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WO2000053811A1 - Puces genomiques et cartographie optique de transcrits - Google Patents

Puces genomiques et cartographie optique de transcrits Download PDF

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WO2000053811A1
WO2000053811A1 PCT/US2000/006342 US0006342W WO0053811A1 WO 2000053811 A1 WO2000053811 A1 WO 2000053811A1 US 0006342 W US0006342 W US 0006342W WO 0053811 A1 WO0053811 A1 WO 0053811A1
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dna
genomic
sequence
clones
array
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John D. Mcpherson
Richard K. Wilson
Xun Meng
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Orion Genomics LLC
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Orion Genomics LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to genomic DNA microarray chips constructed with an ordered tiled array of oligonucleotide genomic DNA clones having minimally overlapping sequences. More specifically, the present invention relates to the use of the genomic DNA microarray chips as a tool for transcription profiling and gene discovery.
  • nucleic acid hybridization technology has evolved from Southern's initial observation that complementary base-pairing could be exploited for the interrogation of nucleic acid molecules immobilized on a solid support by using nucleic acid molecules labeled with a reporter molecule as a probe (Southern, 1975). The technique was extended to the screening of a collection of DNA clones, called a clone library, replicated onto nitrocellulose or nylon filters thereby allowing a direct correlation between clones and observed signals from the hybridizing probes.
  • genomic DNA microarray chips that contain arrays of ordered genomic DNA clones that serve as tools for gene expression analysis.
  • One embodiment of the disclosed invention is a genomic DNA microarray chip, constructed with an array of oligonucleotide sequences, which is immobilized on a solid support. This array is comprised of a group of genomic DNA clones having minimally overlapping sequences and together make up an ordered, tiling path representing at least part of an entire genome with at least one of the oligonucleotide sequences being of an unknown sequence.
  • a second embodiment is a method of determining the genomic location of a labeled known DNA sequence. This is accomplished by hybridizing a known labeled DNA sequence chosen from the group consisting of an EST, cDNA, PCR product or genomic fragment to at least one DNA clone on the immobilized genomic DNA microarray chip.
  • a third embodiment of the disclosed invention is a method of using the genomic DNA microarray chips for initial gene discovery and characterization or visual transcript mapping.
  • a fourth embodiment is a method of preparing the array of DNA clones that will be placed on the genomic DNA microarray chip. This comprises the isolation and fragmentation of the DNA, the cloning of each of these genomic DNA fragments, isolating the individual clones of the genomic DNA fragments, identifying their ordered tiling path and immobilizing the DNA fragments in a sequential alignment representing their original position in the genome
  • Another embodiment is the construction of a genomic DNA microarray chip using an immobilized array of DNA clones, which represent oligonucleotide, sequences of substantially the entire genome.
  • the DNA clones are arranged in an order reflecting the original sequence of oligonucleotides of the genome
  • kits for determining the genomic location of known oligonucleotide sequences that comprises an immobilized ordered array of DNA clones derived from a plurality of genomic DNA fragments and reflects the original sequence of the genome from which it was isolated.
  • the kit would also include a labeled sample of a known oligonucleotide sequence wherein said known oligonucleotide sequence will hybridize to at least one of the immobilized genomic DNA clones on the microarray chip.
  • genomic DNA clone array comprised of a multiwelled plate having one DNA clone in each well of this plate. Each genomic DNA clone was selected from a plurality of cloned genomic DNA fragments and has an overlapping nucleotide sequence in common with the genomic DNA clone in at least one adjacent well. Yet another embodiment of the disclosure is the use of the genomic DNA microarray chip as a tool for the analysis of gene expression, visual transcriptional profiling and visual transcription mapping.
  • Figure 1 Illustrates genomic DNA microarray chips used for transcription profiling.
  • Figure 2A Illustrates a genome containing multiple chromosomes.
  • Figure 2B Illustrates a chromosome and a minimal tiling path for a selected region of that chromosome.
  • Figure 3 Illustrates the alignment of DNA clones on a DNA microarray chip.
  • Figure 4. Illustrates the identification of known genes in a genomic DNA sequence using predicted exons, protein similarities and EST matches.
  • Figure 5. Illustrates the results of hybridized target molecules to minimally overlapping DNA clones.
  • Figure 6. Illustrates visualized genes on individual genomic clones.
  • Figure 7. Illustrates the process of visual transcription profiling.
  • the present invention is directed to the construction and use of genomic DNA microarray chips 10 where the genomic DNA clones are adhered to discrete areas 12 on a solid support surface.
  • the genomic DNA clones are adhered in an ordered array of overlapping, tiled genomic DNA cloned sequences starting at the upper left hand corner of the micro chip and proceeding left to right to the bottom of the chip.
  • the order of the array mimics the order of the original genomic sequence.
  • probe is typically used to describe DNA immobilized onto a solid support, while target is used to describe the labeled molecules that are being queried.
  • a DNA clone is a recombinant DNA molecule that has been replicated autonomously in a suitable host cell.
