Classifications

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  • 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
• • 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
• • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

• the invention relates in part to novel methods for producing cDNA and cRNA from a biological sample. Also included are methods for comparing the presence or amount of mRNA in a biological sample.
• the invention also includes random oligonucleotide primers which comprise an RNA polymerase promoter.
• Gene expression is important for understanding a wide range of biological phenomena, including development, differentiation, senescence, oncogenesis, and many other medically important processes. Recently, changes in gene expression have also been used to assess the activity of new drug candidates and to identify new targets for drug development. The latter objective is accomplished by correlating the expression of a gene or genes known to be affected by a particular drug with the expression profile of other genes of unknown function when exposed to that same drug. Genes of unknown function that exhibit the same pattern of regulation, or signature, in response to the drug are likely to represent novel targets for pharmaceutical development.
• mRNA messenger RNA
• DNA microarrays Randomsay, Nature Biotechnol. 16: 40-44, 1998; Marshall and Hodgson, Nature Biotechnol. 16: 27-31, 1998; Lashkari et al., Proc. Natl. Acad. Sci. (USA) 94: 130-157, 1997; DeRisi et al., Science 278: 680-6, 1997).
• Mammalian cells contain as many as 1 xlO to 3 x 10 different mRNA molecules, each of which varies in abundance (or frequency) within a given cell. The most abundant mRNAs are typically present at many thousands of copies per cell, while others may be present in as few as one copy or less per cell. Techniques for analyzing gene expression at the level of transcription typically require tens to hundreds of micrograms of mRNA, or as much mRNA as might be found in 10 7 -10 9 mammalian cells. Oftentimes, it is impractical to obtain this number of cells from a tissue of interest. For example, a blood sample typically contains 10 6 nucleated cells/ml. Hence, to obtain 10 9 cells for analysis would necessitate taking a 1000 ml blood sample, which is clearly impractical in most instances.
• PCR polymerase chain reaction
• the primers are then extended by a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence.
• a polymerase enzyme to yield extension products that are themselves complementary to the primers and hence serve as templates for synthesis of additional copies of the target sequence.
• Each successive cycle of denaturation, primer annealing, and primer extension essentially doubles the amount of target synthesized in the previous cycle, resulting in exponential accumulation of the target.
• cDNA complementary DNA
• reverse transcriptase reverse transcriptase
• oligo(dT) primer reverse transcriptase
• the products of this reaction can then be amplified directly using random priming (Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York).
• cDNA is a deoxyribonucleic acid that contains the information coding for the synthesis of proteins, but lacks the intervening introns present in genomic DNA.
• cDNA and/or libraries thereof plays a critical role in a variety of different applications in biotechnology and related fields.
• Applications in which cDNAs and/or libraries thereof are employed include gene discovery, differential gene expression analysis, and the like.
• a variety of protocols have been developed to prepare cDNA and libraries thereof, where such methods are continually being modified.
• PCR methodologies in general suffer from several limitations that are well-known in the art. See U.S. Pat. No. 5,716,785 and Innis et al., eds. (1990, PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif.) for review and discussion of limitations.
• One such limitation results from the poor fidelity of commonly used, thermostable polymerase enzymes, such as Taq. This results in nucleotide base misincorporations that are propagated from one cycle to the next. It is estimated that such misincorporations may occur as often as once per one thousand bases of incorporation.
• a second limitation is that different cDNAs are amplified with different efficiencies, resulting in under representation of some cDNA sequences and overrepresentation of others in the amplified product. Even a small difference in efficiency may result in a several-thousand fold differential in the representation of these cDNAs in the product after only 30 cycles of amplification.
• reverse transcription An alternative method of mRNA synthesis and amplification is known as reverse transcription and is described in U.S. Pat. No. 5,716,785, herein incorporated by reference. Unlike PCR, reverse transcription does not result in geometric amplification, but rather in linear amplification.
• an oligo(dT) primer that is extended at the 3'- end with a bacteriophage T7 RNA polymerase promoter is used to prime the poly-A+ mRNA population for cDNA synthesis. After synthesis of the first-strand cDNA, the second-strand cDNA is made using the method of Gubler and Hoffman (Gene 25:263-69, 1983).
• RNA polymerase results in reverse transcription and linear amplification of mRNA that is anti-sense to the poly-A+ RNA.
• This method has several limitations. It is not as sensitive as PCR and requires a much larger sample of mRNA to generate the same amount of material and thus displays low efficiency.
• Microarrays can be divided into two groups based on what is immobilized: A) cDNAs or B) oligodeoxyribonucleotides (oligos).
• oligo-based microarrays one can use sense oligos i.e. oligos having the same polarity as the mRNA/cDNA one wishes to study). This makes oligo design and matching oligos to cDNA records easy.
• the probe mixture then has to be made with anti-sense polarity, so that it can hybridize to the sense oligos.
• the anti-sense probe mixture is generally made as follows: A total RNA (or mRNA) preparation from a given tissue is subjected to reverse transcription using an oligo-dT primer with a dangling T7 promoter in the 5' end, and reverse transcriptase. Second strand cDNA synthesis is performed in the classical way using DNA polymerase I and DNA ligase. Next, the double-stranded cDNA mixture is transcribed in vitro using T7 RNA polymerase, including labeling nucleotides, i.e. biotinylated nucleotides, producing a biotin-labeled, anti-sense, cRNA probe mixture.
• T7 RNA polymerase including labeling nucleotides, i.e. biotinylated nucleotides, producing a biotin-labeled, anti-sense, cRNA probe mixture.
• the cRNA probe mix is hybridized to the oligo microarray, containing sense orientation oligos representing a collection of cDNA sequences.
• a three-step sandwiched detection procedure can be applied, such as one involving streptavidin that binds biotin, a primary antibody to streptavidin, and a fluorophore-labeled secondary antibody, i.e. Cy3, to the primary antibody, with high-stringency washes between each step.
• the fluorescence of specific Cy3 signals are detected by a confocal scanner and digitized. These primary data are then normalized using background controls and internal standards.
• cRNAs directly labeled with Cy3 or other commonly used fluorophore labels In this case, longer oligos (50-60-mers) are used. With the sandwich method described above, sensitivity is greater, and one can use 30- mers.
• the current methods of synthesizing cRNA probes hinges on the use of an oligo-dT- T7 primer for reverse transcription.
• This primer is intended to hybridize to the poly-A tail of most mRNA.
• the problem with this approach is two-fold: 1) some mRNAs don't have a poly-A tail; 2) some mRNA have a long 3' UTR and thus the reverse transcriptase may have difficulties copying the entire mRNA, and may produce truncated cDNAs that mostly represent non-coding sequences.
• the present invention provides a method of production of double- stranded molecules from a single-stranded ribonucleic acid molecule, which involves three steps.
• the first step involves contacting the ribonucleic acid molecule with one or more first random oligonucleotide primers containing an RNA polymerase promoter and reverse transcriptase under conditions sufficient for template-driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a first deoxyribonucleic acid molecule is produced.
• the second step involves contacting this first deoxyribonucleic acid molecule with a second random primer which does not comprise an RNA polymerase promoter, and DNA polymerase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a second deoxyribonucleic acid molecule is produced.
• the third step involves isolating and purifying the resulting double-stranded deoxyribonucleic acid molecule, such that a double-stranded deoxyribonucleic acid molecule is produced.
• the single-stranded ribonucleic acid molecule used to produce double-stranded DNA molecules is an mRNA.
• the molecule produced from the RNA of interest is a cDNA.
• the RNA polymerase used is T3, T7 or SP6 polymerases.
• the DNA polymerase used is E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the double-stranded DNA produced by reverse transcription of the mRNA of interest is introduced into a vector.
• the primers are each 4-50 nucleotides in length.
• Another aspect of the present invention provides a method for synthesizing at least one cRNA from a sample containing a plurality of different mRNAs.
• First strand cDNA is synthesized by contacting at least one mRNA in the sample with one or more random oligonucleotide primers including an RNA polymerase promoter that is sufficiently complementary to a sequence in the mRNA so as to prime first strand cDNA synthesis, and reverse transcriptase under conditions sufficient for reverse transcriptase activity to occur.
• double-stranded cDNA is synthesized by contacting the first strand cDNA with a second random primer that does not contain an RNA polymerase promoter but is sufficiently complementary to a sequence in the first strand cDNA so to prime second strand cDNA synthesis, and with a DNA polymerase under conditions sufficient for RNA polymerase activity to occur.
• the resulting double-stranded cDNA is isolated and purified, and then subjected to in vitro transcription under conditions sufficient for template driven RNA transcription to occur, such that cRNA is produced.
• the single-stranded ribonucleic acid molecule used to produce double-stranded DNA molecules is an mRNA.
