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WO2010077288A2 - Procédés d'identification de différences d'épissage alternatif entre deux échantillons d'arn - Google Patents

Procédés d'identification de différences d'épissage alternatif entre deux échantillons d'arn Download PDF

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WO2010077288A2
WO2010077288A2 PCT/US2009/006450 US2009006450W WO2010077288A2 WO 2010077288 A2 WO2010077288 A2 WO 2010077288A2 US 2009006450 W US2009006450 W US 2009006450W WO 2010077288 A2 WO2010077288 A2 WO 2010077288A2
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rnas
population
subtractive
rna
cdnas
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WO2010077288A3 (fr
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Gene Yeo
Jonathan Scolnick
Fred H. Gage
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Salk Institute for Biological Studies
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Salk Institute for Biological Studies
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

  • the invention relates to molecular methods that can be used in highly parallel genome-wide analyses to identify alternative, e.g., unique and/or previously uncharacterized, splice isoform(s) of one or more genes of interest.
  • AS Alternative splicing
  • probe designs and labeling protocols used for microarray experiments tend to be biased towards the 3' end of the gene, and unless multiple probes are to match each specific exon-exon junction that might be spliced together in an alternative splicing event, certain splice isoforms can remain undetected and uncharacterized.
  • EST databases can be used to compare, e.g., mRNA splice variants across tissues, but splice events in which introns are retained in the mature mRNA are difficult to distinguish from database artifacts that comprise, e.g., pre-mRNA or genomic sequences.
  • compositions and highly parallel methods that can be used for the genome-wide detection of splice isoforms of one or more target gene that are present in, e.g., a tissue of interest at a developmental stage of interest, an organism of interest diagnosed with a disease of interest, and the like.
  • the invention described herein fulfills these and other needs, as will be apparent upon review of the following.
  • the present invention provides methods and related compositions useful for identifying one or more additional splice isoforms of, e.g., a target gene that is present in a sample population of RNAs but not in a subtractive population of RNAs.
  • mRNA species common to both the subtractive and sample RNA populations are removed, leaving mRNA splice isoform(s), e.g., cell type-specific splice isoforms, tissue-specific splice isoforms, disease-specific splice isoforms, and the like, that are unique to the sample population for further manipulation and analysis, e.g., sequencing, e.g., using an automated high-throughput sequencing system.
  • the methods and compositions provided by the invention can advantageously permit the identification of splice isoforms that would be otherwise difficult to isolate using methods that entail, e.g., designing probes specific to all possible exon-exon junctions, a priori knowledge of coding regions in a pre-mRNA, a sequenced genome, etc.
  • the invention provides methods of determining whether a sample population of RNAs includes one or more additional splice isoforms that are not present in a subtractive population of RNAs.
  • the invention provides methods of separating one or more alternate isoform of a target gene from a sample population of RNAs.
  • the methods include providing a subtractive population of RNAs that comprises a first isoform of the target gene and a sample population of RNAs that comprises at least one or more alternate isoform of the target gene.
  • the methods include reverse transcribing the subtractive RNAs to generate a population of subtractive cDNAs, fragmenting the sample RNAs, e.g., via enzymatic digestion, sonication, mechanical shearing, electrochemical cleavage, chemical cleavage, and/or nebulization, and hybridizing the resulting RNA fragments to the subtractive cDNAs.
  • RNA fragments in the sample population that comprise the first isoform of the target gene hybridize to the subtractive cDNAs to produce a subpopulation of RNA: cDNA duplexes, and RNA fragments comprising one or more alternate splice isoform(s) of the target gene comprise a subpopulation of unhybridized RNAs.
  • the methods include removing the RNA: cDNA duplexes from unhybridized RNA fragments, thereby separating the one or more alternate splice isoform of the target gene from the sample population of RNAs.
  • the methods optionally include reverse transcribing the unhybridized RNA fragments, sequencing the resulting cDNAs, e.g., using an automated high-throughput sequencing system, and comparing the sequences of the cDNAs to a sequence of the target gene, e.g., to characterize the additional isoforms of the target gene.
  • Reverse transcribing the unhybridized RNA fragments can optionally include ligating linkers, e.g., linkers that optionally comprise, e.g., a primer hybridization sequence and, e.g., any one or more of the moieties described below, to first ends of the fragments, annealing DNA primers to the linkers, and extending the primers with a reverse transcriptase to produce the cDNAs.
  • linkers e.g., linkers that optionally comprise, e.g., a primer hybridization sequence and, e.g., any one or more of the moieties described below, to first ends of the fragments, annealing DNA primers to the linkers, and extending the primers with a reverse transcriptase to produce the cDNAs.
  • the subtractive and sample populations of RNAs can be derived from any of a variety of cell types in any of a variety organisms, e.g., mammals.
  • the sample population of RNAs can optionally be derived from a first cell type, e.g., a non-disease cell, from an organism, and the subtractive population of RNAs can derived from a second cell type, e.g., a disease cell, from the same organism.
  • the sample population can be derived from a particular cell type in an organism and the subtractive population of RNAs can be derived from the same cell type in a second organism of the same species.
  • a sample population of RNAs can optionally be derived from a cell type in an organism at a first developmental stage, and the subtractive population of RNAs can be derived from the same cell type in the same organism at a second developmental stage.
  • the sample population of RNAs can be derived from a first cell type from a first organism, which cell type has been exposed to a first treatment, and the subtractive population of RNAs can be derived from the same cell type that has been exposed to a second treatment.