  • the term oligonucleotide sequence refers to a fragment of DNA sequence of any length.
  • An oligonucleotide array is defined as an ordered progression of oligonucleotide sequences.
  • a tiled oligonucleotide array is one where the oligonucleotide sequences represent all or some the oligonucleotides of the original selected genomic DNA and are arranged in a manner that represents the exact order of the selected genomic DNA.
  • a contig is a set of overlapping contiguous clones that cover a chromosome region or a whole chromosome.
  • a microarray chip is defined as a miniaturized oligonucleotide array usually adhered to a solid surface such as glass.
  • a microarray chip may be constructed to contain the entire genomic DNA or any portion thereof, even though the example describes the process for the entire genome.
  • genomic DNA oligonucleotide arrays were constructed for the genome of interest using large-insert clones and employing the rapid restriction fingerprinting techniques developed by the present inventors (Marra et al., 1997).
  • the bacterial artificial chromosome (BAC) cloning system developed by Shizuya et al. (PNAS 89, 8794-8797, 1992) was shown to accept and maintain stable human and plant DNA fragments up to a size of 350 kb.
  • BAC bacterial artificial chromosome
  • BACs show several favorable characteristics such as a low frequency of chimeric clones, easy handling of clones and libraries (e.g. propagation, plating, storage, or colony hybridization), and simple purification of the cloned DNA. While YACs due to their large insert sizes (up to more than 1 Mb) are still indispensable for the generation of physical maps of very large (> 500 Mbp) genomes, BACs will serve as preferred resources for map based cloning and large scale genome sequencing. BAC clones are preferred for the physical mapping, in this disclosure although other genomic clones could be used as well with some modifications to the workflow. A BAC library for the probe genome should contain at least 15 genome equivalents.
  • Figure 2A illustrates a target genome 20 that is comprised of multiple chromosomes 24.
  • DNA fragments are generated, cloned and ordered into an ordered genomic array that can represent the coding sequences for all or part of the target genome.
  • DNA is purified from each clone in the BAC library and fingerprinted by restriction digestion. Following agarose gel electrophoresis, the restriction digests are imaged and the fingerprint data used to computationally determine the relationships between all of the clones.
  • the resulting relationship matrix represents the clone-based physical map of the original genome. From this matrix, a minimal set of overlapping, tiled clones, representing the original genome laid end to end can be selected.
  • Figure 2B shows a map consisting of ordered DNA genomic clones from a selected region 26 of a chromosome 24 of a target genome 20.
  • Figure 2B illustrates the ordered array of the entire genomic DNA cloned sequences 22 found in the designated fraction 26 of the chromosome 24. Only a small portion of the total genomic DNA cloned sequences are selected for the final ordered array.
  • the chosen DNA cloned sequences 28 (all the bold lines) mimic the final ordered array of the original genome.
  • These chosen DNA cloned sequences 28 are contigs and are shown in Figure 2B as an ordered overlapping tiled array.
  • a tiled array is made of contigs, each contig containing small regions of overlapping and identical sequences to the contig on its left at its 5' left end and to the contig on its right at its 3' right end.
  • These overlapping sequences for contig 29 are indicated with dotted lines in Figure 2B
  • high molecular weight DNA will be isolated from a genome of interest, randomly sheared or partially cleaved using restriction enzymes and ligated into a suitable vector. After transfer of the ligation products into a suitable bacterial host, individual clones will be isolated and arrayed in microtiter plates to construct a genomic library. The combined inserts of this library will encompass approximately 15-fold DNA coverage of the genome. Each clone from this library will then be grown in culture and its DNA extracted. An aliquot of each DNA will then be digested to completion with a restriction enzyme such as Hind III and the resulting fragments separated according to size by agarose gel electrophoresis.
  • a restriction enzyme such as Hind III
  • An image of the gel will then be captured using a scanning fluorescence detector and the size of all fragments in each lane determined relative to fragments of known size from a marker that is co-electrophoresed with the digested DNA samples.
  • Groups, or contigs of ordered, overlapping clones are then assembled by comparing restriction pattern similarities or fingerprints, assuming that clones sharing a majority of similarly sized fragments originate from the same portion of the genome.
  • a minimally overlapping set of clones can be selected as an ordered tiling path representing the genome. For the human genome, which consists of over 3 billion subunits or base pairs, a tiling path would be comprised of approximately 340,000 BAC clones.
  • A. Preparation of DNA The following is one method of isolation and purification of a recombinant DNA vector from the host organism in which it was placed for amplification.
  • Culture volumes of 1200 ⁇ l of 2X YT (Sambrook et al. 1989) containing 12.5 ⁇ g/ml of chloramphenicol (Sigma; fosmids and bacterial artificial chromosomes (BACs)) or kanamycin (Sigma; PI -derived artificial chromosomes (PACs) clones) or the appropriate quantity of antibiotic for the desired clones are inoculated with a single colony which contains one unique recombinant DNA vector from a freshly streaked plate. If desired, multiple single isolate colonies can be processed individually for comparison.