• the molecule produced from the RNA of interest is a cDNA.
• the RNA polymerase used is T3, T7 or SP6 polymerases.
• the DNA polymerase used is E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the double-stranded DNA produced by reverse transcription of the mRNA of interest is introduced into a vector.
• the primers are each 4-50 nucleotides in length.
• the samples contain total RNA or mRNA from cells.
• the cells are prokaryotic, yeast, eukaryotic, mammalian and/or human cells.
• the sample contains total RNA from 1 x 10 6 cells or less. In another embodiment, the sample contains at least 10,000 different mRNAs.
• Another aspect of the present invention discloses a method for comparing the presence or amount of at least one mRNA of interest in a first sample and in a second sample, where these first and second samples each contain a plurality of different mRNAs from one or more cells.
• First strand cDNA is synthesized by contacting at least one mRNA in the sample with one or more random oligonucleotide primers including an RNA polymerase promoter that is sufficiently complementary to a sequence in the mRNA so as to prime first strand cDNA synthesis, and reverse transcriptase under conditions sufficient for reverse transcriptase activity to occur.
• double-stranded cDNA is synthesized by contacting the first strand cDNA with a second random primer that does not contain an RNA polymerase promoter but is sufficiently complementary to a sequence in the first strand cDNA so to prime second strand cDNA synthesis, and with a DNA polymerase under conditions sufficient for RNA polymerase activity to occur.
• the resulting double-stranded cDNA is isolated and purified, and then subjected to in vitro transcription under conditions sufficient for template driven RNA transcription to occur, such that cRNA is produced.
• the cRNA from the first sample is labeled with a first label; the cRNA produced using the second sample is labeled with a second label distinguishable from the first label.
• the mRNA of interest in the first sample is detected or measured by contacting the first cRNA labeled with the first label with a polynucleotide probe capable of hybridizing to the first cRNA of the mRNA of interest under conditions conducive to hybridization; and detecting any hybridization that occurs between said probe and said first cRNA.
• the mRNA of interest in the second sample is detected or measured by contacting the second cRNA, labeled with second label, with polynucleotide probe capable of hybridizing to second cRNA of the mRNA of interest under conditions conducive to hybridization; and detecting any hybridization that occurs between probe and second cRNA.
• the single-stranded ribonucleic acid molecule used to produce double-stranded DNA molecules is an mRNA.
• the molecule produced from the RNA of interest is a cDNA.
• the RNA polymerase used is T3, T7 or SP6 polymerases.
• the DNA polymerase used is E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the double-stranded DNA produced by reverse transcription of the mRNA of interest is introduced into a vector.
• the primers are each 4-50 nucleotides in length.
• the samples contain total RNA or mRNA from cells.
• the cells are prokaryotic, yeast, eukaryotic, mammalian and/or human cells.
• the sample contains total RNA from 1 x 10 6 cells or less. In another embodiment, the sample contains at least 10,000 different mRNAs.
• the present invention provides a method for producing a 5' enriched cDNA library from a sample of mRNA molecules.
• the mRNA is contacted with one or more first random oligonucleotide primers including an RNA polymerase promoter, and reverse transcriptase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a first population of cDNA molecules is produced.
• This first population of cDNA molecules is contacted with a second random primer not comprising an RNA polymerase promoter, and a DNA polymerase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a second population of cDNA molecules is produced.
• the mRNA contains a poly-adenylated tail.
• the mRNA is contacted with a primer mixture that includes one or more first random oligonucleotide primers containing an RNA polymerase promoter and oligo (dT) primers containing an RNA polymerase promoter.
• the oligo (dT) primer is stably associated with the surface of a solid support.
• the first and second labels are fluorescent, radioactive, enzymatic, hapten, biotin or digoxygenin labels.
• the fluorescent label is fluorescein isothiocyanate, lissamine, Cy3, Cy5, or rhodamine 110.
• a first aliquot of the first cRNA is labeled with a first fluorophore having a first emission spectrum
• a second aliquot of the second cRNA is labeled with a second fluorophore with a second emission spectrum differing from that of the first emission spectrum.
• the first fluorophore is Cy3 and the second fluorophore is Cy5.
• kits including, in one or more containers a mixture of first random oligonucleotide primers, where each first primer contains a 3' end sequence of 10-50 nucleotides that contains an A, a G, a T, or a C nucleotide randomly present in each position, and a 5' end sequence of 10-50 nucleotides that includes an RNA polymerase promoter, a mixture of oligo (dT) primers, where each oligo (dT) primer contains a 3' end sequence of 10-50 nucleotides that includes a T nucleotide present in each position; and also includes a 5' end sequence of 10-50 nucleotides containing an RNA polymerase promoter and a mixture of second random oligonucleotide primers, each second primer containing a sequence of 10-50 nucleotides that comprises an A, a G, a T, or a C nucleotide randomly present in each position.
• the present invention provides a method capable of reverse transcription of mRNA over its full length, particularly the 5' region of the cDNA, and hence capable of providing a full length cDNA even if a long chain mRNA is used as a template.
• the present invention also provides a method for in vitro transcription of double-stranded cDNA molecule to produce a labeled cRNA molecule for use in various methodologies including microarray technology.
• the present invention relates to random oligonucleotide primers that contain an RNA polymerase promoter for synthesis of nucleic acids.
• the invention further relates to methods of use of the oligonucleotide primers of the invention for the synthesis of nucleic acid sequences in a population of cells, which methods preserve fidelity with respect to sequence and transcript representation.
• the present invention incorporates a change in the methods used for priming of the mRNA.
• random oligonucleotide primers of varying lengths (i.e. hexamer or nanomer) are employed that have RNA polymerase promoters in their 5' ends.
• Using these random primers containing the RNA polymerase promoters, alone or in combination with an oligo-dT primer, will increase probe coverage towards the 5' end of the template mRNA, as compared to methods using only an oligo-dT primer.
• RNA polymerase promoter Use of the random primers of the invention, comprising an RNA polymerase promoter, and subsequent second strand cDNA synthesis generates a library of cDNAs of different lengths and different start and stop positions. This results in the generation of a tiled network, completely covering the mRNA population; in particular the cDNAs generated provide coverage across the full length of most of the individual mRNA sequences.
• the ensuing in vitro transcription of these cDNAs generates a complete, full-coverage set of either directly labeled or biotin-tagged cRNA probes.
• the present invention does not necessitate any changes in existing chip oligo design, hybridization, or detection protocols. Thus it is a stream-lined improvement to existing procedures.
• oligonucleotide primers for use in the methods of the invention can be of any suitable size, and are preferably 4-50 nucleotides in length.
• oligonucleotide and oligo as used herein denotes single stranded nucleotide multimers of from about 10 to about 100 nucleotides in length.
• polynucleotide as used herein refers to a single- or double-stranded polymer composed of nucleotide monomers of greater than about 100 nucleotides in length up to about 1000 nucleotides in length.
• an oligo (dT) primer for use in the subject methods is sufficiently long to provide for efficient hybridization to the polyA tail of mRNA.
• the length of the oligo (dT) primer ranges from about 10 to 50 nt in length, preferably from about 10 to 30 nt in length, and more preferably from about 20 to 25 nt length.
• the oligo (dT) primer contains a T nucleotide present in each position of the oligo (dT) primer sequence.
• the oligo (dT) primer is stably attached to the surface of a solid support.
• the solid support may be any convenient solid support known to those skilled in the art.
• solid-phases are suitable for use in the subject methods, such phases being known in the art and commercially available.
• Specific solid-phases of interest include polymeric supports including, but not limited to, polystyrene, pegs, sheets, beads, magnetic beads, and the like.
• stably attached is meant that the oligo (dT) primer remains associated with the surface of the solid support under at least conditions of enzymatic template driven nucleic acid synthesis.
• the oligo (dT) primer may be covalently or non-covalently bonded to the solid phase.
• the oligo (dT) primer is contacted with the mRNA under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, where the mRNA molecule serves as the template molecule.
• a random oligonucleotide primer for use in the subject methods is sufficiently long to provide for efficient hybridization to a complementary nucleic acid.
• random oligonucleotide primer or “random primer” is meant a primer of random base sequence relative to the single-stranded RNA of the first hybrid, i.e. a primer that is not known with certainty to hybridize to the mRNA component of the first hybrid or duplex prior to use.
• the random primer has a sequence that is not known for certainty to have a complementary region in the single-stranded portion of the RNA of the hybrid prior to contact with the random primer.
• the random primer is sufficiently long to provide for efficient hybridization to a complementary region of the mRNA component, where the random primer typically ranges from about 4 to 50 nt, preferably from about 10 to 30 nt and more preferably from about 20 to 30 nt in length.