  • the sample population of RNAs can be derived from a cell, and the subtractive RNAs can be derived from a synthetic source, e.g., a population of oligos comprising a defined set of known splice isoforms. This particular embodiment can be of beneficial use in diagnostic and prognostic assays.
  • Reverse transcribing the subtractive population of RNAs can optionally include annealing tagged DNA primers to 3' ends of the RNAs in the subtractive population and extending the tagged primers with a reverse transcriptase.
  • the tags on the DNA primers can optionally include any of a variety of moieties, e.g., one or more ligand, fluorescent label, blocking group, phosphorylated nucleotide, nucleotide analog, fluorinated nucleotide, nucleotide comprising a heavy atom, biotinylated nucleotide, methylated nucleotide, uracil, sequence capable of forming hairpin secondary structure, oligonucleotide hybridization site, restriction site, DNA promoter, protein binding site, sample or library identification sequence, a thiol linker, a phosphorothioated nucleotide, an amine-reactive nucleotide, and/or cis regulatory sequence.
  • Removing the subpopulation of RNA: cDNA duplexes from the unhybridized RNAs, e.g., to isolate the alternate isoforms(s) of a target gene can optionally include, e.g., electrophoresis or digesting the RNA: cDNA duplexes with RNAseH and a DNAse.
  • the methods can further include annealing tagged DNA primers to 3' ends of the RNAs in the subtractive population, extending the tagged primers with a reverse transcriptase to produce tagged cDNAs.
  • RNA fragments can be hybridized to the population of tagged cDNAs to produce a subpopulation of RNA: tagged cDNA duplexes and a subpopulation of unhybridized RNAs; and the RNA: tagged cDNA duplexes can be separated from the unhybridized RNA fragments via affinity purification.
  • the subtractive and sample populations of RNAs can be reversed.
  • cDNAs which are produced by reverse transcribing sample RNAs, e.g., using any of the methods described above, can be hybridized to RNA fragments produced from a subtractive population of RNAs, e.g., using any of the methods described above.
  • the RNA: cDNA duplexes can then be removed from unhybridized RNA fragments to identify one or more splice isoform, e.g., that is present in the subtractive population of RNAs and absent from the sample population of RNAs.
  • These RNA fragments can be optionally be reverse transcribed and analyzed, as described above. Accordingly, the methods provided by the invention can be used to determine whether there are any additional splice isoforms of a target gene in the subtractive population of RNAs not present in the sample population of RNAs.
  • compositions that include a subpopulation of RNA: cDNA duplexes and a subpopulation of unhybridized RNA fragments that have been produced by the methods described above. It will be apparent to those of skill in the art that the methods and compositions provided by the invention can optionally be used alone or in combination.
  • Kits are also a feature of the invention.
  • the present invention provides kits that include useful reagents, e.g., tagged DNA primers, affinity columns, and/or one or more enzymes that are used in the methods, e.g., a reverse transcriptase, a DNA polymerase, etc.
  • useful reagents e.g., tagged DNA primers, affinity columns, and/or one or more enzymes that are used in the methods, e.g., a reverse transcriptase, a DNA polymerase, etc.
  • Such reagents are most preferably packaged in a fashion to enable their use.
  • kits of the invention optionally include additional reagents, such as a control target nucleic acids, buffer solutions and/or salt solutions, including, e.g., divalent metal ions, i.e., Mg ++ , Mn ++ and/or Fe ++ , nucleic acid adapter tags, e.g., to prepare unhybridized RNA fragments for sequencing, e.g., using a currently available or future automated high-throughput sequencing system.
  • additional reagents such as a control target nucleic acids, buffer solutions and/or salt solutions, including, e.g., divalent metal ions, i.e., Mg ++ , Mn ++ and/or Fe ++ , nucleic acid adapter tags, e.g., to prepare unhybridized RNA fragments for sequencing, e.g., using a currently available or future automated high-throughput sequencing system.
  • kits also typically include a container to hold the kit components, instructions for use of the compositions, e.g., to practice the methods, and other reagents in accordance with the desired application methods, e.g., identifying exon-exon junctions, or other characteristics of alternately spliced mRNA isoforms of a target gene.
  • derived from is used to refer to the original source organism, tissue, cells, etc. from which, e.g., a population of RNAs to be used with the methods of the invention, was obtained.
  • populations of RNAs can be derived from, e.g., a cell line or a eukaryotic organism, including, but not limited to, mammals, nematodes, insects, etc.
  • Linker As used herein, a linker is a short, single-stranded nucleic acid 2-20 nucleotides in length that can be attached to a single stranded nucleic acid, e.g., an RNA, via ligation or by extending the linker, e.g., with a reverse transcriptase.
  • a nucleic acid linker can include any one or more of an oligonucleotide hybridization site, a restriction site, a DNA promoter, a protein binding site, a sample or library identification sequence, a thiol linker, a phosphorothioated nucleotide, an amine-reactive nucleotide, a cis regulatory sequence, modified nucleotide or nucleotide analog, and/or the like.
  • Subtractive cDNAs are the cDNAs that are produced from the reverse transcription of a subtractive population of RNAs.
  • subtractive cDNAs are hybridized to a sample population of RNAs, and, thus, species common to both the subtractive and sample RNA populations form RNA: cDNA duplexes.
  • these RNA: cDNA duplexes are removed from or "subtracted" from the hybridization reaction.
  • Subtractive cDNAs can optionally comprise a tag that includes, e.g., any one or more of the moieties described herein, and such tags can permit the removal/separation of the resulting RNA: cDNA hybrids from unhybridized RNA fragments, e.g., fragments derived from sample RNAs that comprise additional isoform(s) of one or more target gene.