  • Cultures are grown in 2-ml 96-well blocks (Beckman; part 140504) for 24 hr at 37°C with agitation at 300 rpm in a Labline incubator shaker. After growth, glycerol stocks in 96-well format are prepared by combining 50 ⁇ l of 80% glycerol with 100 ⁇ l of culture and mixing with a 12- channel pipettor. The microplates are sealed with Scotch-brand heavy-duty aluminum foil tape and stored at -80°C.
  • Bacterial cell cultures (96-well, 1.9 ml from above) are pelleted by centrifugation at 2700 rpm for 15 min in a Jouan model GR-422 floor centrifuge fitted with microplate carriers. The supernatant is decanted away from the pellet, and the 96-well block inverted on a paper towel for 5 min to drain excess culture media. The inverted block is rapped vigorously on a fresh paper towel until excess culture media is removed and then placed immediately on ice. Alternatively, after removal of the culture media, blocks are sealed with foil tape and stored at -80°C until DNA preparation can be performed.
  • DNA preparation is performed using a modified alkaline lysis procedure (Sambrook et al. 1989).
  • the cell pellet is resuspended by addition of 50 ⁇ l of chilled GET/RNase buffer [50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 0.12 mg/ml RNase
  • Qiafilter 96 filter (Qiagen, part 19663) is placed on top of the manifold in preparation for filtration of the supernatant-containing DNA.
  • the block is then tightly sealed with foil tape and inverted rapidly three times to mix the supernatant and isopropanol Precipitation of the DNA is achieved by room temperature incubation for 15 min followed by a 30-min centrifugation at 4000 rpm.
  • the foil tape is removed and the block inverted to remove the supernatant.
  • the DNA pellet is then washed with 200 ⁇ l of 80% ethanol added to the side of the well, and then collected in the bottom of the well by a 10-min centrifugation at 4000 rpm after sealing the block with foil tape.
  • the tape is removed and the block inverted on paper towels for 5 min to drain excess ethanol away from the pellet.
  • the block is then placed in a Savant DNA 110 SpeedVac set at medium heat for 5 min to dry the DNA.
  • the dried pellet is resuspended in 30 ⁇ l of TE [10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)] in the case of fosmid, BAC, PAC PI clones, or 150 ⁇ l of TE for cosmid clones. Resuspension of the DNA is achieved by incubating the sealed block for 30 min in a 37"C water bath followed by brief vortexing.
  • TE 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA (pH 8.0)
  • DNA is collected in the bottom of the wells by a brief centrifugation and transferred to a nontissue culture-treated microplate that is sealed with foil tape for storage at 20 ° C.
  • DNA is prepared by serial addition of 150 ⁇ l each of GET/RNase, SDS/NaOH, and KAc pH 5.5 as described above. After addition of KAc, the sealed block is inverted gently three times and then placed in ice water for at least 10 min. The block is inverted twice vigorously before centrifugation, as described. While samples are undergoing centrifugation, 330 ⁇ l of 100% ethanol is aliquoted into each well of a 96-well polystyrene Uni-Filter 800 receiver plate (Polyfiltronics). A 0.45 ⁇ M cellulose acetate 96-well filter plate (Polyfiltronics) is then mounted on top of the receiver plate and taped securely in place.
  • Uni-Filter 800 receiver plate Polyfiltronics
  • a 12-channel pipette (Costar) is used to transfer 400 ⁇ l of supernatant- containing DNA to the 96-well filter plate mounted on top of the receiver plate.
  • the assembly consisting of filter plate and receiver plate, is then subjected to an additional centrifugation at 4000 rpm for 15 min. After centrifugation, the filter plate assembly is dismantled and the ethanol decanted.
  • the DNA pellet is washed with 250 ⁇ l of 80% ethanol, dried, and resuspended in the appropriate volume of 10 mM Tris-HCl, 0.1 mM EDTA.
  • This alternative procedure has the advantage of being somewhat more rapid and substantially less expensive due to the use of Polyfiltronics plasticware.
  • Restriction Enzyme Digestion Restriction endonucleases are used to cut the genomic DNA sequences, which were inserted into the vector and amplified, into smaller fragments based on the location of the specified restriction sites of the chosen enzyme.
  • individual restriction digests should consist of 3.75 ⁇ l of ddH20, 1 ⁇ l of 10X buffer B (Boehringer-Mannheim), 0.25 ⁇ l of Hindlll (40 U/ ⁇ l; Boehringer-Mannheim), and 5 ⁇ l of DNA.
  • each digest should contain 6.75 ⁇ l of ddH20, 1 ⁇ l 10X Buffer B (Boehringer-Mannheim), 0.125 ⁇ l of Hindlll (40 U/ ⁇ l; Boehringer-Mannheim), 0.1 ⁇ l of Pstl (100 U/ ⁇ l; NEB), and 6 ⁇ l of DNA.
  • each digest should contain 6.75 ⁇ l of ddH20, 1 ⁇ l 10X buffer B, 0.25 ⁇ l of Hindlll (40 U/ ⁇ l; Boehringer-Mannheim), and 2 ⁇ l of DNA.
