WO2000003037A1 - Ensembles de nucleotides de criblage permettant de determiner la correspondance avec les proprietes de sequence et physiques d'une sonde - Google Patents

Ensembles de nucleotides de criblage permettant de determiner la correspondance avec les proprietes de sequence et physiques d'une sonde Download PDF

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WO2000003037A1
WO2000003037A1 PCT/US1999/015355 US9915355W WO0003037A1 WO 2000003037 A1 WO2000003037 A1 WO 2000003037A1 US 9915355 W US9915355 W US 9915355W WO 0003037 A1 WO0003037 A1 WO 0003037A1
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array
mrna
polynucleotide
membrane
fraction
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Patrick O. Brown
Maximillian Diehn
Michael Eisen
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • mRNAs microbial messenger RNAs
  • ESTs Expressed Sequence Tags
  • nucleic acid array A tool showing considerable promise for parallel analysis of multiple sequences is the nucleic acid array, reviewed by Ramsay (1998) Nat. Biotech. 16:40-44, and in a collection of reviews in Nature Genetics (1999) 21(1 supplement): 1-60.
  • These arrays contain dense collections of nucleic acids, either PCR products or polynucleotides, usually of known sequence, that have been either synthesized or printed at fixed spatial locations on suitable substrates, such as nylon filters or glass slides.
  • suitable substrates such as nylon filters or glass slides.
  • the abundance of specific sequences in solution can be quantitated based on the fluorescent or radioactive signal intensity at the position of the complementary probe. While recent interest has been directed toward the use of arrays for monitoring global gene expression, arrays can also be used for rapid detection of sequence variation.
  • therapeutics that have been successfully developed by biotechnology companies have been naturally secreted proteins, or modified versions thereof, e.g. insulin, human growth factor, erythropoietin, tissue plasminogen activator, DNAse, etc., or agents that bind to cell surface molecules, e.g. herceptin.
  • a large fraction of conventional therapeutics are also directed at cell-surface molecules such as receptors, channels and transporters.
  • Secreted proteins are also of particular utility in the development of clinical diagnostic tests. Identification of secreted proteins specific to tumors would allow the development of non-invasive blood based assays along the lines of the PSA screen for prostate cancer, that could be used to screen high risk populations. Early detection of the disease should allow early treatment leading to better survival statistics. Furthermore, recent advances in monoclonal antibody therapy call for the identification of new tumor-specific membrane associated markers that could serve as targets for monoclonal antibody therapies. Also, antibodies against secreted mediators of inflammation or other pathological processes, e.g. cytokines, chemokines, ef ⁇ , could neutralize these offending agents.
  • cytokines e.g. cytokines, chemokines, ef ⁇
  • sequence of transmembrane regions and signal sequences are helpful, but not decisive in identifying proteins that are membrane bound or secreted.
  • the information in EST databases is typically partial sequences corresponding to the 3' region of an mRNA, which may not include the sequences of transmembrane regions or signal peptides that could be recognized using sequence based rules.
  • sequence-prediction methods are very unreliable in predicting whether a given protein is secreted or membrane bound
  • a high through-put method for determining which gene products fall into defined functional classes based on properties of the pro important class would therefore be of great value.
  • the probes are labeled nucleic acids from a source sample comprising a complex mixture of different nucleic acids.
  • the source sample is fractionated prior to labeling based on a physical attribute, e.g. association with a membrane bound ribosome, association with multiple polysomes, associiation with specific molecules, e.g. proteins; subcellular localization, etc. Each fraction of interest is labeled.
  • the probe or probes is hybridized to an array, and the labels present on the probe or probes is detected. Nucleic acids present on the array are scored for the presence of the labels. Based on this information, target nucleic acids present on the array are characterized as to their correspondence with a desired physical attribute.
  • the methods provide rapid differentiation between sequences that code for soluble intracellular proteins, and proteins that are either secreted or associated with cellular membrane structures such as the plasma membrane.
  • Probe fractionation for these sequences relies the physical association of membrane bound polysomes with mRNA encoding secreted or membrane bound proteins.
  • Figure 1 shows a schematic of polysome isolation and DNA microarray hybridization.
