WO2000026406A1 - Procede permettant d'indexer et de determiner la concentration relative d'arn messagers exprimes - Google Patents

Procede permettant d'indexer et de determiner la concentration relative d'arn messagers exprimes Download PDF

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Publication number
WO2000026406A1
WO2000026406A1 PCT/US1999/023655 US9923655W WO0026406A1 WO 2000026406 A1 WO2000026406 A1 WO 2000026406A1 US 9923655 W US9923655 W US 9923655W WO 0026406 A1 WO0026406 A1 WO 0026406A1
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sequence
pcr
restriction endonuclease
cdna
vector
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PCT/US1999/023655
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English (en)
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Karl W. Hasel
Brian S. Hilbush
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Digital Gene Technologies, Inc.
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Priority to IL14296599A priority Critical patent/IL142965A0/xx
Priority to AU11089/00A priority patent/AU1108900A/en
Priority to JP2000579778A priority patent/JP2002528135A/ja
Priority to CA002350168A priority patent/CA2350168A1/fr
Priority to MXPA01004550A priority patent/MXPA01004550A/es
Priority to EP99954838A priority patent/EP1127159A1/fr
Priority to KR1020017005872A priority patent/KR20010092721A/ko
Priority to EA200100490A priority patent/EA200100490A1/ru
Publication of WO2000026406A1 publication Critical patent/WO2000026406A1/fr
Priority to US09/775,217 priority patent/US20020012922A1/en
Priority to NO20012203A priority patent/NO20012203L/no

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • This invention is directed to methods for simultaneous identification of differentially expressed mRNAs, as well as measurements of their relative concentrations.
  • a complete characterization of the protein molecules that make up an organism would be useful, e.g. for the improved design of drugs, the selection of optimal treatment of individual patients, and for the development of more compatible biomaterials.
  • Such a characterization of expressed proteins would include their identification, sequence determination, demonstration of their anatomical sites of expression, elucidation of their biochemical activities, and understanding of how these activities determine organismic physiology.
  • the description should also include information about how the concentration of each protein changes in response to pharmaceutical or toxic agents.
  • RNA complexity studies analog measurements (measurements in bulk) based on observations of mixed populations of RNA molecules with different specificities in abundances.
  • analog measurements measured in bulk
  • RNA complexity studies were distorted by hidden complications of the fact that the molecules in each tissue that make up most of its mRNA mass comprise only a small fraction of its total complexity.
  • cDNA cloning allowed digital measurements (i.e., sequence-specific measurements on individual species) to be made; hence, more recent concepts about mRNA expression are based upon actual observations of individual RNA species.
  • RNA complexity measurements Brain, liver, and kidney are the mammalian tissues that have been most extensively studied by analog RNA complexity measurements. The lowest estimates of complexity are those of Hastie and Bishop (N.D. Hastie & J. B. Bishop, "The Expression of Three Abundance Classes of Messenger RNA in Mouse Tissues," Cell 9:761-774 (1976)), who suggested that 26x10 6 nucleotides of the 3xl0 9 base pair rodent genome were expressed in brain, 23x10 6 in liver, and 22x10 6 in kidney, with nearly complete overlap in RNA sets. This indicates a very minimal number of tissue-specific mRNAs.
  • mRNA differential display In the study of Liang and Pardee, this method, called mRNA differential display, was used to compare the population of mRNAs expressed by two related cell types, normal and tumorigenic mouse A31 cells. For each experiment, they used one arbitrary 10-mer as the 5'-primer and an oligonucleotide complementary to a subset of poly A tails as a 3' anchor primer, performing PCR amplification in the presence of 35 S-dNTPs on cDNAs prepared from the two cell types. The products were resolved on sequencing gels and 50-100 bands ranging from 100-500 nucleotides were observed.
  • the bands presumably resulted from amplification of cDNAs corresponding to the 3'-ends of mRNAs that contain the complement of the 3' anchor primer and a partially mismatched 5' primer site, as had been observed on genomic DNA templates.
  • the pattern of bands amplified from the two cDNAs was similar, with the intensities of about 80% of the bands being indistinguishable. Some of the bands were more intense in one or the other of the PCR samples; a few were detected in only one of the two samples.
  • mismatched priming must be highly reproducible under different laboratory conditions using different PCR machines, with the resulting slight variation in reaction conditions.
  • this is a drawback of building a database from data obtained by the Liang & Pardee differential display method.
  • U.S. Patents Numbers 5,459,037 (O37) and 5,807,680 ('680) describe an improved method of differential display of mRNA species that reduces the uncertain aspect of 5'-end generation and allows data to be absolutely reproducible in different settings.
  • the method does not depend on potentially irreproducible mismatched priming, reduces the number of PCR panels and gels required for a complete survey, and allows double-strand sequence data to be rapidly accumulated.
  • the improved method also reduces the number of concurrent signals obtained from the same species of mRNA.
  • the '037 and '680 patents are hereby incorporated by reference as part of this disclosure.
  • the specificity of the method could be improved by decreasing mispriming during the synthesis of complimentary DNA molecules and during PCR reactions.
  • the technique could be further refined so that it is more reproducible, more sensitive and easier to use.
  • the technique would provide the ability to use sequences obtained to form databases, and to scan nucleotide data bases such as GenBank to recognize sequence identities and similarities using computer programs such as BLASTN and BLASTX.
  • the improved method sorts mRNAs on the basis of an identity or address determined by 1) a partial nucleotide sequence of length a + b, where a is the length in bases of the restriction endonuclease recognition site and b is the number of parsing bases, where 6 > b ⁇ 3, and 2) the distance of that partial sequence from the poly(A) tail.
  • identity or address is determined by a partial sequence that includes a four base recognition site for a restriction endonuclease and four parsing bases.
  • the recognition site for a restriction endonuclease is Mspl.
  • the method can account for all mRNAs present at concentrations above its detection threshold. In contrast to differential display and RAP-PCR methodologies, there is no uncertain aspect to the generation of 5' ends.
  • the cDNA libraries produced from each of the mRNA samples contain copies of the extreme 3' ends, from the most distal site for Mspl to the beginning of the poly(A) tail, of nearly all poly(A) + mRNAs in the starting RNA sample approximately according to the initial relative concentrations of the mRNAs. Because both ends of the inserts for each species are exactly defined by the sequence of the mRNAs themselves, the fragment lengths are uniform for each species, allowing their later visualization as discrete bands on gels. These lengths are constant regardless of the tissue source of the mRNA, an important fundamental concept of the approach. Messenger RNAs lacking Mspl-recognition sequences are not represented, but these are relatively rare. These mRNAs are captured by applying the method using a different restriction endonuclease that recognizes a different four base recognition sequence.
  • Another aspect of such embodiments of the present invention is the use of sequences adjacent to the 3' restriction endonuclease site, in one preferred embodiment, a Mspl site, to sort the cDNAs in at least two successive PCR steps.
  • the first PCR step utilizes a primer that anneals with sequences derived from the vector, e.g., pBC SK + , but extends across the CGG of the non-regenerated Mspl site to include the first adjacent nucleotide (N,) of the insert.
  • This step segregates the starting population of mRNAs into 4 subpools.
  • each of the 4 subpools produced by the first PCR step is further segregated by division into 64 for a total of 256 subsubpools by using more insert-invasive primers (N,N 2 N 3 N 4 ).
  • a fluorescent label is incorporated into the products for their detection by laser-induced fluorescence by using fluorescent labeled 3'PCR primers in the final PCR step.
  • a separation technique such as electrophoresis is used to resolve the labeled molecules of the PCR product into distinct bands of measurable intensities and corresponding to measurable lengths.
  • Suitable separation techniques include gel electrophoresis, capillary electrophoresis, HPLC, MALDI mass spectroscopy and other suitable separations techniques known in the art that are capable of single base resolution over the range of 50 - 500 bases are encompassed by the present invention.
  • each final PCR reaction product is thus assigned an identity or address based upon an 8-nucleotide sequence including the four base restriction endonuclease site plus four parsing bases (e.g., C-C-G-G-N,-N 2 - N 3 -N 4 ) and the distance of that sequence from the junction between the end of the message and the first A of the polyA tail at the 3' end of the mRNA.
  • a digital sequence tag DST: that is, a 3 '-end EST (expressed sequence tag) derived by the method of the present invention.
  • the intensity of the separated band of labeled PCR product fragments, detected using an appropriate method, preferably laser-induced fluorescence (but radioactive or magnetic labeling and detection may be used) is quantified and stored for each PCR product fragment in a database with the address assigned for that PCR product fragment.
  • the intensity of the separated band of labeled PCR product fragments is proportional to the starting amount of mRNA corresponding to that PCR product fragment.
  • the method of the present invention comprises:
  • each anchor primer having a 5' terminus and a 3' terminus and including: (i) a tract of from 7 to 40 T residues; (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located towards the 5'-terminus relative to the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first sniffer segment being located towards the 5'-terminus relative to the site for cleavage by the first restriction endonuclease; (iv) a second stuffer segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phasing residues located at the 3' terminus of each of the anchor primers selected from the group
  • step (c) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is antisense with respect to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules, thereby defining 5' and 3' flanking vector sequences adjacent to the 5' terminus of the sense strand of the inserted cDNA and the 3' terminus of the sense strand respectively, and said constructs having a 3' flanking vector sequence at least 15 nucleotides in length between said first restriction endonuclease site and a site defining transcription initiation in said promoter;
  • step (e) generating linearized fragments containing the inserted cDNA molecules by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the inserted cDNA molecules or in the bacteriophage-specific promoter, but does recognize sequences in the vector, such that the resulting linearized fragments have a 5' flanking vector sequence of at least 15 nucleotides into the vector 5' to the double-stranded cDNA molecule's second terminus;
  • a biotin moiety is conjugated to the anchor primers, preferably to the 5' terminus of the anchor primers.
  • the first restricted cDNA is separated from the remainder of the cDNA in step (b) by contacting the first restricted cDNA with a streptavidin-coated substrate.
  • streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles.
  • a preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY).
  • the 3 nucleotides at the 3' end of the first 5' PCR primer are joined by phosophodiesterase-resistant linkages, preferably phosphorothioate linkages.
  • the 3 nucleotides at the 3' end of the second 5' PCR primer are joined by phosophodiesterase-resistant linkages, preferably phosphorothioate linkages.
  • the 3 nucleotides at the 3' end of both the first and second 5' PCR primers are joined by phosphorothioate linkages.
  • one of the primers for the second PCR reaction is conjugated to a fluorescent label.
