ratio > 3.0 or ⁇ 0.33. In this particular contrast 9% ofthe array features are identified- as polymorphic.
  • Diversity array technology is also suitable for detecting polymorphisms resulting from insertions in the genome. Since transposable elements are among the primary source of this type of DNA polymorphism, amplification of transposons is used as a method of generating diversity panels for probing rice diversity arrays. This example presents polymorphisms due to the transposon, called Stowaway (Bureau et al., Proc Natl Acad Sci USA 93: 8524-8529, 1996), which is a member ofthe MITE (Miniature Inverted Repeat Transposable Elements) class of mobile elements. Diversity arrays are generated by amplifying sequences that direct adjoin the Stowaway VII subfamily of MITE transposable elements in the rice genome, cloning the amplification products and applying the cloned inserts to an array as described in Examples above.
  • genomic DNA of four rice cultivars Azucena, IR64, Millin, Nipponbare (500 ng in total, 125 ng mixed from each cultivar) is digested with Msel restriction enzyme, and Msel adapters (shown below) are ligated to the restriction fragments. Amplification is carried out using the Internal Primer Right and/or Left (below) and Msel adapter Primer 1. After 25 cycles of amplification 1 ⁇ l of amplified product is used as a template for another round of amplification using Inverted Repeat Primer and Msel adapter Primer 1.
  • AmpUfication products from this reaction are cloned using a TopoTM cloning kit.-
  • the clone inserts are amplified, purified and arrayed on glass slides as in Example 2, resulting in a diversity array comprising 384 clones ready for polymorphism detection.
  • the slides are processed as described in Example 3.
  • Diversity panels are generated from each cultivar separately using the method above and are labeled with a fluorescent dye (Cy3 for Azucena and Cy5 for IR64). Labeled panels are hybridized to the diversity arrays and washed. Fluorescent images are captured using GMS 418 Scanner (Affymetrix, CA USA) and analyzed using the methods described above. Based on other experimental data, about 17% ofthe features are expected to be polymorphic.
  • diversity panels can be generated without the need for a restriction digestion and adapter ligation step. This offers the possibility of a complete automation of this invention.
  • diversity panels are generated by a semi- random, two-step amplification protocol (ST-PCR; Chun et al., Yeast 15: 233-40, 1997).
  • ST-PCR requires only genomic DNA and two pairs of amplification primers used in two successive amplification reactions.
  • Genomic DNA (300 ng total) from two rice cultivars, Azucena and IR64, is used as a template for amplification using two primers: Internal Stowaway VII Right Primer (see table above for sequence) and ST-PCRld Primer (5'-
  • GGCCACGCGTCGACTAGTACN 10 TCGAG-3' (SEQ ID No: 20).
  • Amplification is performed using 0.5 unit Red TaqTM polymerase (Sigma) and using a hot start program in which the polymerase is added after the first step of the program.
  • the program uses the following steps: (1) 95°C for 3 min; 80°C for 2 min; (2) 94°C for 30 s; (3) 2°C for 30 s and -1.0°C for each subsequent cycle; (4) 72°C for 3 min; (5) repeat steps 2-4 five times; (6) 94°C for 30s; (7) 65°C for 30s; (8) 72°C for 3 min; (9) repeat steps 6-8 for 24 more times; (10) hold at 4°C.
  • the product is diluted 1:4 with water and l ⁇ l is removed for a second amplification.
  • Inverted Repeat Primer see Table above
  • ST-PCR2 primer 5'- GGCCACGCGTCGACTAGTAC-3' SEQ ID No: 21
  • the amplification products are cloned.
  • Diversity Panels are scaled up as described in Example 1 and diversity arrays are prepared as in Example 2.
  • Diversity panels are generated from each cultivar separately using the method above and are labeled with fluorescent dye (Cy3 for Azucena and Cy5 for IR64). Labeled panels are hybridized to diversity arrays slides and washed. Fluorescent images are captured using GMS 418 Scanner, and images are analyzed using the methods described above. Based on other experimental data, about 17% ofthe features are expected to be polymorphic.