• Primers contain random arrangements of A, T, C, G, U or I nucleotides, such that each random primer contains an A, a G, a T, a C or an I nucleotide randomly present in each position of the sequence.
• the nucleotides are deoxyribonucleotide triphosphates (dNTPs) i.e.
• primers of interest include those that have from about 5 to 15, preferably from about 6 to 9 nt random nts fused in one embodiment to a restriction enzyme cut site or more preferably fused to an RNA polymerase promoter from about 5 to 20 nt, preferably from about 8 to 16 nt, e.g. 3'(N) 9 GCGGCCGCGCGGCCGC5', where N is any nucleotide. Hexamer or nanomer oligonucleotides are preferred embodiments for random primers. In another preferred embodiment, the primers of the present invention are present in a kit.
• the oligonucleotide primers can be DNA, RNA, chimeric mixtures or derivatives or modified nucleic acid versions thereof, so long as it is still capable of priming the desired reaction.
• the oligonucleotide primer can be modified at the base moiety, sugar moiety, or phosphate backbone, and may include other appending groups or labels, so long as it is still capable of priming the desired amplification reaction.
• nucleic acid as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides.
• ribonucleic acid and "RNA” as used herein mean a polymer composed of ribonucleotides.
• RNA can be single-stranded or in a duplex.
• single-stranded RNA is message RNA (mRNA).
• mRNA message RNA
• deoxyribonucleic acid and DNA as used herein mean a polymer composed of deoxyribonucleotides.
• Deoxyribonucleic acid can be single-stranded or double-stranded.
• DNA is complementary DNA (cDNA).
• the random oligonucleotide primer contains an RNA polymerase promoter.
• the RNA polymerase promoter can be at the 5' end of the random oligonucleotide primer.
• the RNA polymerase promoter can be any RNA polymerase promoter sequence known in the art.
• the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, or a SP6 RNA polymerase promoter sequence; T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase can be used to initiate RNA synthesis from these promoters, respectively.
• a random oligonucleotide primer contains a 3' end sequence of 10-50 nucleotides, said 3' end sequence having an A, a G, a T, a C, or a I nucleotide randomly present in each position of said 3' end sequence; and contains a 5' end sequence of 10-50 nucleotides, said 5' end sequence having an RNA polymerase promoter (i.e. any RNA polymerase promoter sequence known in the art). More preferably, the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, or a SP6 RNA polymerase promoter sequence.
• an oligo (dT) primer contains a 3' end sequence of 10-50 nucleotides, said 3' end sequence having a T nucleotide present in each position of said 3' end sequence; and contains a 5' end sequence of 10-50 nucleotides, said 5' end sequence having an RNA polymerase promoter (i.e. any RNA polymerase promoter sequence known in the art). More preferably, the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence, or a SP6 RNA polymerase promoter sequence.
• a random oligonucleotide primer contains a sequence of 10- 50 nucleotides, said sequence having an A, a G, a T, a C, or a I nucleotide randomly present in each position of said sequence.
• the oligonucleotide primer may contain at least one modified base moiety, including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- ⁇ iethylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosy
• the oligonucleotide primer contains at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2- fluoroarabinose, xylulose, and hexose.
• the oligonucleotide primer contains at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• oligonucleotide primers of the present invention may be derived by cleavage of a larger nucleic acid fragment using non-specific nucleic acid cleaving chemicals or enzymes or site-specific restriction endonucleases; or synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems) and standard phosphoramidite chemistry.
• Phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res.
• methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
• the desired oligonucleotide is synthesized, it is cleaved from the solid support on which it was synthesized and treated, by methods known in the art, to remove any protecting groups present.
• the oligonucleotide may then be purified by any method known in the art, including extraction and gel purification.
• concentration and purity of the oligonucleotide may be determined by examining oligonucleotide that has been separated on an acrylamide gel, or by measuring the optical density at 260 nm in a spectrophotometer.
• Nucleic acid synthesis or amplification products such as synthesize RNA may be labeled with any art-known detectable marker, including radioactive labels such as 32 P, 35 S, 3 H, and the like; fluorophores; chemiluminescers; or enzymatic markers.
• the label is fluorescent.
• fluorophore moieties that can be used as labels are selected from the group including but not limited to: 4-acetamido-4'-isothiocvanatostilbene-2,2'disulfonic acid acridine and derivatives: acridine, acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), -(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151), Cy3, Cy5, cyanosine, 4',6-di
• rhodamine and derivatives 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 110, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N'N'-tetramethyl-6-carboxyrhodamine
• synthesized cRNA is labeled with a label, i.e., any label known in the art and as described herein.
• the synthesized cRNA is labeled with a label wherein the label is a fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label, more preferably the label is fluorescent.
• the preferred method of labeling in this invention is with a fluorophore, such as fluorescein isothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularly preferred.
• chemiluminescers and enzymes may also be used as labels.
• the oligonucleotide may be labeled with an enzymatic marker that produces a detectable signal when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase.
• an enzymatic marker that produces a detectable signal when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase.
• Such enzymatic markers are preferably heat stable, so as to survive the denaturing steps of the amplification process.
• Oligonucleotides may also be indirectly labeled by incorporating a nucleotide linked covalently to a hapten or to a molecule such as biotin, to which a labeled avidin molecule or streptavidin may be bound, or digoxygenin, to which a labeled anti-digoxygenin antibody may be bound.
• Oligonucleotides of the invention may be labeled with labeling moieties during chemical synthesis or the label may be attached after synthesis by methods known in the art.
• the synthesized RNA of the invention (i.e. cRNA) is labeled during synthesis to facilitate its detection in subsequent steps.
• the RNA may be labeled with any art-known detectable marker, including but not limited to radioactive labels such as 32 P, 35 S, 3 H, and the like; fluorophores; chemiluminescers; or enzymatic markers.
• Labeling of RNA is preferably accomplished by including one or more labeled NTPs in the in vitro transcription reaction mixture (See, Example 1).
• NTPs may be directly labeled with a radioisotope, such as 32 P, 35 S, 3 H. Radiolabeled NTPs are available from several sources, including New England Nuclear (Boston, Mass.) and Amersham. NTPs may be directly labeled with a fluorescent label such as Cy3 or Cy5.
• biotinylated or allylamine-derivatized NTPs are incorporated during the in vitro transcription reaction and the resultant cRNAs thereafter labeled indirectly, for example, by the addition of fluorophore- conjugated avidin (in the case of biotin) or the NHS ester of a fluorophore (in the case of allylamine).
• fluorescently labeled NTPs may be incorporated during the in vitro transcription reaction, which fluorescently labels the resultant cRNAs directly.
• fluorophore moieties that can be used as labels are set forth herein.
• the preferred label in the methods of this invention is a fluorophore, such as fluorescein isothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularly preferred.
• the RNA may be labeled with an enzymatic marker that produces a detectable signal when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase.
• an enzymatic marker that produces a detectable signal when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase.
• Such enzymatic markers are preferably heat stable, so as to survive the denaturing steps of the amplification process.
• RNA may also be indirectly labeled by incorporating a nucleotide linked covalently to a hapten or to a molecule such as biotin, to which a labeled avidin molecule may be bound, or digoxygenin, to which a labeled anti-digoxygenin antibody may be bound.
• RNA may be labeled with labeling moieties during chemical synthesis or the label may be attached after synthesis by methods known in the art.
• Labeling of RNA is preferably accomplished by preparing cRNA that is fluorescently labeled with NHS-esters. Most preferably, labeling of RNA is accomplished in a two-step procedure in which allylamine-derivatized UTP is incorporated during in vitro transcription. Following the in vitro transcription reaction, unincorporated nucleotides are removed and the allylamine-containing RNAs are conjugated to the N-hydroxysuccinimide (NHS) esters of Cy3 or Cy5.
• NHS N-hydroxysuccinimide
• This two-step method of preparing fluorescent-labeled cRNA is relatively inexpensive and suitable for use in two color hybridizations to DNA microarrays.
• aminoallyl (AA)-labeled nucleic acids are prepared by incorporation of AA-nucleotides.
• AA- UTP Sigma A-5660
• AA-cRNA is prepared using the Ambion MegaScript T7 RNA polymerase in vitro transcription kit, with AA-UTP substituted at 50-100% of the total UTP concentration. It is essential to remove all traces of amine- containing buffers such as Tris prior to derivatizing the AA-nucleic acids.
• AA-nucleic acids prepared in enzymatic reactions are preferably cleaned up on appropriate QIAGEN columns: RNeasy Mini kit (for RNA) or QIAquick PCR Purification kit (for DNA) (QIAGEN Inc- USA, Valencia, Calif.).
• QIAGEN columns samples are applied twice.
• 80% EtOH is preferably substituted for the buffer provided with the QIAGEN kit.
