WO2009130480A1 - Analyse d’acides nucléiques - Google Patents

Analyse d’acides nucléiques Download PDF

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Publication number
WO2009130480A1
WO2009130480A1 PCT/GB2009/001057 GB2009001057W WO2009130480A1 WO 2009130480 A1 WO2009130480 A1 WO 2009130480A1 GB 2009001057 W GB2009001057 W GB 2009001057W WO 2009130480 A1 WO2009130480 A1 WO 2009130480A1
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Prior art keywords
nucleic acid
probe
sequence
identity
sample nucleic
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PCT/GB2009/001057
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English (en)
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Graham John Speight
Edwin Mellor Southern
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Oxford Gene Technology Ip Limited
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Publication of WO2009130480A1 publication Critical patent/WO2009130480A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • C12Q1/6855—Ligating adaptors

Definitions

  • This invention is in the field of nucleic acid analysis, in particular analysis of biological samples comprising nucleic acids.
  • nucleic acids is important in many areas of technology, including biological research. Nucleic acid analysis can be difficult, because sometimes only a few copies of each different nucleic acid are present in a sample, so very sensitive analysis methods are required. Analysis of short nucleic acids (e.g. nucleic acids less than 100 nucleotides in length) can be particularly difficult, because the majority of the common nucleic acid analysis methods are designed and optimised for analysis of long nucleic acids (e.g. nucleic acids 100 nucleotides or longer). Analysis of nucleic acids 50 nucleotides or less in length can be especially difficult.
  • the invention provides processes, devices and related products for analysing nucleic acids, including short nucleic acids.
  • the methods of the invention use a hybridisation step to facilitate discrimination of nucleic acids of interest from other nucleic acids.
  • the methods of the invention use an extension tag for analysis of nucleic acids of interest. This combination of hybridisation and analysis using an extension tag facilitates analysis of nucleic acids, and is particularly advantageous for analysis of short nucleic acids. It enables nucleic acids, particularly short nucleic acids, to be readily detected and manipulated.
  • the invention provides various processes for analysing nucleic acids.
  • the invention provides a process for analysing a nucleic acid, comprising the steps: a) providing a sample nucleic acid; b) extending the 5' and/or 3' end of the sample nucleic acid, to generate an extended nucleic acid having an extension tag at its 5' and/or 3' end; c) providing a probe comprising a probe sequence; d) contacting the extended nucleic acid with the probe, under conditions that allow the extended nucleic acid to hybridise to the probe if the sample nucleic acid comprises a sequence complementary to the probe sequence; and e) using the extension tag to analyse the sample nucleic acid if the extended nucleic acid hybridises to the probe.
  • step b) comprises extending the 5' end of the sample nucleic acid, to generate a 5' extended nucleic acid.
  • step b) comprises extending the 3' end of the sample nucleic acid, to generate a 3' extended nucleic acid. This general approach is illustrated schematically in Fig. 1 B.
  • step b) comprises extending both the 5' and 3' ends of a sample nucleic acid, to generate an extended nucleic acid having extension tags at both its 5' and 3' ends.
  • This general approach is illustrated in Fig. 9. Methods in which multiple extension tags are used are thought to be advantageous, as described elsewhere herein.
  • the invention provides a process for analysing a nucleic acid, comprising the steps: a) providing a sample nucleic acid; b) providing a probe comprising a probe sequence, the probe further comprising an extension tag at its 5' and/or 3' end; c) contacting the sample nucleic acid with the probe, under conditions that allow the sample nucleic acid to hybridise to the probe if the sample nucleic acid comprises a sequence complementary to the probe sequence; and d) using the extension tag to analyse the sample nucleic acid if the sample nucleic acid hybridises to the probe.
  • the probe comprises an extension tag at its 5' end. This general approach is illustrated schematically in Fig. 2A. In other methods, the probe comprises an extension tag at its 3' end. This general approach is illustrated schematically in Fig. 2B.
  • the probe comprises extension tags at both its 5' and 3' ends. This general approach is illustrated schematically in Fig. 10. Methods in which multiple extension tags are used are thought to be advantageous, as described elsewhere herein.
  • the invention also provides processes wherein both the sample nucleic acid and the probe have extension tags at their 5' and/or 3' ends. Accordingly, the invention provides a process for analysing a nucleic acid, comprising the steps: a) providing a sample nucleic acid; b) extending the 5' end of the sample nucleic acid, to generate an extended nucleic acid having a first extension tag at its 5' end; c) providing a probe comprising a probe sequence, the probe further comprising a second extension tag at its 5' end; d) contacting the extended nucleic acid with the probe, under conditions that allow the extended nucleic acid to hybridise to the probe if the sample nucleic acid comprises a sequence complementary to the probe sequence; and e) using the first and/or second extension tags to analyse the sample nucleic acid if the extended nucleic acid hybridises to the probe.
  • the invention also provides a process for analysing a nucleic acid, comprising the steps: a) providing a sample nucleic acid; b) extending the 3' end of the sample nucleic acid, to generate an extended nucleic acid having a first extension tag at its 3' end; c) providing a probe comprising a probe sequence, the probe further comprising a second extension tag at its 3' end; d) contacting the extended nucleic acid with the probe, under conditions that allow the extended nucleic acid to hybridise to the probe if the sample nucleic acid comprises a sequence complementary to the probe sequence; and e) using the first and/or second extension tags to analyse the sample nucleic acid if the extended nucleic acid hybridises to the probe.
  • the sample nucleic acid or extended nucleic acid may be immobilised on a solid support via its 5' or 3' end, or via an internal nucleotide, as described elsewhere herein.
  • the probe may be immobilised on a solid support via its 5' or 3' end, or via an internal nucleotide, as described elsewhere herein.
  • the order in which some steps of the methods of the invention are performed is not critical, provided that a sample nucleic acid or an extended nucleic acid and a probe are used in the hybridisation and analysis steps.
  • the step of providing a sample nucleic acid may be performed before or after the step of providing a probe.
  • the step of extending a sample nucleic acid can be performed before or after the step of providing a probe.
  • the step of extending a sample nucleic acid (if part of the method) may be performed before or after the hybridisation step.
  • the analysis step may be performed before or after the hybridisation step, but will normally be performed after the hybridisation step, to facilitate discrimination of nucleic acids of interest from other nucleic acids.
  • the invention also provides devices and kits useful in the methods of the invention.
  • the invention provides a device for analysing a nucleic acid, the device comprising a support to which a probe comprising a probe sequence is immobilised, and having an extended nucleic acid hybridised to the probe, wherein the extended nucleic acid comprises a sequence complementary to the probe sequence, and further comprises an extension tag at its 5' and/or 3' end.
  • a device for analysing a nucleic acid comprising a support to which a probe comprising a probe sequence is immobilised, the probe further comprising an extension tag at its 5' and/or 3' end, and having a sample nucleic acid hybridised to the probe.
  • a device for analysing a nucleic acid comprising a support to which a sample nucleic acid comprising a sequence complementary to a probe sequence is immobilised, and having a probe hybridised to the sample nucleic acid, wherein the probe comprises the probe sequence and further comprises an extension tag at its 5' and/or 3' end.
  • a device for analysing a nucleic acid comprising a support to which an extended nucleic acid is immobilised, the extended nucleic acid comprising a sequence complementary to a probe sequence and further comprising an extension tag at its 5' and/or 3' end, and having a probe comprising the probe sequence hybridised to the extended nucleic acid.
  • a kit for use in the methods of the invention may comprise:
  • kits for use in the methods of the invention may comprise:
  • a support to which a probe is immobilised, the probe comprising a probe sequence and further comprising an extension tag at its 5' and/or 3' end;
  • a reagent for using the extension tag to analyse the sample nucleic acid if the sample nucleic acid hybridises to the probe The dimensions and parameters of the various features of the devices and kits of the invention can vary according to particular needs and applications. Likewise, the precise steps of the methods of the invention can vary according to particular needs and applications. Different analyses can require different devices or processes within the scope of the invention. For instance, different sample types may require devices with different dimensions, or may require different sample preparation steps or different extension tag analysis methods. Moreover, devices can be designed and used based on previous experimental data. For example, if a device fails to give useful data in an initial experiment, variables such as the type of probe, type of extension tag, temperature of operation, buffers, timings etc. can be altered in further experiments.
  • a sample nucleic acid analysed in the methods of the invention will generally be a nucleic acid 10-3000 nucleotides in length, but may be longer.
  • a sample nucleic acid may, for example, be 10-2500, 10-2000, 10-1500, 10-1000, 10-750, 10-500, 10-400, 10-300, 10- 250, 10-200, 10-150, 10-100 or 10-75 nucleotides in length.
  • a sample nucleic acid analysed in the methods of the invention is a short nucleic acid.
  • a sample nucleic acid may be 10-50 nucleotides in length.
  • a sample nucleic acid may be 10-40, 10-30, 10-25, 10- 20, 10-15, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30 or 20-25 nucleotides in length.
  • the sample nucleic acid will be an RNA. In other embodiments, the sample nucleic acid will be a DNA.
  • the sample nucleic acid may comprise a coding sequence (i.e. a nucleic sequence that is normally translated into an amino acid sequence in vivo) or a non-coding sequence.
  • the sample nucleic acid may be a fragment of genomic DNA.
  • the sample nucleic acid may be a messenger RNA (mRNA), or a fragment of an mRNA.
  • mRNA messenger RNA
  • the sample nucleic acid may be a non-coding RNA (ncRNA).