  • the DNA prepared as described above is not quantitated. Yields are usually uniform and the volumes indicated for digestion are adequate.
  • Components of the digestion cocktail are assembled in 96-well thin wall cycle plates (Robbins Scientific). Digestion is achieved by incubation of the cycle plates at 37° C for 4.5 hr in a 96-well thermocycler (MJ Research). After digestion a brief centrifugation collects the DNA in the bottom of the wells and 2 ⁇ l of 6x loading dye (0.25% bromophenol blue, 0.25% xylene cyanol FF., 15% Ficoll; Sa brook et al. 1989) is added to each well. Cycle plates are sealed with foil tape and stored at 4 ° C overnight before agarose gel electrophoresis.
  • 6x loading dye 0.25% bromophenol blue, 0.25% xylene cyanol FF., 15% Ficoll; Sa brook et al. 1989
  • the restricted genomic DNA fragments were separated by agarose gel electrophoresis stained, scanned and visualized for contig sizing.
  • One-percent agarose (SeaKem LE; FMC BioProducts) gels are prepared in IX TAE (Sambrook et al. 1989). Molten agarose is cooled to 46 ° C in a water bath with occasional stirring and then poured into 20 by 25 -cm UN transparent trays (Life Technologies) resting on a level surface. The comb is then inserted. For each gel, 150 ml of molten agarose is used, resulting in a gel thickness of approximately 3.5 mm. The comb used should form 51 wells with the following dimensions: 2 mm wide by 1 mm thick by 3 mm deep, where thick is the dimension in the direction of D ⁇ A migration.
  • the gel After the gel solidifies the comb is removed, the gel is wrapped in Saran Wrap and stored at 4°C until electrophoresis. Typically this storage time period should not exceed 3 days. Gels are removed from 4 ° C storage and placed into electrophoresis units containing buffer at the desired electrophoresis temperature for at least 10-min before sample loading.
  • the restriction enzyme digestion/loading dye mixture (1.75 ⁇ l) is loaded into each well. In the first well and every fifth well thereafter, 1 ⁇ l of a standard marker D ⁇ A sample is loaded.
  • Marker D ⁇ A should be a mixture of 1 kb ladder (Life Technologies) and both Marker II and Marker III (Boehringer-Mannheim) in the following proportions: 0.83 ⁇ l (1 ⁇ g/ ⁇ l) 1 kb ladder, 3.33 ⁇ l (250 ng/ ⁇ l) Marker II, 3.33 ⁇ l (250 ng/ ⁇ l) Marker III, 92 51 ⁇ l TE [10 mM Tris (pH 8.0), 0.1 mM EDTA (pH 8.0)], 25 ⁇ l 6X loading dye. Immediately before electrophoresis, 20 ⁇ l of this mixture is removed to a separate tube, diluted by the addition of 17 ⁇ l of TE and 3 ⁇ l of 6X loading dye and incubated at 60 ° C for 5 min
  • Gel images are first cropped and then converted from the proprietary 16-bit Molecular Dynamics format to 8-bit TIFF images, and transferred by ftp to Unix workstations for band calling and contig building
  • the Molecular Dynamics Fluorlmager is also used to measure the yield of D ⁇ A, prepared as described above, using protocols and Pico Green stain obtained from Molecular Dynamics.
  • restriction digests are imaged and the fingerprint data used to computationally determine the relationships between all of the clones.
  • the resulting relationship matrix represents the clone-based physical map of the genome From this matrix, a minimal set of overlapping clones, or tiled clones, representing the genome laid end to end can be selected and placed adjacent to each other in the multi- well plates.
  • Identification of restriction fragment bands is preferably performed interactively using an unmodified implementation of the program Image 2.0 (F. Wobus and R. Durbin, unpubl.) and subsequently Image 3.3 (D. Platt, F. Wobus, and R. Durbin, unpubl ), suitably modified to accept gel images generated as described above.
  • Band call data are collected and used to perform contig assembly in the program FPC (C. Soderlund and I. Longden, Sanger
  • tolerance refers to a window size; for example, if tolerance is set at 7, then two restriction fragments occurring in different fingerprints must have relative mobilities within seven-tenths of a millimeter to be considered equivalent fragments. A decrease in tolerance decreases the window size and therefore, increases the stringency of the comparison. It is important to note that all of the calculations performed in FPC have used the relative mobilities of the restriction fragments and not the sizes of the restriction fragments.
  • the cutoff score is a threshold value representing the maximum allowable probability of a chance match between any two clones (the Sulston score).
  • clone 2 Select the clone (clone 2) exhibiting the best match (i.e., the matching clone exhibiting the smallest Sulston score) to clone 1 and manually compare, using a fingerprint viewing tool provided by FPC, its fingerprint to that of clone 1 to determine the number of shared fragments. The overlap between the clones can then be drawn manually in FPC. If the clone 2 fingerprint exhibits no unique restriction fragments, bury (hide) clone 2 within clone 1. If unique fragments are observed in clone 2, repeat the entire procedure-using clone 2 for the next search against the FPC database.
