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WO2000070039A1 - METHODE DE SOUSTRACTION D'ADNc PAR SUPPRESSION DE LA SYNTHESE D'ARNm CIBLES SPECIFIQUEMENT - Google Patents

METHODE DE SOUSTRACTION D'ADNc PAR SUPPRESSION DE LA SYNTHESE D'ARNm CIBLES SPECIFIQUEMENT Download PDF

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WO2000070039A1
WO2000070039A1 PCT/CA2000/000586 CA0000586W WO0070039A1 WO 2000070039 A1 WO2000070039 A1 WO 2000070039A1 CA 0000586 W CA0000586 W CA 0000586W WO 0070039 A1 WO0070039 A1 WO 0070039A1
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mrna
oligonucleotide
rna
duplex
sample
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Jerry Pelletier
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McGill University
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McGill University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Definitions

  • the present invention relates to genetic engineering and moie particularly to a method to specifically modify a mRNA population
  • the present invention can therefore be applied to the field of cDNA synthesis and cDNA cloning More specifically, the invention relates to methods of customizing an mRNA population by selectively eliminating chosen mRNAs
  • the invention further relates to customized cDNA libraries generated from such a customized mRNA population
  • the invention also relates to a method of specifically targeting and cleaving and/or degrading mRNAs in order to customize the mRNA population
  • the invention relates to a method for subtracting cDNAs by suppressing the synthesis of specifically targeted mRNAs
  • cDNAs complementary DNAs
  • a full length cDNA allows one to predict transcription initiation start sites, translation initiation start sites, deduce certain protein characteristics based on primary ammo acid sequences, predict transcription termination sites, and visually inspect the 5' and 3' untranslated regions for elements which may be nvolved in post-transc ⁇ ptional regulation of gene expression
  • the analysis of several complete cDNAs of a given gene enables one to gather information on alternative splicing, alternative promoter usage, and alternative polyadenylation signals - all events knownto be important in gene expression regulation
  • the comparison of genomic and cDNA sequence is essential to determine exon-intron structure and document the occurrence of RNA editing - a post-transcriptional regulatory mechanism on which only little information is available
  • the cloning of mRNA into cDNA for the purposes of functional studies is a complex interrelated series of enzyme-catalyzed reactions involving the in vitro synthesis of a DNA copy of mRNA, its subsequent conversion to duplex cDNA and insertion into an appropriate prokaryotic vector
  • the procedure usually involves the following series of steps
  • Reverse transc ⁇ ptase is then utilized to prime from the DNA primer and copy the RNA template into cDNA
  • Second strand synthesis is performed utilizing RNAse H, DNA polymerase I, and DNA ligase
  • reassociation-kinetic analysis indicates that the population of mRNAs of a typical cell can be subdivided into three fequency classes (ref 2)
  • ref 2 fequency classes
  • the invention relates to a method which enables a versatile customization of an mRNA population
  • the invention relates to a method of customizing an mRNA population which comprises a targeting of a chosen mRNA or mRNAs with at least one oligonucleotide, under conditions which favor the formation of a duplex between the targeted mRNA or mRNAs and at least one oligonucleotide
  • the method then comprises a separation of the duplexed mRNA from non-duplexed mRNA, and/or the use of the properties of duplexed versus non-duplexed mRNA (e g RT processivity inhibition, differential substrate forcleavage/degradation) thereby yielding a customized mRNA population
  • the invention also relates to methodsbased on the use of specific o gonucleotides to customize an mRNA population
  • a method targets high abundance mRNAs, and is coupled to a method of generating cDNA libraries, the frequency of re-isolati ⁇ i of cDNAs can then be reduced and, as a corollary, favor the isolation of novel cDNAs
  • the invention also concerns methods to subtract targeted cDNAs by suppressing their synthesis
  • the present invention also relates to a simple method for normalizing mRNAs which does not compromise the generation of a full length cDNA library therefrom
  • the present invention also concerns methods of controlling the time of annealing in order to remove abundant mRNAs or cDNAs from libraries while maintaining less abundant mRNAs or cDNAs in the library
  • the invention relates to a procedure of suppressing the synthesis of target transcripts during the first strand cDNA synthesis, based on the ability of a duplex specific RNAse to specifically target and degrade the RNA moiety of a RNA/DNA duplex
  • the RNAse is RNAse H
  • the mRNA population is customized using an annealing of a complementary DNA oligonucleotide to a given RNA to obtain a RNA/DNA duplex, treating the duplex with RNAse H, thereby resulting in a specific cleavage of only the target RNA at the site of hybridization