  • a suitable fluorescent label is selected from the group consisting of spiro(isobenzofuran- 1 (3H),9'-(9H)-xanthen)-3-one, 6-carboxylic acid,
  • 3',6'-dihydroxy-6-carboxyfluorescein (6-FAM, ABI); spiro(isobenzofuran-l(3H),9'-(9H)-xanthen)-3-one, 5-carboxylic acid, 3',6'- dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro(isobenzofuran-l(3H), 9'-(9H)-xanthen)-3-one, 3',6'-dihydroxy- fluorescein (FAM, Molecular Probes);
  • BODIPY FL 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propanoic acid
  • fluorescent labels including 4, 7, 2', 4', 5', 7' hexachloro 6-carboxyfluorescein (“HEX,” ABI), “NED” (ABI) and 4, 7, 2', 7' tetrachloro 6-carboxyfluorescein (“TET,” ABI) are known in the art.
  • the phasing residues in step (a) have a 3' terminus of -V-N-N. In other embodiments, the phasing residues in step (a) have a 3' terminus of-V or -V-N.
  • the "x" in step (i) is 3.
  • the phasing residues in step (a) are -V-N-N and the "x" in step (i) is 3.
  • the anchor primers each have from 8 to 18 T residues in the tract of T residues. In one preferred embodiment, the anchor primers each have 18 T residues in the tract of T residues. In other embodiments, the anchor primers each have from 8 to 18 T residues, preferably from 8 to 16 T residues, more preferably from 8 to 14 T residues, most preferably from 8 to 12 T residues, in the tract of T residues. In another preferred embodiment, the anchor primers each have 12 T residues in the tract of T residues.
  • the first stuffer segment of the anchor primers is 14 residues in length.
  • the first stuffer segment has the nucleotide sequence A-A- C-T-G-G-A-A-G-A-A-T-T-C (SEQ ID NO: 1).
  • the first stuffer segment has the nucleotide sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A (SEQ ID NO: 2).
  • the bacteriophage-specific promoter is selected from the group consisting of T3 promoter, T7 promoter and SP6 promoter.
  • the bacteriophage-specific promoter is T3 promoter.
  • the primer for priming of transcription of cDNA from cRNA has the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO: 14). In another embodiment, the primer for priming of transcription of cDNA from cRNA has the sequence A-G-C-T-C-T-G-T-G-G-T-G-A-G-G-A-T-C (SEQ ID NO: 28). In further embodiment, the primer for priming of transcription of cDNA from cRNA has the sequence T-C-G-A-C-T-G-T-G-G-T-G-A-G-C-A-T-G (SEQ ID NO: 35).
  • the vector is the plasmid pBC SK+ cleaved with C and Notl and the 3' PCR primer in steps (h) and (i) is G-A-G-C-T-C-C-A-C-C-G-C-G-T (SEQ ID NO: 47).
  • the vector is the plasmid pBC SK+ cleaved with CJal and Notl and the 3' PCR primer in steps (h) and (i) is G-A-G-C-T- C-G-T-T-T-C-C-C-C-A-G (SEQ ID NO: 48).
  • the first restriction endonuclease that recognizes more than six bases is selected from the group consisting of Ascl. Bael. Fsel. Notl. Pad. Pmel
  • PpuMI RsrII, Sapl, SexAI. Sffl, Sgfl, SerAI. Srfl, Sse8387I and Swal.
  • a preferred first restriction endonuclease that recognizes more than six bases is Notl.
  • the second restriction endonuclease recognizing a four-nucleotide sequence is selected from the group consisting of Mbol. Dpnll, Sau3AI. Tsp509I. Hpall. Bfal. Csp6I. Msel. Hhal. NlaTfl. Taql. Mspl. Maell and HinPlI.
  • Preferred second restriction endonucleases recognizing a four-nucleotide sequence are Mspl. Sau3AI and Nlalll.
  • the restriction endonuclease used in step (e) has a nucleotide sequence recognition that includes the four-nucleotide sequence of the second restriction endonuclease used in step (b).
  • the second restriction endonuclease is Mspl and the restriction endonuclease used in step (e) is Sma I.
  • the second restriction endonuclease is Taql and the restriction endonuclease used in step (e) is Xhol.
  • the second restriction endonuclease is HinPlI and the restriction endonuclease used in step (e) is Narl.
  • the second restriction endonuclease is Maell and the restriction endonuclease used in step (e) is Aatll.
  • the vector of step (c) is in the form of a circular DNA molecule having first and second vector restriction endonuclease sites flanking a vector stuffer sequence, and further comprising the step of digesting the vector with restriction endonucleases that cleave the vector at the first and second vector restriction endonuclease sites.
  • the vector stuffer sequence includes an internal vector stuffer restriction endonuclease site between the first and second vector restriction endonuclease sites.
  • One suitable host cell is Escherichia coli.
  • step (e) includes digestion of the vector with a restriction endonuclease which cleaves the vector at the internal vector stuffer restriction endonuclease site.
  • the restriction endonuclease used in step (e) also cleaves the vector at the internal vector stuffer restriction endonuclease site.
  • a general scheme for linearizing a pSK vector without a suitable restriction endonuclease having a six base recognition site containing an internal four base recognition site comprises: (i) dividing the plasmid containing the insert into two fractions, a first fraction cleaved with the restriction endonuclease Xhol and a second fraction cleaved with the restriction endonuclease Sail: (ii) recombining the first and second fractions after cleavage; (iii) dividing the recombined fractions into thirds and cleaving the first third with the restriction endonuclease Hindlll, the second third with the restriction endonuclease BarnHI.
  • the mRNA population has been enriched for polyadenylated mRNA species.
  • the resolving of the amplified fragments in step (j) is conducted by electrophoresis to display the products.
  • the intensity of products displayed after electrophoresis is about proportional to the abundances of the mRNAs corresponding to the products in the original mixture.
  • the method further comprises a step of determining the relative abundance of each mRNA in the original mixture from the intensity of the product corresponding to that mRNA after electrophoresis.
  • the step of resolving the polymerase chain reaction amplified fragments by electrophoresis comprises electrophoresis of the fragments on multiple gels.
  • the method further comprises the steps of:
  • each anchor primer having a 5' terminus and a 3' terminus and including: (i) a tract of from 7 to 40 T residues; (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located towards the 5'-terminus relative to the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first stuffer segment being located towards the 5 '-terminus relative to the site for cleavage by the first restriction endonuclease; (iv) a second stuffer segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phasing residues -V-N-N located at the 3' terminus of each of
  • step (d) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is sense with respect to a T3 promoter within the vector to form a population of constructs containing the inserted cDNA molecules, thereby defining 5' and 3' flanking vector sequences adjacent to the 5' terminus of the sense strand of the inserted cDNA and the 3' terminus of the sense strand respectively, and said constructs having a 5' flanking vector sequence at least 15 nucleotides in length between said second restriction endonuclease site and a site defining transcription initiation in said promoter: (e) transforming Escherichia coli with the vector into which the cleaved cDNA has been inserted to produce vectors containing cloned inserts;
  • step (f) generating linearized fragments containing the inserted cDNA molecules by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the inserted cDNA molecules or in the T3 promoter;
  • the mixture of 48 anchor primers has the sequence A-A-C-T-G-G- A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T- T-T-T-T-T- V-N-N (SEQ ID NO: 5).
  • the mixture of 48 anchor primers has the sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C- C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8).
  • the mixture of 12 anchor primers has the sequence A-A-C-T-G-G- A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4).
  • the mixture of 12 anchor primers has the sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C- C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7).
  • the mixture of 3 anchor primers has the sequence A-A-C-T-G-G-A- A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3).
  • the mixture of 3 anchor primers has the sequence G- A-A-T-T-C- A- A-C-T-G-G- A- A-G-C-G-G-C-C-G-C- A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ LD NO: 6).
  • the first restriction endonuclease is Mspl and the second restriction endonuclease is Notl.
  • the first 5' PCR-primer is G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22).
  • the 3 'PCR primer in the second polymerase chain reaction is the nucleotide of SEQ ID NO: 47 conjugated to a fluorescent label, more preferably, the nucleotide of SEQ ID NO: 47 conjugated to 6-FAM.
  • Suitable values of "x" in step (i) are integers from 1 to 5.
  • the "x" in step (i) is 3.
  • a method for detecting a change in the pattern of mRNA expression in a tissue associated with a physiological or pathological change comprising the steps of:
  • samples are compared.
  • samples are taken at multiple times and compared.
  • the physiological or pathological change is selected from the group consisting of Alzheimer's disease, parkinsonism, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder.
  • the physiological or pathological change is associated with learning or memory, emotion, glutamate neurotoxicity, feeding behavior, olfaction, vision, movement disorders, viral infection, electroshock therapy, the administration of a drug or the toxic side effects of drugs.
  • the physiological or pathological change is selected from the group consisting of circadian variation, aging, and long term potentiation.
  • the physiological or pathological change is selected from processes mediated by transcription factors, intracellular second messengers, hormones, neurotransmitters, growth factors and neuromodulators.
  • the physiological or pathological change is selected from processes mediated by cell-cell contact, cell-substrate contact, cell-extracellular matrix contact and contact between cell membranes and cytoskeleton.
  • the normal or neoplastic tissue comprises cells taken or derived from an organ or organ system selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system, the central nervous system, the enteric nervous system, the endocrine system, the integument (including skin, hair and nails), the skeletal system (including bone and muscle), the urinary system and the reproductive system.
  • an organ or organ system selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system, the central nervous system, the enteric nervous system, the endocrine system, the integument (including skin, hair and nails), the skeletal system (including bone and muscle), the urinary system and the reproductive system.
  • the normal or neoplastic tissue comprises cells taken or derived from the group consisting of epithelia, endothelia, mucosa, glands, blood, lymph, connective tissue, cartilage, bone, smooth muscle, skeletal muscle, cardiac muscle, neurons, glial cells, spleen, thymus, pituitary, thyroid, parathyroid, adrenal cortex, adrenal medulla, adrenal cortex, pineal, skin, hair, nails, teeth, liver, pancreas, lung, kidney, bladder, ureter, breast, ovary, uterus, vagina, testes, prostate, penis, eye and ear.
  • the normal or neoplastic tissue is derived from a structure within the central nervous system selected from the group consisting of retina, cerebral cortex, olfactory bulb, thalamus, hypothalamus, anterior pituitary, posterior pituitary, hippocampus, nucleus accumbens, amygdala, striatum, cerebellum, brain stem, suprachiasmatic nucleus, and spinal cord.