• Samples are eluted twice with 50 ⁇ l volumes of 70° C. H 2 O.
• samples may be cleaned up by repeated cycles of dilution and concentration on Microcon-30 filters.
• AA-nucleic acids are derivatized with NHS-esters, preferably Cy 3 or Cy 5.
• NHS-esters preferably Cy 3 or Cy 5.
• 2-6 ⁇ g of AA-labeled nucleic acid are aliquoted into a microfuge tube, adjusting the total volume to 12 ⁇ l with H 2 O.
• the NHS-ester is dissolved at a concentration of -15 mM in anhydrous DMSO (-200 nmoles in 13 ⁇ l). 27 ⁇ l of 0.1 M sodium carbonate buffer, pH 9, are added. 12 ⁇ l of the dye mix (containing -60 nmoles dye-NHS ester) are then immediately added to the AA-labeled nucleic acid (-6-20 pmoles of a 1 kb molecule).
• the spectrum of the labeled nucleic acid is preferably measured from 220 nm-700 nm.
• the percent recovery of nucleic acid and molar incorporation of dye is calculated from extinction coefficients and absorbance values at l m a x - Recovery of nucleic acid is typically ⁇ 80%.
• the mole percent of dye incorporated per nucleotide ranges from 1.5-5% of total nucleotides.
• in vitro transcribed RNA is labeled with biotin (See, Example 1) or other labels, as described above.
• biotin See, Example 1
• the label is fluorescent and the label is incorporated by procedures commonly used in the art, as well as by the procedure described in Example 1 for a biotin label.
• RNAs from two populations of cells perhaps derived from different tissues or perhaps the same tissue exposed to different stimuli (i.e. comparison of a diseased tissue sample with a normal tissue sample or the comparison of a drug-treated tissue with a untreated tissue).
• Such comparisons are facilitated by labeling the RNAs from one population with a first fluorophore and the RNAs from the other population, with a second fluorophore, where the two fluorophores have distinct emission spectra.
• Cy3 and Cy5 are particularly preferred fluorophores for use in comparing gene expression between two different populations of cells.
• the methods of the invention are applicable to nucleic acid sequences derived from both eukaryotic and prokaryotic cells (i.e. yeast cells), although they are preferably used with eukaryotic cells, more preferably, with mammalian cells and most preferably, with human cells.
• eukaryotic and prokaryotic cells i.e. yeast cells
• cells that may serve as sources of DNA or RNA are nucleated blood cells, established cell lines, tumor cells, and tissue biopsy specimens, among others that will be readily apparent to those of skill in the art.
• RNA i.e. cRNA
• Total cellular RNA, cytoplasmic RNA, mRNA, or poly(A)+ RNA may be used.
• Methods for preparing total and poly(A)+ RNA are well known and are described generally in Sambrook et al. (1989, Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York).
• RNA may be isolated from eukaryotic cells by procedures that involve lysis of the cells and denaturation of the proteins contained therein.
• Cells of interest include wild-type cells, drug-exposed wild-type cells, diseased cells, modified cells, and drug-exposed modified cells.
• RNA is extracted from cells of the various types of interest using guanidinium thiocyanate lysis followed by CsCl centrifugation to separate the RNA from DNA (Chirgwin et al., 1979, Biochemistry 18:5294-5299).
• Poly(A)+ RNA is selected by selection with oligo-dT cellulose (see Sambrook et al., 1989, Molecular Cloning ⁇ A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
• separation of RNA from DNA can be accomplished by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.
• RNase inhibitors may be added to the lysis buffer.
• mRNAs such as transfer RNA (tRNA) and ribosomal RNA (rRNA).
• tRNA transfer RNA
• rRNA ribosomal RNA
• Most mRNAs contain a ⁇ oly(A) tail at their 3' end. This allows them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or SephadexTM (see Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York).
• poly(A)+ mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.
• the sample of RNA can contain a plurality of different mRNA molecules, each different mRNA molecule having a different nucleotide sequence.
• the mRNA molecules in the RNA sample contain at least 100 different nucleotide sequences. More preferably, the mRNA molecules of the RNA sample contain at least 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000 or 100,000 different nucleotide sequences.
• the RNA sample is a mammalian RNA sample (i.e. human RNA sample), the mRNA molecules of the mammalian RNA sample containing about 20,000 to 30,000 different nucleotide sequences.
• total RNA or mRNA from cells are used in the methods of the invention.
• the source of the RNA can be cells of a plant or animal, human, mammal, primate, non-human animal, dog, cat, mouse, rat, bird, yeast, eukaryote, prokaryote, etc.
• the method of the invention is used with a sample containing total mRNA or total RNA from 1 x 10 6 cells or less.
• the subject invention is directed to methods of preparing double-stranded nucleic acid molecules from single-stranded nucleic acid molecules, and specifically to methods of preparing double-stranded deoxyribonucleic acid molecules from single-stranded ribonucleic acid molecules.
• the single-stranded ribonucleic acid molecule is an mRNA molecule having a polyA tail while the double-stranded deoxyribonucleic acid molecule is a cDNA molecule.
• the mRNA molecule need not contain a polyA tail.
• the subject invention is directed to methods of producing double-stranded cDNA molecules that include the sequence information from the 5' end of the template mRNA from which they are prepared.
• the length of the cDNA molecules prepared by the subject methods typically ranges from about 0.5 to 3.0 kb, usually from about 1.0 to 2.0 kb and more usually from about 1.0 to 1.5 kb.
• the first step in the subject methods is to prepare a first deoxyribonucleic acid strand (i.e. cDNA).
• This first DNA strand is prepared from the initial single-stranded RNA molecule, e.g. the mRNA. This step is generally accomplished by contacting the initial RNA with a primer and a reverse transcriptase under conditions sufficient for template driven enzymatic DNA synthesis to occur.
• this first step involves contacting a single stranded RNA molecule, i.e. mRNA, with one or more first random primers containing an RNA polymerase promoter and reverse transcriptase under conditions sufficient for template driven enzymatic DNA synthesis to occur, where the mRNA molecule serves as the template molecule.
• the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence or a SP6 RNA polymerase promoter sequence and T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase are used to initiate RNA or DNA synthesis from these promoters, respectively.
• the initial single-stranded RNA molecule contains a polyadenylated tail
• one or more random primers containing an RNA polymerase promoter contacts the mRNA in combination with an oligo (dT) primer containing an RNA polymerase promoter under conditions for template driven enzymatic DNA synthesis to occur.
• the oligo (dT) primer is stably associated with the surface of a solid support.
• RNA deoxyribonucleic acid molecule i.e. first strand cDNA
• the primers used in the method of the invention are each 4-50 nucleotides in length. Suitable oligonucleotide primers are described herein and can be modified as necessary by the skilled artisan.
• the initial mRNA that serves as template in the first step may be present in a variety of different samples, where the sample will typically be derived from a physiological source.
• the physiological source may be derived from a variety of eukaryotic sources, with physiological sources of interest including sources derived from single celled organisms such as yeast and multicellular organisms, including plants and animals, particularly mammals, where the physiological sources from multicellular organisms may be derived from particular organs or tissues of the multicellular organism, or from isolated cells derived therefrom.
• the physiological source may be subjected to a number of different processing steps, where such processing steps might include tissue homogenization, cell isolation and cytoplasmic extraction, nucleic acid extraction and the like, where such processing steps are known to those of skill in the art.
• processing steps might include tissue homogenization, cell isolation and cytoplasmic extraction, nucleic acid extraction and the like, where such processing steps are known to those of skill in the art.
• Methods of isolating RNA from cells, tissues, organs or whole organisms are known to those of skill in the art and are described herein and in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press)(1989).
• RNA polymerase is any know RNA polymerase but most preferably the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
• a reverse transcription or first strand DNA synthesis buffer, dithiothreitol (DTT), a mixture of deoxyribonucleotide triphosphates (dNTPs) i.e. dATP, dTTP, dCTP, dGTP, or dlTP, and nuclease-free H 2 O are present.
• DTT dithiothreitol
• dNTPs deoxyribonucleotide triphosphates
• the order in which the reagents are combined may be modified as desired, and the entire illustrative procedure described therein can be modified by the skilled artisan.
• Second strand cDNA synthesis is carried out by any method known in the art, as illustrated in Example 1.
• the second step of the subject methods is to contact the first deoxyribonucleic acid molecule with a second random primer which does not contain an RNA polymerase promoter, and DNA polymerase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a second deoxyribonucleic acid molecule is produced resulting in a double-stranded deoxyribonucleic acid molecule (cDNA).
• the double-stranded deoxyribonucleic acid molecule can be isolated and purified according to any method known in the art and illustrated in Example 1.