  • a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation ofa duplex between the oligonucleotide and the mRNA molecule and submitting the mRNA-o gonucleotide mixture to a purification step which enables a separation of duplexed mRNA from non-duplexed mRNA thereby yielding a customized mRNA population
  • a method for suppressing the synthes ⁇ of target transcripts during first strand cDNA synthesis comprising the steps of selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule and submitting the mRNA-oligonucleotide mixture to a duplex specific RNAse which specifically targets the RNA moiety of the duplex and performing first strand cDNA synthess on the RNAse-treated duplexes of the previous step
  • the present invention also provides a method of subtracting cDNAs by suppressing their synthesis comprising the steps of annealing a set of complementary DNA oligonucleotides to a given mRNA sample under conditions enabling a formation of mRNA-oligonucleotide duplexes, and performing first strand cDNA synthesis on non-duplexed mRNAs
  • a method of targeting a chosen mRNA or mRNA population for degradation using ohgos which are specific for the chosen mRNA or mRNA population to generate duplexes of oligos and mRNA, submitting the duplex to a duplex-directed degradation or cleavage which leaves non-duplexed mRNA intact and purifying the non-duplexed mRNAs
  • the invention enaues the targeting of any chosen mRNA population, provided that sequence data fr ⁇ n the chosen mRNA population is available
  • sequence data fr ⁇ n the chosen mRNA population is available
  • the use of oligos to target an mRNA or mRNA population is a versatile and simple way to apply the customizing method of the present invention to any chosen mRNA or mRNA population
  • the present invention is not limited to selective elimination of only abundant transcripts
  • the present invention provides a means of selecting any characteristic of a targeted mRNA which can be used to reduce and/or eliminate same from an mRNA population, provided that oligos whichare specific and complementary thereto are available
  • the method of customizing an mRNA population in accordance with the present invention therefore enables the customizing of the cDNA library
  • the duplexed mRNAs can then be separated from non-duplexed mRNAs in order to yield the customized mRNA population
  • the present invention is described in more detail using a purification through the cap structuie to selectively purify such flagged mRNAs, the invention is not so limited Indeed a number of methods can be used to separate the oligonucleotide-mRNA duplexes from non-duplexed mRNA, in order to yield the customized mRNA population
  • Non-limiting examples of such methods include hydroxyapatite, which selectively binds double-stranded duplexes, streptavidin/biotin methods, based on the use of biotinylated ohgonucleotides, duplex-dependent mRNA cleavage and/or degradation (enzymatic or non-enzymatic directed cleavage/degradation)
  • a number of methods known to a skilled artisan include hydroxyapatite, which selectively binds double-
  • a targeting of specific mRNAs or RNAs can be done by a number of methods and that the oligos exemplified herein are but a specific embodiment of targeting of specific mRNAs
  • One preferred utility of the method of customizing an mRNA population is in the generation of customized cDNA libraries
  • the method of customizing of an mRNA population in accordance with the present invention can be incorporated in an "improved" method to make cDNA libraries
  • one envisaged application of the invention is during first strand cDNA synthesis to remove abundant genes from libraries This results in a subtraction process and will facilitate the rate of gene discovery
  • the cDNA of any gene can be specifically inhibited utilizing an approach described herein While the targeting and specific degradation of mRNA has been demonstrated with an RNAse H-dependent (duplex-dependent) degradation of the RNA template, the present invention should not be so limted Indeed, the present invention extends to any method involving enzymatic or non-enzy
  • any modified oligonucleotide as known in the art can be used for this purpose, provided that a cleavage of the RNA template occurs, resulting in a physical separation of the 5' and 3' ends of the targeted mRNA template
  • RNAse H and Reverse Transc ⁇ ptase activities are separated in two different steps (Steps I and II in Fig 3), the instant invention should not be so limited Tte most common reverse transc ⁇ ptases in use, MMLV SuperscriptTM, does not contain RNase H activity Hence, in accordance with an embodiment based on an enzymatic-directed cleavage of the mRNA, a separate RNAseH activity needs to be added
  • RT reverse transcnptases
  • AMV or HIV reverse transc ⁇ ptase contain both activities (reverse transcriptase and RNAse H) in one complex.
  • RNA cleavage/degradation and cDNA synthesis could be carried out in accordance with the present invention.
  • DNA oligonucleotides are annealed to a given RNA template, followed by a direct incubation with the reverse transcriptases containing the RNAse H activity.
  • the RT could degrade the complementary RNA strand.