  • a method of detecting a difference in action of a drug to be screened and a known compound comprising the steps of- (a) obtaining a first sample of tissue from an organism treated with a compound of known physiological function;
  • the drug to be screened is selected from the group consisting of antidepressants, neuroleptics, tranquilizers, anticonvulsants, monoamine oxidase inhibitors, stimulants, anti-parkinsonism agents, skeletal muscle relaxants, analgesics, local anesthetics, cholinergics, antiviral agents, antispasmodics, steroids, and non- steroidal anti-inflammatory drugs.
  • drug to be screened and “drug to be tested” are used herein to refer to a broad class of useful chemical and therapeutic agents including physiologically active steroids, antibiotics, antifungal agents, antibacterial agents, antineoplastic agents, analgesics and analgesic combinations, anorexics, anthelmintics, antiarthritics, antiasthia agents, anticonvulsants, antidepressants, antidiabetic agents, antidiarrheals, antihistamines, anti-inflammatory agents, antimigraine preparations, antimotion sickness preparations, antinauseants, antiparkinsonism drugs, antipruritics, antipsychotics, antipyretics, antispasmodics, including gastrointestinal and urinary; anticholinergics, sympathomimetics, xanthine derivatives, cardiovascular preparations including calcium channel blockers, betablockers, antiarrhythmics, antihypertensives diuretics, vasodilators including general, coronary, peripheral and
  • physiologically active in describing the agents contemplated herein is used in a broad sense to comprehend not only agents having a direct pharmacological effect on the host but also those having an indirect or observable effect which is useful in the medical arts, e.g., the coloring or opacifying of tissue for diagnostic purposes, the screening of ultraviolet radiation from the tissues and the like.
  • typical fungistatic and fungicidal agents include thiabendazole, chloroxine, amphotericin, candicidin, fungimycin, nystatin, chlordantoin, clotrimazole, ethonam nitrate, miconazole nitrate, pyrrolnitrin, salicylic acid, fezatione, ticlatone, tolnaftate, triacetin, zinc, pyrithione and sodium pyrithione.
  • Steroids include cortisone, cortodoxone, fluoracetonide, fludrocortisone, difluorsone diacetate, flurandrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and its esters, chloroprednisone, clorcortelone, descinolone, desonide, dexamethasone, dichlorisone, difluprednate, flucloronide, flumethasone, flunisolide, fluocinonide, flucortolone, fluoromethalone, fluperolone, fluprednisolone, meprednisone, methylmeprednisone, paramethasone, prednisolone and predisone.
  • Antibacterial agents include sulfonamides, penicillins, cephalosporins, penicillinase, erythromycins, linomycins, vancomycins, tetracyclines, chloramphenicols, streptomycins, and the like.
  • antibacterials include erythromycin, erythromycin ethyl carbonate, erythromycin estolate, erythromycin glucepate, erythromycin ethylsuccinate, erythromycin lactobionate, lincomycin, clindamycin, tetracycline, chlortetracycline, demeclocycline, doxycycline, methacycline, oxytetracycline, minocycline, and the like.
  • Peptides and proteins include, in particular, small to medium-sized peptides, e.g., insulin, vasopressin, oxytocin, growth factors, cytokines as well as larger proteins such as human growth hormone.
  • Other agents encompass a variety of therapeutic agents such as the xanthines, triamterene and theophylline, the antitumor agents, 5-fluorouridinedeoxyriboside, 6-mercaptopurinedeoxyriboside, vidarabine, the narcotic analgesics, hydromorphone, cyclazine, pentazocine, bupomo ⁇ hine, the compounds containing organic anions, heparin, prostaglandins and prostaglandin-like compounds, cromolyn sodium, carbenoxolone, the polyhydroxylic compounds, dopamine, dobutamine, 1-dopa, a- methyldopa, angiotensin antagonists, polypeptides such as bradykinin, insulin, ad
  • agents include iododeoxyuridine, podophyllin, theophylline, isoproterenol, triamcinolone acetonide, hydrocortisone, indomethacin, phenylbutazone paraaminobenzoic acid, aminopropionitrile and penicillamine.
  • a database is constructed comprising the data produced by the quantitation of the display of sequence-specific PCR products.
  • the database further comprises data concerning sequence relationships, gene mapping and cellular distributions.
  • the invention provides a method for recognizing sequence identities and similarities between the sequence of 3 '-ends of mRNA molecules present in a sample and a database of sequences, comprising the steps of:
  • each anchor primer having a 5' terminus and a 3' terminus and including: (i) a tract of from 7 to 40 T residues; (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located towards the 5'-terminus relative to the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first stuffer segment being located towards the 5 '-terminus relative to the site for cleavage by the first restriction endonuclease; (iv) a second stuffer segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phasing residues located at the 3' terminus of each of the anchor primers selected from the
  • step (c) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is antisense with respect to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules, thereby defining 5' and 3' flanking vector sequences adjacent to the 5' terminus of the sense strand of the inserted cDNA and the 3' terminus of the sense strand respectively, and said constructs having a 3' flanking vector sequence at least 15 nucleotides in length between said first restriction endonuclease site and a site defining transcription initiation in said promoter; (d) transforming a host cell with the vector into which the cleaved cDNA has been inserted to produce vectors containing cloned inserts;
  • step (e) generating linearized fragments containing the inserted cDNA molecules by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the inserted cDNA molecules or in the bacteriophage-specific promoter, but does recognize sequences in the vector, such that the resulting linearized fragments have a 5' flanking vector sequence of at least 15 nucleotides into the vector 5' to the double-stranded cDNA molecule's second terminus;
  • the method further comprises the step of
  • the method also comprises the steps of
  • the invention provides a method for recognizing sequence identities and similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample and a database of sequences, comprising the steps of: eluting a cDNA fragment corresponding to a mRNA molecule present in a sample; amplifying the eluted cDNA fragment in a polymerase chain reaction to produce an amplified cDNA fragment; cloning the amplified cDNA fragment into a plasmid; producing a DNA molecule corresponding to the cloned cDNA fragment; sequencing the produced DNA molecule, thereby determining the sequence of the eluted cDNA fragment; and comparing the sequence of the eluted cDNA fragment to the sequences in a database thereby recognizing sequence identities and similarities.
  • the step of comparing the sequence of the eluted cDNA fragment to the sequences in a database is performed using a computer.
  • the method also comprises the additional step of displaying the results of the comparison graphically.
  • sequence identities and similarities between the sequence of a cDNA fragment corresponding to a mRNA molecule present in a sample and a database of sequences are recognized by a method comprising the steps of : eluting a cDNA fragment corresponding to a mRNA molecule present in a sample, where the cDNA fragment has a length determined by the position of a restriction endonuclease recognition site and a poly(A) tail of the mRNA molecule; determining a partial sequence of the cDNA fragment by performing a polymerase chain reaction with a 5' PCR primer corresponding to the sequence of the restriction endonuclease recognition site and comparing the determined partial sequence of the eluted cDNA fragment and the length of the cDNA fragment to the sequences in a database thereby recognizing sequence identities and similarities.
  • the present invention provides a method of producing a transformed polynucleotide sequence database entry, comprising the steps of: choosing a source sequence from a polynucleotide sequence database entry; locating a poly(A) tail sequence within the source sequence; locating an endonuclease recognition site sequence within the source sequence that is closest to the first recognition site; determining an index sequence consisting of about two to about six nucleotides adjacent to the endonuclease recognition site; determining a correlate sequence within the source sequence, said correlate sequence including the sequence bounded by the poly(A) tail and the endonuclease recognition site and including at least part of the endonuclease recognition site; determining the length of the correlate sequence; and storing information concerning the location and sequence of the poly(A) tail, the location and sequence of the endonuclease recognition site, and the length of the correlate sequence in relation to the source sequence, thereby producing a transformed database entry.
  • the method includes the step of displaying graphic
  • the invention also provides a method of improving the resolution of the length and amount of PCR products by diminishing background that is due to amplification of untargeted cDNAs comprising the steps of: selecting a sample of a cRNA population, wherein each cRNA molecule comprises insert sequence and vector-derived sequence; performing reverse transcription using a reverse transcription primer that hybridizes to the vector-derived sequence and that extends about five nucleotides to about six nucleotides into the insert sequence to produce a cDNA reverse transcription product; subdividing the cDNA reverse transcription product; performing at least one polymerase chain reaction using the subdivided cDNA reverse transcription product, a 3'PCR primer and a 5' PCR primer that hybridizes to the vector-derived sequence and extends about seven nucleotides to about nine nucleotides into the insert sequence to produce a PCR product, thereby diminishing background that is due to amplification of untargeted cDNAs.
  • Figure 1 is a diagrammatic depiction of the improved method of the present invention showing the various stages of priming, cleavage, cloning, antisense RNA transcription and amplification showing the sequences of anchor and other primers schematically - see text for complete sequences;
  • Figure 2 is a diagrammatic depiction of an embodiment of the improved method using biotinylated anchor primers with streptavidin coated substrate and showing the various stages of priming, cleavage, cloning, antisense RNA transcription and amplification showing the sequences of anchor and other primers schematically - see text for complete sequences;
  • Figure 3 is a plot of relative abundance of labeled PCR products versus product length in base pairs using a fluorescent detection system, showing analysis of PCR products obtained using a 5' PCR primer C-G-A-C-G-G-T-A-T-C-G-G-G-T-G (SEQ ID NO: 42), starting from mRNA samples from serum-starved (A) and serum- added (B) human MG63 cells, data from (A) and (B) were overlaid in the bottom panel (C) using software for comparison of relative expression levels between samples;
  • Figure 4 is a plot comparing the relative abundance of labeled PCR products versus product length in base pairs using a fluorescent detection system for the method employing two PCR steps versus the method employing only one PCR step, showing the results obtained from analysis of mRNA extracted from serum-starved (A and C) and serum-added (B and D) MG63 osteosarcoma cells using either one PCR step (A-D) or two PCR steps (E
  • SEQ ID NO: 44 which differ only at the NI position (in bold), for serum starved (os-) and serum added (os+) samples, showing that the PCR products generated with 109T and 45 A appear to be nearly identical from templates produced by the one PCR step method (A-D), whereas the products detected following PCR from templates produced using the two PCR step method are overall quite distinct (E- H);
  • Figure 5 is a plot comparing the relative abundance of labeled PCR products versus product length in base pairs using a fluorescent detection system for the comparing results obtained using the standard method depicted in Figure 1 and the magnetic bead embodiment of the method depicted in Figure 2, showing that data from the magnetic bead embodiment display a marked increase in reproducibility across samples (similarity of fragments generated and consistency of intensity values) compared to data derived from the standard embodiment of the method;
  • Figure 6 is graph showing a linear relationship between cRNA concentration and the peak amplitude of the resulting PCR product for several different tissues
  • Figure 7 shows the nucleotide sequences and restriction maps of the multiple cloning sites of plasmids pBC SK + /DGT1, pBS SK + /DGT2, pBS SK + /DGT3, pBC SK + /DGT4 and pBS SK + /DGT5;
  • Figure 8 is a diagrammatic depiction of an embodiment of the improved method using biotinylated anchor primers with streptavidin coated substrate and showing the various stages of priming, cleavage, cloning, sense RNA transcription and amplification showing the sequences of anchor and other primers schematically - see text for complete sequences.