  • Subtractive RNAs As used herein, “subtractive RNAs" (or, alternately, a
  • “subtractive population of RNAs” are a reference population of RNAs that are used to remove mRNA species that common to both a subtractive RNA population and the sample RNA population to which the subtractive population is being compared.
  • Subtractive cDNAs are produced from subtractive RNAs, e.g., via reverse transcription.
  • Subtractive RNAs can be derived from any of a number of sources, e.g., cells, tissues, organisms, etc., and typically represent a control population of RNAs to which, e.g., a sample population of RNAs is compared to, e.g., identify one or more additional splice isoforms of a target gene that are present in the sample population of RNAs and absent from the subtractive population of RNAs.
  • Tags refers to a moiety linked to a molecule of interest that can be used as a molecular label to detect the molecule of interest in population and/or as a by which to separate the molecule of interest from the population.
  • tags can be hybridized to the ends of the nucleic acid fragments and extended with a polymerase to produce tagged fragments or ligated to the ends of the nucleic acid fragments with a ligase.
  • Tags can comprise any one or more moieties that include, e.g., a ligand, a fluorescent label, a blocking group, a phosphorylated nucleotide, a nucleotide analog, a fluorinated nucleotide, a nucleotide comprising a heavy atom, a biotinylated nucleotide, a methylated nucleotide, a uracil, a sequence capable of forming hairpin secondary structure, an oligonucleotide hybridization site, a restriction site, a DNA promoter, a protein binding site, a sample or library identification sequence, a thiol linker, a phosphorothioated nucleotide, an amine-reactive nucleotide, and/or a cis regulatory sequence.
  • a tag can comprise a linker, described above.
  • treatment refers to a defined set of experimental conditions to which, e.g., a source organism, a source tissue, a source cell line, etc. was exposed prior to the collection of its RNA.
  • a treatment can include, e.g., exposing a clonal population of undifferentiated T cells to a defined set of cytokines for a defined length of time; exposing a disease cell line to a drug; etc.
  • Figure 1 provides a schematic depiction of a subtractive population of RNAs and a sample population of RNAs.
  • Figure 2 provides a schematic that depicts how methods of the invention can be used to isolate an isoform of a target gene that is found only in the sample population.
  • Figure 2 also illustrates embodiments of the compositions that are provided by the invention. DETAILED DESCRIPTION OF THE INVENTION
  • compositions provided herein can be used in combination for the reliable identification of splice variants of a target gene, e.g., variants that are present in a sample population of RNAs but absent from a second population of RNAs to which the sample population is being compared.
  • the methods are also useful in determining whether a sample population of RNAs includes one or more unique splice isoforms that are not present in the second population of RNAs.
  • the methods of the invention provide several advantages over currently available technologies, e.g., cDNA library screening, northern blotting, RT-PCR, 5' and 3' RACE, cloning, EST sequencing, and microarray technology.
  • conventional cDNA library screening and Northern blotting are often not sensitive enough to detect low abundance alternate splice isoforms, e.g., isoforms that are present in a population in a single copy or very few copies.
  • Molecular techniques such as RT-PCR, cloning, EST sequencing, and 5' or 3' RACE (Rapid Amplification of cDNA Ends), are laborious, and these methods can become impracticable, and costly, if scaled to the degree necessary to perform genome-wide analyses.
  • microarray technologies permit highly parallel analyses of splice isoforms in RNA populations
  • microarray platforms are expensive. Additionally, the ability to identify differential splice isoforms of a target gene using a microarray depends on whether each exon-exon junction that can be produced by a splicing event is represented by a sufficient number of probes in the array. Furthermore, splice isoforms comprising five or fewer exons can be difficult to detect (Robinson et al. (2009) "Differential splicing using whole-transcript microarrays.” BMC Bioinformatics 10: 156).
  • the methods provided herein are cost-effective, highly parallel, and can be used to efficiently identify alternative splice isoforms present in an RNA sample, e.g., regardless of their copy number or the number of exons they comprise.
  • the detailed description is organized to first elaborate the methods and compositions provided by the invention for isolating, e.g., unique or previously uncharacterized RNA splice isoforms of one or more target genes of interest that are present in a sample population of RNAs and absent from a subtractive population of RNAs. Next, details regarding alternative splicing are described. Details regarding sequencing reactions and high-throughput sequencing systems are then provided. Kits, systems, and broadly applicable molecular biological techniques that can be used to perform any of the methods are described thereafter.
  • the methods provided herein can be used to determine whether one population of RNAs includes one or more different RNA splice isoforms that are not present in a second population. Accordingly, the methods can be performed to determine whether the first population of RNAs includes a particular RNA splice isoform, or any additional RNA splice isoforms, not present in a second population of RNAs.
  • two populations of RNA are provided: subtractive population 100 and sample population 125 (see Figure 1).
  • Subtractive RNA population 100 which comprises splice isoform 101 of a target gene, is reverse transcribed to produce subtractive population of cDNAs 115.
  • the cDNAs in population 115 are synthesized by annealing tagged primers to subtractive RNAs 100 and extending the primers, e.g., with a reverse transcriptase, such that each cDNA 110 in population 115 comprises tag 105.
  • Sample RNA population 125 which comprises splice isoforms 101 and 102 of the target gene, is fragmented, e.g., using any one or more of the methods described herein, to produce population of RNA fragments 130.