  • Target sequences are identified based on their hybridization to a probe.
  • the probes are labeled in order to provide information about a physical attribute, e.g. length of mRNA, association with ribosomes, sub-cellular localization, etc.
  • the probes are obtained from a source sample that is fractionated based on such a physical attribute. The fractions are then differentially labeled.
  • the probes hybridize to an array, and the label present on the probes is detected. Nucleic acids present on the array are scored for hybridization with a probe having particular label characteristics. Based on this information, target nucleic acids are identified that correspond to the desired physical attribute.
  • the source nucleic acid may be fractionated to give a single fraction of interest, which is then labeled for use as a probe.
  • the hybridization pattern of the probe is usually compared to the hybridization pattern of a comparison probe labeled with a spectrally resolvable label.
  • the comparison probe may be another fraction from the separation, the unfractionated material, or any other suitable reference sequence. It will be understood by one of skill in the art that many detectable labels useful in conjugating nucleic acids are available, and can be used to provide combinatorial diversity in the subject methods.
  • the source nucleic acid may separated into two or more fractions, where the label is used to distinguish between fractions.
  • a fractionation that yields two fractions of material may be labeled as follows: fraction (1) with fluorochrome X; and fraction (2) with fluorochrome Y.
  • Analysis of the hybridized array for relative intensities of the X and Y fluorochrome allows identification of a target nucleic acid as corresponding to fraction (1) or (2).
  • a selected fraction may be labeled with fluorochrome X; and a sample of the unfactionated source nucleic acid labeled with fluorochrome Y.
  • the source sample provides the basis for the labeled probe.
  • the source sample is mRNA from a cell population, which is fractionated based on physical properties, e.g. association of the mRNA with sub- cellular organelles or proteins; sub-cellular localization of the mRNA; nuclear transport; size of the mRNA species, etc.
  • the fractionated mRNA may be directly labeled, indirectly labeled, or amplified prior to labeling.
  • the isolation procedure will be performed in conjunction with the fractionation for a desired physical property, as the association of mRNA with proteins and organelles in the intact cell provides useful information about the encoded protein.
  • the source sample is derived from a species where a significant amount of genomic or cDNA sequence information is available. A number of organisms have sufficient sequence information to meet these requirements, including organisms with complete known genome sequences, e.g. Aquifex aeolicus;
  • Methanococcus jannaschii Mycoplasma genitalium; Mycoplasma pneumoniae; Saccharomyces cerevisiae; Synechocystis PCC6803; and organisms with substantial sequence and mapping information known, e.g. Arabidopsis thaliana; Caenorhabditis elegans; Drosophila melanogaster; Homo sapiens; Leishmania major; Mus musculus;
  • Oryza sativa Zea mays, etc.
  • the source sample is fractionated according to a physical property.
  • the physical property may provide information about the encoded polypeptide.
  • secreted and membrane bound mRNA species are typically associated with membrane bound polysomes.
  • the polysomes may be fractionated by precipitation with antibodies, e.g. pan-specific antibodies that recognize a peptide motif or domain of interest.
  • Subcellular localization, association with specific proteins, nuclear transport, and translational regulation are all properties that can be measured by physical fractionation of mRNAs.
  • the size of the mRNA represented by each unknown gene represented in a microarray can be determined.
  • cytoplasmic and membrane bound polysomes are separated. This can be accomplished via various subcellular fractionation protocols, including ones that are detergent or density gradient centrifugation based. A description of the density gradient based isolation processes can be found in Mechler (1987) Meth. Enzymol. 152:241-247. The mRNA molecules of secreted or membrane-bound proteins are translated by ribosomes that are found in the membrane-bound polysomal fraction.
  • Polysome precipitation with antibody species uses a two step fraction, and may be performed in combination with the subcellular fractionation.
  • the polysome complexes that are isolated by the above methods may further comprise nascent polypeptide chains encoded by the mRNA.
  • An immunoprecipitation is performed to further fractionate the polysomes into those that bind to an antibody, and those that do not.
  • polysome immunoprecipitation are antibodies that are pan-specific for a particular motif; class of proteins; domain; amino acid residue, e.g.
  • polysomes are separated by density according to the size of the polysomal complex.