  • tissue of rice cultivar Millin are collected. These tissues are: (1) 4-week old seedling leaves, (2) 4-week old seedling roots, (3) mature pollen and anther, (4) immature pollen and anther, (5) fertilized ovary and stigma, (6) unfertilized ovary and stigma, (7) mature embryo, (8) immature embryo, (9) immature endosperm, (10) flag leaves and (11) 3-week callus.
  • Genomic DNA is isolated from these tissues and a mixed sample of DNA is completely digested with Mspl or HpaH, both methylation sensitive.
  • Diversity panels from Mspl-digested and HpaH -digested DNA are prepared using the methods described in Example 1 (using Mspl adapter and Mspl primer sequences presented in Table I). The diversity panels are scaled up as described in Example 1 and diversity arrays are prepared as in Example 2.
  • Differences in methylation patterns among the tissues analyzed are also identified through comparison of normalized ratios of signal intensity for a specific tissue.
  • the signal is normalized to the signal obtained from hybridization with labeled TOPO vector sequence.
  • Statistical methods described herein are used to identify the features with developmentally- regulated pattern of cytosine methylation.
  • a number of tissue specific CpG methylation patterns at CCGG sites are confirmed by Southern analysis in which DNA from the diversity panels are hybridized with labeled insert from a clone identified as differentially methylated in fertilized ovary and stigma.
  • Fig 14 The absence of hybridization in lane 5 confirms the low value of hybridization obtained from the normalized data ( Figure 14).
  • DNA sequences are determined for 20 of the tissue methylation polymorphic fragments.
  • One of the fragments has high sequence identity with the rice chloroplast genome and the rest of the fragments are derived from the nuclear genome.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés permettant de déterminer le génotype d'organismes par analyse d'hybridation et en particulier en établissant la parenté des organismes individuels à l'intérieur d'une espèce. L'invention concerne également des jeux d'échantillons ordonnés adressables, comportant des panneaux divers de molécules d'acide nucléique, dans lesquels les molécules sur les jeux d'échantillons ordonnés sont adressables ou uniquement identifiables d'une certaine manière. Un panneau de diversité résulte d'un procédé pouvant distinguer les différences de séquence entre les échantillons d'acide nucléique. Comme nous l'avons déjà mentionné, divers procédés peuvent être utilisés afin de générer le panneau de diversité. Après la production dudit panneau de diversité, les produits d'acide nucléique du panneau de diversité sont séparés pour être appliqués sur un jeu d'échantillons ordonné. Le panneau de diversité séparé est ensuite déposé sur un substrat afin de créer un jeu d'échantillons ordonné adressable et hybridé doté d'acides nucléiques marqués. Le génotype d'un organisme est déterminé par le motif d'hybridation.
EP01934221A 2000-03-29 2001-03-29 Procedes de genotypage par analyse d'hybrydation Withdrawn EP1268859A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006256A2 (fr) * 1995-08-09 1997-02-20 Institut National De La Sante Et De La Recherche Medicale Molecules isolees d'acides nucleiques utiles comme marqueurs de la leucemie et pour pronostiquer un cancer du sein
WO1999023256A1 (fr) * 1997-10-30 1999-05-14 Cold Spring Harbor Laboratory Ensembles de sondes et procedes d'utilisation de ces sondes pour detecter l'adn
WO2000034518A1 (fr) * 1998-12-04 2000-06-15 Keygene N.V. Arrangement et procede d'analyse de sequences d'acides nucleiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006256A2 (fr) * 1995-08-09 1997-02-20 Institut National De La Sante Et De La Recherche Medicale Molecules isolees d'acides nucleiques utiles comme marqueurs de la leucemie et pour pronostiquer un cancer du sein
WO1999023256A1 (fr) * 1997-10-30 1999-05-14 Cold Spring Harbor Laboratory Ensembles de sondes et procedes d'utilisation de ces sondes pour detecter l'adn
WO2000034518A1 (fr) * 1998-12-04 2000-06-15 Keygene N.V. Arrangement et procede d'analyse de sequences d'acides nucleiques

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Title
See also references of WO0173119A3 *

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