  • RNA populations 100 and 125 can be derived from any of a variety of sources, e.g., cells, tissues, organs, etc, in which differences in AS patterns may be of interest to the practitioner. For example, where differences in splicing patterns between cell types are to be examined, subtractive RNA population 100 can be derived from a first cell type in an organism, and sample RNA population 125 can be derived from a second cell type in the same organism. Alternately, subtractive RNA population 100 can be derived from a first cell type in a first organism, and sample RNA population 125 can be derived from a first cell type in a second organism of the same species as the first organism.
  • RNA populations 100 and 125 can be obtained from the same tissue at different developmental stages or, e.g., from a disease cell and a non-disease cell.
  • subtractive RNA population 100 can be derived from a cell
  • sample RNA population 125 can be derived from a synthetic source, e.g., a population of oligos comprising a defined set of known splice isoforms, e.g., isoforms 101 and 102.
  • RNA fragments 130 can be produced from RNA population 100, and cDNAs 115 can be derived from RNA population 125.
  • composition 200 which is provided by the invention, includes a subpopulation of RNA: cDNA duplexes 210, which subpopulation comprises cDNAs from population 115 and the complementary RNA fragments from population 130 to which they hybridize. Also included in composition 200 are unhybridized RNA fragments 215, e.g., RNA fragments from population 130 for which there are no complementary cDNA sequences in population 115.
  • an RNA: cDNA duplex 210 can comprise cDNA 250 which includes, e.g., tag 105, and encodes the reverse transcript of splice isoform 101, and RNA fragments 245, which comprise subsequences of splice isoforms 101 and 102 that can hybridize to cDNA 250.
  • RNA fragments 215 can comprise subsequences 240 of splice isoform 102, e.g., RNA subsequences that do not hybridize to any cDNA sequences in population 115.
  • invention also provides methods of determining whether a particular splice isoform, e.g., isoform 101 and/or 102, are present in a population of RNAs, e.g., population 100.
  • RNA: cDNA duplexes 210 are then removed from population 200, e.g., via enzymatic digestion, electrophoresis, affinity purification, or the like (see Figure 2B).
  • DNAse and RNAse H can be added to population 200 to digest the cDNAs and the RNA fragments hybridized to the cDNAs, respectively, in duplexes 210.
  • population 200 can be run over an affinity column that comprises a moiety that binds tag 105. Only unhybridized RNA fragments 215 will be eluted from the column.
  • the RNA: cDNA duplexes (e.g., duplexes 210) which are typically a higher molecular weight than the unhybridized RNA fragments, e.g., fragments 215, can be separated from the unhybridized RNA fragments via electrophoresis.
  • the methods facilitate the removal of mRNA species common to both subtractive RNA population 100 and sample RNA population 125, while mRNA fragments 215, e.g., cell type-specific splice isoforms, tissue-specific splice isoforms, disease-specific splice isoforms, and/or the like, that are unique to sample population 125, remain and can be further characterized.
  • Such analyses can include, e.g., ligating linkers to the RNA fragments in preparation for reverse transcription. The reverse transcribed fragments can then be sequenced, optionally using any of a variety of high- throughput sequencing platforms described below.
  • the methods of the invention can also used to determine whether the sample population of RNAs comprises any splice isoforms of a target gene that are not present in the subtractive population, and/or vice versa.
  • AS pre-mRNA splicing
  • AS is a precisely regulated process in which a pre-mRNA's exons are separated and reconnected in different combinations to produce alternative mature mRNA species that encode multiple protein isoforms.
  • alternative splicing can alter the function of proteins by removing or adding specific domains, e.g., nuclear localization signals, transcription activation domains, DNA or RNA binding domains, trans-membrane domains, phosphorylation sites, and/or post- translation modification sites.
  • alternative splicing can cause substantial changes in protein structure by altering even just a few residues at a splice site (Davletov, et al.
  • AS can also generate variability within untranslated regions of mRNAs which affect gene expression by adding or removing mRNA elements that, e.g., regulate translation efficiency, mRNA stability, or intracellular localization.
  • AS has been observed in nearly all multicellular organisms.
  • bioinformatic analyses based on EST sequences and exon-exon junction microarray studies estimate that 59%-74% of human genes are alternatively spliced (Johnson, et al. (2003) "Genome-wide array of human alternative pre-mRNA splicing with exon junction microarrays.” Science 302: 2142-2144; Kan, et al. (2001) "Gene structure prediction and alternative splicing analysis using genomically aligned ESTs.” Genome Res 11: 889-900), indicating that AS is one major source of protein diversity in humans.
  • the methods and compositions provided by the invention can permit the identification of splice isoforms that would be otherwise difficult to isolate using currently available methods that entail, e.g., designing probes specific to all possible exon-exon junctions, having a priori knowledge of coding regions in a pre-mRNA, knowing the complete sequence of, e.g., a large mammalian genome, etc.
  • AS events can undergo regulation in which splicing pathways are modulated according to, e.g., cell type (see, e.g., Cooper, T. A. (2005) "Alternative splicing regulation impacts heart development.” Cell 120: 59-72), developmental stage (see, e.g., Barberan- Soler, et al. (2008) "Alternative Splicing Regulation During C. elegans Development: Splicing Factors as Regulated Targets.” PIoS 4: elOOOOOl), gender (see, e.g., Chang, et al.
  • spliceosome a large complex comprising over 100 core proteins and 5 small nuclear RNAs (snRNAs) (described in, e.g., Smith, et al.
  • RNA splicing is dependent upon the identity of nucleotide sequences, or "core splicing signals" at the 5' splice site, the 3' splice site, and the branch point.