  • ncRNA non-coding RNA
  • Types of ncRNA that can be analysed in the methods of the invention include but are not limited to microRNAs (miRNAs), ribosomal RNAs (rRNAs), small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), small Cajal Body specific RNAs (scaRNAs) and transfer RNAs (tRNAs).
  • miRNAs microRNAs
  • rRNAs ribosomal RNAs
  • siRNAs small interfering RNAs
  • snRNAs small nuclear RNAs
  • piRNAs small nucleolar RNAs
  • piRNAs piwi-interacting RNAs
  • scaRNAs small Cajal Body specific RNAs
  • the methods of the invention may comprise a step of separating a sample nucleic acid from a complementary strand before using the sample nucleic acid in the methods of the invention, e.g.
  • the methods of the invention may comprise extending the 5' and/or 3' end of the sample nucleic acid before or after separating the sample nucleic acid from the complementary strand.
  • sample nucleic acid is initially hybridised to a complementary strand
  • the methods of the invention may be used to analyse the complementary strand in addition to the sample nucleic acid, if desired.
  • sample nucleic acid may be obtained from any appropriate source.
  • the sample nucleic acid might be obtained from a biological sample, such as a biological sample comprising material derived from cells.
  • the methods and devices of the invention may thus be used to analyse a biological sample comprising a sample nucleic acid.
  • Biological samples can be derived from a variety of organisms and cell types, including both eukaryotes and prokaryotes. Accordingly, in some embodiments a biological sample is a sample obtained from eukaryotic cells. In other embodiments, a biological sample is a sample obtained from prokaryotic cells.
  • the invention can be used to analyse samples obtained from bacteria, including, but not limited to: E.coli; B.subtilis; N. meningitidis; N. gonorrhoeae; S. pneumoniae; S.mutans; S.agalactiae; S. pyogenes; P. aeruginosa; H. pylori; M.catarrhalis; H. influenzae; B. pertussis; C.diphtheriae; C.tetani; etc.
  • the invention can be used to analyse samples obtained from animal cells, plant cells, fungal cells (particularly yeasts), etc.
  • Preferred biological samples of interest are those derived from mammalian cells, such as human cells, mouse cells or rat cells.
  • Specific cell types of interest include but are not limited to: blood cells, such as lymphocytes, natural killer cells, leukocytes, neutrophils, monocytes platelets, etc.; tumour cells, such as carcinomas, lymphomas, leukaemic cells; gametes, including ova and spermatozoa; heart cells; kidney cells; pancreas cells; liver cells; brain cells; skin cells; stem cells, including adult stem cells and embryonic stem cells; etc. Cell lines can also be analysed.
  • blood cells such as lymphocytes, natural killer cells, leukocytes, neutrophils, monocytes platelets, etc.
  • tumour cells such as carcinomas, lymphomas, leukaemic cells
  • gametes including ova and spermatozoa
  • heart cells such as lymphocytes, natural killer cells, leukocytes, neutrophils, monocytes platelets, etc.
  • tumour cells such as carcinomas, lymphomas, leukaemic cells
  • gametes including ova and spermatozoa
  • heart cells such as lymphocytes
  • a biological sample may comprise material derived from multiple cells, such that the invention is used for analysis of nucleic acids obtained from a cell population.
  • each biological sample may comprise material derived from an individual cell, such that the invention is used for analysis of nucleic acids obtained from an individual cell.
  • a biological sample may comprise: material derived from less than 1x10 8 cells, material derived from less than 1x10 7 cells, material derived from less than 1 x10 6 cells, material derived from less than 1x10 5 cells, material derived from less than 1x10 4 cells, material derived from less than 1x10 3 cells, material derived from less than 100 cells, material derived from less than 50 cells, material derived from less than 25 cells, material derived from less than 20 cells, material derived from less than 15 cells, material derived from less than 10 cells, material derived from less than 5 cells, material derived from less than 3 cells, or material derived from less than 2 cells (i.e. material derived from an individual cell).
  • a biological sample may comprise: material derived from more than 2 cells, material derived from more than 3 cells, material derived from more than 5 cells, material derived from more than 10 cells, material derived from more than 15 cells, material derived from more than 20 cells, material derived from more than 25 cells, material derived from more than 50 cells, material derived from more than 100 cells, material derived from more than 1 x10 3 cells, material derived from more than 1x10 4 cells, material derived from more than 1 x10 5 cells, material derived from more than 1 x10 6 cells, material derived from more than 1x10 7 cells, or material derived from more than 1 x10 8 cells.
  • the methods of the invention may include a sample preparation step that permits separation of the components of interest (i.e. nucleic acids) from other components of samples.
  • the other components of the samples i.e. those components from which the nucleic acids were separated
  • the well known Boom method may be used to obtain sample nucleic acids (reference 4).
  • sample preparation step that permits generation of components of interest (i.e. nucleic acids) from precursors in a sample.
  • sample nucleic acids of a desired length may be generated from longer nucleic acids by an appropriate form of fragmentation e.g. sonication, endonuclease cleavage (including both random endonuclease cleavage and restriction endonuclease cleavage), exonuclease cleavage, chemical cleavage, heat treatment or mechanical shearing.
  • a polymerase is used to extend the 3' end of a sample nucleic acid or a probe, as described in more detail elsewhere herein.
  • the methods of the invention may therefore include a step to ensure that a suitable underhanging 3' end is present when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • the methods may comprise obtaining an initial nucleic acid and synthesising the reverse complement of the initial nucleic acid to generate a sample nucleic acid suitable for analysis using a polymerase in the methods of the invention.
  • a step of synthesising the reverse complement of an initial nucleic acid may be performed after extending the 5' and/or 3' end of the initial nucleic acid, as illustrated schematically in Fig. 6. In other embodiments, a step of synthesising the reverse complement of an initial nucleic acid may be performed before extending the 5' and/or 3' end of the initial nucleic acid.
  • the methods of the invention involve synthesising the complement of an initial nucleic acid, and the initial nucleic acid is an RNA
  • the methods of the invention may include a step of reverse transcription of the initial nucleic acid (or of an extended version of the initial nucleic acid, if synthesising the reverse complement is performed after extending the initial nucleic acid).
  • the methods of the invention involve synthesising the complement of an initial nucleic acid
  • the sample nucleic acid is a DNA
  • the initial nucleic acid is a RNA
  • the initial RNA may be degraded with an RNase (e.g. RNase H) leaving the sample DNA for analysis. Removal of the initial nucleic acid may be useful to avoid it interfering with hybridisation of the probe to the sample nucleic acid or extended nucleic acid in the hybridisation step.
  • RNase e.g. RNase H
  • the methods of the invention can be used to analyse a single sample nucleic acid alone or when present in a mixture containing further nucleic acids.
  • the methods of the invention can also be used to analyse multiple sample nucleic acids alone or when present in a mixture containing further nucleic acids.
  • the methods of the invention can be used to analyse a single sample nucleic acid or multiple sample nucleic acids in a biological sample comprising material derived from cells.
  • a biological sample comprises material derived from cells
  • the sample may be obtained by any suitable method. Both mechanical and chemical methods are envisaged; exemplary methods are described below.
  • a lysis solution can be applied to cells.
  • a lysis solution can be applied to cells on the support, and the cells lysed in situ.
  • Typical lysis solutions that can be used may comprise components such as: a surfactant e.g. an ionic detergent such as sodium dodecyl sulphate (SDS); an enzyme to digest proteins e.g. proteinase K; an enzyme to digest nucleic acids e.g. a DNase and/or RNase; an enzyme to digest saccharides (e.g.
  • a chaotrope to inactivate enzymes and solubilise cellular components e.g. a guanidine salt, such as guanidinium isothiocyanate; an organic solvent (e.g. toluene, ether, phenylethyl alcohol, dimethyl sulfoxide (DMSO), benzene, methanol, or chloroform); an antibiotic; a thionin; a chelating agent (e.g. ethylenediaminetetraacetic acid, EDTA); a basic protein (e.g. protamine, or chitosan) etc.
  • a guanidine salt such as guanidinium isothiocyanate
  • an organic solvent e.g. toluene, ether, phenylethyl alcohol, dimethyl sulfoxide (DMSO), benzene, methanol, or chloroform
  • an antibiotic e.g. toluene, ether, phenyle
  • reagents are commonly used in existing techniques for bulk cell lysis.
  • the choice of reagent(s) will depend on the nature of the analytes of interest e.g. if the aim is to analyse RNA then proteases and reagents that degrade DNA may be included in the lysis solution, but not reagents that degrade RNA. Alternatively, if the aim is to analyse DNA then proteases and reagents that degrade RNA may be included in the lysis solution, but not reagents that degrade DNA.
  • Reference 5 discloses a method for fast lysis of a single cell (or cellular component thereof) by generating a shock wave, and to minimise manipulation trauma the cell is either positioned by laser tweezers or is cultured as an adhered cell. Ultrasonic vibration can also be applied to a device in order to lyse cells, as can laser light, which has previously been used to lyse single cells, as in reference 6. Lysis of single cells in a microfluidic device by osmotic shock is reported in reference 7.
  • Reference 8 describes navigation and steering of single cells with optical tweezers to different areas of a microfluidic network where the flow properties can be controlled by electrophoresis and electroosmosis. A cell is captured between two electrodes where it can be lysed by an electric pulse.
  • Electroporation may be used to lyse cells. Depending on the magnitude of the electric field used for electroporation, a membrane may simply be opened, allowing access to a cell's contents, or may rupture, leading to cell lysis (see reference 9). A field strong enough to cause lysis is preferred.