  • clone 3 The best match (clone 3) is identified, and its fingerprint is compared manually against the fingerprints of clone 1 and clone 2.
  • RFLP restriction fragment length polymorphism
  • the clone in question should be labeled with a tag in FPC so that it will not be selected for other manipulations including DNA sequencing.
  • FPC vector-insert junction fragments
  • this parameter can, in practice, often be relaxed provided the constraint of internal consistency within the contig is met and new bands evident in a pair- wise comparison between two clones are confirmed by the next clone entering the contig.
  • a minimal tiling path is selected from the contig such that the selected clones encompass all restriction fragments across the contig with minimal duplication of coverage.
  • genomic DNA cloned sequences from the selected area 26 of the genomic DNA or chromosome 24 are used to construct the genomic DNA microarray chip 30.
  • Each (bolded) genomic DNA cloned sequence 32 in the tiling path of a genome will be arrayed 36 in the appropriate area 37 on the solid surface 34, such as a standard glass microscope slide, using conventional techniques.
  • Current arraying technology such as that developed by Molecular Dynamics, Inc. of Sunnyvale, CA, provides for the deposition of over 9,000 DNA samples on a microscope slide. Thus, the tiling path for a large size genome is easily contained on such arrays.
  • Genomic DNA microarray chips allow the construction of useful arrays for complete and comprehensive gene expression analysis without the need for costly, inaccurate and time-consuming characterization of all genes within the genome of interest.
  • the ability to efficiently interrogate all of the genes of a genome of interest has not yet been realized using the conventional current chip technology and configurations currently in use for gene expression analysis.
  • a unique feature of employing the genomic DNA microarray chips is that the hybridization of labeled targets can be observed at more than one microscopic optical level. Using a scanning confocal fluorescence microscope, the observation of the hybridized- labeled targets is seen as a single fluorescent spot on the solid surface. When using a phase fluorescent microscope on the same genomic DNA microarray chip, individual DNA molecules can be observed in the field of each spot that have been hybridized with labeled targets. Using the high range of microscopic objectives, differences in the hybridization patterns of exons or other probed areas can be seen on the individual DNA molecules.
  • a glass microscope slide is used as the solid support surface onto which the arrayed genomic DNA clones are placed.
  • the glass microscope slide is prepared using the following method.
  • the purified genomic D ⁇ A clones are restricted, fixed and aligned in ordered square arrays that mimic the clone locations set up originally in the multi-well plates. This matched array is used for comparing the hybridization patterns with the proper clonal recognition. This array may represent a part of or the total genome.
  • D ⁇ A molecules are first linearized by digestion with a suitable rare cutter restriction enzyme that has a recognition site in the multiple cloning site of the vector.
  • D ⁇ A molecules are elongated and aligned in square arrays by spotting droplets of D ⁇ A solution onto the derivatized glass surfaces, followed by air drying, using an Eppendorf micro manipulator in combination with a x-y table (interfaced to a computer) controlled by microstepper motors.
  • a glass capillary tube (500 ⁇ m, i.d.) can be used to draw D ⁇ A samples and then spot onto derivatized glass surfaces by simple contact. Each spot will be typically 900 ⁇ m with a spot to spot variation of ⁇ 100 ⁇ m. A center to center spacing between spots of 1.5 mm is controlled by computer program settings of the micromanipulator, and x-y table combination.
  • grids can be generated by using a modified commercially available laboratory automation robot equipped with a 500 ⁇ m ID stainless steel capillary pipetting tool, and a specialized workspace deck capable of holding multiple 96 well microtiter plates and up to 12 optical mapping surfaces in a vacuum chuck.
  • Fluid droplets (5-50 pg/ ⁇ l of D ⁇ A in Tris EDTA buffer) of 10-20 nl are spotted onto open glass surfaces that had been derivatized with APTES or [3-triethoxysilyl-propyl]trimethylammonium chloride (TESP), using customized robots for deposition of spots as described in Jing et al., 1998. 2. Genome Mapping
  • genomic D ⁇ A sequence 40 can be hybridized with known ESTs, cD ⁇ As, PCR products or genomic fragments that are labeled with reporter molecules
  • This genomic D ⁇ A cloned sequence 40 was derived from a selected area 26 of the genomic D ⁇ A or chromosome 24. In this case the labeled mR ⁇ A or D ⁇ A sequences are referred to as the probe and the genomic DNA cloned sequence 40 is the target. In Figure 4A, the genomic location of Gene A 42, Gene B 44, and Gene C 46 is being sought using the labeled probes mentioned above.
  • Gene B 44 should have five exons 45b which correlates with the five exons 43 a that were seen with the hybridization of known mRNAs and cDNAs.
  • the three exons 41b located with the known ESTs in Gene B 44 correlates well with these predictions also.
  • the predicted four exons 45c of Gene C 46 do not match completely with the hybridization pattern of the known mRNAs or cDNAs 43b and no known EST hybridization was seen at all within Gene C 46.