  • a method according to the present invention based on the polymerase chain reaction (PCR) technique, provides means for visualization of nearly every mRNA expressed by normal or neoplastic eukaryotic cells or tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA.
  • the method is based on the observation that virtually all mRNAs conclude with a 3'-poly (A) tail but does not rely on the specificity of primer binding to the tail.
  • the improved method comprises:
  • each anchor primer having a 5' terminus and a 3' terminus and including: (i) a tract of from 7 to 40 T residues; (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located towards the 5'-terminus relative to the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first stuffer segment being located towards the 5 '-terminus relative to the site for cleavage by the first restriction endonuclease; (iv) a second stuffer segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of
  • T residues and (v) phasing residues located at the 3' terminus of each of the anchor primers selected from the group consisting of -V, -V-N, and -V-N-N, preferably -V-
  • V is a deoxyribonucleotide selected from the group consisting of A, C, and G
  • N is a deoxyribonucleotide selected from the group consisting of A, C, G, and T, the mixture including anchor primers containing all possibilities for V and N;
  • step (c) inserting each double-stranded cDNA molecule from step (b) into a vector in an orientation that is antisense with respect to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules, thereby defining 5' and 3' flanking vector sequences adjacent to the 5' terminus of the sense strand of the inserted cDNA and the 3' terminus of the sense strand respectively, and said constructs having a 3' flanking vector sequence at least 15 nucleotides in length between said first restriction endonuclease site and a site defining transcription initiation in said promoter; (d) Transforming a host cell with the vector into which the cleaved cDNA has been inserted to produce vectors containing cloned inserts;
  • step (e) generating linearized fragments containing the inserted cDNA molecules by digestion of the constructs produced in step (c) with at least one restriction endonuclease that does not recognize sequences in either the inserted cDNA molecules or in the bacteriophage-specific promoter, but does recognize sequences in the vector, such that the resulting linearized fragments have a 5' flanking vector sequence of at least 15 nucleotides into the vector 5' to the double-stranded cDNA molecule's second terminus;
  • step (c) above comprises inserting each double- stranded cDNA molecule from step (b) into a vector in an orientation that is sense with respect to a bacteriophage-specific promoter within the vector to form a population of constructs containing the inserted cDNA molecules ( Figure 8).
  • the first step in the method requires an mRNA population.
  • Methods of extraction of RNA are well-known in the art and are described, for example, in J. Sambrook et al., "Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), vol. 1, ch. 7, “Extraction, Purification, and Analysis of Messenger RNA from Eukaryotic Cells,” incorporated herein by this reference.
  • Other isolation and extraction methods are also well-known. Typically, isolation is performed in the presence of chaotropic agents such as guanidinium chloride or guanidinium thiocyanate, although other detergents and extraction agents can alternatively be used.
  • the mRNA is isolated from the total extracted RNA by chromatography over oligo(dT)-cellulose or other chromatographic media that have the capacity to bind the polyadenylated 3'-portion of mRNA molecules.
  • total RNA can be used. However, it is generally preferred to isolate poly(A) + RNA.
  • Double-stranded cDNAs are then prepared from the mRNA population using a mixture of anchor primers to initiate reverse transcription.
  • Each anchor primer has a 5' terminus and a 3' terminus and including: (i) a tract of from 7 to 40 T residues; (ii) a site for cleavage by a first restriction endonuclease that recognizes more than six bases, the site for cleavage being located towards the 5'-te ⁇ ninus relative to the tract of T residues; (iii) a first stuffer segment of from 4 to 40 nucleotides, the first stuffer segment being located towards the 5'-terminus relative to the site for cleavage by the first restriction endonuclease; (iv) a second stuffer segment interposed between the site for cleavage by a first restriction endonuclease that recognizes more than six bases and the tract of T residues, and (v) phasing residues located at the 3' terminus of each of the
  • the mixture comprises a mixture of three anchor primers. Where the anchor primers have phasing residues of- V-N, the mixture comprises a mixture of twelve anchor primers. Where the anchor primers have phasing residues of -V-N-N, the mixture comprises a mixture of 48 anchor primers.
  • the anchor primers each have 18 T residues in the tract of T residues, end in -V-N-N, and have a first stuffer segment of 14 residues in length.
  • Preferred sequences of the first stuffer segment are selected from the group consisting of A-A-C-T-G-G-A-A-G-A-A-T-T-C (SEQ ID NO: 1) and G- A-A-T-T-C- A-A-C-T- G-G-A-A (SEQ ID NO: 2).
  • the site for cleavage by a restriction endonuclease that recognizes more than six bases is the Notl cleavage site.
  • One preferred set of three anchor primers has the sequence A-A-C-T-G-G-A- A-G-A-A-T-T-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3).
  • Another preferred set of twelve anchor primers has the sequence A-A-C-T-G-G-A-A-G-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T- T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4).
  • a further preferred set of 48 anchor primers has the sequences A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C- C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 5).
  • the set of 3 anchor primers has the sequence G-A-
  • the set of 12 anchor primers has the sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G- C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7).
  • the set of 48 anchor primers has the sequence G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T- T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ LD NO: 8).
  • One member of this mixture of anchor primers initiates synthesis at a fixed position at the 3'-end of all copies of each mRNA species in the sample, thereby defining a 3'-end point for each species.
  • Suitable reverse transcriptases include those from avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV).
  • a preferred reverse transcriptase is the MMLV reverse transcriptase.
  • magnetic beads are used to improve the preparation of the cDNA population ( Figures 2 and 8).
  • the biotin moiety is conjugated to the 5' terminus of the anchor primer and the first restricted cDNA is separated from the remainder of the cDNA by contacting the first restricted cDNA with a streptavidin-coated substrate, such as number of streptavidin coated magnetic beads.
  • the cDNA sample is cleaved with two restriction endonucleases.
  • the first restriction endonuclease recognizes a site having more than six bases and cleaves at a single site within each member of the mixture of anchor primers.
  • the second restriction endonuclease is an endonuclease that recognizes a 4-nucleotide sequence.
  • Such endonucleases typically cleave at multiple sites in most cDNAs.
  • the first restriction endonuclease is Notl and the second restriction endonuclease is Mspl.
  • the enzyme Notl does not cleave within most cDNAs. This is desirable to minimize the loss of cloned inserts that would result from cleavage of the cDNAs at locations other than in the anchor site.
  • the second restriction endonuclease can be Taql. Maell or HinPlI.
  • the use of the above three restriction endonucleases can detect rare mRNAs that are not cleaved by Mspl.
  • the second restriction endonuclease generates a 5'- overhang compatible for cloning into the desired vector, as discussed below.
  • This cloning, for the vector chosen from the group consisting of pBC SK + , pBS SK + , pBC SK7DGT1, pBS SK7DGT2 and pBS SK7DGT3 is into the Oal site, as discussed below.
  • the second restriction endonuclease can be Sau3AI.
  • restriction endonuclease can also detect rare mRNAs that are not cleaved by Mspl.
  • the second restriction endonuclease generates a 5'-overhang compatible for cloning into the desired vector, as discussed below. This cloning for the vector pBC SK7DGT4 is into the BamHI site, as discussed below.
  • the second restriction endonuclease can be Nlalll.
  • the use of this restriction endonuclease can also detect rare mRNAs that are not cleaved by Mspl.
  • the second restriction endonuclease generates a 5'-overhang compatible for cloning into the desired vector, as discussed below. This cloning for the vector pBS SK7DGT5, is into the Sphl site, as discussed below.
  • Suitable restriction endonucleases can be used to detect cDNAs not cleaved by the above restriction endonucleases.
  • Suitable second restriction endonucleases recognizing a four-nucleotide sequence are Mbol. Dpnll. Sau3AI, TSD509I, Hpall. Bfal. Csp ⁇ l. Msel. Hhal. Nlalll. Taql. Mspl. Maell and HinPlI.
  • Suitable first restriction endonucleases that recognize more than six bases are
  • a suitable vector includes a multiple cloning site having a Notl restriction endonuclease site.
  • a suitable vector is the plasmid pBC
  • the vector contains a bacteriophage-specific promoter.
  • the promoter is a T3 promoter, a SP6 promoter, or a T7 promoter.
  • a preferred promoter is a bacteriophage T3 promoter.
  • the cleaved cDNA is inserted into the promoter in an orientation that is antisense with respect to the bacteriophage-specific promoter ( Figures 1 and 2). In another preferred embodiment, the cleaved cDNA is inserted into the promoter in an orientation that is sense with respect to the bacteriophase- specific promoter ( Figure 8).
  • the vector includes a multiple cloning site having a nucleotide sequence chosen from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • Preferred vectors are based on the plasmid vector pBluescript (pBS or pBC) SK+ (Stratagene) in which a portion of the nucleotide sequence from positions 656 to 764 was removed and replaced with a sequence of at least 110 nucleotides including a Notl restriction endonuclease site.
  • This region designated the multiple cloning site (MCS), spans the portion of the nucleotide sequence from the Sad site to the Kpnl site.
  • a suitable plasmid vector such as pBC SK + or pBS SK + (Stratagene) was digested with suitable restriction endonuclease to remove at least 100 nucleotides of the multiple cloning site.
  • suitable restriction endonucleases for removing the multiple cloning site are Sad and Kpnl.
  • a cDNA portion comprising a new multiple cloning site, having ends that are compatible with Notl and C after digestion with first and second restriction endonucleases was cloned into the vector to form a suitable plasmid vector.
  • Preferred cDNA portions comprising new multiple cloning sites include those having the nucleotide sequences described in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
  • cDNA clones are linearized by digestion with a single restriction endonuclease that recognizes a sequence having more than six bases that includes the four nucleotide sequence of the second restriction endonuclease site.
  • a preferred plasmid vector referred to herein as pBC SK7DGT1, comprises the MCS of SEQ ID NO:9.
  • the pairs for second restriction endonuclease and linearization restriction endonuclease are, respectively: Mspl and Smal: HinPlI and Narl; Tagl and Xhol: Maell and Aatll.
  • pBS SK7DGT2 Another preferred plasmid vector, refe ⁇ ed to herein as pBS SK7DGT2, comprises the MCS of SEQ ID NO: 10, and was prepared as described above for pBC SK7DGT1.