• the DNA polymerase used for second strand cDNA synthesis is any DNA polymerase known in the art and more preferably E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the primers used in the method of the invention are each 4-50 nucleotides in length. Suitable oligonucleotide primers are described herein and can be modified by the skilled artisan.
• a second strand DNA synthesis buffer, DNA ligase, a mixture of deoxyribonucleotide triphosphates (dNTPs) i.e. dATP, dTTP, dCTP, dGTP, or dlTP, RNase H (to degrade any remaining RNA molecules), and nuclease- free H 2 O are present (See, Example 1).
• double-stranded cDNA molecules with different lengths and different start and stop positions.
• double-stranded cDNA molecules in which the cDNA molecule includes the genetic information of the 5' terminus of the RNA, e.g. mRNA.
• the length of the double-stranded cDNA molecule typically ranges from about 500 to 3000 nt, preferably from about 1000 to 2000 nt and more preferably from about 1500 to 2000 nt.
• the present invention is directed to a method for producing a 5' enriched cDNA library from a sample of mRNA molecules, said method containing: (a) contacting said mRNA with (i) one or more first random oligonucleotide primers containing an RNA polymerase promoter, and (ii) reverse transcriptase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a first population of cDNA molecules is produced; (b) contacting said first population of cDNA molecules (i) with a second random primer not containing an RNA polymerase promoter, and (ii) DNA polymerase under conditions sufficient for template driven enzymatic deoxyribonucleic acid synthesis to occur, whereby a second population of cDNA molecules is produced; and (c) isolating said second population of double-stranded cDNA molecules; such that said 5' enriched library is produced.
• 5' enriched is meant that a significant proportion of the cDNAs in the library contain the nucleotide sequence information of the 5' end of the mRNAs from which the cDNAs are derived.
• the 5' enriched cDNA libraries of the subject invention contain at least 5, usually at least 50 and more usually at least 100 distinct cDNAs (i.e. cDNAs that differ in sequence from each other), where the number of distinct cDNAs in the library may be as high as 10,000 or higher.
• a significant portion of the cDNA constituents of the library include the 5' sequence information of their corresponding mRNA from which they were derived, where the percentage of distinct cDNAs in the library that include 5' sequence information from their corresponding mRNAs is at least about 10%, usually at least about 20% and more usually at least about 25%, where the percentage may be as high as 30% or higher, including in certain embodiments, 40%, 50%, 60% 70% 80%, 90% or higher.
• the initial mRNA that serves as template in the first step may be present in a variety of different samples, where the sample will typically be derived from a physiological source.
• the physiological source is prokaryotic tissue or cell (i.e. yeast cells) and eukaryotic tissue or cell.
• the sample source is a mammalian tissue or cell and most preferably the sample source is a human tissue or cell.
• the sample contains total RNA or total mRNA from the tissue or cells described herein.
• the sample contains total RNA from 1 x 10 6 cells or less and the sample contains at least 10,000 different mRNAs.
• the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence or a SP6 RNA polymerase promoter sequence and T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase are used to initiate RNA or DNA synthesis from these promoters, respectively.
• the DNA polymerase used for second strand cDNA synthesis is any DNA polymerase known in the art and more preferably E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the primers used in the method of the invention are each 4-50 nucleotides in length. Suitable oligonucleotide primers are described herein and can be modified by the skilled artisan.
• one or more random primers containing an RNA polymerase promoter contacts the mRNA in combination with an oligo (dT) primer containing an RNA polymerase promoter under conditions for template driven enzymatic DNA synthesis to occur.
• the oligo (dT) primer is stably associated with the surface of a solid support.
• the second population of double-stranded cDNA molecules can be introduced into a vector as described below.
• the present invention provides a method for synthesizing at least one cRNA from a sample containing a plurality of different mRNAs, said method containing: (a) synthesizing first strand cDNA by contacting at least one mRNA in said sample with (i) one or more random oligonucleotide primers containing an RNA polymerase promoter that is sufficiently complementary to a sequence in the mRNA so as to prime first strand cDNA synthesis, and (ii) reverse transcriptase under conditions sufficient for reverse transcriptase activity to occur; (b) synthesizing double-stranded cDNA by contacting the first strand cDNA with (i) a second random primer not containing an RNA polymerase promoter wherein said second random primer is sufficiently complementary to a sequence in the first strand cDNA so to prime second strand cDNA synthesis, and (ii) a DNA polymerase under conditions sufficient for RNA polymerase activity to occur; (c) iso
• In vitro transcription can be accomplished by any method well known in the art, i.e., Example 1; Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York. Prior to subjecting double-stranded cDNA to in vitro transcription, it may be necessary to introduce the double-stranded cDNA molecule into a vector using any methods well know to those of skill in the art (Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York) and as described herein.
• the physiological source is prokaryotic tissue or cell (i.e. yeast cell) and eukaryotic tissue or cell.
• the sample source is a mammalian tissue or cell and most preferably the sample source is a human tissue or cell.
• the sample contains total RNA or total mRNA from the tissue or cells described herein. In a preferred embodiments the sample contains total RNA from up to 1 x 10 cells and the sample contains at least 10,000 different mRNAs.
• the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence or a SP6 RNA polymerase promoter sequence and T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase are used to initiate RNA or DNA synthesis from these promoters, respectively.
• the DNA polymerase used for second strand cDNA synthesis is any DNA polymerase known in the art and more preferably E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the primers used in the method of the invention are each 4-50 nucleotides in length.
• oligonucleotide primers are described herein and can be modified by the skilled artisan.
• one or more random primers containing an RNA polymerase promoter contacts the mRNA in combination with an oligo (dT) primer containing an RNA polymerase promoter under conditions for template driven enzymatic DNA synthesis to occur.
• the oligo (dT) primer is stably associated with the surface of a solid support.
• synthesized cRNA is labeled with a label, i.e., any label known in the art and as described herein.
• the synthesized cRNA is labeled with a label, wherein the label is a fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label. More preferably, the label is fluorescent.
• the preferred label in the methods of this invention is a fluorophore, such as fluorescein isothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularly preferred.
• the present invention provides a method for determining the presence or absence of a target mRNA from a sample and determining the effect of drug treatment on a target mRNA in a sample following the in vitro transcription step and production of cRNA
• the present invention is directed to a method wherein the mRNA is extracted from at least one cell of interest, and further containing (e) contacting the cRNA produced in step (d) described above with an array containing one or more species of polynucleotide positioned at preselected sites on the array, under conditions conducive to hybridization; and (f) detecting any hybridization that occurs between said one or more species of polynucleotide and said cRNA.
• the invention is directed to a method for comparing the presence or amount of at least one mRNA of interest in a first sample and in a second sample, said first sample and said second sample each containing a plurality of different mRNAs from one or more cells, said method containing: (a) synthesizing first strand cDNA by contacting at least one mRNA in said sample with (i) one or more first random oligonucleotide primers containing an RNA polymerase promoter that is sufficiently complementary to a sequence in the mRNA so as to prime first strand cDNA synthesis, and (ii) reverse transcriptase under conditions sufficient for reverse transcriptase activity to occur; (b) synthesizing double- stranded cDNA by contacting the first strand cDNA with (i) a second random primer not containing an RNA polymerase promoter that is sufficiently complementary to a sequence in the first strand cDNA so to prime second strand cDNA synthesis, and (ii) a
• the initial mRNA that serves as template in the first step may be present in a variety of different samples, where the sample will typically be derived from a physiological source.
• the physiological source is prokaryotic tissue or cell (i.e. yeast cell) and eukaryotic tissue or cell.
• the sample source is a mammalian tissue or cell and most preferably the sample source is a human tissue or cell.
• the sample contains total RNA or total mRNA from the tissue or cells described herein. In a preferred embodiments the sample contains total RNA from 1 x 10 6 cells or less and the sample contains at least 10,000 different mRNAs.
• the RNA polymerase promoter is a T7 RNA polymerase promoter sequence, a T3 RNA polymerase promoter sequence or a SP6 RNA polymerase promoter sequence and 17 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase are used to initiate RNA or DNA synthesis from these promoters, respectively.
• the DNA polymerase used for second strand cDNA synthesis is any DNA polymerase known in the art and more preferably E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the primers used in the method of the invention are each 4-50 nucleotides in length.
• oligonucleotide primers are described herein and can be modified by the skilled artisan.
• one or more random primers containing an RNA polymerase promoter contacts the mRNA in combination with an oligo (dT) primer containing an RNA polymerase promoter under conditions for template driven enzymatic DNA synthesis to occur.
• the oligo (dT) primer is stably associated with the surface of a solid support
• synthesized first and second cRNAs is labeled with a label, i.e., any label known in the art and described herein.
• the synthesized first and second cRNAs is labeled with a label wherein the label is a fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label.