  • ESEs exonic splice enhancers
  • ESSs exonic splice silencers
  • ISEs intronic splice enhancers
  • ISSs intronic splice silencers
  • the methods and compositions of the invention can be beneficially used to determine which splice isoforms are present in, e.g., a cell or tissue, e.g., at specific developmental stages or in response to particular environmental stimuli, and such data can inform drug discovery and diagnostics efforts.
  • protein-rich target genes of interest whose splice variants can be further characterized using the invention include, e.g., cadherins, which play roles in cell adhesion.
  • Cadherins are involved in morphogenesis of tissues such as the neural tube, and their misexpression has been implicated in human malignancies (Wheelock et al.
  • AS and its disruption can also influence the susceptibility of an individual to a disease and/or the disease's severity (Wang, et al. (2007) "Splicing in disease: disruption of the splicing code and the decoding machinery.” Nat Rev Genet 8: 749-761; Srebow, et al. (2006) “The connection between splicing and cancer.” Journ Cell Sci 119: 2635-2641; Faustino, et al. (2003) "Pre-mRNA splicing and human disease.” Genes Dev 17: 419-437).
  • splicing defects have been identified as the cause of numerous diseases including ⁇ -thalassemia, cystic fibrosis, and premature aging (Faustino, et al. (2003) "Pre-mRNA splicing and human disease.” Genes Dev 17: 419-437). Determining how the splicing of, e.g., a target gene, is altered in, e.g., a disease cell, can inform strategies directed at reversing or circumventing misregulated splicing events.
  • the methods provided herein can be used to detect whether alternate RNA splice isoforms are present in a population of RNAs derived from a patient, e.g., as compared to a subtractive population of RNAs, to make a diagnosis, predict a prognosis, or inform a drug regimen.
  • RNAs derived from a patient e.g., as compared to a subtractive population of RNAs
  • Further details regarding spliceosome proteins and splicing mechanism can be found in, e.g., Jurica (2008) "Detailed close-ups and the big picture of spliceosomes.” Curr Opin Struct Biol 18: 315-20; Schellenberg, et al.
  • RNA fragments that are enriched following the removal of cDNA:RNA hybrids can comprise additional splice isoforms of a target gene, e.g., that are present in a sample population of RNAs and absent from the subtractive population of RNAs or vice versa (see Figure 2 and corresponding description).
  • RNA fragments can optionally be reverse transcribed, according to methods described elsewhere herein, and sequenced using, e.g., any of a variety of high-throughput DNA sequencing systems (reviewed in, e.g., Chan, et al.
  • Affymetrix and Complete Genomics, Inc. rely on indirect methods of determining a DNA's sequence, e.g., sequencing by hybridization (SBH), in which a sequence of a DNA is assembled based on experimental data obtained from hybridization experiments performed to determine the oligonucleotide content of the DNA chain.
  • SBH sequencing by hybridization
  • SBH typically employs an array comprising a known arrangement of short oligonucleotides of known sequence, e.g., oligonucleotides representing all possible sequences of a given length.
  • SoLID a commercial sequencing system available from Applied Science
  • Biosystems is based on "sequencing by ligation" (SBL), in which the mismatch sensitivity of a DNA ligase enzyme is used to determine the underlying sequence of the target nucleic acid molecule.
  • SBL sequencing by ligation
  • one or more sets of encoded adaptors is ligated to the terminus of a target polynucleotide, e.g., a single-stranded DNA of unknown sequence.
  • Encoded adaptors whose protruding strands form perfectly matched duplexes with the complementary protruding strands of the target polynucleotide are ligated, and the identity of the nucleotides in the protruding strands is determined by an oligonucleotide tag carried by the encoded adaptor. Such determination, or "decoding” is carried out by specifically hybridizing a labeled tag complementary to its corresponding tag on the ligated adaptor.
  • SBS sequencing by synthesis
  • 454 Sequencing a technology available from 454 Life Sciences, is a massively-parallellized, multiplex pyrosequencing system (Nyren (2007) "The History of Pyrosequencing.” Methods MoI Biol 373: 1-14; Ronaghi (2001) "Pyrosequencing sheds light on DNA sequencing.” Genome Res 11: 3-11; and Wheeler, et al. (2008) "The complete genome of an individual by massively parallel DNA sequencing.” Nature 452: 872-876) that relies on fixing nebulized, adapter-ligated single-stranded DNA fragments to small DNA- capture beads.
  • Single molecule real-time sequencing is another massively parallel sequencing technology that can be compatible with the high-throughput resequencing of target nucleic acids isolated isolated from a sample, e.g., by using capture probes synthesized according to any of the methods described previously.
  • SMRT technology relies on arrays of multiplexed zero-mode waveguides (ZMWs) in which, e.g., thousands of sequencing reactions can take place simultaneously.
  • ZMWs multiplexed zero-mode waveguides
  • the ZMW is a structure that creates an illuminated observation volume that is small enough to observe, e.g., the template-dependent synthesis of a single single-stranded DNA molecule by a single DNA polymerase (See, e.g., Levene, et al. (2003) "Zero Mode Waveguides for Single Molecule Analysis at High Concentrations," Science 299: 682-686).
  • cDNAs derived from unhybridized RNA fragments can be sequenced using systems that include bridge amplification technologies, e.g., in which primers bound to a solid phase are used in the extension and amplification of solution phase target nucleic acid acids prior to SBS.