  • a cleavage/degradation of the targeted mRNA is not necessary in all conditions to enable the construction of a customized cDNA library. Indeed, an inhibition of the processivity of the RT would be sufficient to enable the generation of a customized library.
  • a mRhA or mRNAs would be targeted with oligonucleotides and the processivity of the RT would be inhibited by the duplex, thereby reducing or abrogating the generation of cDNAs from the targeted mRNA templates.
  • the cDNAs can then be separated by a variety of means favoring the purification of full-length cDNAs according to physical feature of the mRNAs (cap-dependent purification, size-dependent separation, poly A- dependent purification).
  • the oligonucleotides are blocked at the 3' end (as commonly known) to block the priming of the RT from the oligo. Since the blocking oligonucleotide could also be used as a primer by the RT, in one embodiment, oligonucleotides which contain a modified 3' end (which does not serve as primer for the RT) are used. Such oligonucleotides which cannot act as primers (6) were used in Fig.
  • the targeted mRNAs are degraded by RNAse H and full-length mRNAs are selected and purified using oligo dT priming, reverse transcriptase and CAPtureTM. It will be recognized therefore that the present invention enables a "flagging" of specific mRNAs (e.g. mRNA/oligo duplexes). These duplexes can serve as targets for cleavage or degradation or can block the processivity of a DNA polymerase (e.g.
  • the instant invention further extends to PCR analysis of gene products generated by RT of mRNA into cDNA
  • primers flanking both sides of the duplex could be used
  • RNA is the preferred targeted nucleic acid in accordance with the methods of the present invention
  • RNA in general can be targeted wth oligonucleotides in order to customize the RNA population
  • the methods of the present invention can be adapted to target, for example, uncapped mRNAs (and to generate a cDNA library therefrom)
  • RNA oligonucleotides are selected via a cap structure-dependent purification step using a cap column (CAPtureTM) It shall be recognized that other cap-dependent purification methods can be used Non- hmiting examples thereof include anti-cap antibodies and direct oxydization of the cap structure (refs 9, 10, 1 1 )
  • a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in ai mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule, and submitting the mRNA-oligonucleotide mixture to a purification step which enables a
  • the mRNA oligonucleotide mixture enables a flagging of specific mRNAs, such that this flagging enables a selective purification or targeting of chosen mRNAs from the oligonucleotide mRNA mixture
  • a method for suppressing the synthesis of target transcripts during first strand cDNA synthesis comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oligonucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture, under conditions which enable a formation of a duplex between the oligonucleotide and the mRNA molecule submitting the mRNA-oligonucleotide mixture to a duplex-specific RNAse which specifically targets the RNA moiety of the duplex, and performing first strand cDNA synthes ⁇ on the RNAse-treated duplexes, thereby suppressing the synthesis of RNAse- cleaved duplexes
  • a method of subtracting cDNAs by suppressing their synthesis comprising the steps of annealing a set of complementary DNA oligonucleotides to a given mRNA sample under conditions enabling a formation of mRNA-oligonucleotide duplexes, and performing first strand cDNA synthesis on non-duplexed mRNAs
  • a method of customizing an mRNA population comprising selecting an oligonucleotide which is complementary to an mRNA molecule in an mRNA sample, incubating the oiigpnucleotide with the mRNA sample to obtain an mRNA oligonucleotide mixture under conditions which enable a formation of a duplex betweenthe oligonucleotide and the mRNA molecule, and submitting the mRNA-oligonucleotide mixture to a duplex- dependent
  • the RNA or mRNA oligonucleotide mixture instead of being submitted to the duplex-dependent cleavage and/or degradation, is submitted to a purification step enabling the separation of duplexed RNA or duplexed mRNA from non-duplexed RNA or mRNA
  • Nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission
  • nucleic acids refers to a number of rouinely used recombinant DNA (rDNA) technology terms Nevertheless, definitions of selected examples of such rDNA terms are provided for clarity and consistency For certainty, it is emphasized that the present invention finds utility with nucleic acids in general
  • nucleic acids which can be used in accordance with the teachings of the present invention include that from eukaryotic cells such as that of animal cells, plant cells, or microorganisms as well as that from prokaryotic cells and viruses