  • the size is correlated with the number of ribosomes present on each mRNA molecule, and is indicative of the level of translation of that mRNA, where a greater number of ribosomes are present when translation levels are high.
  • Probes derived from these fractions will therefore identify sequences whose corresponding mRNAs have different levels of translation in the cell type, or in the physiologic condition, of interest. Any suitable method may be used to fractionate the polysomes, such as sucrose gradients and the like. mRNAs may be fractionated according to length on gels, gradients, etc. as known in the art.
  • fractions identify sequences in an array based on the length of the mRNAs that they represent.
  • Amplification The fractionated nucleic acid sample may be labeled directly, or it may first be amplified in order to provide larger amounts, increased signal, etc.
  • mRNA may be amplified by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences, or by joining oligonucleotides to the ends of the cDNAs, and amplifying the entire cDNA sample by using primers specific for these added oligonucleotides.
  • mRNA may be amplified by the methods described in U.S. Patent no.
  • cDNA is synthesized from a ribonucleic acid (RNA) sequence using a complementary primer linked to an RNA polymerase promoter region.
  • Anti-sense RNA is transcribed from the cDNA by an RNA polymerase capable of binding to the promoter region.
  • a detectable label may be included in such amplification reactions, or may be conjugated to the fractionated nucleic acid after, or in the absence of, amplification reactions.
  • Standard labeling protocols for nucleic acids are described in Sambrook et al:, Kambara et al. (1988) BioTechnolo ⁇ v 6:816-821; Smith et al. (1985) Nuc. Acids Res. 13:2399-2412.
  • Suitable labels include fluorochromes, e.g.
  • fluorescein isothiocyanate FITC
  • rhodamine Texas Red
  • phycoerythrin allophycocyanin
  • 6-carboxyfluorescein 6-FAM
  • 2 l ,7 l -dimethoxy-4',5'- dichloro-6-carboxyfluorescein JOE
  • 6-carboxy-X-rhodamine ROX
  • HEX ⁇ -carboxy ⁇ ' ⁇ 'J' ⁇ J- hexachlorofluorescein
  • 5-carboxyfluorescein 5-FAM
  • TAMRA N.N.N'.N'-tetramethyl- ⁇ - carboxyrhodamine
  • fluorochromes suitable for multicolor analysis are known in the art. For example, Haddad ef al. (1998) Hum Genet 103(5):619-25 discuss the number of spectrally distinguishable fluorochromes or fluorochrome combinations, and the simultaneous visualization of probes in 24 different colors.
  • Other fluorochromes are known in the art, for examples see Wiegant ef al. (1996) J Histochem Cytochem 44(5):525-9; Vaandrager et al. (1996) Blood 88(4): 1177-82; van Gijlswijk ef al. (1997) Histochem Cvtochem 45(3):375-82; and others.
  • the label may also be an indirect system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label.
  • the label may be conjugated to one or both of the primers in an amplification.
  • the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
  • a quickly and easily detectable signal is preferred, and fluorescent tagging of the probe is preferred.
  • Other suitable labels include heavy metal labels, magnetic probes, chromogenic labels, e.g. phosphorescent labels, dyes, and fluorophores, spectroscopic labels, enzyme linked labels, radioactive labels, and labeled binding proteins. The label will be selected such that at least two different labels can be simultaneously measured in one hybridization.
  • each fraction will be labeled in such a way that it is distinguished from the other fractions or from a reference nucleic acid sample. This can be as simple as labeling two fractions, each with a different fluorochrome.
  • each fraction can be compared to the same common reference by labeling each individual fraction , in turn, with fluor 1 , and labeling a reference mixture, which might be a selected fraction, or composed of samples of each fraction, with fluor 2, and then for each fraction hybridizing the mixture of fluor 1 -labelled fraction with fluor 2-labeled reference mixture to an array, and then measuring the fluorescence ratio of the two fluors at each array element, (see DeRisi ef al.
  • array as used herein is intended to refer to high density collections of nucleic acid sequences. Arrays may be gridded on a planar surface, or may be attached to particles. High density microarrays of polynucleotides are known in the art and are commercially available.