  • solution hybridization of labeled probe DNA derived from coding or non-coding sequences can be used to localize the target within the complete array or tiling path.
  • a scan of the array using an appropriate conventional imaging system e.g., fluorescence detection
  • targets contain the probe sequence. See Heiskanen et al. (1994).
  • These target clones can then be the focus of additional experiments (e.g., DNA sequencing) to further characterize the genomic sequence identified by the probe.
  • the labeled probe need not be derived from the same genome as the arrayed oligonucleotide DNAs but could be from another species. If an analogous gene or homologue is present in the target genome, the clone or clones in which it is contained will be identified with this procedure. In this manner, known markers are assigned to the tiling path to anchor the contigs to an existing map with new markers concomitantly assigned to a map position. Hybridization of the probe to immobilized DNA is detected as a specific signal associated with a clone or overlapping clones. This feature will be advantageous in the application of data from model organism genomes to commercially important genomes such as that of human or food crops and animals.
  • the foregoing method of making a genomic DNA microarray chip is preferably modified by placing an entire ordered set of genomic clones on the array, in order to increase the resolution of the map position obtained following hybridization of the probe.
  • the overlap of clones that are identified as containing the target can be used to define precisely the map position to the minimal region in common between all of these clones.
  • Genomic DNA Microarray Chips for Transcriptional Profiling or Gene Expression Analysis
  • Conventional gene chip technology is directed towards detecting changes in expression levels of specific genes in mRNA populations derived from different tissues, developmental stages or from different environmentally challenged organisms.
  • the probe population is derived solely from known genes.
  • the genomic DNA microarray chip teaches that the whole or a partial genome of an organism can be used as the probe in the form of arrayed, ordered, tiled genomic DNA clones.
  • One important advantage of these genomic DNA arrays over currently used gene chips is that all or part of the coding segments of the genome is represented without prior knowledge of their existence.
  • genomic DNA microarray chip By substituting a genomic DNA microarray chip for a conventional gene chip in a typical gene expression analysis or transcriptional profiling procedure, previously unknown genes can be identified, since they will hybridize to target molecules in a mRNA population.
  • the probe and target nomenclature as it is applied to conventional gene chip technology constitutes somewhat of a departure from conventional thinking in that the newly discovered gene probe was previously unknown. Rather, the labeled molecule that detects the gene will be acting as a probe.
  • the genomic DNA clones that are immobilized on the array have been characterized as to their location in a particular genome, the standard chip nomenclature will be applied and the immobilized DNA will be referred to herein as the probe. 2.
  • Fluorescent signal saturation will obscure the differential expression of genes within a clone by saturating the reporter signal observed. Fluorescent signal saturation on the genomic DNA microarray chip can occur for several reasons. For example, a high gene density in the genome of small model organisms will often yield multiple genes within any one large-insert clone and their combined signals will cause signal saturation. Likewise, when two neighboring genes are expressed at high levels, their combined signals may obscure the detection of differential expression of the two genes.
  • one approach to overcoming the problem of signal saturation is to use a genomic DNA microarray chip 50 containing a highly redundant set of partially overlapping clones as described in Example 1. Because the arrayed clones have a large staggered overlap (the overlapping region shown between the solid lines 57 and 58 in Figure 5), probes containing the differentially expressed gene in target A 53 and target B 55, but not the constitutively expressed gene or genes, will be present. In this manner, the differential expression will not be obscured in all clones.
  • a 2.7 kb C21orf3 cDNA 62 and an 956 bp EST 7 64 is hybridized to a PAC 92C23 DNA sequence 60 (L Peltonin et al., NPffl-Helsinki). Individual exons can be observed when the cDNA 62 and the EST 64 are detected via their fluorescent reporter molecules. Using readily available commercial microscopes, images of the elongated DNA molecules can be captured.
  • Optical mapping methods allow the deposition of thousands of copies of a DNA clone to be examined in each spot. Hundreds of spots have been placed on a single glass slide, with even higher-density achievable.
  • An imaging system can be used to capture an image of elongated probes having distinct regions that hybridize to the target. By using different labels for each of the target populations, differentially expressed sequences can be observed in the presence of constitutively expressed neighboring sequences. More specifically, individual genes contained within each of the immobilized clones will be directly visualized. Furthermore, the individual exons of each gene will be resolved, allowing the detection of differential or alternative splicing. Conventional DNA chips cannot detect this important component of gene regulation, unless all exons of a gene are individually represented in the probe array. Once again, this would require that the complete gene structure of every gene on a conventional chip be known in advance.
  • Amounts of 100-200 ng of each cDNA (EST) are directly labeled with fluor-12-dUTP (Stratagene) by use of random primer labeling (Stratagene; Prime-It Fluor). Large insert clones, including cDNAs, are labeled with fluor-12-dUTP by nick translation according to standard protocols (Boehringer Mannheim, Nick Translation Mix).