  • processivity of a polymerase refers to its property to continue to act on a substrate instead of dissociating therefrom
  • nucleic acid molecule refers to a polymer of nucleotides
  • Non-limiting examples thereof include DNA (e g genomic DNA, cDNA) and RNA molecules (e g mRNA)
  • the nucleic acid molecule can be obtained by cloning techniques or synthesized DNA can be double-stranded or single-stranded (coding strand or non-coding strand [anthozoans])
  • recombinant DNA refers to a DNA molecule resulting from the joining of DNA segments This is often eferred to as genetic engineering
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, whch are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction Other types of amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below As commonly known in the art, the oligonucleotides are designed to bind to a complementary sequence under selected conditions
  • the nucleic acid (i e DNA or RNA) for practsing the present invention may be obtained according to well known methods
  • Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assayformat and the particular needs and targeted genomes employed In general, the oligonucleotide probes or primers are at least 10 nucleotides in length, preferably between 12 and 24 molecules and they may be adapted to be especiallysuited to a chosen nucleic acid amplification system
  • the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybnzidation thereof with its targeted sequence (see below and in Sambrook et al 1989 Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories, Ausubel et al 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc , N Y )
  • oligonucleotide or "DNA” molecule or sequence refers to a molecule comprised of the deoxy ⁇ bonucleotides adenine (A), guanine (G), thymine (T) and/or cytosine (C), in a double-stranded form
  • oligonucleotide or “DNA” can be found in linear DNA molecules or fragments, viruses, plasmids vectors chromosomes orsynthetically derived DNA
  • particular double-stranded DNA sequences may be described according to the normal convention of giving only the sequence in the 5' to 3' direction
  • oligonucleotides or DNA can also be found as single-stranded molecules
  • "oligonucleotides” or “oligos” define a molecule having two or more nucleotides ( ⁇ bo or deoxy ⁇ bonucleo
  • Probes and oligonucleotides of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann Reports Med Chem 23 295 and Moran et al , 1987, Nucleic acid molecule Acids Res , 14 5019 Probes of the invention can be constructed of either ⁇ bonucleic acid (RNA) or deoxy ⁇ bonucleic acid (DNA), and preferably of DNA General teachings on the synthesis of oligonucleotides and substituents and modifications thereof can be found for example in US 5,438, 131 The selection of the best suited synthesis pathway of an oligonucleotide and of the appropriate modifications, and substituentsto be used, may be selected accordingly by the person of ordinary skill to which the instant invention pertains
  • a primer' defines an oligonucleotide which is capable of annealing to a target sequence thereby creating a double stranded region or duplex which can serve as an initiation point for DNA synthesis under suitable conditions 'Nucleic acid hybridization refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al 1989, supra and Ausubel et al , 1997, supra) and are commonly known in the art In the case of a hybridization toa nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt (5 x SSC or 5 x SSPE), 5 x Denhardt's solution 1 % SDS and 100
  • radioactive nucleotides can be incorporated into oligonucleotides of the invention by several methods
  • Non- limiting examples thereof include kinasing the 5' ends of the probes using v- 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E coli in the presence of radioactive dNTP (i e uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like
  • the oligos of the present invention can incorporate a radiolabel by T4 DNA polynucleotide kinase or can be synthesized with fluorescently labelled bases
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods See generally Kwoh et al , 1990, Am Biotechnol Lab 8 14-25 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill Non-limiting examples of amplification techniques include polymerase chain reaction (PCR), hgase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ rep case system and NASBA (Kwoh et al 1989 Proc Natl Acad Sci U3A 86, 1173-1 177, ⁇ zardi et al , 1988, BioTechnology ⁇ 1197-1202, Malek et al , 1994, Methods Mol Biol , 28 253-260, and Sambrook et al , 1989, supra)
  • amplification will be carried out using PCR Polymerase chain reaction (PCR) is carried out in accordance with known techniques See, e e
  • the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or poiypeptide.