  • bridge amplification technologies e.g., in which primers bound to a solid phase are used in the extension and amplification of solution phase target nucleic acid acids prior to SBS.
  • RNA fragments e.g., derived from a sample population of RNAs, that encode one or more splice isoform of one or more target gene, e.g., that is present in a sample population of RNAs but not in a subtractive population of RNAs (see Figure 1 and corresponding description).
  • the principle of this approach relies on the removal of mRNA species common to both the subtractive and sample RNA populations, leaving behind RNA fragments that comprise splice isoform(s) unique to the sample population of RNAs.
  • Such splice isoforms can optionally include, e.g., cell type-specific splice isoforms, tissue- specific splice isoforms, disease-specific splice isoforms, etc.
  • the alternative splice isoforms are thus isolated from the sample RNA population and can be subject to further analysis, e.g., sequencing using an automated high-throughput sequencing system.
  • the methods of the invention can also optionally be used to identify mRNA species that are present in the sample population of RNAs in higher abundance relative to the subtractive population of RNAs.
  • the subtractive and sample populations of RNAs can be reversed.
  • cDNAs that are produced by reverse transcribing a sample population of RNAs can be hybridized to RNA fragments derived from a subtractive population of RNAs.
  • the RNA: cDNA duplexes can then be removed from unhybridized RNA fragments, e.g., fragments derived from subtractive RNAs, to identify one or more splice isoform that is both present in the subtractive population of RNAs and absent from the sample population of RNAs.
  • These RNA fragments can be optionally be reverse transcribed and further characterized, as described above.
  • Subtractive cDNAs e.g., derived from reverse transcription of the subtractive RNAs
  • RNA fragments e.g., produced from a sample population of RNAs
  • the results of subtractive hybridization are validated using additional techniques that are well known in the art, e.g., northern blot, in situ hybridization, RT-PCR, and the like. These techniques are described in detail in, e.g., e.g., Sambrook et al., Molecular Cloning - A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 (“Sambrook”); and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2007) ("Ausubel”).
  • RNAs and cDNAs [0052] The methods described herein include providing two distinct populations of
  • RNAs e.g., a subtractive population of RNAs and a sample population of RNAs.
  • the subtractive RNAs used to generate subtractive cDNAs, and the sample RNAs are fragmented and hybridized to the subtractive cDNAs.
  • Hybridization produces a subpopulation of unhybridized RNA fragments and a subpopulation of RNA: cDNA duplexes, which are then removed from the unhybridized RNA fragments.
  • Determining the sequences of the unhybridized RNA fragments can be useful, e.g., in identifying one or more splice variants of one or more target gene or in comparing the differential expression of, e.g., splice isoforms of a target gene, e.g., between different tissue types, between different treatments to the same tissue type, or between different developmental stages of the same tissue type.
  • mRNA can typically be isolated from almost any source using protocols and methods described in, e.g., Sambrook and Ausubel.
  • the yield and quality of the isolated mRNA can depend on how a tissue is stored prior to RNA extraction, the means by which the tissue is disrupted during RNA extraction, or on the type of tissue from which the RNA is extracted, and RNA isolation protocols can be optimized accordingly.
  • mRNA isolation kits are commercially available, e.g., the mRNA-ONLYTM Prokaryotic mRNA Isolation Kit and the mRNA-ONLYTM Eukaryotic mRNA Isolation Kit (Epicentre Biotechnologies), the FastTrack 2.0 mRNA Isolation Kit (Invitrogen), and the Easy-mRNA Kit (BioChain).
  • mRNA from various sources e.g., bovine, mouse, and human
  • tissues e.g. brain, blood, and heart
  • BioChain Hayward, CA
  • Ambion Austin, TX
  • Clontech Mesountainview, CA
  • reverse transcriptase is used to generate cDNAs from the mRNA templates.
  • Methods and protocols for the production of cDNA from mRNAs e.g., harvested from prokaryotes as well as eukaryotes, are elaborated in cDNA Library Protocols, I. G. Cowell, et al., eds., Humana Press, New Jersey, 1997, Sambrook, and Ausubel.
  • kits are commercially available for the preparation of cDNA, including the Cells-to-cDNATM II Kit (Ambion), the RETROscriptTM Kit (Ambion), the CloneMinerTM cDNA Library Construction Kit (Invitrogen), and the Universal RiboClone ® cDNA Synthesis System (Promega).
  • Many companies e.g., Agencourt Bioscience and Clontech, offer cDNA synthesis services.
  • RNA fragments are generated from a sample population of RNAs, i.e., in preparation for hybridization to subtractive cDNAs derived from the subtractive population of RNAs.
  • RNA fragments There exist a plethora of ways of producing such RNA fragments. These include, but are not limited to, mechanical methods, such as sonication, mechanical shearing, nebulization, hydroshearing, and the like; enzymatic methods, such as exonuclease digestion, endonuclease digestion, and the like; chemical cleavage, and electrochemical cleavage. These methods are further explicated in Sambrook and Ausubel. In preferred embodiments, chemical cleavage is used to fragment RNAs, as detailed in the example below.
  • tagged subtractive cDNAs are produced from the subtractive population of RNAs via reverse transcription.
  • the tags can permit the detection of RNA: cDNA duplexes, e.g., in a population of nucleic acids that comprises a subpopulation of RNA: cDNA duplexes and a subpopulation of unhybridized RNA fragments, e.g., following hybridization of subtractive cDNAs to a population of RNA fragments derived from a sample population of RNAs.