  • Purified mRNA is isolated using a commercial kit (e.g. FastTrack mRNA isolation kit) according to the manufacturer's protocol. Purified mRNA from target tissues is used to prepare fluorescently labeled cDNA for hybridization to the microarrays. Cy3-dUTP or Cy5- dUTP (Amersham) is incorporated during reverse transcription of 1.25 ⁇ g of polyadenylated [poly(A)+] RNA, primed by a dT(16) oligomer. This mixture is heated to 70 ° C for 10 min, and then transferred to ice.
  • a commercial kit e.g. FastTrack mRNA isolation kit
  • a premixed solution consisting of 200 U of Superscript II (Gibco), buffer, deoxyribonucleoside triphosphates, and fluorescent nucleotides, is added to the RNA. Nucleotides are used at these final concentrations: 500 ⁇ M for dATP, dCTP, and dGTP and 200 ⁇ M for dTTP. Cy3-dUTP and Cy5-dUTP are used at a final concentration of 100 ⁇ M. The reaction is then incubated at 42 ° C for 2 hours.
  • Purified, labeled DNA is resuspended in 11 ⁇ l of 3.5X SSC, 0.3% SDS, and 10 ⁇ g of polydeoxyadenylic acid.
  • Blocking DNA e.g., 20 ⁇ g of human CoTl DNA (Gibco-BRL) for microarrays with human DNA clones
  • Slides with genomic DNA clones are denatured for 2 minutes in 70% formamide, 0.6XSSC, pH 7.0 at 72°C then put through an ice cold ethanol series (70%, 90%, 100%) for 2 minutes each and air dried.
  • a 25 ⁇ l solution containing 20 ng of labeled probe, 30% Formamide, 1XSSC and 10% dextran Sulfate is denatured at 75 ° C for 5 minutes and added to the above-prepared slide.
  • a coverslip is placed on the top of the slide and slide is sealed with rubber cement.
  • Hybridization is carried out at 37 ° C for 24 hours in a humidify chamber. After the hybridization, the slide is washed with 50% formamide and 2XSSC 3X5 minutes each at at at at
  • Automatic imaging workstations are built around a Zeiss 135 (or equivalent) inverted microscopes equipped for epifluorescence, with 100X Zeiss plan-neofluor oil immersion objectives, numerical aperture 1.3, and multiband-pass filter pack (suitable for fluorescent labels and DNA count réelleain).
  • Preferred microscopes are equipped with a Dage SIT68GL low light-level video camera for acquiring focus, and a Princeton Instruments cooled charge- coupled device digital camera (1,316 X 1,032 pixels, KAF 1400 chip, 12-bit digitization) for high-resolution imaging and photometry.
  • a Ludl Electronics x-y microscope stage with 0.1- ⁇ m resolution is used for translation.
  • DNA molecules are imaged using a software package that integrates all of the workstation functions such as movement of the microscope stage, focus, and image collection. Control of light path actuators, video auto-focus, and sample translation (x-y stage) is accomplished by a Ludl Electronics MAC 2000 interface bus with the following modules installed. PSSYST 200, MCMSE 500, MDMSP 503, AFCMS 801, FWSC 800, and
  • the Ludl MAC 2000 is interfaced via RS232 serial connection to a Sun Microsystems SPARC 20 dual-processor computer workstation.
  • the Princeton Instruments charge-coupled device camera also is interfaced, via a Pentium-based microcomputer controller and distributed network, to a Sun workstation. Software for control of the above peripherals is written in the C programming language.
  • Digital images are acquired by the workstation and stored on hard-disk arrays for image processing and extraction of transcript mapping data.
  • the system runs on a network of Sun workstations with a networked file system.
  • Images are analyzed by locating specific hybridization signals from the labeled molecules on the elongated DNA molecules.
  • the positions of the specific signals are measured from each end of the elongated DNA molecule.
  • the average measurement from multiple DNA molecules showing hybridization is used to position the point of signal hybridization.
  • VTP Visual Transcriptional Profiling
  • VTP Visual Transcription Profiling
  • a genomic DNA microarray chip 70 from the selected area 26 of the genomic DNA or chromosome 24.
  • Different colored labels can be assigned for each plant cell mRNA populations to be compared, (e.g., Target A a normal mRNA population 72 or Target B mutant phenotype mRNA population 74). These two populations of labeled targets are hybridized to the DNA on the same chip or different chips.
  • Changes in the gene expression between these two mRNA populations can be detected with colored fluorescent reporter labels that are attached to the mRNA molecules. Differential expression is observed by evaluating the hybridization signals obtained for the unique hybridization of the target molecules to an arrayed DNA or variations in the co- localization of the reporter molecules when compared to other mRNA targets. This evaluation of signals performed using the same techniques as employed with standard DNA microarrays, See Duggan et al. (1999).