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific ammo acid sequence thereby giving rise the a specific poiypeptide or protein It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art
  • the term ' vector is commonly known in the art and defines a plasmid DNA phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned Numerous types of vectors exist and are well known in the art
  • the term "allele” defines an alternative form of a gene which occupies a given locus on a chromosome
  • a “mutation” is a detectable change in the genetic material which can be transmitted to pursehter cell
  • a mutation can be, for example, a detectable change in one or more deoxy ⁇ bonucleotide
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position Spontaneous mutatior ⁇ and experimentally induced mutations exist
  • the res ⁇ t of a mutations of nucleic acid molecule is a mutant nucleic acid molecule
  • a mutant poiypeptide can be encoded from this mutant nucleic acid molecule
  • the term “purified” refers to a molecule having been separated from a cellular component
  • a “purified protein” has been purified to a level not found in nature
  • a "substantially pure” molecule is a molecule that is lacking in substantially all other cellular components
  • the term “purified” is used to relate to a non
  • kits comprising an oligonucleotide or kit thereof of the present invention specifically designed to target (or "flag") a chosen mRNA or mRNAs
  • a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers
  • Such containers include small glass containers, plastic containers or strips of plastic or paper
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-cortam mated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another
  • such containers will include for example a container which wil accept the test sample (e g mRNA), a container which contains the oligonucleotides used in the assay and containeis which contain reagents used to separate o go-mRNA duplexes from non- duplexed mRNA
  • such containers will include, for example, a container which will accept the test sample (
  • Figure 1 shows an example of the steps involved in generating cDNA libraries from mRNA
  • a number of strategies can be used for cDNA library generation, of which only one is shown above, all libraries require as a first step, a primer which Reverse Transcriptase (RT) can prime from
  • RT Reverse Transcriptase
  • representation of mRNA abundance is maintained overall during construction, such that abundant mRNA species end up-being the most abundant cDNAs in the libraries, this is an impediment to novel gene discovery since random isolation of cDNAs from libraries will often lead to re-isolation of more abundantly expressed genes
  • Figure 2 is a schematic diagram illustrating the specificity of cleavage of targeted RNA/DNA duplex by RNAse H
  • FIG. 3 is a schematic outline which shows an incorporation of a subtraction of cDNAs by suppression of cDNA synthesis with an affinity selection step to remove cDNAs which correspond to RNAse H-targeted mRNAs using affinity selection of mRNA/cDNA duplexes with 5' m7G structures (5,7)
  • the designation of mRNAs as species l-lll relate to their classification with respect to their frequency of distribution (see text for further details)
  • Figure 4 shows an embodiment of the subtraction by suppression of synthesis according to one aspect of the present invention using the WT1 and D75 mRNAs
  • A) is a schematic diagram illustrating WT1 and D75 mRNAs, showing the sizes of the expected cDNA products resulting from p ⁇ mi ⁇ g with oligo d(T)
  • For WT1 one product of 1500 nucleotides (nts) is expected whereas for D75 two products are expected, one resulting from priming from tie poly (A) tail ( 2300 n
  • RNAse H is an enzyme which specifically targets and degrades the RNA moiety of a RNA/DNA duplex Therefore annealing of a complementary DNA oligonucleotide to a given RNA followed by treatment with RNAse H, results in a specific cleavage of only the target RNA at the site of hybridization (Fig 2) This has the effect of physically separating the RNA targets into two portions, one half containing the 3' end, and another half containing the 5' m7G cap structure As outlined in Fig 3, if cDNAs are generated from these twoportions, utilizing for example reverse transcriptase and an oligo d(T) primer, a cDNA molecule originating from the targeted mRNA (mRNA species I, in Figs 2 and 3)
  • EXAMPLE 1 Validation of one embodiment of the method of subtracting cDNAs using WT1 or D75 mRNAs.
  • WT1 and D75 two mRNA species, WT1 and D75, were pretreated with 4 complementary DNA oligonucleotides specific to WT1 (l-IV) and one control oligonucleotide (SP6) which does not hybridize to either WT1 or D75 (Fig 4B)
  • the RNAs were incubated with RNAse H Following RNAseH treatment, cDNA reactions were performed with MMLV Superscript IITM and oligo d(T) (Fig 4C)
  • a cDNA product originating from WT1 has the expected size of 1 ,500 nts
  • cDNA synthesized from D75 mRNA generates products of 2,000 and 2,300 rts, due to priming from the poly (A) tail and
  • Figure 4 shows that indeed, oligonucleotides with different 3' ends can be used to target a chosen mRNA for degradation
  • oligos #1 and 4 which can prime cDNA synthesis

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Abstract

La présente invention concerne le génie génétique et plus particulièrement une méthode de modification spécifique d'une population d'ARNm. La présente invention peut par conséquent être appliquée au domaine de la synthèse de l'ADNc et du clonage de l'ADNc. Plus spécifiquement, l'invention concerne des méthodes de personnalisation d'une population d'ARNm par élimination sélective d'ARNm choisis. L'invention concerne également des banques d'ADNc personnalisées produites à partir de cette population d'ARNm personnalisée. L'invention concerne aussi une méthode de ciblage et de clivage et/ou dégradation spécifique d'ARNm afin de personnaliser la population d'ARNm. De plus, l'invention a trait à une méthode de soustraction d'ADNc par suppression de la synthèse d'ARNm ciblés spécifiquement.
PCT/CA2000/000586 1999-05-17 2000-05-17 METHODE DE SOUSTRACTION D'ADNc PAR SUPPRESSION DE LA SYNTHESE D'ARNm CIBLES SPECIFIQUEMENT Ceased WO2000070039A1 (fr)

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