  • Biochemical analysis is often preceded by such purification or modification steps to remove substances which may interfere, either in terms of a target analyte's interaction with an analytical component, or in terms of accessing or interpreting results.
  • sample nucleic acid is an RNA then DNA and/or protein may be removed before analysis, as mentioned above. Suitable sample processing steps will be evident to the skilled person, in light of the sample nucleic acid to be analysed.
  • Protocols for preparing samples for analysis by microarray are well known in the art, and may be used in conjunction with this invention e.g. protocols for cell disruption, for total RNA purification, for mRNA purification, for cDNA preparation, for genomic DNA purification, for disrupting secondary structure in a nucleic acid prior to hybridisation, for labelling, etc.
  • the sample nucleic acid is a ncRNA, such as a miRNA
  • methods including steps to purify and/or enrich the ncRNA and/or to disrupt any secondary structure in the ncRNA prior to hybridisation may be preferred.
  • Sample nucleic acids in a biological sample may be associated with proteins so one or more steps may be included in the methods of the invention to separate a sample nucleic acid from a protein, e.g. heat treatment, treatment with SDS or treatment with pronase.
  • samples e.g. cell lysates
  • cells may be lysed in situ on a support, as described elsewhere herein.
  • the invention is particularly relevant to analysis of non-coding RNAs (ncRNAs).
  • the methods of the invention may thus involve extending a ncRNA, to generate a ncRNA having an extension tag at its 5' and/or 3' end.
  • the invention therefore provides an extended ncRNA, comprising a ncRNA sequence, the extended ncRNA further comprising an extension tag at its 5' and/or 3' end.
  • the invention also provides an extended ncRNA, comprising a ncRNA sequence and further comprising an extension tag at its 5' and/or 3' end, wherein the extended ncRNA is hybridised to a probe comprising a probe sequence that is complementary to the ncRNA sequence.
  • the invention also provides a probe comprising a probe sequence that is complementary to a ncRNA sequence, the probe further comprising an extension tag at its 5' and/or 3' end.
  • the invention also provides a probe comprising a probe sequence that is complementary to a ncRNA sequence, the probe further comprising an extension tag at its 5' and/or 3' end, hybridised to the ncRNA sequence.
  • the ncRNA sequence may be a sequence found in the NONCODE database, the RNA db database, or the miRBase database (see elsewhere herein). miRNA analysis
  • miRNAs are short single-stranded RNA molecules.
  • miRNAs are involved in the regulation of gene expression in eukaryotes. They are not translated into proteins, i.e. they are non-coding RNAs. They regulate gene expression at the level of transcription (e.g. by promoting cleavage of mRNAs) or translation (e.g. by inhibiting translation of mRNAs), generally by binding to the 3'-UTR of an mRNA.
  • Each miRNA is thought to regulate expression of multiple genes.
  • miRNAs have only relatively recently been identified, they have already been identified in many organisms. miRNAs are implicated in a variety of cellular processes, including development, differentiation, proliferation and programmed cell death.
  • miRNAs are thought to play a role in diseases, including chronic lymphocytic leukemia, colonic adenocarcinoma, Burkitt's Lymphoma and viral infection. miRNA expression patterns can therefore be used to distinguish between different cell types, such as between different tumour cell types.
  • the sample nucleic acid is a microRNA (miRNA).
  • miRNAs and/or their precursors i.e. pri-miRNAs and/or pre-miRNAs.
  • miRNA hairpin precursors give rise to two mature miRNAs, one from each of the 5' and 3' arms of the hairpin. These two forms are sometimes referred to as the '5p' and '3p' forms, respectively (e.g. miRNA-140-5p and miRNA-140-3p; see reference 3).
  • the less predominant of the two mature miRNAs is also sometimes referred to as the 'star' form (miRNA * ) (e.g. miRNA-140 and miRNA140 * ; see reference 3).
  • the processes and devices of the invention may be used to analyse either or both of the mature miRNAs formed from a precursor miRNA of interest, as desired (i.e.
  • the processes and devices of the invention may be used to analyse either or both of miRNA-5p and miRNA-3p, or either or both of miRNA and miRNA * , for a precursor miRNA of interest).
  • the sample nucleic acid may be the predominant mature miRNA formed from a precursor miRNA of interest.
  • the sample nucleic acid may be the less predominant mature miRNA (miRNA * ) formed from a precursor miRNA of interest.
  • the sample nucleic acid may be the 5p mature miRNA formed from a precursor miRNA of interest.
  • the sample nucleic acid may be the 3p mature miRNA formed from a precursor miRNA of interest.
  • a sample miRNA is obtained from an animal (such as a human) or from a plant.
  • Specific organisms from which a sample miRNA may be obtained for analysis according to the invention include Homo sapiens, Mus musculus, Danio rerio, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and Populus trichocarpa.
  • nucleotide sequences of known miRNAs can be identified from databases (e.g. miRBase at http://microrna.sanqer.ac.uk/).
  • a sample nucleic acid may comprise a sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • a sample nucleic acid may consist of a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6396.
  • a sample nucleic acid may consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • Identity indicates that at any particular position in two aligned sequences, the nucleotide residue is identical between the sequences. Percentage identity between two sequences can be readily calculated using well-known methods, e.g. using a BLAST algorithm with the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). As used herein, the percentage identity shared by two nucleotide sequences is the percentage of nucleotide residues in the two sequences that are identical after aligning the sequences to achieve the maximum percent sequence identity.
  • a sample nucleic acid is a miRNA
  • the methods of the invention may include one or more steps for enriching and/or purifying miRNAs from an initial sample.
  • miRNA isolation methods and kits are known to the skilled person and are commercially available (e.g. the m/ ⁇ /anaTM miRNA isolation kit from Ambion, Inc. and the miRNeasy Mini Kit from QIAGEN).
  • the methods of the invention may involve extending a miRNA, to generate a miRNA having an extension tag at its 5' and/or 3' end.
  • the invention therefore provides an extended miRNA, comprising a miRNA sequence, the extended miRNA further comprising an extension tag at its 5' and/or 3' end.
  • the invention also provides an extended miRNA, comprising a miRNA sequence and further comprising an extension tag at its 5' and/or 3' end, wherein the extended miRNA is hybridised to a probe comprising a probe sequence that is complementary to the miRNA sequence.
  • the miRNA sequence may be a sequence 15-30 nucleotides in length, 15-25 nucleotides in length, or 20-25 nucleotides in length.
  • the miRNA sequence may be a sequence found in the miRBase database or any other miRNA database.
  • the miRNA sequence may comprise a sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with a nucleotide sequence selected from the group consisting of SEQ ID NOs:1- 6396.
  • the miRNA sequence may consist of a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with a nucleotide sequence selected from the group consisting of SEQ ID NOs:1- 6396.
  • the miRNA sequence may consist of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • SEQ ID Nos: 1-6396 SEQ ID Nos: 2086-2763 are preferred when studying human miRNA.
  • the invention also provides a probe comprising a probe sequence that is complementary to a miRNA sequence, the probe further comprising an extension tag at its 5' and/or 3' end.
  • the invention also provides a probe comprising a probe sequence that is complementary to a miRNA sequence, the probe further comprising an extension tag at its 5' and/or 3' end, wherein the probe is hybridised to the miRNA sequence.
  • the sequence that is complementary to a miRNA sequence may be a sequence 15-30 nucleotides in length, 15-25 nucleotides in length, or 20-25 nucleotides in length.
  • the sequence that is complementary to a miRNA sequence may be a sequence complementary to a sequence found in the miRBase database.
  • the sequence that is complementary to a miRNA sequence may comprise a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • the sequence that is complementary to a miRNA sequence may consist of a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • the sequence that is complementary to a miRNA sequence may consist of the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • the methods of the invention involve use of a probe comprising a probe sequence.
  • the probe interacts with a sample nucleic acid (or the sample nucleic acid portion of an extended nucleic acid) by hybridisation.
  • the methods of the invention may involve using a single probe to analyse a sample nucleic acid, or using different probes to analyse different sample nucleic acids.
  • the probes may be immobilised on one or more supports, as described elsewhere herein.
  • a device comprising different probes immobilised on a single support allows parallel analysis of multiple sample nucleic acids using the same support.
  • the methods of the invention do not involve use of a support, and the devices of the invention do not comprise a support i.e. in some embodiments the methods of the invention are carried out in solution.
  • the probes used will generally be chosen in order to give analytical data of interest. In particular, the length and sequence of the probes will be chosen according to the sample nucleic acids of interest.
  • the probes are single-stranded, and may be DNA.
  • a probe may be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycerol nucleic acid (GNA) or a threose nucleic acid (TNA).
  • a probe comprises a probe sequence that is hybridisable to a sample nucleic acid (or to the sample nucleic acid portion of an extended nucleic acid).
  • the probe sequence in the probe may be 10-3000, 10-2500, 10-2000, 10-1500, 10-1000, 10-750, 10-500, 10-400, 10-300, 10-250, 10-200, 10-150, 10-100, 10-75, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30 or 20-25 nucleotides in length.
  • a suitable length probe sequence can readily be selected depending on the type of analysis required.
  • a probe sequence may be the same length as a sample nucleic acid (or the sample nucleic acid portion of an extended nucleic acid), or a different length.
  • the length of the probe sequence will be at least 10% of the length of a sample nucleic acid (or of the length of the sample nucleic acid portion of an extended nucleic acid).
  • the length of the probe sequence may be at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the length of a sample nucleic acid (or of the length of the sample nucleic acid portion of an extended nucleic acid).