  • sequence of polynucleotides on the array will correspond to cDNA or genomic sequences, usually obtained from the species that is the source of the probe.
  • Arrays of interest for the subject methods will generally comprise at least about 10 2 different sequences, usually at least about 10 3 different sequences, and may comprise 10 4 , 10 5 , or more different sequences. This number of sequences may be deposited on a small planar surface of about 1-10 cm 2 .
  • the length of polynucleotide present on the array is an important factor in how sensitive hybridization will be to the presence of a mismatch.
  • polynucleotides will be at least about 12 nt in length, more usually at least about 15 nt in length, preferably at least about 20 nt in length or longer.
  • Patent no. 5,134,854 (Pirrung etal , and U.S. Patent no. 5,445,934 (Fodor etal.) using light-directed synthesis techniques.
  • the polynucleotides are synthesized stepwise on a substrate at positionally separate and defined positions.
  • Use of photosensitive blocking reagents allows for defined sequences of synthetic steps over the surface of a matrix pattern.
  • the surface of the substrate can be positioned to generate a desired pattern of regions, each having a defined sequence polynucleotide synthesized and immobilized thereto.
  • microarrays are generated by deposition of pre-synthesized polynucleotides onto a solid substrate, for example as described in U.S. Patent no. 5,807,522, issued September 15, 1998.
  • the production of the collection of specific polynucleotides may be produced in at least two different ways. Present technology certainly allows production of ten nucleotide oligomers on a solid phase or other synthesizing system. See e.g., instrumentation provided by Applied Biosystems, Foster City, Calif.
  • amplification reactions are used to generate specific fragments of DNA, or clones contained inserts of the desired sequences may be grown by conventional techniques. Once the desired repertoire of possible oligomer sequences of a given length have been synthesized, this collection of reagents may be individually positionally attached to a substrate. This attachment could be automated in any of a number of ways.
  • each polynucleotide may be attached to a single bead or substrate.
  • the beads may be encoded to indicate the specificity of attached polynucleotide.
  • the probe is then bound to the whole collection of beads and those beads that have appropriate complementary sequence will hybridize to the probe.
  • a sorting system may be utilized to sort those beads that actually bind the target from those that do not. This may be accomplished by presently available cell sorting devices or a similar apparatus. After the relatively small number of beads which have bound the probe are collected, the encoding scheme may be read off to determine the specificity.
  • An encoding system may include a magnetic system, a shape encoding system, a color encoding system, or a combination of any of these, or any other encoding system.
  • Hybridization The hybridization conditions between probe and target is selected such that the hybridization of the two molecules is both sufficiently specific and sufficiently stable. See Hames and Higgins (1985) Nucleic Acid Hybridisation: A Practical Approach, IRL Press, Oxford. These conditions will be dependent both on the specific sequence and often on the guanine and cytosine (GC) content of the complementary hybrid strands. The conditions may often be selected to be universally equally stable independent of the specific sequences involved. This typically will make use of a reagent such as an alkylammonium buffer (see Wood et al. (1985) P.N.A.S. 82:1585-1588; and Krupov ef al. (1989) FEBS Letters 256:118-122).
  • a reagent such as an alkylammonium buffer (see Wood et al. (1985) P.N.A.S. 82:1585-1588; and Krupov ef al. (1989) FEBS Letters 256:118-122).
  • alkylammonium buffer tends to-minimize differences in hybridization rate and stability due to GC content. By virtue of the fact that sequences then hybridize with approximately equal affinity and stability, there is relatively little bias in strength or kinetics of binding for particular sequences.
  • the different probes may be combined in a single hybridization reaction, or each probe may be applied to a separate array substrate. In a preferred embodiment, two or more differently labeled probes are combined in the hybridization reaction.
  • Data analysis includes the steps of determining fluorescent intensity as a function of substrate position from the data collected, correcting for background, removing technically deficient data, i.e. data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the targets from the remaining data.
  • the resulting data may be displayed as an image with the intensity or color in each region varying according to the binding affinity between targets and probes.
  • Arrays can be scanned to detect hybridization of the labeled probes.
  • Methods and devices for detecting fluorescently marked targets on devices are known in the art.
  • Such detection devices often include a microscope and light source for directing light at a substrate.