  • the multiple cloning site does not accept cDNA inserts produced using Maell.
  • the pairs for second restriction endonuclease and linearization restriction endonuclease are, respectively: Mspl and Smal: HinPlI and Narl: and Tagl and Xhol.
  • pBS SK7DGT3 Another preferred plasmid vector, referred to herein as pBS SK7DGT3, comprises the MCS of SEQ ID NO: 11.
  • the pairs for second restriction endonuclease and linearization restriction endonuclease (of step E, below) are, respectively: Mspl and Smal; HinPlI and Narl: Taql and Xhol; Maell and Aatll.
  • pBC SK7DGT4 Another preferred plasmid vector, referred to herein as pBC SK7DGT4, comprises the MCS of SEQ ID NO: 12.
  • the pair of second restriction endonuclease and linearization restriction endonuclease (of step E, below) enzymes suitable for use with this vector are, respectively, Sau3 Al and Bglll.
  • pBS SK7DGT5 Another preferred plasmid vector, referred to herein as pBS SK7DGT5, comprises the MCS of SEQ ID NO: 13.
  • the pair of second restriction endonuclease and linearization restriction endonuclease (of step E, below) enzymes suitable for use with this vector are, respectively, NMII and Ncol.
  • the vector includes a vector stuffer sequence that comprises an internal vector stuffer restriction endonuclease site between the first and second vector restriction endonuclease sites.
  • the linearization step includes digestion of the vector with a restriction endonuclease which cleaves the vector at the internal vector stuffer restriction endonuclease site.
  • the restriction endonuclease used in the linearization step also cleaves the vector at the internal vector stuffer restriction endonuclease site.
  • Suitable host cells for cloning are described, for example, in Sambrook et al, "Molecular Cloning: A Laboratory Manual," supra.
  • the host cell is prokaryotic.
  • a particularly suitable host cell is a strain of K coli.
  • a suitable E. coli strain is MCI 061.
  • a small aliquot is also used to transform E. coli strain XL 1 -Blue so that the percentage of clones with inserts is determined from the relative percentages of blue and white colonies on X-gal plates. Only libraries with in excess of 5x10 5 recombinants are typically acceptable.
  • Plasmid preparations are then made from each of the cDNA libraries. Linearized fragments are then generated by digestion with at least one restriction endonuclease.
  • vector is the plasmid pBC SK + and Mspl is used both as the second restriction endonuclease and as the linearization restriction endonuclease.
  • vector is the plasmid pBC SK +
  • the second restriction endonuclease is chosen from the group consisting of Mspl, Maell.
  • Taql and HinPlI and the linearization is accomplished by a first digestion with Smal followed by a second digestion with a mixture of Kpnl and Apal
  • the vector is chosen from the group consisting of pBC SK + /DGT1, pBS SK + /DGT2, pBS SK + /DGT3, pBC SK + /DGT4 and pBS SK + /DGT5.
  • one suitable enzyme combination is provided where the second restriction endonuclease is Mspl and the restriction endonuclease used in the linearization step is Sma I.
  • Another suitable combination is provided where the second restriction endonuclease is Taql and the restriction endonuclease used in the linearization step is Xhol.
  • a further suitable combination is provided where the second restriction endonuclease is HinPlI and the restriction endonuclease used in the linearization step is Narl. Yet another suitable combination is provided where the second restriction endonuclease is Maell and the restriction endonuclease used in the linearization step is Aatll. If the vector is pBC SK + /DGT4, another suitable combination is provided by Sau3AI as the second restriction endonuclease and Bglll as the restriction endonuclease used in the linearization step. If the vector is pBS SK + /DGT5, another suitable combination is provided by Nlalll as the second restriction endonuclease and Ncol as the restriction endonuclease used in the linearization step.
  • any plasmid vector lacking a cDNA insert was cleaved at the 6-nucleotide recognition site (underlined in Figure 7A) for Smal.
  • Narl. Xhol. or Aat ⁇ found between the Notl site and the C site and the recognition site having more than six bases for Smal.
  • Narl. Xhol or Aatll sites found 3' to the Clal site.
  • plasmid vectors containing inserts would be cleaved at the 6-nucleotide recognition site for Smal. Narl. Xhol or Aatll sites found 3' to the Clal site.
  • the next step is a generation of a cRNA preparation of antisense cRNA transcripts. This is performed by incubation of the linearized fragments with an RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter.
  • an RNA polymerase capable of initiating transcription from the bacteriophage-specific promoter.
  • the promoter is a T3 promoter, and the polymerase is therefore T3 RNA polymerase.
  • the polymerase is incubated with the linearized fragments and the four ribonucleoside triphosphates under conditions suitable for synthesis (Ambion, Austin, TX).
  • First-strand cDNA is transcribed using Moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies, Gaithersburg, MD). With this reverse transcriptase annealing is performed at 42°C, and the transcription reaction at 42°C.
  • the reaction uses a primer which is 15 to 30 nucleotides in length and complementary to the 5' flanking vector sequence.
  • the cRNA is transcribed using a thermostable reverse transcriptase and a primer as described below.
  • a preferred transcriptase is the avian recombinant reverse transcriptase, known as ThermoScript RT, available from Life Technologies (Gaithersburg, MD).
  • the primer used is at least 15 nucleotides in length, corresponding in sequence to the 3'-end of the bacteriophage-specific promoter.
  • Another suitable transcriptase is the recombinant reverse transcriptase from
  • Thermus thermophilus known as rTth. available from Perkin-Elmer (Norwalk, CT).
  • the primers typically have the sequence A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-G-T (SEQ ID NO: 14) or G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO: 47).
  • the next step is the use of the product of transcription as a template for a polymerase chain reaction with a first set of primers as described below to produce polymerase chain reaction amplified fragments.
  • the product of first-strand cDNA transcription is used as a template for a polymerase chain reaction with a first 3' PCR primer and a first 5' PCR primer to produce polymerase chain reaction amplified fragments.
  • the first 3' PCR primer typically is 15 to 30 nucleotides in length, and is complementary to 3' flanking vector sequences between the first restriction endonuclease site and the site defining transcription initiation by the bacteriophage-specific promoter.
  • the first 5'-PCR primers have a 3' terminus consisting of -N, where "N,” is one of the four deoxyribonucleotides A, C, G, or T, the primer being 15 to 30 nucleotides in length and complementary to the 5' flanking vector sequence with the primer's complementarity extending into one nucleotide of the insert-specific nucleotides of the cRNA, wherein a different one of the first 5' PCR primers is used in each of four different subpools.
  • a suitable 3'-PCR primer is selected from the group consisting of G-A-G-C-T-C-C-A- C-C-G-C-G-G-T (SEQ ID NO: 47) and G-A-G-C-T-C-G-T-T-T-C-C-C-A-G (SEQ ID NO: 48).
  • a suitable 5'-PCR primer can have the sequence G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22) where in a given reaction N is either A, G, C, or T.
  • PCR is performed using a PCR program of 15 seconds at 94°C for denaturation, 15 seconds at 50°C - 65°C for annealing, and 30 seconds at 72°C for synthesis on a suitable thermocycler such as the PTC-200 (MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus, Norwalk, CT).
  • a suitable thermocycler such as the PTC-200 (MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus, Norwalk, CT).
  • the annealing temperature is optimized for the specific nucleotide sequence of the primer, using principles well known in the art.
  • the high temperature annealing step minimizes artifactual mispriming by the first 5'-PCR primer at its 3'-end and promotes high fidelity copying.
  • the next step is the use of the products of the first PCR reaction as templates for a second polymerase chain reaction with a second set of primers as described below to produce a second set of polymerase chain reaction amplified fragments.
  • the product of first PCR reaction is used as a template for a polymerase chain reaction with a second 3' PCR primer and a second 5'-PCR primer to produce polymerase chain reaction amplified fragments.
  • the second 3' PCR primer typically is 15 to 30 nucleotides in length, and is complementary to 3' flanking vector sequences between the first restriction endonuclease site and the site defining transcription initiation by the bacteriophage-specific promoter.
  • the second 5' PCR primer is defined as having a 3'-terminus consisting of-N,-N x , wherein N, is identical to the N, used in the first polymerase chain reaction for that subpool, "N” is as is step (H), and "x" is an integer from 1 to 5, the primer being 15 to 30 nucleotides in length and complementary to the 5' flanking vector sequence with the primer's complementarity extending across into the insert-specific nucleotides of the cRNA in a number of nucleotides equal to "x" + 1, wherein a different one of the second 5' PCR primers is used in different subpools of the second series of subpools and wherein there are 4 X subpools in the second series of subpools for each of the subpools in the first set of subpools.
  • the primers used are: (a) a second 3' PCR primer that corresponds in sequence to a sequence in the vector adjoining the site of insertion of the cDNA sample in the vector; and (b) a 5'-PCR primer selected from the group consisting of: (i) the first 5' PCR primer which was used in the first PCR reaction for that subpool; (ii) the first 5' PCR primer from which the first-strand cDNA was made for that subpool extended at its3 '-terminus by an additional residue -N; (iii) the first 5' PCR primer used for that subpool extended at its 3' terminus by two additional residues -N-N, (iv) the first 5' PCR primer used for that subpool extended at its 3' terminus by three additional residues -N-N-N; and (v) the first 5' PCR primer used for that subpool extended at its 3' terminus by four additional residues -N-N-N, wherein N can be any of A, C
  • Suitable 3' PCR primers are selected from the group consisting of G-A-G-C-T- C-C-A-C-C-G-C-G-G-T (SEQ ID NO: 47) and G-A-G-C-T-C-G-T-T-T-C-C-C-A- G (SEQ ID NO: 48).
  • bacteriophage-specific promoter is the T3 promoter
  • PCR primer is chosen from the group consisting of the sequences: A-G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 16);
  • C-G-A-C-G-G-T-A-T-C-G-N-N-N-N-N (SEQ ID NO: 25); G-A-C-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO: 26); A-C-G-T-A-T-C-G-G-N-N-N-N-N-N (SEQ ID NO: 16);
  • PCR is performed using a PCR program of 15 seconds at 94°C for denaturation, 15 seconds at 50°C - 65°C for annealing, and 30 seconds at 72°C for synthesis on a suitable thermocycler such as the PTC-200 (MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus, Norwalk, CT).
  • a suitable thermocycler such as the PTC-200 (MJ Research) or the Perkin-Elmer 9600 (Perkin-Elmer Cetus, Norwalk, CT).
  • the annealing temperature is optimized for the specific nucleotide sequence of the primer, using principles well known in the art.
  • the high temperature annealing step minimizes artifactual mispriming by the 5'-primer at its 3'-end and promotes high fidelity copying.