  • Methods and devices in which the length of the probe sequence is similar to the length of a sample nucleic acid (or the length of the sample nucleic acid portion of an extended nucleic acid) may be preferred, particularly for analysis of short nucleic acids, because they may improve discrimination of nucleic acids of interest from other nucleic acids by making use of the available nucleotide residues in the sample nucleic acid during the hybridisation step.
  • a probe sequence is complementary to a genomic DNA or mRNA sequence.
  • a probe sequence is complementary to a ncRNA sequence, e.g. a microRNA (miRNA) sequence, a ribosomal RNA (rRNA) sequence, a small interfering RNA (siRNA) sequence, a small nuclear RNA (snRNA) sequence, a small nucleolar RNA (snoRNA) sequence, a piwi-interacting RNA (piRNA) sequence, a small Cajal Body specific RNA (scaRNA) sequence, or a transfer RNA (tRNA) sequence.
  • miRNA microRNA
  • rRNA ribosomal RNA
  • siRNA small interfering RNA
  • snRNA small nuclear RNA
  • piRNA piwi-interacting RNA
  • scaRNA small Cajal Body specific RNA
  • tRNA transfer RNA
  • a probe sequence may be complementary to a sequence from a prokaryote or a eukaryote, such as a mammal (e.g. a human, mouse or rat) or a plant.
  • a probe sequence may be selected that is completely complementary to a known nucleotide sequence (e.g. a known miRNA sequence).
  • a probe sequence may be selected that is not completely complementary to a known nucleotide sequence.
  • a probe may be selected that shares at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the complement of a known nucleotide sequence.
  • Probes that are not fully complementary to a known sequence might be selected to allow analysis of multiple homologous sample nucleic acids using a single probe, such as homologous sequences from a single organism, or homologous sequences from different organisms (e.g. humans and mice).
  • a probe sequence will normally be selected taking into account the length and sequence of the sample nucleic acid, the intended hybridisation conditions, and in light of the type of analysis required.
  • a probe sequence may comprise a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • a probe sequence may consist of a nucleotide sequence that shares at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • a probe sequence may consist of the complement of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-6396.
  • identity indicates that at any particular position in two aligned sequences, the nucleotide residue is identical between the sequences. Percentage identity between two sequences can be readily calculated using well-known methods, e.g. using a BLAST algorithm with the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). As used herein, the percentage identity shared by two nucleotide sequences is the percentage of nucleotide residues in the two sequences that are identical after aligning the sequences to achieve the maximum percent sequence identity.
  • extension tags in the methods of the invention.
  • An appropriate type of extension tag can be selected by the skilled person, based on the type of analysis desired. In each case, an extension tag will be selected that facilitates analysis of the nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • the use of an extension tag provides a convenient means for detection or manipulation of a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • Exemplary types of extension tags for use in the methods of the invention are described herein. Other appropriate tags can be selected by the skilled person.
  • An extension tag may comprise a nucleotide sequence.
  • an extension tag may comprise a nucleotide sequence capable of acting as a template for polymerase-mediated extension of a probe if an extended nucleic acid hybridises to the probe.
  • the methods may comprise using an extension tag as a template for polymerase-mediated extension of a probe. This general approach is illustrated schematically in Fig. 3A (the stars in Fig. 3A and the other figures represent detectable labels).
  • an extension tag may comprise a nucleotide sequence capable of acting as a template for polymerase-mediated extension of a sample nucleic acid if the sample nucleic acid hybridises to a probe.
  • the methods of the invention may comprise using an extension tag as a template for polymerase-mediated extension of a sample nucleic acid. This general approach is illustrated schematically in Fig. 3B.
  • an extension tag may comprise a nucleotide sequence capable of acting as a template for ligase-mediated extension of a probe if an extended nucleic acid hybridises to the probe.
  • the methods may comprise using the extension tag as a template for ligase-mediated extension of the probe. This general approach is illustrated in Fig. 4A.
  • an extension tag may comprise a nucleotide sequence capable of acting as a template for ligase-mediated extension of a sample nucleic acid if the sample nucleic acid hybridises to a probe.
  • the methods may comprise using the extension tag as a template for ligase-mediated extension of a sample nucleic acid. This general approach is illustrated schematically in Fig. 4B.
  • extension tag comprises a nucleotide sequence
  • the extension tag preferably comprises a known nucleotide sequence.
  • an extension tag comprises a nucleotide sequence
  • the extension tag may be a DNA or an RNA.
  • an extended sample nucleic acid may be a chimeric RNA-DNA molecule, or a non-chimeric RNA-RNA molecule.
  • an extended sample nucleic acid may be a chimeric RNA-DNA molecule that comprises a miRNA sequence linked to a DNA extension tag, or may be a non-chimeric RNA-RNA molecule that comprises a miRNA sequence linked to an RNA extension tag.
  • an extended sample nucleic acid may be a chimeric DNA-RNA molecule, or a non- chimeric DNA-DNA molecule.
  • an extension tag comprises a nucleotide sequence
  • the extension tag may be obtained by a de novo synthesis method, or by purification.
  • An extension tag may comprise both DNA and RNA nucleotides, if desired.
  • An extension tag may comprise one or more modified bases or analogs, if desired.
  • an extension tag may be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycerol nucleic acid (GNA) or a threose nucleic acid (TNA).
  • extension tag comprises a nucleotide sequence
  • the extension tag nucleotide sequence is preferably heterologous to the probe sequence and its complement.
  • a heterologous extension tag sequence is preferred, as it reduces the likelihood of false positive and/or false negative results, which might arise if the extension tag nucleotide sequence is complementary to the probe sequence or its complement.
  • the extension tag may comprise a repetitive nucleotide sequence, or a homopolymer sequence.
  • An extension tag nucleotide sequence may be 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more, nucleotides in length.
  • An extension tag nucleotide sequence may be fewer than 50, fewer than 45, fewer than 40, fewer than 35, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 5, fewer than 4, fewer than 3, or fewer than 2, nucleotides in length.
  • an extension tag may comprise a nucleotide sequence, or consist of a nucleotide sequence, that is 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10- 40, 10-30 or 10-20 nucleotides in length.
  • a sample nucleic acid analysed in the methods of the invention may be a nucleic acid 10-3000, 10-2500, 10-2000, 10-1500, 10-1000, 10-750, 10-500, 10- 400, 10-300, 10-250, 10-200, 10-150, 10-100, 10-75, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30 or 20-25 nucleotides in length.
  • an extended nucleic acid comprising a sample nucleic acid and an extension tag may be 1 1-3050, 1 1-2550, 11-2050, 11-1550, 1 1-1050, 1 1 -800, 1 1-550, 11-450, 1 1-350, 1 1-300, 1 1-250, 11-200, 1 1-150, 1 1-125, 1 1-100, 11-90, 1 1-80, 1 1-75, 1 1-70, 1 1-65, 1 1-60, 1 1-55, 1 1-50, 1 1-45, 1 1-40, 11-35, 11-30 or 11-26 nucleotides in length, including any extension tag(s).
  • a probe for use in the methods of the invention may comprise a probe sequence that is 10-3000, 10-2500, 10-2000, 10-1500, 10-1000, 10-750, 10-500, 10- 400, 10-300, 10-250, 10-200, 10-150, 10-100, 10-75, 10-50, 10-40, 10-30, 10-25, 10-20, 10-15, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30 or 20-25 nucleotides in length.
  • a probe may be 11-3050, 11-2550, 1 1-2050, 1 1-1550, 1 1-1050, 1 1- 800, 11-550, 1 1-450, 1 1-350, 11-300, 11-250, 1 1-200, 1 1-150, 11-125, 1 1-100, 1 1-90, 1 1- 80, 1 1-75, 11-70, 11-65, 1 1-60, 11-55, 11-50, 11-45, 11-40, 1 1-35, 1 1-30 or 1 1-26 nucleotides in length, including any extension tag(s).
  • an extension tag nucleotide sequence may be shorter than the sample nucleic acid.
  • the extension tag nucleotide sequence may be less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, less than 85%, less than 90%, less than 95%, less than 96%, less than 97%, less than 98% or less than 99%, of the length of a sample nucleic acid.
  • an extension tag nucleotide sequence may be longer than the sample nucleic acid.
  • the extension tag nucleotide sequence may be at least 1.05 times, at least 1.10 times, at least 1.15 times, at least 1.20 times, at least 1.25 times, at least 1.30 times, at least 1.40 times, at least 1.50 times, at least 1.75 times, at least 2.00 times, at least 2.50 times, at least 3.00 times, at least 3.50 times, at least 4.00 times, at least 4.50 times or at least 5.00 times, the length of the sample nucleic acid.
  • the extension tag nucleotide sequence may be at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 28, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 nucleotides in length.
  • Methods and devices in which the extension tag nucleotide sequence is longer than the sample nucleic acid may be preferred, because they may provide greater sensitivity by allowing generation of a greater signal for detection in the analysis step. This concept of using a longer extension tag nucleotide sequence is illustrated schematically in Fig. 13.
  • An extension tag may comprise a moiety capable of being recognised by a specific binding reagent. Accordingly, the methods of the invention may comprise contacting the extension tag with a specific binding reagent, and detecting binding of the specific binding reagent to the extension tag. This general approach is illustrated schematically in Figs. 5A and 5B.
  • an extension tag may comprise an antigen specifically recognised by an antigen-binding reagent, such as an antibody, or the extension tag may comprise an antigen-binding reagent.
  • the extension tag may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20, amino acid residues.
  • An extension tag may comprise a biotin group capable of binding to a specific binding reagent comprising a streptavidin or avidin group (or the extension tag may comprise a streptavidin or avidin group capable of binding to a specific binding reagent comprising a biotin group).