  • a photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate.
  • a confocal detection device that may be used in the subject methods is described in U.S. Patent no. 5,631 ,734.
  • a scanning laser microscope is described in Shalon ef al. (1996) Genome Res. 6:639.
  • a scan, using the appropriate excitation line, is performed for each fluorophore used.
  • the digital images generated from the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescent signal from one probe is compared to the fluorescent signal from the other probe(s), and the relative signal intensity determined.
  • Other devices may use a CCD camera to image the entire array or segments of the array.
  • the initial data resulting from the detection system is an array of data indicative of fluorescent intensity versus location on the substrate.
  • the data are typically taken over regions substantially smaller than the area in which the polynucleotide was attached. For example, with a "spot" of 500 microns by 500 microns, the data may be taken over regions having dimensions of 5 microns by 5 microns. Within any "spot", a large number of fluorescence data points may be collected.
  • the detection method provides a positional localization of the region where hybridization has taken place, and this position is then correlated with the corresponding nucleic acid attached to that position.
  • nucleic acid sequences are obtained from partial cDNA clones (or ESTs), it is desirable to obtain the corresponding full-length sequence.
  • Libraries of cDNA are made from selected tissues. Preferably, the tissue is the same as the tissue from which the EST or the probe was obtained.
  • the choice of cell type for library construction can be made after the identity of the protein encoded by the gene corresponding to the nucleic acid of the invention is known. This will indicate which tissue and cell types are likely to express the related gene, and thus represent a suitable source for the mRNA for generating the cDNA.
  • cDNA can be prepared by using primers based on sequence from the partial cDNA, or using poly- T primers.
  • RNA protection experiments are performed as follows. Hybridization of a full- length cDNA to an mRNA will protect the RNA from RNase degradation. If the cDNA is not full length, then the portions of the mRNA that are not hybridized will be subject to RNase degradation. This is assayed, as is known in the art, by changes in electrophoretic mobility on polyacrylamide gels, or by detection of released monoribonucleotides.
  • 5' RACE PCR Protocols: A Guide to Methods and Applications, (1990) Academic Press, Inc.
  • PCR methods may be used to amplify the members of a cDNA library that comprise the desired insert.
  • the desired insert will contain sequence from the full length cDNA that corresponds to the EST sequence.
  • Such PCR methods include gene trapping and RACE methods.
  • Rapid amplification of cDNA ends or RACE, is a PCR method of amplifying cDNAs from a number of different RNAs. The cDNAs are ligated to an oligonucleotide linker, and amplified by PCR using two primers.
  • One primer is based on sequence from the instant nucleic acids, for which full length sequence is desired, and a second primer comprises sequence that hybridizes to the oligonucleotide linker to amplify the cDNA.
  • Another PCR-based method generates full- length cDNA library with anchored ends without needing specific knowledge of the cDNA sequence.
  • the method uses lock-docking primers (l-VI), where one primer, poly TV (l-lll) locks over the polyA tail of eukaryotic mRNA producing first strand synthesis and a second primer, polyGH (IV-VI) locks onto the polyC tail added by terminal deoxynucleotidyl transferase (TdT). This method is described in WO 96/40998.
  • the subject nucleic acid compositions can be used to, for example, produce polypeptides, as probes for the detection of mRNA in biological samples, e.g., extracts of cells, to generate additional copies of the nucleic acids, to generate ribozymes or antisense oligonucleotides, and as single stranded DNA probes or as triple-strand forming oligonucleotides.
  • kits may be supplied which provide the necessary reagents in a convenient form and together.
  • kits could be provided that include chips containing an appropriate microarray for the subject to be analyzed, labels and fractionation reagents.
  • Other components such as automated systems for determining and interpreting the hybridization results, software for analyzing the data, or other aids may also be included depending upon the particular protocol which is to be employed.
  • PNAS 89:3010 was used to amplify mRNA sequences present in fractionated polysome samples from Jurkat cells while mRNA samples from yeast cells were analyzed without amplification.
  • the array used for the Jurkat experiment contained approximately 5500 human cDNAs while the array used in the yeast experiment contained almost every known putative protein-encoding gene in the yeast genome (approximately 6500 open reading frames).