• the label is fluorescent.
• the preferred label in the methods of this invention is a fluorophore, such as fluorescein isothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularly preferred.
• a first aliquot of the first cRNA is labeled with a first fluorophore having a first emission spectrum
• a second aliquot of the second cRNA is labeled with a second fluorophore with a second emission spectrum differing from that of the first emission spectrum; more preferably the first fluorophore is Cy3 and the second fluorophore is Cy5.
• the present invention is directed to a method wherein in steps (h) and (i) as described above, the steps of contacting the first cRNA labeled with said first label with said polynucleotide probe, and contacting the second cRNA labeled with said second label with said polynucleotide probe, are carried out concurrently or sequentially.
• steps (h) and (i) as described above the steps of contacting the first cRNA labeled with said first label with said polynucleotide probe, and contacting the second cRNA labeled with said second label with said polynucleotide probe, are carried out concurrently or sequentially.
• concurrently refers to two events occurring at the same time and the term “sequentially” and refers to succeeding or following in a particular order.
• the first sample contains mRNAs from cells that are diseased and wherein said second sample contains mRNAs from normal cells; or first sample contains mRNAs from drug-treated cells and wherein said second sample contains mKNAs from untreated cells, wherein said levels of said mRNAs from these cells can be compared.
• the present invention is directed to a method wherein in steps (h) and (i) are carried out by a method containing contacting said first and second cRNAs with an array containing one or more species of polynucleotide probe positioned at preselected sites on the array, under conditions sufficient for hybridization to occur; and detecting any hybridization that occurs between said polynucleotide probes and said cRNAs.
• steps (h) and (i) are carried out by a method containing contacting said first and second cRNAs with an array containing one or more species of polynucleotide probe positioned at preselected sites on the array, under conditions sufficient for hybridization to occur; and detecting any hybridization that occurs between said polynucleotide probes and said cRNAs.
• the invention utilizes the ability to measure the responses of a biological system to a large variety of perturbations. Exemplary methods for measuring biological responses are described herein. One of skill in the art will appreciate that this invention is not limited to the following specific methods for measuring the responses of a biological system.
• the presence of cRNA(s) of interest can be detected or measured by procedures including but not limited to Northern blotting or using bead-bound oligonucleotides as probes, or the use of polynucleotide microarrays.
• the synthesized labeled cRNA probes of the invention can be utilized for various other methodologies, including but not limited to dot blot analysis, differential hybridization, RT-PCR, and anti-sense RNA studies.
• Dot blot analysis For any type of dot blot experiment that employs cDNA, genomic DNA, mRNA, or oligos, one skilled in the art can use the invention to synthesize full-length coverage cRNA probes.
• genomic DNA this method can be used to ask evolutionary-type questions such as: "are there exons present in this organism that are similar to exons from this other organism, but less so for this one?"
• Differential hybridization When studying expression differences between cell types or tissues, it is sometimes useful to employ differential hybridization.
• a cDNA library displayed as bacteriophage plaques may be hybridized with the cRNA probes, synthesized and labeled by the methods of the present invention, from the cell type or tissue one wants to compare.
• a reverse transcription reaction of an mRNA pool from a certain tissue can be primed using the random oligonucleotide primer of the present invention containing an RNA polymerase promoter.
• the next step, PCR one would use a gene- specific upper primer directed at the 5 prime end of the desired mRNA, and a 17 promoter- complementary primer. If used during stringent conditions (a long (27-30-mer) upper primer, high annealing temperature, or preferably a touch-down PCR protocol) one can isolate the full-length cDNA of a gene of choice.
• this cDNA is ready to be in vitro transcribed in the anti-sense direction using T7 RNA polymerase.
• the anti-sense RNA can be used in anti-sense RNA studies for a) use in secondary structure determination of the anti- sense RNA; b) use directly labeled with radioactivity or biotin as an anti-sense RNA probe for in situ hybridization; c) use in RNA Interference experiments in duplex with the corresponding sense mRNA, produced by conventional means; d) other anti-sense RNA experiments.
• a single reverse transcription reaction can be used for multiple different genes only by changing the upper primer. This is particularly useful if the tissue or cell type under study is scarce.
• one or more labels is introduced into the RNA during the in vitro transcription step, as described herein, to facilitate gene expression profiling.
• Gene expression can be profiled in any of several ways. The preferred method is to probe a DNA microarray with the labeled RNA transcripts generated as described above.
• a DNA microarray, or chip is a microscopic array of DNA fragments or synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (Schena, BioEssays 18: 427, 1996).
• the DNA in a microarray may be derived from genomic or cDNA libraries, from fully sequenced clones, or from partially sequenced cDNAs known as expressed sequence tags (ESTs). Methods for obtaining such DNA molecules are generally known in the art (See, e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, vol. 2, Current Protocols Publishing, New York). Alternatively, oligonucleotides may be synthesized by conventional methods, such as phosphoramidite-based synthesis.
• Gene expression profiling can be done for purposes of screening, diagnosis, staging of a disease, monitoring response to therapy, as well as for identifying genetic targets of drugs and of pathogens.
• DNA microarrays are typically probed using mRNA, extracted and synthesized to cRNA from the cells whose gene expression profile it is desired to analyze, using the methods of the invention (i.e. producing double-stranded DNA, synthesizing at least one cRNA molecule, labeling at least one synthesized cRNA molecule, producing 5' enriched cDNA library).
• the mRNAs are typically labeled separately with fluorescent dyes that emit at different wavelengths, as described above.
• Some embodiments of this invention are based on measuring the transcriptional rate of genes. Transcriptional rates can be measured by techniques of hybridization to arrays of nucleic acid or nucleic acid mimic probes. However measured, the result is either the absolute, relative amounts of transcripts or response data, including values representing RNA abundance ratios, which usually reflect DNA expression ratios (in the absence of differences in RNA degradation rates).
• aspects of the biological state other than the transcriptional state such as the translational state, the activity state, or mixed aspects can be measured.
• measurement of the transcriptional state is made by hybridization to transcript arrays, which are described herein. Certain other methods of transcriptional state measurement are also described herein.
• Transcript arrays also referred to herein as “microarrays”
• Transcript arrays can be employed for analyzing the transcriptional state in a biological sample and especially for measuring the transcriptional states of a biological sample exposed to graded levels of a drug of interest or to graded perturbations to a biological pathway of interest.
• transcript arrays are produced by hybridizing detectably labeled polynucleotides representing the mRNA transcripts present in a cell (e.g., fluorescently labeled cRNA that is synthesized by the methods of the present invention) to a microarray.
• a microarray is a surface with an ordered array of binding (e.g., hybridization) sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes.
• Microarrays can be made in a number of ways. However produced, microarrays share certain preferred characteristics. Specifically, the arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.
• the microarrays are small, usually smaller than 5 cm 2 , and they are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions.
• a given binding site or unique set of binding sites in the microarray will specifically bind the product of a single gene in the cell.
• site physical binding site
• the microarray is an array of polynucleotide probes, the array containing a support with at least one surface and at least 100 different polynucleotide probes, each different polynucleotide probe containing a different nucleotide sequence and being attached to the surface of the support in a different location on the surface.
• the nucleotide sequence of each of the different polynucleotide probes is in the range of 40 to 80 nucleotides in length. More preferably, the nucleotide sequence of each of the different polynucleotide probes is in the range of 50 to 70 nucleotides in length. Even more preferably, the nucleotide sequence of each of the different polynucleotide probes is in the range of 50 to 60 nucleotides in length.
• the array contains polynucleotide probes of at least 2,000, 4,000, 10,000, 15,000, 20,000, 50,000, 80,000, or 100,000 different nucleotide sequences.
• the nucleotide sequence of each polynucleotide probe in the array is specific for a particular target polynucleotide sequence.
• the target polynucleotide sequences contain expressed polynucleotide sequences of a cell or organism.
• the cell or organism is a mammalian cell or organism.
• the cell or organism is a human cell or organism.
• the nucleotide sequences of the different polynucleotide probes of the array are specific for at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the genes in the genome of the cell or organism. Most preferably, the nucleotide sequences of the different polynucleotide probes of the array are specific for all of the genes in the genome of the cell or organism.
• the polynucleotide probes of the array hybridize specifically and distinguishably to at least 10,000, to at least 20,000, to at least 50,000, different polynucleotide sequences, to at least 80,000, or to at least 100,000 different polynucleotide sequences.
• the polynucleotide probes of the array hybridize specifically and distinguishably to at least 90%, at least 95%, or at least 99% of the genes or gene transcripts of the genome of a cell or organism. Most preferably, the polynucleotide probes of the array hybridize specifically and distinguishably to the genes or gene transcripts of the entire genome of a cell or organism.