  • the tags permit the RNA: cDNA duplexes to be separated, e.g., via affinity purification or the like, from the subpopulation of unhybridized RNA fragments, e.g., RNA fragments that represent splice isoforms of one or more target gene that are present in a sample population of RNAs but not present in the subtractive population of RNAs.
  • Nucleic acid tags e.g., such as those optionally present on the subtractive cDNAs, can comprise any of a plethora of ligands, such as high-affinity DNA-binding proteins; modified nucleotides, such as methylated, biotinylated, or fluorinated nucleotides; and nucleotide analogs, such as dye-labeled nucleotides, non-hydrolysable nucleotides, or nucleotides comprising heavy atoms.
  • ligands such as high-affinity DNA-binding proteins
  • modified nucleotides such as methylated, biotinylated, or fluorinated nucleotides
  • nucleotide analogs such as dye-labeled nucleotides, non-hydrolysable nucleotides, or nucleotides comprising heavy atoms.
  • tags can optionally comprise one or more fluorescent label, blocking group, phosphorylated nucleotide, thiol linker, phosphothiorated nucleotide, amine-reactive nucleotide, uracil, and/or the like.
  • fluorescent label for example, a fluorescent label, blocking group, phosphorylated nucleotide, thiol linker, phosphothiorated nucleotide, amine-reactive nucleotide, uracil, and/or the like.
  • reagents are widely available from a variety of vendors, including Perkin Elmer, Jena Bioscience and Sigma-Aldrich.
  • Nucleic acid tags can also include oligonucleotides that comprise specific sequences, such as restriction sites, cis regulatory sites, nucleotide hybridization sites, protein binding sites, sequences capable of forming hairpin secondary structures, DNA promoters, sample or library identification sequences, and the like. Such sequences can be of advantageous use in, e.g., sequencing cDNAs derived from unhybridized sample RNA fragments that have been reverse transcribed using tagged primers. Linkers that are attached to unhybridized RNA fragments in preparation for reverse transcription can also beneficially include any one or more of the sequences listed above.
  • Oligonucleotide tags can be custom synthesized by commercial suppliers such as Operon (Huntsville, AL), IDT (Coralville, IA) and Bioneer (Alameda, CA). Any of a number of methods that are well known in the art can be used to join tags to nucleic acids of interest, include chemical linkage, ligation, and extension of a primer comprising a tag by a polymerase or reverse transcriptase. Further details regarding nucleic acid tags and the methods by which they are attached to nucleic acids of interest are elaborated in Sambrook and Ausubel.
  • RNA hybrids comprise additional mRNA splice isoforms and not, e.g., RNA species that are expressed at higher levels in the sample population.
  • a variety of nucleic acid amplification and/or copying methods are known in the art and can be implemented to, e.g., amplify subtractive cDNAs and/or cDNAs derived from the reverse transcription of unhybridized RNA fragments, e.g., RNA fragments from a sample population of RNAs.
  • the most widely used in vitro technique among these methods is polymerase chain reaction (PCR), which requires the addition of nucleotides, oligonucleotide primers, buffer, and an appropriate polymerase to the amplification reaction mix.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • RCA rolling-circle amplification
  • MDA multiple- displacement amplification
  • Kits are also a feature of the invention.
  • the present invention provides kits that include useful reagents, e.g., tagged DNA primers, affinity columns, and/or one or more enzymes that are used in the methods, e.g., a reverse transcriptase, a DNA polymerase, etc.
  • useful reagents e.g., tagged DNA primers, affinity columns, and/or one or more enzymes that are used in the methods, e.g., a reverse transcriptase, a DNA polymerase, etc.
  • Such reagents are most preferably packaged in a fashion to enable their use.
  • kits of the invention optionally include additional reagents, such as a control target nucleic acids, buffer solutions and/or salt solutions, including, e.g., divalent metal ions, i.e., Mg + *, Mn ++ and/or Fe ++ , nucleic acid adapter tags, e.g., to prepare unhybridized RNA fragments for sequencing, e.g., using a currently available or future automated high-throughput sequencing system.
  • additional reagents such as a control target nucleic acids, buffer solutions and/or salt solutions, including, e.g., divalent metal ions, i.e., Mg + *, Mn ++ and/or Fe ++ , nucleic acid adapter tags, e.g., to prepare unhybridized RNA fragments for sequencing, e.g., using a currently available or future automated high-throughput sequencing system.
  • kits also typically include a container to hold the kit components, instructions for use of the compositions, e.g., to practice the methods, and other reagents in accordance with the desired application methods, e.g., identifying exon-exon junctions, or other characteristics of alternately spliced mRNA isoforms of a target gene.
  • the methods and compositions provided by the invention can advantageously be integrated with systems that can, e.g., automate and/or multiplex the steps of the methods described herein, e.g., methods for separating one or more alternate isoform of a target gene from a sample population of RNAs.
  • Systems of the invention can include one or more modules, e.g., that automate a method herein, e.g., for high-throughput sequencing applications.
  • Such systems can include fluid-handling elements and controllers that move reaction components into contacts with one another, signal detectors, and system software/instructions.
  • Systems of the invention can optionally include modules that provide for detection or tracking of products, e.g., unhybridized RNAs that comprise sequences that correspond to a splice isoform of a target gene that is present in a sample population of RNAs but not in a subtractive population of RNAs. Additionally or alternatively, the systems can monitor the synthesis of cDNAs from such unhybridized RNAs and/or detect the nucleotide sequence of such cDNAs, e.g., produced during a sequencing reaction. Detectors can include spectrophotometers, epifluorescent detectors, CCD arrays, CMOS arrays, microscopes, cameras, or the like.