  • An extension tag that comprises a moiety capable of being recognised by a specific binding reagent may be preferable depending on the type of analysis required (e.g. if use of a specific detection method is desired in the analysis step).
  • an extension tag may additionally facilitate downstream processing or analysis of the nucleic acid-probe hybrid.
  • the extension tag may contain one or more restriction endonuclease cleavage sites, one or more sequences that facilitate recombination (e.g. loxP sequences), or one or more sequences that facilitate cloning (e.g. a Gateway cloning sequence), for further processing or analysis of the nucleic acid-probe hybrid.
  • any suitable method can be used to generate the extended nucleic acid.
  • a sample nucleic acid may be extended by ligation, e.g. by ligation to an extension tag comprising a nucleotide sequence.
  • ligation e.g. by ligation to an extension tag comprising a nucleotide sequence.
  • phosphorylation or de-phosphorylation of the sample nucleic acid may be required before ligation, depending on the source of the sample nucleic acid.
  • phosphorylation or de-phosphorylation of the extension tag may be required before ligation, depending on the source of the extension tag. Suitable methods for ligation of nucleic acids, and suitable methods for phosphorylation or de-phosphorylation of nucleic acids, are known in the art.
  • a sample nucleic acid may be extended using a polymerase enzyme.
  • Enzymes that can extend nucleic acids without requiring a template can be used, such as a terminal transferase, a polyA polymerase, or a polynucleotide phosphorylase.
  • a sample nucleic acid may be extended by a non-enzymatic (chemical) method.
  • Probes used in the methods of the invention will generally be obtained by a de novo synthesis method, rather than by purification. Thus, a physical step of extending the 5' and/or 3' end of a probe will not normally be required, because the probe will be designed to include an extension tag ab initio, if appropriate.
  • the methods of the invention may comprise a step of extending the 5' and/or 3' end of a probe to form an extended probe comprising an extension tag at its 5' and/or 3' end, if a probe does not include an extension tag ab initio.
  • a probe can be extended using any suitable method, such as those mentioned herein for extension of a sample nucleic acid.
  • a probe may comprise both DNA and RNA nucleotides, if desired.
  • a probe may comprise one or more modified bases or analogs, if desired.
  • a probe may be a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a glycerol nucleic acid (GNA) or a threose nucleic acid (TNA).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • GNA glycerol nucleic acid
  • TAA threose nucleic acid
  • the methods of the invention involve hybridisation of a sample nucleic acid or an extended nucleic acid to a probe.
  • the hybridisation step is performed under conditions that allow the sample nucleic acid or extended nucleic acid to hybridise to the probe if the sample nucleic acid comprises a sequence complementary to the probe sequence.
  • Sample nucleic acids or extended nucleic acids are analysed if they hybridise to the probe under these conditions.
  • conditions may be selected that allow hybridisation only if a sample nucleic acid comprises a sequence that is completely complementary to a probe sequence. In other embodiments, conditions may be selected that allow hybridisation if a sample nucleic acid comprises a sequence that is partially complementary to a probe sequence.
  • the degree of specificity required may vary according to the needs of an individual experiment e.g. in some experiments it may be desirable to analyse nucleic acids with nucleotide mismatch(es) relative to a probe, but other experiments may require absolute stringency.
  • the conditions used for hybridisation may allow a sample nucleic acid or an extended nucleic acid to hybridise to a probe only if the sample nucleic acid comprises a nucleotide sequence that shares at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity, with the probe sequence.
  • the conditions used for hybridisation may allow a sample nucleic acid or an extended nucleic acid to hybridise to a probe only if the sample nucleic acid comprises a nucleotide sequence that shares 100% identity with the probe sequence (or as near as possible to 100%, allowing for experimental limitations e.g. 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity).
  • the conditions used for hybridisation may allow a sample nucleic acid or an extended nucleic acid to hybridise to a probe only if the sample nucleic acid comprises a nucleotide sequence that differs by only 1 residue, by up to 2 residues, by up to 3 residues, by up to 4 residues, by up to 5 residues, by up to 6 residues, by up to 7 residues, or by up to 8 residues, from the probe sequence.
  • Stringency is a term commonly used in molecular biology that refers to conditions in a hybridisation reaction that favour the association of very similar molecules over association of molecules that differ. In the methods of the invention, higher stringency conditions will be used when it is desired to allow hybridisation only if a sample nucleic acid comprises a sequence that is completely complementary to a probe sequence. Lower stringency conditions will be used when it is desired to allow hybridisation if a sample nucleic acid comprises a sequence that is partially complementary to a probe sequence.
  • a support may be used to facilitate analysis of a nucleic acid.
  • supports can be used to facilitate nucleic acid analysis will be understood by the skilled person. Supports are commonly used in the microarray field.
  • a support is used in the methods of the invention.
  • the particular arrangement that should be used will be determined from (a) whether a sample nucleic acid, extended nucleic acid or probe is to be immobilised on a support, (b) the orientation of the immobilisation, i.e. whether it is via the 5' or 3' end of the nucleic acid, extended nucleic acid or probe, or via an internal nucleotide, and (c) the location of the extension tag(s).
  • Figs. 7A-7H for probe immobilisation
  • Figs. 8A-8H for sample nucleic acid or extended nucleic acid immobilisation
  • Various other arrangements are also envisaged, e.g. where a sample nucleic acid, extended nucleic acid or probe is immobilised on a support via an internal nucleotide, or where multiple extension tags are used (see elsewhere herein).
  • a support a sample nucleic acid, extended nucleic acid or probe should be immobilised on the support in a hybridisable format.
  • the skilled person will readily be able to design an appropriate arrangement depending on the type of analysis desired.
  • a probe is immobilised on a support.
  • a probe may be immobilised on a support via its 5' or 3' end, or via an internal nucleotide.
  • a probe immobilised on a support via its 5' end may be used according to the invention as summarised in the following table.
  • a probe is immobilised on a support via its 5' end, and an extended nucleic acid comprises an extension tag at its 5' end (as in Fig. 7A).
  • the probe is immobilised on a support via its 5' end and the extended nucleic acid comprises an extension tag at its 3' end (as in Fig. 7B).
  • the probe is immobilised on a support via its 5' end and the probe comprises an extension tag at its 5' end (as in Fig. 7C).
  • the probe is immobilised on a support via its 5' end and the probe comprises an extension tag at its 3' end (as in Fig. 7D).
  • a probe immobilised on a support via its 3' end may be used according to the invention as summarised in the following table.
  • the probe is immobilised on a support via its 3' end and the extended nucleic acid comprises an extension tag at its 5' end (as in Fig. 7E).
  • the probe is immobilised on a support via its 3' end and the extended nucleic acid comprises an extension tag at its 3' end (as in Fig. 7F).
  • the probe is immobilised on a support via its 3' end and the probe comprises an extension tag at its 5' end (as in Fig. 7G).
  • the probe is immobilised on a support via its 3' end and the probe comprises an extension tag at its 3' end (as in Fig. 7H).
  • Methods in which a probe is immobilised on a support are preferred, because it will generally be easier to immobilise a probe on a support than to immobilise a sample nucleic acid or extended nucleic acid on a support (a probe will normally be synthesised de novo rather than purified, and can be pre-synthesised then applied to the support, or synthesised in situ on the support).
  • a probe will normally be synthesised de novo rather than purified, and can be pre-synthesised then applied to the support, or synthesised in situ on the support.
  • methods in which a sample nucleic acid or extended nucleic acid are immobilised on a support are also envisaged (see elsewhere herein).
  • a single probe may be immobilised on a support, or a series of different probes may be immobilised on a support.
  • the probe When a single probe is immobilised on a support, the probe is preferably arranged in a discrete patch on the support, to facilitate data analysis.
  • the different probes are preferably arranged in discrete patches on the support, to facilitate data analysis. If different probes are not separate then it may not be clear which of the different sample nucleic acids or extended nucleic acids gives rise to an observed signal. It is possible, however, for neighbouring patches of different probes to overlap slightly, or not to have tight boundaries, provided that the signal arising from one patch can be distinguished from the signal arising from a different patch. In some embodiments, it may be advantageous for patches to overlap, or even for different probes to be immobilised on a single patch.
  • the different probes are preferably immobilised on a substantially planar surface (e.g. a glass microscope slide).
  • a substantially planar surface e.g. a glass microscope slide
  • devices having different probes immobilised in patches on different parts of a substantially non-planar surface are also envisaged.
  • Methods for immobilising probes onto supports in a hybridisable format are well known from the microarray field e.g. attachment via linkers, to a matrix on the support, to a gel on the support, etc.
  • the best-known method is the photolithographic masking method used by Affymetrix for in situ synthesis of nucleotides on a glass support, but electrochemical in situ synthesis methods are also known, as are inkjet deposition methods.
  • a probe may thus comprise a linker for attachment of the probe to a solid support, in addition to the probe nucleotide sequence.
  • a linker may be located at the 5' and/or 3' end of a probe, depending on the immobilisation desired.
  • immobilised probes can be pre-synthesised and then attached to a support, or can be synthesised in situ on a support by delivering precursors to a growing chain. Either of these methods can be used to construct a device of the invention.
  • Preferred immobilised probes are formed by in situ synthesis using electrochemical deprotection of a growing nucleic acid chain (as described in references 10, 11 & 12).
  • a probe may be immobilised via its 5' end or via an internal nucleotide, such that it has a free 3' end, in order to facilitate chain extension.
  • the devices of the invention may contain P different probes, wherein P is selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500 or more.