  • one of the primers for the second PCR reaction is preferably conjugated to a fluorescent label.
  • a suitable fluorescent label is selected from the group consisting of spiro(isobenzofuran- 1 (3H),9'-(9H)-xanthen)-3-one, 6-carboxylic acid,
  • 3',6'-dihydroxy-6-carboxyfluorescein (6-FAM, ABI); spiro(isobenzofuran-l(3H),9'-(9H)-xanthen)-3-one, 5-carboxylic acid, 3',6'- dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro(isobenzofuran-l(3H), 9'-(9H)-xanthen)-3-one, 3',6'-dihydroxy- fluorescein (FAM, Molecular Probes);
  • fluorescent labels including 4, 7, 2', 4', 5', 7' hexachloro 6-carboxyfluorescein (“HEX,” ABI), 4, 7, 2', T tetrachloro 6- carboxyfluorescein (“TET,” ABI) and “NED” (ABI) are known in the art.
  • a prefe ⁇ ed fluorescent label is spiro(isobenzofuran-l(3H),9'-(9H)-xanthen)- 3-one, 6-carboxylic acid, 3',6'-dihydroxy-6-carboxyfluorescein (6-FAM).
  • autoradiographic detection methods can be used.
  • the PCR is performed in the presence of 35 S-dATP
  • the PCR amplification can be carried out in the presence of a radionuclide labeled deoxyribonucleoside triphosphate, such as [ 32 P]dCTP or [ 33 P]dCTP.
  • a radionuclide labeled deoxyribonucleoside triphosphate such as [ 32 P]dCTP or [ 33 P]dCTP.
  • it is generally prefe ⁇ ed to use a 35 S-labeled deoxyribonucleoside triphosphate for maximum resolution.
  • the detection method employs oligonucleotides that are labeled with magnetic particles that are used and detected as described in U.S. Patent No. 5,656,429, the teachings of which are inco ⁇ orated by reference.
  • the 3 nucleotides at the 3' end of the first or second 5' PCR primer are joined by phosphorothioate linkages. See, Mullins, J. I., de Noronha, C. M. Amplimers with 3 '-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming. PCR Methods Appl 1992 2(2):131-136; Ott, J. and Eckstein, F. Protection of oligonucleotide primers against degradation by DNA polymerase I. Biochemistry 1987 26(25):8237-8241; Uhlmann, E., Ryte, A., and Peyman, A.
  • the polymerase chain reaction amplified fragments are then resolved by a separation method such as electrophoresis to display bands representing the 3'-ends of mRNAs present in the sample.
  • Electrophoretic techniques for resolving PCR amplified fragments are well- understood in the art and need not be further recited here in detail.
  • the corresponding PCR products are resolved in denaturing DNA sequencing gels and visualized by laser induced fluorescence.
  • the corresponding PCR products are resolved using capillary electrophoresis and visualized by laser induced fluorescence.
  • one of the primers for the second PCR reaction is conjugated to a fluorescent label.
  • a suitable fluorescent label is selected from the group consisting of spiro(isobenzofuran-l(3H),9'-(9H)-xanthen)-3-one, 6-carboxylic acid, 3',6'-dihydroxy-6-carboxyfluorescein (6-FAM, ABI); spiro(isobenzofuran-l(3H),9'-(9H)-xanthen)-3-one, 5-carboxylic acid, 3',6'- dihydroxy-5-carboxyfluorescein (5-FAM, Molecular Probes); spiro(isobenzofuran- 1 (3H), 9'-(9H)-xanthen)-3-one, 3',6'-dihydroxy- fluorescein (FAM, Molecular Probes); 9-(2,5-dicarboxyphenyl)-3,6- bis(dimethylamino)-xanthylium
  • TAMRA 9-(2,4(or 2,5)-dicarboxyphenyl)-3,6- bis(dimethylamino)- xanthylium, inner salt
  • TAMRA Molecular Probes
  • Other suitable fluorescent labels including 4, 7, 2', 4', 5', T hexachloro 6-carboxyfluorescein ("HEX,” ABI), NED (ABI) and 4, 7, 2*, T tetrachloro 6-carboxyfluorescein (“TET,” ABI) are known in the art.
  • fluorescence is used to detect the resolved cDNA species.
  • other detection methods such as phosphorimaging or autoradiography, or magnetic detection, can also be used.
  • the cDNA libraries produced from each of the mRNA samples contain copies of the extreme 3'-ends from the most distal site for Mspl to the beginning of the poly(A) tail of all poly(A) + mRNAs in the starting RNA sample approximately according to the initial relative concentrations of the mRNAs. Because both ends of the inserts for each species are exactly defined by sequence, their lengths are uniform for each species allowing their later visualization as discrete bands on a gel, regardless of the tissue source of the mRNA.
  • the intensity of products displayed after electrophoresis is about proportional to the abundances of the mRNAs co ⁇ esponding to the products in the original mixture.
  • the method further comprises a step of determining the relative abundance of each mRNA in the original mixture from the intensity of the product corresponding to that mRNA after electrophoresis.
  • this method comprises:
  • the comparison is made in adjacent lanes of a single gel.
  • a database comprising the data produced by the quantitation of the display of sequence-specific products is constructed and maintained using suitable computer hardware and computer software.
  • a database further comprises data concerning sequence relationships, gene mapping and cellular distributions.
  • the length and at least part of the nucleotide sequence of the PCR products are compared to expected values determined from a database of nucleotide sequences.
  • the tissue can be derived from the central nervous system.
  • the central nervous system can be derived from a structure within the central nervous system that is the retina, cerebral cortex, olfactory bulb, thalamus, hypothalamus, anterior pituitary, posterior pituitary, hippocampus, nucleus accumbens, amygdala, striatum, cerebellum, brain stem, suprachiasmatic nucleus, or spinal cord.
  • the tissue is derived from the central nervous system
  • the physiological or pathological change can be any of Alzheimer's disease, parkinsonism, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder.
  • the method of the present invention can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus. Additionally, particularly with reference to mRNA species occurring in particular structures within the central nervous system, the method can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders.
  • This method can also be used to study the results of the administration of drugs and/or toxins to an individual by comparing the mRNA pattern of a tissue before and after the administration of the drug or toxin. Results of electroshock therapy can also be studied.
  • the tissue can be from an organ or organ system that includes the cardiovascular system, the pulmonary system, the digestive system, the peripheral nervous system, the liver, the kidney, skeletal muscle, and the reproductive system, or from any other organ or organ system of the body.
  • mRNA patterns can be studied from liver, heart, kidney, or skeletal muscle.
  • samples can be taken at various times so as to discover a circadian effect of mRNA expression.
  • this method can ascribe particular mRNA species to involvement in particular patterns of function or malfunction.
  • the normal or neoplastic tissue comprises cells taken or derived from an organ or organ system selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system, the central nervous system, the enteric nervous system, the endocrine system, the integument (including skin, hair and nails), the skeletal system (including bone and muscle), the urinary system and the reproductive system.
  • an organ or organ system selected from the group consisting of the cardiovascular system, the lymphatic system, the respiratory system, the digestive system, the peripheral nervous system, the central nervous system, the enteric nervous system, the endocrine system, the integument (including skin, hair and nails), the skeletal system (including bone and muscle), the urinary system and the reproductive system.
  • the normal or neoplastic tissue comprises cells taken or derived from the group consisting of epithelia, endothelia, mucosa, glands, blood, lymph, connective tissue, cartilage, bone, smooth muscle, skeletal muscle, cardiac muscle, neurons, glial cells, spleen, thymus, pituitary, thyroid, parathyroid, adrenal cortex, adrenal medulla, adrenal cortex, pineal, skin, hair, nails, teeth, liver, pancreas, lung, kidney, bladder, ureter, breast, ovary, uterus, vagina, testes, prostate, penis, eye and ear.
  • the mRNA resolution method of the present invention can be used as part of a method of screening for a side effect of a drug.
  • a method of screening for a side effect of a drug comprises:
  • this method can be used for drugs affecting the central nervous system, such as antidepressants, neuroleptics, tranquilizers, anticonvulsants, monoamine oxidase inhibitors, and stimulants.
  • this method can in fact be used for any drug that may affect mRNA expression in a particular tissue.
  • the effect on mRNA expression of anti-parkinsonism agents, skeletal muscle relaxants, analgesics, local anesthetics, cholinergics, antispasmodics, steroids, non- steroidal anti-inflammatory drugs, antiviral agents, or any other drug capable of affecting mRNA expression can be studied, and the effect determined in a particular tissue or structure.
  • a further application of the method of the present invention is in obtaining the sequence of the 3'-ends of mRNA species that are displayed.
  • a method of obtaining the sequence comprises:
  • the cDNA that has been excised can be amplified with the primers previously used in the second PCR step.
  • the cDNA can then be cloned into pCR II (Invitrogen, San Diego, CA) by TA cloning and ligation into the vector.
  • Minipreps of the DNA can then be produced by standard techniques from subclones and a portion denatured and split into two aliquots for automated sequencing by the dideoxy chain termination method of S anger.
  • a commercially available sequencer can be used, such as a ABI sequencer, for automated sequencing.
  • the cDNA sequences obtained can then be used to design primer pairs for semiquantitative PCR to confirm tissue expression patterns. Selected products can also be used to isolate full-length cDNA clones for further analysis. Primer pairs can be used for SSCP-PCR (single strand conformation polymo ⁇ hism-PCR) amplification of genomic DNA. For example, such amplification can be carried out from a panel of interspecific backcross mice to determine linkage of each PCR product to markers already linked. This can result in the mapping of new genes and can serve as a resource for identifying candidates for mapped mouse mutant loci and homologous human disease genes.
  • SSCP-PCR single strand conformation polymo ⁇ hism-PCR
  • SSCP-PCR uses synthetic oligonucleotide primers that amplify, via PCR, a small (100-200 bp) segment.
  • M. Orita et al. "Detection of Polymo ⁇ hisms of Human DNA by Gel Electrophoresis as Single-Strand Conformation Polymo ⁇ hisms," Proc. Natl. Acad. Sci. USA 86: 2766-2770 (1989); M. Orita et al., “Rapid and Sensitive Detection of Point Mutations in DNA Polymo ⁇ hisms Using the Polymerase Chain Reaction," Genomics 5: 874-879 (1989)).
  • the excised fragments of cDNA can be radiolabeled by techniques well- known in the art for use in probing a northern blot or for in situ hybridization to verify mRNA distribution and to learn the size and prevalence of the corresponding full- length mRNA.
  • the probe can also be used to screen a cDNA library to isolate clones for more reliable and complete sequence determination.
  • the labeled probes can also be used for any other pu ⁇ ose, such as studying in vitro expression.