• the array has at least 100, at least 250, at least 1,000, or at least 2,500 probes per 1 cm 2 , preferably all or at least 25% or 50% of which are different from each other.
• the array is a positionally addressable array (in that the sequence of the polynucleotide probe at each position is known).
• the nucleotide sequence of each polynucleotide probe in the array is a DNA sequence.
• the DNA sequence is a single-stranded DNA sequence.
• the DNA sequence may be, e.g., a cDNA sequence, or a synthetic sequence.
• the nucleotide sequence of each polynucleotide probe in the array is a oligodeoxyribonucleotides sequence.
• the site on the array corresponding to a gene i.e., capable of specifically binding the product of the gene
• a gene for which the encoded mRNA is prevalent will have a relatively strong signal.
• cRNAs from two different cells are hybridized to the binding sites of the microarray.
• one biological sample is exposed to a drug and another biological sample of the same type is not exposed to the drug.
• one cell is exposed to a pathway perturbation and another cell of the same type is not exposed to the pathway perturbation.
• the cRNA derived from each of the two cell types are differently labeled so that they can be distinguished.
• cRNA from a cell treated with a drug is synthesized using a fluorescein-labeled NTP
• cRNA from a second cell, not drug-exposed is synthesized using a rhodamine-labeled NTP.
• the cRNA from the drug-treated (or pathway perturbed) cell will fluoresce green when the fluorophore is stimulated and the cRNA from the untreated cell will fluoresce red.
• the drug treatment has no effect, either directly or indirectly, on the relative abundance of a particular mRNA in a cell
• the mRNA will be equally prevalent in both cells and, upon reverse transcription, red-labeled and green- labeled cRNA will be equally prevalent.
• the binding site(s) for that species of RNA will emit wavelengths characteristic of both fluorophores (and appear brown in combination).
• the drug-exposed cell is treated with a drug that, directly or indirectly, increases the prevalence of the mRNA in the cell, the ratio of green to red fluorescence will increase. When the drug decreases the mRNA prevalence, the ratio will decrease.
• cRNA from a single cell, and compare, for example, the absolute amount of a particular mRNA in, e.g., a drug-treated or pathway-perturbed cell and an untreated cell.
• the target nucleic acid can be genomic or cDNA or mRNA or synthetic, human or animal, or of a microorganism, etc.
• the target sequence is a wild type human genomic or RNA or cDNA sequence, mutation of which is implicated in the presence of a human disease or disorder, or alternatively, can be the mutated sequence.
• the mutation can be an insertion, substitution, and/or deletion of one or more nucleotides, or a translocation.
• Microarrays are known in the art and consist of a surface to which probes that correspond in sequence to gene products (e.g., cDNAs, mRNAs, cRNAs, polypeptides, and fragments thereof), can be specifically hybridized or bound at a known position.
• the microarray is an array (i.e., a matrix) in which each position represents a discrete binding site for a product encoded by a gene (e.g., a protein or RNA), and in which binding sites are present for products of most or almost all of the genes in the organism's genome.
• the "binding site” is a nucleic acid or nucleic acid analogue to which a particular cognate cRNA can specifically hybridize.
• the nucleic acid or analogue of the binding site can be, e.g., a synthetic oligomer, a full-length cRNA, a less-than full length cRNA, or a gene fragment.
• the microarray contains binding sites for products of all or almost all genes in the target organism's genome. This microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often at least about 75%, more often at least about 85%, even more often more than about 90%, and most often at least about 99%.
• the microarray contains binding sites for products of human genes.
• This microarray will have binding sites corresponding to at least about 5-10% of the genes in the genome, preferably at least about 10-15%, and more preferably at least about 40%.
• the microarray has binding sites for genes relevant to the action of a drug of interest, disease of interest, or in a biological pathway of interest.
• a "gene” is identified as an open reading frame (ORF) of preferably at least 50, 75, or 99 amino acids from which a mRNA is transcribed in the organism (e.g., if a single cell) or in some cell in a multicellular organism.
• ORF open reading frame
• the number of genes in a genome can be estimated from the number of mRNAs expressed by the organism, or by extrapolation from a well-characterized portion of the genome. When the genome of the organism of interest has been sequenced, the number of ORFs can be determined and mRNA coding regions identified by analysis of the DNA sequence.
• ORFs open reading frames
• Analysis of these ORFs indicates that there are 5885 ORFs that are likely to specify protein products (Goffeau et al., 1996, Science 274:546-567, which is incorporated by reference in its entirety).
• the human genome is estimated to contain approximately 10 5 genes.
• the "binding site" to which a particular cognate cRNA specifically hybridizes is usually a nucleic acid or nucleic acid analogue attached at that binding site.
• the binding sites of the microarray are DNA polynucleotides corresponding to at least a portion of each gene in an organism's genome. These DNAs can be obtained by, e.g., polymerase chain reaction (PCR) amplification of gene segments from genomic DNA, cDNA (e.g. , by RT-PCR), or cloned sequences.
• PCR polymerase chain reaction
• PCR primers are chosen, based on the known sequence of the genes or cDNA, that result in amplification of unique fragments (i.e., fragments that do not share more than bases of contiguous identical sequence with any other fragment on the microarray).
• Computer programs are useful in the design of primers with the required specificity and optimal amplification properties. See, e.g., Oligo version 5.0 (National Biosciences).
• Oligo version 5.0 National Biosciences.
• it will sometimes be desirable to amplify segments near the 3' end of the gene so that when oligo-dT primed cDNA probes are hybridized to the microarray, less-than-full-length probes will bind efficiently.
• each gene fragment on the microarray will be between about 50 bp and about 2000 bp, more typically between about 100 bp and about 1000 bp, and usually between about 300 bp and about 800 bp in length.
• PCR methods are well known and are described, for example, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif., which is incorporated herein by reference in its entirety. It will be apparent that computer controlled robotic systems are useful for isolating and amplifying nucleic acids.
• nucleic acid for the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (e.g., Froehler et al., 1986, Nucleic Acid Res 14:5399-5407). Synthetic sequences are between about 15 and about 100 bases in length, preferably between about 20 and about 50 bases. In some embodiments, synthetic nucleic acids include non-natural bases, e.g., inosine. Where the particular base in a given sequence is unknown or is polymorphic, a universal base, such as inosine or 5-nitroindole, may be substituted.
• nucleic acid analogues may be used as binding sites for hybridization.
• An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., 1993, Nature 365:566-568 and U.S. Pat. No. 5,539,083).
• the binding (hybridization) sites are made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209).
• the polynucleotide of the binding sites is RNA.
• the nucleic acid or analog is attached to a solid support, which may be made from glass, silicon, plastic (e.g., polypropylene, nylon, polyester), polyacrylamide, nitrocellulose, cellulose acetate or other materials. In general, non-porous supports, and glass in particular, are preferred.
• the solid support may also be treated in such a way as to enhance binding of oligonucleotides thereto, or to reduce non-specific binding of unwanted substances thereto.
• the glass support is treated with polylysine or silane to facilitate attachment of oligonucleotides to the slide.
• Methods of immobilizing DNA on the solid support may include direct touch, micropipetting (Yershov et al., Proc. Natl. Acad. Sci. USA (1996) 93(10):4913-4918), or the use of controlled electric fields to direct a given oligonucleotide to a specific spot in the array (U.S. Pat. No. 5,605,662,).
• DNA is typically immobilized at a density of 100 to 10,000 oligonucleotides per cm 2 and preferably at a density of about 1000 oligonucleotides per cm 2 .
• a preferred method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., 1995, Science 270:467-470. This method is especially useful for preparing microarrays of cDNA. See also DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena et al., Proc. Natl. Acad. Sci.
• oligonucleotides directly on a solid support particularly preferred methods include photolithography (see Fodor et al., Science 251: 767-73, 1991; McGall et al., Proc. Natl. Acad. Sci. (USA) 93: 13555-60, 1996) and piezoelectric printing (Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20- 4), with the piezoelectric method being the most preferred.
• a high-density oligonucleotide array is employed.
• Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al., 1996, Nature Biotechnol. 14:1675-80; U.S. Pat. No. 5,578,832,; U.S. Pat. No. 5,556,752; and U.S. Pat. No.
• oligonucleotides e.g., 20-mers
• the array produced contains multiple probes against each target transcript.
• Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs or to serve as various type of control.
• Another preferred method of making microarrays is by use of an inkjet printing process to synthesize oligonucleotides directly on a solid phase, as described, e.g., in U.S. Pat. No. 6,419,883, which is incorporated by reference herein in its entirety.
• microarrays e.g., by masking
• any type of array for example, dot blots on a nylon hybridization membrane (see Sambrook et al., 1989, Molecular Cloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), could be used, although, as will be recognized by those of skill in the art, very small arrays will be preferred because hybridization volumes will be smaller.