  • the devices may contain at least 10 w different probes, wherein N is selected from 0, 1 , 2, 3, 4, 5 or more. Immobilisation of at least 10 6 different probes onto a single support is well known in the field of microarrays.
  • the P or 10 w different probes will typically be arranged in P or 10 w different patches on the support, respectively.
  • the devices may contain two or more patches of a single probe, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more patches of the same probe.
  • a patch preferably has an area of at least 10* m 2 , where X is selected from -2, -3, -4, -5, -6, -7, -8, -9, -10, -1 1 , -12, etc.
  • Microarrays with patch sizes in the order of 10 ⁇ m x 10 ⁇ m are readily prepared using current technology.
  • Patches within devices of the invention may have the same size, or different sizes.
  • the edge-to-edge separation of patches is preferably at least 10 y m, where Y is selected from -3, -4, -5, etc. Adjacent patches may abut or may overlap, but it is preferred that adjacent patches are separated by a gap.
  • a patch preferably has a rectangular or square shape, but may also have a circular shape.
  • the shape and size of the patches will be determined by the characteristics of the support (e.g. when the support is a microtiter plate, the size and shape of the patches may match the size and shape of the well bases). Patches within devices of the invention may have the same shape, or different shapes.
  • reagents comprising different extension tags may be applied to a single probe patch on a support.
  • sample nucleic acids from two different sources e.g. from two different cell types
  • two different extension tags e.g. distinguishing between the two extension tags using dyes of different colours.
  • Using two extensions on a single target permits increased signal. It also facilitates determination of end structures and/or the integrity of targets. Either end of the target can be used, thus giving full length confirmation.
  • a sample nucleic acid or an extended nucleic acid is immobilised on a support.
  • a sample nucleic acid or an extended nucleic acid may be immobilised via its 5' or 3' end, or via an internal nucleotide.
  • a sample nucleic acid or an extended nucleic acid immobilised on a support via its 5' end may be used according to the invention as summarised in the following table.
  • a sample nucleic acid is immobilised on a support via its 5' end and the probe comprises an extension tag at its 5' end (as in Fig. 8A).
  • a sample nucleic acid is immobilised on a support via its 5' end and the probe comprises an extension tag at its 3' end (as in Fig. 8B).
  • an extended nucleic acid is immobilised on a support via its 5' end and the extended nucleic acid comprises an extension tag at its 5' end (as in Fig. 8C).
  • an extended nucleic acid is immobilised on a support via its 5' end and the extended nucleic acid comprises an extension tag at its 3' end (as in Fig. 8D).
  • a sample nucleic acid or an extended nucleic acid immobilised on a support via its 3' end may be used according to the invention as summarised in the following table.
  • a sample nucleic acid is immobilised on a support via its 3' end and a probe comprises an extension tag at its 5' end (as in Fig. 8E).
  • a sample nucleic acid is immobilised on a support via its 3' end and a probe comprises an extension tag at its 3' end (as in Fig. 8F).
  • an extended nucleic acid is immobilised on a support via its 3' end and the extended nucleic acid comprises an extension tag at its 5' end (as in Fig. 8G).
  • an extended nucleic acid is immobilised on a support via its 3' end and the extended nucleic acid comprises an extension tag at its 3' end (as in Fig. 8H).
  • sample nucleic acid or extended nucleic acid may be immobilised on a support, or a series of different sample nucleic acids or extended nucleic acids may be immobilised on a support.
  • sample nucleic acids or extended nucleic acids may be arranged on a support as described elsewhere herein for probe immobilisation.
  • Figs. 7A, 7C, 7G, 8A, 8C and 8G may be preferred when a polymerase-based detection method is to be used in the analysis step (see elsewhere herein), because they involve generation of an underhanging 3' end.
  • Figs. 7B, 7E, 8B and 8E may be less preferred, because an extension tag is ideally directed away from the surface of the support after hybridisation, to facilitate its use in the analysis step.
  • the inventors envisage that arrangements of the type shown in Figs. 7B, 7E, 8B and 8E might require longer probes or the use of linkers to ensure that the extension tag is available for use in the analysis step (see Fig. 14, which is based on Fig. 7B; corresponding arrangements are envisaged for Figs. 7E, 8B and 8E).
  • the probe or extended nucleic acid might be sufficiently flexible to ensure that the extension tag is available for use in the analysis step (see Fig. 15 which is based on Fig. 7B; again corresponding arrangements are envisaged for Figs. 7E, 8B and 8E).
  • a support may be constructed of any suitable material.
  • the choice of materials for the support is influenced by a number of design considerations, and suitable materials can readily be selected by the skilled person based on the requirements of a particular device. For example, the material(s) should be stable to the reagents applied to the device during use, and compatible with the methods used for analysing the nucleic acid using the extension tag(s).
  • the invention provides a device for analysing a nucleic acid, comprising a support permeable to the reagents that are applied to the device during use.
  • Such devices may comprise means for applying reagents to one or both faces of the support and/or means for removing reagents from one or both faces of the support.
  • the device may comprise one or more inlet(s) that permit reagents to be applied to one or both faces of the permeable support and/or one or more outlet(s) that permit reagents to be removed from one or both faces of the permeable support.
  • a permeable support may, for instance, be constructed from Nylon, nitrocellulose, a polyvinylidene fluoride (PVDF) membrane, such as a GVHP membrane (Millipore), Immobilon-P (Millipore) or Immobilon-FL (Millipore).
  • PVDF polyvinylidene fluoride
  • a suitable material should be selected by the skilled person.
  • a hard material For some applications it will be desirable to use a hard material; other applications may need a flexible material.
  • fluorescence is to be used in the analysis step, then the material should be transparent to the excitation and emission wavelengths, and also have low intrinsic fluorescence at these wavelengths. Materials that can propagate an illuminating evanescent wave (by total internal reflection) may be preferred for use with certain analysis techniques.
  • supports used in the invention can be made from a variety of materials, including but not limited to silicon oxides, polymers, ceramics, metals, etc.
  • Specific materials that can be used include, but are not limited to: glass; polyethylene; polydimethylsiloxane (PDMS); polypropylene; and silicon.
  • the invention may be used to analyse a plurality of different sample nucleic acids using a method or device as described in co-pending patent application no. PCT/GB2007/004961 (filed 21 st December 2007) published as WO 2008/075086.
  • the methods and devices of the invention may be used to analyse a plurality of different sample nucleic acids by a process that comprises the steps of: a) applying the sample nucleic acids (or extended nucleic acids generated from the sample nucleic acids as described herein) to a support, to which support a probe is immobilised; and b) allowing the sample nucleic acids (or extended nucleic acids) to interact with the probe, wherein the sample nucleic acids (or extended nucleic acids) are applied in step a) to different areas of the support to produce a spatial arrangement of samples on the support, and the spatial arrangement of the samples is maintained in step b), thus permitting the results of the analysis to be matched to individual samples.
  • This general approach is illustrated schematically in Fig. 1
  • the devices of the invention may also include:
  • Electrodes can be used to generate an electrical potential across a device, to cause sample transfer etc.
  • a light source e.g. a laser.
  • a laser can be used for data collection.
  • a detector e.g. a mass spectrometer.
  • the methods of the invention may comprise extending the 5' and 3' ends of a sample nucleic acid, to generate an extended nucleic acid having extension tags at both its 5' and 3' ends (as illustrated in Fig. 9).
  • the methods of the invention may comprise use of a probe that comprises extension tags at both its 5' and 3' ends (as illustrated in Fig. 10).
  • the invention also provides processes wherein both the sample nucleic acid and the probe have an extension tag at their 5' and/or 3' ends (as illustrated in Figs. 11 and 12). All of these methods involve the use of multiple extension tags.
  • extension tags are envisaged for use in combination with a support as described elsewhere herein.
  • these different uses of multiple extension tags are envisaged for use in the types of arrangements illustrated schematically in Figs. 7A-7H and Figs. 8A-8H.
  • the skilled person will readily be able to design an appropriate combination of extension tags and immobilisation depending on the type of analysis desired.
  • Figs. 9 and 10 might be analysed by ligating nucleotide sequences to both the 5' and 3' ends of the probe (Fig. 9), or to both the 5' and 3' ends of the sample nucleic acid (Fig. 10), if the extension tags both comprise nucleotide sequences.
  • the arrangement illustrated in Fig. 9 might be analysed by ligating nucleotide sequences to both the 5' and 3' ends of the probe (Fig. 9), or to both the 5' and 3' ends of the sample nucleic acid (Fig. 10), if the extension tags both comprise nucleotide sequences.
  • Fig. 9 the arrangement illustrated in Fig. 9
  • 1 1 might be analysed by ligating nucleotide sequences to both the 3' end of the probe and the 3' end of the extended nucleic acid, or by polymerase extension of both the 3' end of the probe and the 3' end of the extended nucleic acid (if the extension tags both comprise nucleotide sequences).
  • the arrangement illustrated in Fig. 12 might be analysed by ligating nucleotide sequences to both the 5' end of the probe and the 5' end of the extended nucleic acid, if the extension tags both comprise nucleotide sequences.
  • nucleic acid-probe hybrids comprising multiple extension tags using a combination of analysis methods is envisaged, such as using both polymerase-mediated and ligase-mediated detection methods (e.g. for the arrangement shown in Figs. 9 and 10, if the extension tags both comprise nucleotide sequences).
  • extension tags may be the same or different. Using the same extension tag may be preferred in some embodiments, as it may simplify the analysis step. In other embodiments, using different extension tags may be preferred (e.g. a biotin tag at one end, and a nucleotide sequence tag at the other end, of an extended sample nucleic acid), because it may increase sensitivity or increase specificity, or may provide greater flexibility for processing and analysis of the nucleic acid-probe hybrid.