  • panels of primers and degenerate mixtures of primers suitable for the practice of the present invention are panels of primers and degenerate mixtures of primers suitable for the practice of the present invention. These include: (1) a panel of primers comprising 16 primers of the sequence A-G-G-T-C-G- A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 16), wherein N is one of the four deoxyribonucleotides A, C, G, or T; (2) a panel of primers comprising 64 primers of the sequences A-G-G-T-C-G-
  • a panel of primers comprising 256 primers of the sequences A-G-G-T-C- G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 18);
  • a panel of primers comprising 1024 primers of the sequences A-G-G-T-C- G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N-N (SEQ ID NO: 19);
  • a panel of primers comprising 4096 primers of the sequences A-G-G-T-C- G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N (SEQ ID NO: 20);
  • a panel of primers comprising 3 primers of the sequences A-A-C-T-G-G- A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 3);
  • a panel of primers comprising 12 primers of the sequences A-A-C-T-G-G- A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 4), wherein V is a deoxyribonucleotide selected from the group consisting of A, C, and G; (8) a panel of primers comprising 48 primers of the sequences A-A-C-T-G-G-
  • a panel of primers comprising 3 primers of the sequences G-A-A-T-T-C- A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6);
  • a panel of primers comprising 12 primers of the sequences G-A-A-T-T- C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7); (11) a panel of primers comprising 48 primers of the sequences G-A-A-T-T- C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T- T-T-T-T-T-T-V-N (SEQ ID NO: 7); (11) a panel of primers comprising 48 primers of the sequences G-A-A-T-T- C-A-A
  • a panel of primers comprising 4 different oligonucleotides each having the sequence G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22);
  • a panel of primers comprising 16 different oligonucleotides each having the sequence G-T-C-G-A-C-G-G-T-A-T-C-G-G-N-N (SEQ ID NO: 23);
  • a panel of primers comprising 64 different oligonucleotides each having the sequence T-C-G-A-C-G-G-T-A-T-C-G-G-N-N-N (SEQ ID NO: 24); (15) a panel of primers comprising 256 different oligonucleotides each having the sequence C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 25);
  • a panel of primers comprising 1024 different oligonucleotides each having the sequence G-A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N-N (SEQ ID NO: 26);
  • a panel of primers comprising 4096 different oligonucleotides each having the sequence A-C-G-G-T-A-T-C-G-G-N-N-N-N-N-N-N-N-N-N-N (SEQ ID NO: 27);
  • a degenerate mixture of primers comprising a mixture of 3 primers of the sequences A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T- T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 2), each of the 3 primers being present in about an equimolar quantity; (19) a degenerate mixture of primers comprising a mixture of 12 primers of the sequences A-A-C-T-G-G-A-A-A-T-T-C-G-G-C-C-G-C-A-G-G-A-A-T- T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T
  • a degenerate mixture of primers comprising a mixture of 48 primers of the sequences A-A-C-T-G-G-A-A-G-A-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-T-
  • a degenerate mixture of primers comprising a mixture of 3 primers of the sequences G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-T- T-T-T-T-T-T-T-T-T-T-T-T-V (SEQ ID NO: 6), each of the 3 primers being present in about an equimolar quantity;
  • a degenerate mixture of primers comprising a mixture of 12 primers of the sequences G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A- T-T-T-T-T-T-T-T-T-T-T-T-T-V-N (SEQ ID NO: 7), each of the 12 primers being present in about an equimolar quantity; and
  • a degenerate mixture of primers comprising a mixture of 48 primers of the sequences G-A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-G-C-A-G-G-A-A- T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO: 8), each of the 48 primers being present in about an equimolar quantity.
  • Example 1 Application of the Improved Method.
  • the improved method of the present invention is based upon the observation that virtually all eukaryotic mRNAs conclude with a poly(A) tail, but, unlike differential display (Liang, P. and A.B. Pardee (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967-971), the method of the present invention uses the specificity of primer binding to the tail only to fix a site on each mRNA, not to subdivide mRNAs into pools.
  • the improved method is illustrated in three embodiments in Figures 1, 2 and 8.
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture of all 48 5 '-biotinylated anchor primers of a set to initiate reverse transcription ( Figures 2 and 8) (Gubler, U. and B. Hoffman (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263-269) (Schibler, K., M. Tosi, A.C. Pittet, L. Fabiani and P.K. Wellauer (1980) Tissue-specific expression of mouse amylase genes. J. Mol. Biol. 142:93-116).
  • One such suitable set is A-A-C-T- G-G-A-A-G-A-T-T-C-G-C-G-G-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T- T-T-T-T-T-T-T- V-N-N (SEQ ID NO: 5), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double stranded cDNA.
  • Each biotinylated double stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG.
  • the 3' fragments of cDNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles.
  • a preferred streptavidin-coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY).
  • the cDNA fragment product was released by digestion with Notl. which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • the 3' Mspl-Notl fragments which are of uniform length for each mRNA species, were directionally ligated into Clal-. Notl- cleaved plasmid pBC SK + (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli
  • SURE cells (Stratagene). The ligation regenerates the Notl site, but not the Mspl site.
  • Plasmid preps (Qiagen) were made from the cDNA library of each sample under study.
  • each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3' cDNA inserts and their flanking sequences, including the T3 promoter, intact.
  • the product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.
  • T3 RNA polymerase MEGAscript kit, Ambion
  • the polylinker region of the parent vector contains a site for Mspl between its Clal and Notl sites and, therefore, the Mspl digestion step eliminated the 5' tag from cRNAs transcribed from insertless plasmids, rendering them inert in the product amplification steps described below. Plasmid DNA was removed from the mixture of antisense cRNA transcripts by incubation with RNase-free DNase.
  • each of the cRNA preparations was processed in a three-step fashion.
  • 250ng of cRNA was converted to first-strand cDNA using the 5' RT primer (5PRIMER in Figures 1 and 2 and 8) A-G-G-T-C-G-A-C-G-G-T-A-T-C- G-G, (SEQ ID NO: 14).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO: 22), each paired with an "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-G-T (SEQ ID NO: 47), using the program
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction, with 100 ng each of the fluoresceinated "universal" 3' PCR primer, the oligonucleotide G-A-G-C-T-C-C-A-C- C-G-C-G-G-T (SEQ ID NO: 47) conjugated to 6-FAM and the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO:25), using the program 94 degrees Celsius, 15 seconds;
  • the final PCR was carried out for 30 cycles using 2ng of DNA template and 1 OOng of each 5PRIMER 3 N 1 N 2 N 3 N 4 primer (SEQ ID NO: 25) and 3' PCR primer (SEQ ID NO:47) conjugated to 6-FAM.
  • the major application of the present invention is for comparing mRNA expression profiles for two or more tissue samples.
  • oligonucleotides were synthesized corresponding to the 5PRIMER 3 N 1 N 2 N 3 N 4 (SEQ LD NO: 25) for each candidate extended at the 3' end with an additional 14 nucleotides from the sequences adjacent to the terminal Mspl sites in the GenBank sequences. These were paired with the fluorescent 3PRLMER (SEQ LD NO: 47) in PCRs using the N, cDNA as substrate.
  • reverse transcriptase was used to generate 4 cDNA subpools from cRNA by initiating transcription with one of the four NI primers of the form 5PRIMERN1 (SEQ ID NO: 22).
  • the final PCR was carried out for 30 cycles using 2ng of DNA template and lOOng of each 5' PCR primer (SEQ ID NO: 25) and 6-FAM labeled 3' PCR primer (SEQ ID NO:47).
  • PCR products generated with 109T and 45 A appear to be nearly identical from templates produced by the one PCR step variant (compare Fig. 4A to Fig. 4C, and Fig. 4B to Fig. 4D).
  • the products detected following PCR from templates produced using the two PCR step method are overall quite distinct (compare Fig. 4E to Fig. 4G, and Fig. 4F to Fig. 4H).
  • the two PCR step embodiment of the method thus provides a substantial improvement over the closest previously available method.
  • the method of the present invention was performed on serum-starved and serum- treated MG63 cells using either the one PCR step (Table I) or two PCR step (Table II) embodiments.
  • Table I reverse transcriptase was used to generate four cDNA subpools from cRNA by initiating transcription with one of the set of four NI 5' PCR primers (SEQ ID NO: 22).
  • SEQ ID NO: 22 NI 5' PCR primers
  • Taq DNA polymerase was used in PCR (20 cycles) to generate double stranded cDNA subpools with 5' PCR primer (SEQ ID NO: 22) and as 3' PCR primer (SEQ LD NO: 47).
  • the final PCR in both Table I and Table II was performed identically with the complete series of 256 5'-PCR primers paired (SEQ LD NO: 25) with 6FAM-labeled 3' PCR primer (SEQ LD NO: 47) using 2ng input cDNA template. From the PCR reaction displays, differentially regulated molecules were identified and isolated for cloning and sequencing pu ⁇ oses.
  • DNA sequence data was obtained for individual clones and gene identification determined following database searches using the BLAST algorithm.
  • clones found to be exact matches to known human genes are listed by gene name and GenBank locus ID.
  • the fidelity of the parsing step using 5PRIMERN1 (SEQ ID NO: 22) in either reverse transcription (Table I) or PCR reactions (Table II) was assessed by tabulating the sequence match of the clone at the NI position to the GenBank sequence.
  • 5PRIMERN1 SEQ ID NO: 22
  • anchor primers are biotinylated at their 5' end (compare Figures 1 and 2).
  • Biotinylated cDNA fragments can be captured using a streptavidin-coated substrate, preferably streptavidin-coated paramagnetic beads (Dynal).
  • Figure 5 compares the results from the standard basic method to those obtained using anchor primers labeled with magnetic beads.
  • cDNA libraries were constructed using the standard technique (as outlined in Figure 1) and the magnetic bead alternative embodiment (see Figure 2) from 2 ⁇ g mRNA aliquots from five separate samples of striatum from haloperidol treated mice taken in a time series (0, 0.75, 7 hours, 10 and 14 days).
  • Example 5 Demonstration of linearity in the three-step method: Relationship of PCR product peak height to input cRNA concentration.
  • a Sall-Notl cDNA fragment (SEQ ID NO: 51) was cloned into the library vector pBCSK+, linerarized and cRNA produced by transcription from the T3 promoter synthetic cRNA was constructed to give rise to a peak of known size (492b ⁇ ) in PCR. Varying amounts of cRNA (0, 25, 100, or 250pg) were introduced into a 250ng pool of cRNA prior to reverse transcription with the N 0 primer (SEQ ID NO: 14). 400pg of cDNA was used as template for PCR reactions with 5' PCR primer (SEQ ID NO: 22) and 3' PCR primers (SEQ ID NO:47), respectively.