  • Optical labeling is particularly useful because of the sensitivity and ease of detection of these labels, as well as their relative handling safety, and the ease of integration with available detection systems (e.g., using microscopes, cameras, photomultipliers, CCD arrays, CMOS arrays and/or combinations thereof).
  • High- throughput analysis systems using optical labels include DNA sequencers, array readout systems, cell analysis and sorting systems, and the like.
  • fluorescent products and technologies see, e.g., Sullivan (ed) (2007) Fluorescent Proteins, Volume 85, Second Edition (Methods in Cell Biology) (Methods in Cell Biology) ISBN-10: 0123725585; Hof et al.
  • System software e.g., instructions running on a computer can be used to track and inventory reactants or products, and/or for controlling robotics/ fluid handlers to achieve transfer between system stations/modules.
  • the overall system can optionally be integrated into a single apparatus, or can consist of multiple apparatus with overall system software/instructions providing an operable linkage between modules.
  • RNAs are prepared from cells and enriched for polyadenylated mRNAs using methods well known to one of skill in the art.
  • EDTA is then added to a final concentration of 6OmM to quench the reactions, and the fragmented mRNAs are run on a 15% acrylamide/7M urea gel.
  • a fragment of the gel that corresponds to the position on the gel at which 25-50 base pair-long fragments migrate is excised, and the gel slice is placed in an 0.5ml microfuge tube that has had holes punched through the bottom with a 21 gauge syringe needle.
  • the 0.5 ml tube is placed in 1.5ml microfuge tube and spun briefly until pieces of the gel slice are pushed into the 1.5 ml tube through the holes in the 0.5 ml tube.
  • RNAse free tubes Following the incubation, the 1.5 ml microfuge tube is spun for 15 minutes at 7500, and the supernatant is transferred to a new tube. The supernatant is filtered through a Spin-X centrifuge filter (available from Corning) to remove any gel pieces that may have been transferred. After all gel pieces have been removed, the RNA fragments are ethanol precipitated and pelletted, a technique well known by those of skill in the art. The preceding steps are performed in RNAse free tubes with RNAse-free reagents.
  • Subtractive RNAs are prepared by harvesting total RNA from cells and purifying polyadenylated mRNAs from the total RNA using any of a variety of methods known to those of skill in the art. The polyadenylated RNAs are then reverse transcribed into cDNA with biotin-conjugated oligo dT primers. The resulting RNAxDNA duplexes are treated with RNAse H to hydrolyze the RNA strands of the duplexes. It is assumed that cDNAs are produced from the RNAs at a 1:1 ratio.
  • RNA cDNA duplexes are purified from the population of unhybridized
  • RNA with excess Streptavidin beads (1 mg beads can hold lOOpmol of Biotin). Beads are added to the hybridization mix and incubated at 4°C for 5 hours. The tube containing the beads and hybridization mix is spun, pelletting the subtractive biotin-conjugated cDNAs and the RNAs to which they have hybridized. The supernatant, which contains the unhybridized RNAs, e.g., RNAs that are unique to the sample population, is transferred to a new 1.5 ml microfuge tube, and the RNAs are ethanol precipitated and pelletted, according to methods well known in the art.
  • RNA fragments are then resolved on a 15% acrylomide/7M urea gel, and fragment of the gel that corresponds to the position on the gel at which 25-50 base pair-long fragments migrate is excised.
  • the RNA fragments are eluted from the gel slice, as described above.
  • RNA fragments for sequencing [0070]
  • Solexa adaptors are ligated to the RNA fragments, which are then gel purified as described above. 5' linkers are ligated to the purified fragment, and the ligation products are subject to a second round of gel purification.
  • the fragments to which the Solexa adaptors and 5' linkers have been attached treated with DNAs and extracted with phenol/chloroform.
  • the adaptor-ligated fragments are then precipitated with 1:1 ethanol: isopropanol and pelletted. The pellet is resuspended, and a reverse transcriptase reaction is performed with Solexa 3' primers to produce cDNAs from the RNAs.
  • the cDNAs are amplified via PCR, and the PCR products run on a gel. DNA fragments between 60 and 100 base pairs in size are extracted from the gel.

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Abstract

La présente invention concerne des procédés de séparation d'une ou plusieurs isoformes alternées d'un gène cible, laquelle isoforme alternée est présente dans un échantillon de population d'ARN mais pas dans une population d'ARN soustractive. La présente invention concerne également des compositions comprenant une sous-population de duplex ARNxADN et une sous-population de fragments d'ARN non hybridés.
PCT/US2009/006450 2008-12-09 2009-12-08 Procédés d'identification de différences d'épissage alternatif entre deux échantillons d'arn Ceased WO2010077288A2 (fr)

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US20150337364A1 (en) * 2014-01-27 2015-11-26 ArcherDX, Inc. Isothermal Methods and Related Compositions for Preparing Nucleic Acids
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JP2004187606A (ja) * 2002-12-12 2004-07-08 Institute Of Physical & Chemical Research 核酸アイソフォームの同定、分析および／またはクローニング方法
US7919238B2 (en) * 2003-08-08 2011-04-05 Albert J. Wong Method for rapid identification of alternative splicing
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US11390905B2 (en) 2016-09-15 2022-07-19 Archerdx, Llc Methods of nucleic acid sample preparation for analysis of DNA
US11795492B2 (en) 2016-09-15 2023-10-24 ArcherDX, LLC. Methods of nucleic acid sample preparation

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