  • the methods of the invention involve using an extension tag to analyse a sample nucleic acid if the sample nucleic acid or an extended version of the sample nucleic acid hybridises to a probe.
  • extension tag to analyse the sample nucleic acid may comprise using the extension tag to detect the presence or absence or amount of a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • using the extension tag to analyse the sample nucleic acid may comprise using the extension tag to manipulate a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • detection methods can be used in the analysis step of the methods of the invention.
  • the detection method to be used depends on the nature of the extension tag.
  • Various detection methods are known for use with microarrays, and may be suitable for use in conjunction with the present invention; some such methods are described below.
  • an extension tag may act as a template for polymerase- mediated extension of a probe or sample nucleic acid.
  • the analysis step may comprise contacting a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe with a polymerase (and an appropriate polymerase reaction mixture), and using the polymerase to extend the 3' end of the sample nucleic acid or probe.
  • polymerase-mediated extension of a nucleic acid normally requires an underhanging 3' end, on which the polymerase enzyme can act.
  • some arrangements encompassed by the invention are potentially not ideally suited to analysis by polymerase extension (e.g. see the arrangements illustrated schematically in Figs. 1 B, 2B, 7B, 7D, 7E, 7F, 7H, 8B, 8D, 8E, 8F and 8H).
  • Other arrangements envisaged by the inventors are more clearly suited to analysis by polymerase extension, as explained below.
  • the orientation of the probe and sample (or extended) nucleic acid, and the way in which any support is used, can be designed to ensure that an underhanging 3' end is present in the arrangement when a sample nucleic acid or extended nucleic acid hybridises to a probe (e.g. see the arrangements illustrated schematically in Figs. 1A, 2A, 3A, 3B, 7A, 7C, 7G, 8A, 8C, 8G and 9-12).
  • a sample nucleic acid, extended nucleic acid or probe may be immobilised via its 5' end or via an internal nucleotide, such that it has a free 3' end.
  • the methods of the invention may include a step to ensure that a suitable 3' end is present when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • the methods may comprise obtaining an initial nucleic acid and synthesising the reverse complement of the initial nucleic acid to generate a suitable sample nucleic acid or extended nucleic acid for analysis using a polymerase, as described elsewhere herein (see Fig. 6).
  • an extension tag may comprise a nucleotide sequence capable of acting as a template for polymerase-mediated extension of a primer when a sample nucleic acid or extended sample nucleic acid hybridises to a probe.
  • the primer will be partially or completely complementary to the extension tag nucleotide sequence.
  • the primer comprises a sequence that is partially or completely complementary to the 3' end of the extension tag, so that when the primer hybridises to the extension tag its 3' end can be extended by a polymerase using the extension tag as a template.
  • the methods of the invention may therefore comprise using an extension tag as a template for polymerase-mediated extension of a primer e.g.
  • Fig. 17 which shows an embodiment where the sample nucleic acid is extended using an extension tag: a corresponding embodiment is envisaged where the probe comprises the extension tag).
  • Polymerase extension of a primer using an extension tag as a template may be preferable where the probe or sample nucleic acid does not have an underhanging 3' end on which a polymerase can act.
  • the polymerase enzyme used to extend the sample nucleic acid or probe may be a DNA-dependent DNA polymerase or a DNA-dependent RNA polymerase.
  • the polymerase enzyme used to extend the sample nucleic acid or probe may be an RNA-dependent DNA polymerase.
  • a suitable polymerase enzyme may readily be selected by the skilled person from those commonly used in molecular biology.
  • Polymerase enzymes that may be suitable for use in the invention include, but are not limited to the Klenow fragment of DNA polymerase I, Bst DNA Polymerase, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Vent DNA polymerase, Pfu DNA polymerase.
  • a combination of different polymerases may be used in the analysis step.
  • the analysis step may generally comprise contacting a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe with an enzyme (and an appropriate enzyme reaction mixture), and using the enzyme to extend the sample nucleic acid or probe.
  • Non-enzymatic (chemical methods) may be used to extend a sample nucleic acid or probe in the analysis step, if desired.
  • the analysis step may even more generally comprise contacting a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe with one or more reagents, whereby the reagents extend the sample nucleic acid or probe.
  • an extension tag may act as a template for ligase-mediated extension of the probe.
  • the arrangements shown in Figs. 7A, 7C, 7D, 7F, 7G, 7H, 8A, 8C, 8D, 8F, 8G, 8H can readily be used in conjunction with ligase-mediated analysis methods.
  • a suitable ligase may readily be selected by the skilled person from those commonly used in molecular biology (e.g. E. coli DNA ligase, T4 DNA ligase or Pfu DNA ligase), for example taking into account the nature of the substrates to be ligated. In some embodiments, a combination of different ligases may be used in the analysis step.
  • a T4 RNA ligase may be used in the analysis step.
  • T4 RNA ligase 2 may be used in the analysis step.
  • T4 RNA ligase 2 has a high rate of nick joining for substrates containing various combinations of DNA and RNA (e.g. see reference 13).
  • the nucleotide sequence ligated to the sample nucleic acid or the probe may be longer than the extension tag nucleotide sequence, to further facilitate analysis of the sample nucleic acid (see Fig. 16, which is based on Fig. 4A; a corresponding arrangement is envisaged based on Fig. 4B).
  • the methods of the invention may involve covalent modification of the sample nucleic acid or probe, e.g. using a polymerase or ligase as shown in Figs. 3A-4B. Methods which result in covalent modification of the substrate nucleic acid may be particularly advantageous when used in conjunction with a support, because they allow devices to be washed without loss of signal. Accordingly, in some embodiments, the methods of the invention comprise analysing a sample nucleic acid by using an extension tag as a template for covalent modification of a sample nucleic acid or probe.
  • the methods of the invention may involve hybridising a detectable label to the extension tag and detecting the label, without covalently linking the label to the sample nucleic acid or probe.
  • the methods of the invention may involve arrangements similar to those shown in Figs. 4A and 4B, but without actual ligation. Accordingly, in some embodiments, the methods of the invention do not comprise analysing a sample nucleic acid by using an extension tag as a template for covalent modification of a sample nucleic acid or probe.
  • ligase-mediated extension of a nucleic acid normally requires a phosphate at the 5' end of a nucleic acid, on which the ligase enzyme can act.
  • the methods of the invention may include a step to ensure that a suitable 5' phosphate is present when a sample nucleic acid or extended nucleic acid hybridises to a probe. Such steps may comprise phosphorylating the 5' end of a sample nucleic acid or a probe.
  • the methods of the invention may include a step to remove a terminal phosphate group before of after a nucleic acid or extended nucleic acid hybridises to a probe.
  • steps may comprise de-phosphorylating a sample nucleic acid or a probe (e.g. by alkaline phosphatase treatment).
  • steps may be particularly desirable for some nucleic acid samples, if those samples are known to contain phosphorylated nucleic acids.
  • the molecule ligated to the sample nucleic acid or probe may comprise a repetitive nucleotide sequence, or a homopolymer sequence.
  • the ligated molecule will generally comprise a nucleotide sequence that is partially or completely complementary to the extension tag nucleotide sequence.
  • the ligated molecule may comprise a nucleotide sequence that is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more, nucleotides in length.
  • the ligated molecule may comprise a nucleotide sequence that is fewer than 50, fewer than 45, fewer than 40, fewer than 35, fewer than 30, fewer than 25, fewer than 20, fewer than 15, fewer than 10, fewer than 5, fewer than 4, fewer than 3, or fewer than 2, nucleotides in length.
  • the ligated molecule may comprise a nucleotide sequence, or consist of a nucleotide sequence, that is 2-50, 2-40, 2-30, 2-20, 2- 10, 2-5, 5-50, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30 or 10-20 nucleotides in length.
  • the methods of the invention may involve qualitative and/or quantitative analysis of a sample nucleic acid. Quantitative detection methods are preferred.
  • an extension tag to detect a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe may involve using the extension tag to (covalently or non-covalently) label the nucleic acid-probe hybrid with a detectable label.
  • detectable labels can be selected by the skilled person, depending on the type of analysis required and the visualisation method to be used. Fluorescent labels are preferred for use with the invention.
  • Intercalating dyes may be used for detection of a sample nucleic acid or an extended nucleic acid after hybridisation to a probe. Fluorescence can be excited using an evanescent wave.
  • a device of the invention may include a laser source (and/or a laser detector).
  • Other sources of light for excitation can also be used e.g. lamps, LEDs, etc.
  • Fluorescence resonance energy transfer (FRET) detection methods may also be used. FRET detection methods may in some embodiments increase the signal-to-noise ratio.
  • detection can be achieved by incorporating labelled nucleotides, e.g. fluorescent nucleotides (see for example Figs. 3A, 3B, 4A, 4B, 13, 17 and 18, where the stars represent detectable labels).
  • the methods of the invention may incorporate at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14 or at least 15, labelled nucleotides in the analysis step.
  • Labelled dideoxynucleotides ddNTPs
  • ddNTPs may be used for incorporation of a single labelled nucleotide, if desired.
  • dNTPs with suitable fluorophores attached may be used. Unlike a sequencing reaction, it is not necessary to use different coloured fluorophores for different nucleotides, because individual nucleotides do not need to be distinguished. Similarly, there is no need to label every nucleotide, and so 1 , 2, 3 or 4 of dATP, dCTP, dGTP and dTTP may be labelled, and a mixture of labelled and unlabelled dNTPs can be used. Incorporation of a large number of fluorophores (e.g.