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Abstract

Procédé amélioré pour l'identification simultanée, spécifique de séquences, d'ARNm dans une population d'ARNm, qui permet la visualisation de presque chaque ARNm exprimé par un tissu sous forme de bande distincte sur un gel dont l'intensité correspond en gros à la concentration de l'ARNm. En général, ledit procédé consiste à former de l'ADNc à l'aide d'amorces d'ancrage destinées à se fixer sur une terminaison 3', à produire des segments d'insertion clonés à partir de l'ADNc dans un vecteur contenant un promoteur spécifique de bactériophage pour la synthèse subséquente de l'ARN, à produire des fragments linéarisés des segments d'insertion clonés, à préparer l'ARNc, à transcrire l'ADNc à partir de l'ARNc et à effectuer deux amplifications par PCR, spécifiques de séquences, de l'ADNc. Dans des modes de réalisation préférés, ledit procédé consiste à comparer la longueur et au moins une partie de la séquence nucléotidique des produits de PCR à des valeurs attendues déterminées à partir d'une base de données de séquences nucléotidiques. Ledit procédé permet d'identifier des modifications de l'expression de l'ARNm associées à l'administration de médicaments ou à des états physiologiques ou pathologiques. Des vecteurs et des amorces utiles pour la mise en oeuvre dudit procédé sont également décrits.
PCT/US1999/023655 1998-11-04 1999-10-14 Procede permettant d'indexer et de determiner la concentration relative d'arn messagers exprimes WO2000026406A1 (fr)

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Application Number Priority Date Filing Date Title
IL14296599A IL142965A0 (en) 1998-11-04 1999-10-14 METHOD FOR INDEXING AND DETERMINING THE RELATIVE CONCENTRATION OF EXPRESSED MESSSENGER RNAs
AU11089/00A AU1108900A (en) 1998-11-04 1999-10-14 Method for indexing and determining the relative concentration of expressed messenger rnas
JP2000579778A JP2002528135A (ja) 1998-11-04 1999-10-14 発現されたメッセンジャーＲＮＡの相対濃度の指数化（ｉｎｄｅｘ）及び決定方法
CA002350168A CA2350168A1 (fr) 1998-11-04 1999-10-14 Procede permettant d'indexer et de determiner la concentration relative d'arn messagers exprimes
MXPA01004550A MXPA01004550A (es) 1998-11-04 1999-10-14 Metodo para clasificar y determinar la concentracion relativa de los arn mensajeros expresados.
EP99954838A EP1127159A1 (fr) 1998-11-04 1999-10-14 Procede permettant d'indexer et de determiner la concentration relative d'arn messagers exprimes
KR1020017005872A KR20010092721A (ko) 1998-11-04 1999-10-14 발현된 메신저 rna의 상대적인 농도를 산출하고 결정하는방법
EA200100490A EA200100490A1 (ru) 1998-11-04 1999-10-14 Способ индексации и определения относительной концентрации экспрессирующихся информационных рнк
US09/775,217 US20020012922A1 (en) 1998-11-04 2001-02-01 Simplified method for indexing and determining the relative concentration of expressed messenger RNAs
NO20012203A NO20012203L (no) 1998-11-04 2001-05-03 Fremgangsmåte for å indeksere og bestemme den relative konsentrasjon av uttrykt mRNA

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EP1092045A1 (fr) * 1998-06-30 2001-04-18 The Scripps Research Institute Procede de differentiation simultanee d'arn messagers exprimes de maniere differentiee et procede de mesure des concentrations relatives
EP1092044A1 (fr) * 1998-06-30 2001-04-18 The Scripps Research Institute PROCEDE AMELIORE D'IDENTIFICATION SIMULTANEE D'UNE PLURALITE D'ARNm EXPRIMES DE FA ON DIFFERENTIELLE ET DE MESURE DES CONCENTRATIONS RELATIVES
WO2003064689A2 (fr) * 2002-01-29 2003-08-07 Global Genomics Ab Procedes et dispositifs pour l'identification de caracteristiques geniques
WO2003105761A2 (fr) * 2002-06-12 2003-12-24 Research Development Foundation Immunotoxine servant d'agent therapeutique et ses utilisations
US7807352B2 (en) 2001-06-30 2010-10-05 Enzo Life Sciences, Inc. Process for producing two or more copies of nucleic acids in a library, and process for detecting or quantifiying more than one nucleic acid in a library
CN101538606B (zh) * 2009-02-19 2012-03-21 上海浩源生物科技有限公司 检测一种或多种靶核酸的方法及其试剂盒
US9068948B2 (en) 2002-03-12 2015-06-30 Enzo Life Sciences, Inc. Processes for detection of nucleic acids
US9353405B2 (en) 2002-03-12 2016-05-31 Enzo Life Sciences, Inc. Optimized real time nucleic acid detection processes
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids

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EP1092045A1 (fr) * 1998-06-30 2001-04-18 The Scripps Research Institute Procede de differentiation simultanee d'arn messagers exprimes de maniere differentiee et procede de mesure des concentrations relatives
EP1092044A1 (fr) * 1998-06-30 2001-04-18 The Scripps Research Institute PROCEDE AMELIORE D'IDENTIFICATION SIMULTANEE D'UNE PLURALITE D'ARNm EXPRIMES DE FA ON DIFFERENTIELLE ET DE MESURE DES CONCENTRATIONS RELATIVES
EP1092044A4 (fr) * 1998-06-30 2002-10-30 Scripps Research Inst PROCEDE AMELIORE D'IDENTIFICATION SIMULTANEE D'UNE PLURALITE D'ARNm EXPRIMES DE FA ON DIFFERENTIELLE ET DE MESURE DES CONCENTRATIONS RELATIVES
EP1092045A4 (fr) * 1998-06-30 2003-05-28 Scripps Research Inst Procede de differentiation simultanee d'arn messagers exprimes de maniere differentiee et procede de mesure des concentrations relatives
US9279147B2 (en) 2001-06-30 2016-03-08 Enzo Life Sciences, Inc. Processes for detecting or quantifying analytes of interest
US9637778B2 (en) 2001-06-30 2017-05-02 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
US9873956B2 (en) 2001-06-30 2018-01-23 Enzo Biochem, Inc. Compositions and processes for analyte detection, quantification and amplification
US9790621B2 (en) 2001-06-30 2017-10-17 Enzo Life Sciences, Inc. Composition of matter comprising library of first nucleic acid analyte copies
US7807352B2 (en) 2001-06-30 2010-10-05 Enzo Life Sciences, Inc. Process for producing two or more copies of nucleic acids in a library, and process for detecting or quantifiying more than one nucleic acid in a library
US9777312B2 (en) 2001-06-30 2017-10-03 Enzo Life Sciences, Inc. Dual polarity analysis of nucleic acids
US8557522B2 (en) 2001-06-30 2013-10-15 Enzo Life Sciences, Inc. Processes for detecting or quantifying more than one nucleic acid in library
US8597888B2 (en) 2001-06-30 2013-12-03 Enzo Life Sciences, Inc. Processes for detecting or quantifying more than one nucleic acid in a library
US9057100B2 (en) 2001-06-30 2015-06-16 Enzo Life Sciences, Inc. Composition comprising array of nucleic acid primer sets
US9777406B2 (en) 2001-06-30 2017-10-03 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
US9234234B2 (en) 2001-06-30 2016-01-12 Enzo Life Sciences, Inc. Detection and quantification process for more than one nucleic acid in library
US9771667B2 (en) 2001-06-30 2017-09-26 Enzo Life Sciences, Inc. Arrays comprising chimeric compositions
US9745619B2 (en) 2001-06-30 2017-08-29 Enzo Biochem, Inc. Process for detecting or quantifying nucleic acids in a library
US9309563B2 (en) 2001-06-30 2016-04-12 Enzo Life Sciences, Inc. Compositions and processes for analyte detection, quantification and amplification
US9650666B2 (en) 2001-06-30 2017-05-16 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
US9617585B2 (en) 2001-06-30 2017-04-11 Enzo Life Sciences, Inc. Processes for detecting or quantifying more than one nucleic acid in a library
US9428797B2 (en) 2001-06-30 2016-08-30 Enzo Life Sciences, Inc. Nucleic acid detecting or quantifying processes
US9434984B2 (en) 2001-06-30 2016-09-06 Enzo Life Sciences, Inc. Composition comprising an array which further comprises chimeric compositions
US9487821B2 (en) 2001-06-30 2016-11-08 Enzo Life Sciences, Inc. Composition comprising library of double stranded nucleic acids
US9611508B2 (en) 2001-06-30 2017-04-04 Enzo Life Sciences, Inc. Processes for detecting or quantifying nucleic acids in a library
US9617584B2 (en) 2001-06-30 2017-04-11 Enzo Biochem, Inc. Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
WO2003064689A3 (fr) * 2002-01-29 2003-11-13 Global Genomics Ab Procedes et dispositifs pour l'identification de caracteristiques geniques
WO2003064689A2 (fr) * 2002-01-29 2003-08-07 Global Genomics Ab Procedes et dispositifs pour l'identification de caracteristiques geniques
US9353405B2 (en) 2002-03-12 2016-05-31 Enzo Life Sciences, Inc. Optimized real time nucleic acid detection processes
US9316587B2 (en) 2002-03-12 2016-04-19 Enzo Life Sciences, Inc. Processes for quantitative or qualitative detection of single-stranded or double-stranded nucleic acids
US9261460B2 (en) 2002-03-12 2016-02-16 Enzo Life Sciences, Inc. Real-time nucleic acid detection processes and compositions
US9068948B2 (en) 2002-03-12 2015-06-30 Enzo Life Sciences, Inc. Processes for detection of nucleic acids
US10144957B2 (en) 2002-03-12 2018-12-04 Enzo Life Sciences, Inc. Optimized real time nucleic acid detection processes
WO2003105761A3 (fr) * 2002-06-12 2006-02-09 Res Dev Foundation Immunotoxine servant d'agent therapeutique et ses utilisations
WO2003105761A2 (fr) * 2002-06-12 2003-12-24 Research Development Foundation Immunotoxine servant d'agent therapeutique et ses utilisations
CN101538606B (zh) * 2009-02-19 2012-03-21 上海浩源生物科技有限公司 检测一种或多种靶核酸的方法及其试剂盒

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KR20010092721A (ko) 2001-10-26
CA2350168A1 (fr) 2000-05-11
JP2002528135A (ja) 2002-09-03
MXPA01004550A (es) 2002-09-18
EA200100490A1 (ru) 2001-10-22
EP1127159A1 (fr) 2001-08-29

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