• nucleic acid hybridization and wash conditions are optimally chosen so that the probe "specifically binds" or “specifically hybridizes” to a specific array site, i.e., the probe hybridizes, duplexes or binds to a sequence array site with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence.
• one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch.
• the polynucleotides are perfectly complementary (i.e. no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al., 1996, Genome Research 6:639-645, and Chee et al., 1996, Science 274:610-614).
• Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA, DNA, PNA) of labeled probe and immobilized polynucleotide or oligonucleotide.
• length e.g., oligomer versus polynucleotide greater than 200 bases
• type e.g., RNA, DNA, PNA
• the fluorescence emissions at each site of a transcript array can preferably be detected by scanning confocal laser microscopy.
• a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al., 1996, Genome Research 6:639-645, which is incorporated by reference in its entirety for all purposes).
• the arrays are scanned with a laser fluorescent scanner with a computer controlled X-Y stage and a microscope objective.
• Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser and the emitted light is split by wavelength and detected with two photomultiplier tubes.
• Fluorescence laser scanning devices are described in Shalon et al., 1996, Genome Res. 6:639-645 and in other references cited herein.
• the fiber-optic bundle described by Ferguson et al., 1996, Nature Biotechnol. 14: 1681-1684 may be used to monitor mRNA abundance levels at a large number of sites simultaneously.
• Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analog to digital board.
• the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site. If necessary, an experimentally determined correction for "cross talk" (or overlap) between the channels for the two fluors may be made.
• a ratio of the emission of the two fluorophores can be calculated. The ratio is independent of the absolute expression level of the cognate gene, but is useful for genes whose expression is significantly modulated by drug administration, gene deletion, or any other tested event.
• the relative abundance of an mRNA in two biological samples is scored as a perturbation and its magnitude determined (i.e., the abundance is different in the two sources of mRNA tested), or as not perturbed (i.e., the relative abundance is the same).
• a difference between the two sources of RNA of at least a factor of about 25% RNA from one source is 25% more abundant in one source than the other source), more usually about 50%, even more often by a factor of about 2 (twice as abundant), 3 (three times as abundant) or 5 (five times as abundant) is scored as a perturbation.
• two samples are hybridized simultaneously to permit differential expression measurements. If neither sample hybridizes to a given spot in the array, no fluorescence will be seen. If only one hybridizes to a given spot, the color of the resulting fluorescence will correspond to that of the fluor used to label the hybridizing sample (for example, green if the sample was labeled with Cy3, or red, if the sample was labeled with Cy5). If both samples hybridize to the same spot, an intermediate color is produced (for example, yellow if the samples were labeled with fluorescein and rhodamine). Then, applying methods of pattern recognition and data analysis known in the art, it is possible to quantify differences in gene expression between the samples. Methods of pattern recognition and data analysis are described in e.g., U.S. Pat. No. 6,203,569 and U.S. Pat. No. 6,271,002; which are incorporated herein by reference in their entireties.
• kits for carrying out the subject methods contain in one or more containers: (a) a mixture of first random oligonucleotide primers, each first primer containing a 3' end sequence of 10-50 nucleotides, said 3' end sequence having an A, a G, a T, or a C nucleotide randomly present in each position of said 3' end sequence; and each first primer having a 5' end sequence of 10-50 nucleotides, said 5' end sequence containing an RNA polymerase promoter; (b) a mixture of oligo (dT) primers, each oligo (dT) primer containing a 3' end sequence of 10-50 nucleotides, said 3' end sequence having a T nucleotide present in each position of said 3' end sequence; and each oligo (dT) primer having a 5' end sequence of 10- 50 nucleotides, said 5' end sequence containing an RNA polymerase promoter;
• Oligonucleotide primers can be in any form, e.g., lyophilized, or in solution (e.g., a distilled water or buffered solution), etc. Oligonucleotides for use in the same reaction can be combined in a single container or can be in separate containers.
• the kit contains in a separate container an RNA polymerase specific to said RNA polymerase promoter.
• the kit contains and RNA polymerase for in vitro transcription of double-stranded cDNA. More preferably, the RNA polymerase is T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase.
• the kit contains in a separate container a DNA polymerase. More preferably, the DNA polymerase is E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, or T4 DNA polymerase.
• the kit contains a buffer for reverse transcription or first strand DNA synthesis, a buffer for DNA synthesis, a buffer for in vitro transcription of double-stranded cDNA, DNA ligase, an RNase (i.e. RNase H).
• a buffer for reverse transcription or first strand DNA synthesis a buffer for DNA synthesis
• a buffer for in vitro transcription of double-stranded cDNA a buffer for in vitro transcription of double-stranded cDNA
• DNA ligase i.e. RNase H
• the kit contains deoxyribonucleotide triphosphate molecules (dNTPs) and ribonucleotide triphosphate molecules (rNTPs). More preferably the dNTPs are dATP, dCTP, dTTP and dGTP and the rNTPs are rATP, rCTP, rUTP and rGTP.
• the dNTPs and rNTPs are labeled, i.e., any label known in the art and as described herein.
• the label is a fluorescent, radioactive, enzymatic, hapten, biotin, or digoxygenin label, more preferably the label is fluorescent.
• the preferred label is a fluorophore, such as fluorescein isothiocyanate, lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularly preferred.
• the kit optionally further contains a set of directions for carrying out reverse transcription or first strand cDNA synthesis and second strand cDNA synthesis; as well as a set of directions for carrying out the in vitro transcription of double-stranded cDNA into cRNA.
• the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof.
• the kit optionally further contains a control nucleic acid, and/or a microarray, and/or means for stimulating and detecting fluorescent light emissions from fluorescently labeled RNA, and/or expression profile projection and analysis software capable of being loaded into the memory of a computer system.
• kits containing random oligonucleotide primers of the invention containing an RNA polymerase promoter and microarrays contain a solid phase, e.g., a surface, to which probes are hybridized or bound at a known location of the solid phase.
• these probes consist of nucleic acids of known, different sequence, with each nucleic acid being capable of hybridizing to an RNA species or to a cDNA species derived therefrom.
• the probes contained in the kits of this invention are nucleic acids capable of hybridizing specifically to nucleic acid sequences derived from RNA species which are known to increase or decrease in response to perturbations to the particular protein whose activity is determined by the kit.
• the probes contained in the kits of this invention preferably substantially exclude nucleic acids which hybridize to RNA species that are not increased in response to perturbations to the particular protein whose activity is determined by the kit.
• kits of the invention further contains expression profile projection and analysis software capable of being loaded into the memory of a computer system.
• expression profile analysis software capable of being loaded into the memory of a computer system.
• An example of such a system is described in reference in U.S. Pat. No. 6,271,002, which is incorporated herein by reference in its entirety.
• the expression profile analysis software contained in the kits of this invention is essentially identical to the expression profile analysis software 512 described in reference in U.S. Pat. No. 6,271,002.
• RNA can be isolated according to any method well known in the art and as described herein.
• Primers are random oligonucleotide primers containing an RNA polymerase promoter as described herein. Random primers can be used alone or in combination with an oligo (dT) primer containing an RNA polymerase promoter up to 2 ⁇ g total primer. Note that the procedure calls for a Reverse Transcriptase with no RNase H activity.
• 5X First-Strand buffer comprises 250 mM Tris-HCl (pH 8.3), 375 mM KC1, 15 mM MgCl 2 . The volume of water depends on the amount of Superscript II added in Step 6.
• the yield of the second strand reaction is typically > 80% relative to the amount of first strand cDNA synthesized.
• RNA is destroyed by adding 30 ⁇ l of 1 M NaOH / 2 mM EDTA. Incubate at 65 °C for 10 minutes. Neutralize with 30 ⁇ l of IN HC1, and 30 ⁇ l of 1 M Tris, pH 7.
• Gentiy discard the supernatant and completely air-dry the double-strand cDNA pellet at room temperature for 10 minutes in a clean and dust-free environment.
• This protocol produces biotin-labeled cRNA probes from the double strand cDNA template synthesized above.
• a high yield transcription kit such as T7 MEGAscriptTM from Ambion Inc. (cat. #1334). Please see the instructions of the manufacturer.
• Biotin-labeling is meant to be illustrative and not to limit the invention. The procedure can be modified by one of ordinary skill in the art to introduce any label as described herein. Furthermore, it may be necessary to introduce the double-stranded cDNA into a vector suitable for in vitro transcription. This can be accomplished by any suitable method and any suitable vector well known to the skilled artisan or as described herein.
• the final yield with Ambion' s MEGAscriptTM kit is typically 25-35 ⁇ g of cRNA transcript per 1 ⁇ g of mRNA.

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