  • a unique feature of employing the genomic DNA microarray chips is that the hybridization of labeled targets can be observed at more than one microscopic optical level. Using a scanning confocal fluorescence microscope, the observation of the hybridized- labeled targets is seen as a single fluorescent spot on the solid surface. When using a phase fluorescent microscope on the same genomic DNA microarray chip, individual DNA molecules can be observed in the field of each spot that have been hybridized with labeled targets. Using the high range of microscopic objectives, differences in the hybridization patterns of exons or other probed areas can be seen on the individual DNA molecules.
  • VTM Visual Transcript Mapping
  • Visual Transcript Mapping can use the genomic DNA microarray chips to provide a novel approach and technique for initial gene discovery and characterization. Previously undiscovered genes can be found using the genomic DNA microarray chips as the probe for hybridization against total labeled mRNAs.
  • genomic DNA microarray chip After a genomic DNA microarray chip has been used to find and pinpoint a potentially interesting gene or region of the genome, more focused DNA sequencing work can be done to further characterize the gene that has been found within a specific genomic interval. For an investigator interested in such a gene or genomic region, this improved technology would provide considerable savings in terms of both cost and time.
  • Horelli-Kuitunen N, Aaltonen, J., Yaspo, M.L., Eeva, M., Wessman, M., Peltonen, L., Palotie, A. (1999). Mapping ESTs by fiber-FISH. Genome Res. 9,62-71. Jing, J., Reed, J., Huang, J., Hu, X., Clarke, V., Edington, J., Housman, D.,

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Abstract

L'invention concerne une puce génomique d'ADN constituée d'un micro-réseau, construite à l'aide d'un réseau ordonné en mosaïque de clones d'ADN génomique d'oligonucléotide, présentant des séquences de recouvrement minimal dans une séquence qui mime le génome sélectionné. Ces puces génomiques d'ADN constituées d'un micro-réseau sont utilisées comme outil d'établissement de profil visuel et comme cartographie génique visuelle.
PCT/US2000/006342 1999-03-11 2000-03-09 Puces genomiques et cartographie optique de transcrits Ceased WO2000053811A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033123A1 (fr) * 2000-10-18 2002-04-25 Cosmogenome Co., Ltd Jeux ordonnes d'echantillons d'oligonucleotides pour reconnaissance numerique par ordinateur
WO2002018646A3 (fr) * 2000-08-25 2003-08-28 Rosetta Inpharmatics Inc Decouverte de genes a l'aide de microreseaux
EP1453973A4 (fr) * 2001-12-11 2005-04-13 Affymetrix Inc Methodes de determination d'une activite transcriptionnelle
WO2004111267A3 (fr) * 2003-06-12 2005-06-09 Bc Cancer Agency Methodes de preparation d'une bibliotheque de reserves pourvues de plaques ayant une resolution inferieure a une megabase et utilisations
US7013221B1 (en) 1999-07-16 2006-03-14 Rosetta Inpharmatics Llc Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays
US7371516B1 (en) 1999-07-16 2008-05-13 Rosetta Inpharmatics Llc Methods for determining the specificity and sensitivity of oligonucleo tides for hybridization
US7807447B1 (en) 2000-08-25 2010-10-05 Merck Sharp & Dohme Corp. Compositions and methods for exon profiling
WO2014152397A3 (fr) * 2013-03-14 2014-12-04 The Broad Institute, Inc. Purification sélective d'arn et de complexes moléculaires liés à l'arn

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US6054270A (en) * 1988-05-03 2000-04-25 Oxford Gene Technology Limited Analying polynucleotide sequences

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054270A (en) * 1988-05-03 2000-04-25 Oxford Gene Technology Limited Analying polynucleotide sequences
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7013221B1 (en) 1999-07-16 2006-03-14 Rosetta Inpharmatics Llc Iterative probe design and detailed expression profiling with flexible in-situ synthesis arrays
US7371516B1 (en) 1999-07-16 2008-05-13 Rosetta Inpharmatics Llc Methods for determining the specificity and sensitivity of oligonucleo tides for hybridization
WO2002018646A3 (fr) * 2000-08-25 2003-08-28 Rosetta Inpharmatics Inc Decouverte de genes a l'aide de microreseaux
US6713257B2 (en) 2000-08-25 2004-03-30 Rosetta Inpharmatics Llc Gene discovery using microarrays
US7807447B1 (en) 2000-08-25 2010-10-05 Merck Sharp & Dohme Corp. Compositions and methods for exon profiling
WO2002033123A1 (fr) * 2000-10-18 2002-04-25 Cosmogenome Co., Ltd Jeux ordonnes d'echantillons d'oligonucleotides pour reconnaissance numerique par ordinateur
EP1453973A4 (fr) * 2001-12-11 2005-04-13 Affymetrix Inc Methodes de determination d'une activite transcriptionnelle
WO2004111267A3 (fr) * 2003-06-12 2005-06-09 Bc Cancer Agency Methodes de preparation d'une bibliotheque de reserves pourvues de plaques ayant une resolution inferieure a une megabase et utilisations
WO2014152397A3 (fr) * 2013-03-14 2014-12-04 The Broad Institute, Inc. Purification sélective d'arn et de complexes moléculaires liés à l'arn

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