  • Membrane-bound polysomes were purified from yeast cells by a different subcellular fractionation method. Nevertheless, the mRNAs most enriched in the membrane fraction appeared to encode secreted or membrane-associated proteins.
  • the Yeast Protein Database http://quest7.proteome.com/YPDhome.html was used as the source of subcellular localization information of the products of defined yeast genes.
  • YPD Yeast Protein Database
  • YPD categories cytosolic, cytoskeletal, or nuclear
  • YPD categories mitochondrial, vesicles of secretory system, endoplasmic reticulum, Goigi, vacuoiar, iipid particles, plasma membrane, peroxisomal, extracellular, or unspecified membrane
  • % Membrane-associated refers to the percentage of the sum of the "Free” and “Membrane-associated” categories.
  • Polysome isolation Grow up tissue culture cells in roller bottles (5x10 8 cells/gradient). Treat with 50 ⁇ M cycloheximide for 10' at 37°. Pellet cells in 250 ml centrifuge tubes for 10 min at 2800 RPM (4°). After first round of centrifugation, aspirate supernatant and replace with remaining media from roller bottles. Centrifuge as before. Repeat as necessary. Wash cells 2x with PBS supplemented with 50 ⁇ M cycloheximide.
  • RBS 50 ⁇ M cycloheximide, 10 mM KCI, 1.5 mM MgCI 2 , 10 mM Tris-HCI, pH 7.4
  • Harvest gradients by puncturing bottom of centrifuge tubes with an 18-gauge needle and collecting 1 ml fractions in 1.5 ml microcentrifuge tubes. Measure absorbance of fractions at 260 nm to determine presence of nucleic acid. Free ribosomes and free mRNA will be present in the load zone while membrane-associated ribosomes and mRNA will be at the interface between the 1.98 M and 1.3 M sucrose steps. Alternatively, the membrane fraction can be isolated with a pipet from the top of the gradient.
  • RNA and 1.98M/1.3M interface fractions (Membrane-associated RNA) and isolate total RNA from each with TRIZOL Reagent (Life Technologies, Inc.) At this point can either further isolate polyA + RNA or use total RNA as input for labeling reaction. If using total RNA, use 50-100 ⁇ gRNA in normal labeling reaction. If desired, amplify mRNA using the Eberwine RNA amplification protocol (Eberwine, et al., PNAS 89:3010, 1992). Perform DNA microarray hybridization, labeling free RNA with one dye and membrane-associated RNA with a second dye.
  • S. cerevisiae mRNA Fractionation Polysome Isolation: Grow 2 L culture of yeast cells to log phase. Centrifuge at
  • RNA Isolation and Microarray Hybridization Isolate mRNA using TRIZOL

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Abstract

L'invention concerne des techniques de caractérisation de polynucléotides cible dans un ensemble. Cet ensemble est hybridé avec des sondes, lesdites sondes étant fractionnées et marquées au moyen d'étiquettes pouvant être distinguées par voie spectrale afin de fournir des informations relatives à un attribut physique de l'acide nucléique, par exemple, la longueur de l'ARNm, l'association avec les ribosomes, la localisation infracellulaire, etc. On procède à une évaluation des acides nucléiques présents dans l'ensemble en vue de l'hybridation avec une sonde ayant des caractéristiques d'étiquette particulières. A partir de ces informations, on identifie les acides nucléiques cible correspondant à l'attribut physique recherché.
PCT/US1999/015355 1998-07-10 1999-07-07 Ensembles de nucleotides de criblage permettant de determiner la correspondance avec les proprietes de sequence et physiques d'une sonde WO2000003037A1 (fr)

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Cited By (8)

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EP1169477A1 (fr) * 1999-03-24 2002-01-09 Quark Biotech, Inc. Adnc codant pour des proteines secretees ou membranaires
EP1169477A4 (fr) * 1999-03-24 2003-01-02 Quark Biotech Inc Adnc codant pour des proteines secretees ou membranaires
US6599701B1 (en) 1999-08-25 2003-07-29 Clarity Biosciences, Inc. Identifying organisms by detecting intronic nucleic acids
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