  • incorporated dNTPs in at least 5% of incorporated dNTPs, such as >10%, >20%, >30%, >40%, >50%, >75%, or more) means that the labelled strand can readily be detected by any of the familiar means of fluorescence detection, thus revealing a positive signal even for a single hybridisation event. Thus even low-abundance nucleic acids can be detected.
  • fluorophores Rather than incorporate fluorophores directly, it is also possible to incorporate a specific functional group (e.g. biotin, aminoallyl) to which fluorophores can later be coupled ('post- labelling') e.g. after steps such as reverse transcription, washing, etc.
  • a specific functional group e.g. biotin, aminoallyl
  • Sensitive techniques are available for detection of single fluorophores [15,16], so detection of an individual nucleic acid/probe hybrid containing multiple fluorophores is well within current technological capabilities.
  • Current apparatuses that can identify single fluorophores have a pixel resolution of -150 nm.
  • references 17 & 18 describe a single molecule reader (commercially available as the 'CytoScout' from Upper Austrian Research GmbH) in which a CCD detector is synchronized with the movement of a sample scanning stage, enabling continuous data acquisition to collect data from an area 5mm x 5mm within 1 1 minutes at a pixel size of 129 nm.
  • the methods, devices and kits of the invention allow detection of individual sample nucleic acids, such as individual miRNA molecules.
  • the methods of the invention may involve synthesis of a second nucleic acid strand, using the complement of the extension tag nucleotide sequence (generated as described above) as a template for extension of a primer comprising a sequence corresponding to (i.e. partially or completely identical to) at least part of the extension tag nucleotide sequence.
  • the second nucleic acid strand may also be synthesised to incorporate label, and this label can be the same as or different from the label used to synthesise the complement of the extension tag nucleotide sequence.
  • the primer comprises a sequence that corresponds to the 5' end of the extension tag, so that when the primer hybridises to the extension tag complement its 3' end can be extended by a polymerase (or ligase) using the extension tag as a template.
  • a polymerase or ligase
  • Nucleic acid-probe hybrids may also be detected by amplification, for example by rolling circle amplification (RCA, e.g. references 19 and 20, also references 21 and 22) or multiple displacement amplification (MDA; e.g. references 23 and 24).
  • RCA rolling circle amplification
  • MDA multiple displacement amplification
  • Suitable reagents are commercially available (e.g. from Qiagen Ltd., Crawley).
  • Nucleic acid-probe hybrids may also be detected by chemiluminescence methods. Suitable methods have been reported (e.g. references 25 and 26) and suitable reagents are commercially available (e.g. from Applied Biosystems, Foster City, CA). For example, a sample nucleic acid or probe in a nucleic acid-probe hybrid may be extended using biotinylated dNTPs, and the product detected by applying (strept)avidin-horseradish peroxidase (HRP) or (strept)avidin-alkaline phosphatase (AP) followed by a chemiluminescence substrate, and then image capture.
  • HRP streptavidin-horseradish peroxidase
  • AP strept)avidin-alkaline phosphatase
  • a suitable detection method can be selected in light of the specific binding reagent that is to be used.
  • a secondary antibody may be used to detect (and optionally visualise) binding of the first antibody.
  • using an extension tag to analyse a sample nucleic acid may comprise using the extension tag to manipulate a nucleic acid-probe hybrid formed when a sample nucleic acid or extended nucleic acid hybridises to a probe.
  • an extension tag can be used to facilitate downstream processing or analysis of the nucleic acid-probe hybrid.
  • a device of the invention can be interfaced with a mass spectrometer. Integration of microfluidic devices with mass spectrometry is known.
  • the methods of the invention may involve performing one or more control reactions, to confirm that the method is functioning as expected. Negative and/or positive control reactions may be performed. Appropriate controls can readily be selected by the skilled person.
  • the devices and kits of the invention may therefore comprise one or more reagents suitable for use in performing a negative or positive control reaction, e.g. a nucleic acid that is known to hybridise to the probe under the conditions used for the hybridisation step (for a positive control) a nucleic acid that is known not to hybridise to the probe under the conditions used for the hybridisation step (for a negative control).
  • kits for use in the methods of the invention.
  • kits of the invention may comprise apparatus or reagents for obtaining a sample nucleic acid. Suitable apparatus and reagents are known in the art, so the skilled person can readily choose appropriate apparatus or reagents to include in a kit of the invention.
  • a kit may comprise apparatus or reagents for obtaining sample nucleic acids using the Boom method (guanidinium isothiocyanate/silica extraction; reference 4).
  • a kit may also comprise apparatus or reagents for obtaining an RNA by a glass fibre filter (GFF) or silicate adsorption method.
  • GFF glass fibre filter
  • kits of the invention may comprise a reagent for extending the 5' and/or 3' end of a sample nucleic acid, to generate an extended nucleic acid having an extension tag at its 5' and/or 3' end.
  • a reagent for extending the 5' and/or 3' end of a sample nucleic acid to generate an extended nucleic acid having an extension tag at its 5' and/or 3' end.
  • Suitable reagents are known in the art, and the skilled person can easily choose an appropriate reagent for use in a kit of the invention.
  • a kit may comprise a ligase enzyme or a polymerase enzyme, as described elsewhere herein.
  • kits may also comprise a support (as described elsewhere herein), to which a probe (as described elsewhere herein) is optionally immobilised.
  • kits may comprise a reagent for using the extension tag to analyse a sample nucleic acid if the sample nucleic acid or extended nucleic acid hybridises to the probe.
  • Suitable reagents are known in the art, and the skilled person can easily choose an appropriate reagent for use in a kit of the invention in light of the disclosure herein. Uses
  • the devices and methods of the invention can be used to perform various analyses.
  • the invention therefore provides, in general, the use of a method or device as disclosed herein to analyse a nucleic acid.
  • the invention provides the use of a method or device as disclosed herein to identify the type of cell(s) present in a biological sample (such as sample taken from a patient, or a food sample).
  • a biological sample such as sample taken from a patient, or a food sample.
  • the invention provides a method for identifying the type of cell(s) present in a biological sample, the method comprising analysing a sample nucleic acid obtained from the biological sample using an analysis method as described herein, and using the results of the analysis to identify the type of cell(s) present in the biological sample.
  • the invention provides the use of a method or device as disclosed herein to determine the presence or absence of a diagnostic or prognostic marker in a patient sample.
  • the invention provides a method for determining the presence or absence of a diagnostic or prognostic marker in a patient sample, comprising analysing a sample nucleic acid obtained from the patient sample using an analysis method as described herein, and using the results of the analysis to determine the presence or absence of a diagnostic or prognostic marker in the patient sample. These methods may be performed in vitro on a sample taken from a patient.
  • the invention provides the use of a method or device as disclosed herein to diagnose a disease or condition in a patient (e.g. a human patient).
  • a patient e.g. a human patient
  • the invention provides a method for diagnosing a disease or condition in a patient, comprising analysing a sample nucleic acid obtained from the patient using an analysis method as described herein, and using the results of the analysis to diagnose a disease or condition in the patient.
  • the disease or condition may be a cancer or a viral infection.
  • methods of diagnosis as described herein are performed in vitro on a sample taken from a patient.
  • the invention provides the use of a method or device as disclosed herein to identify a diagnostic or prognostic marker for a disease or condition.
  • the invention provides a method for identifying a diagnostic or prognostic marker for a disease or condition, comprising (i) analysing a sample nucleic acid obtained from patient having the disease or condition using an analysis method as described herein, (ii) analysing a sample nucleic acid obtained from a healthy patient using an analysis method as described herein, (iii) comparing the results of steps (i) and (ii) to identify a diagnostic or prognostic marker for the disease or condition.
  • These methods may be performed in vitro on a sample taken from a patient.
  • the invention provides the use of a method or device as disclosed herein to select a therapeutic strategy or treatment regimen for treating a disease or condition in a patient.
  • the invention provides a method for selecting a therapeutic strategy or treatment regimen for treating a disease or condition in a patient (e.g. a human patient), comprising analysing a sample nucleic acid obtained from the patient using an analysis method as described herein, and using the results of the analysis to select a therapeutic strategy or treatment regimen for treating the disease or condition.
  • a patient e.g. a human patient
  • these methods may be performed in vitro on a sample taken from a patient.
  • the invention provides the use of a method or device as disclosed herein to analyse stem cells, such as human embryonic stem (hES) cells.
  • stem cells such as human embryonic stem (hES) cells.
  • the methods and devices of the invention may be used to determine whether a given cell is a stem cell of a desired type (e.g. to determine whether a cell is a hES cell), or to determine the differentiation state of a stem cell (e.g. to determine whether a cell is an undifferentiated hES cell).
  • the invention provides the use of a method or device as disclosed herein to monitor progression or status of a disease or condition in a patient, e.g. to monitor a patient's response to treatment.
  • the invention provides the use of a method or device as disclosed herein to analyse a sample taken from a human patient, such as a blood sample, a plasma sample, a serum sample, a tissue sample, or a saliva sample.
  • the invention also provides the use of a method or device as disclosed herein for biosurveillance, e.g. to detect pathogens in samples, such as water, food or soil samples.
  • the invention also provides the use of a method or device as disclosed herein to detect the presence or absence of a nucleic acid in a sample, e.g. to detect the presence or absence of a miRNA in a biological sample.
  • the invention also provides the use of a method or device as disclosed herein to analyse a population of cells.
  • the invention also provides the use of a method or device as disclosed herein to analyse a single cell.
  • the invention may be used to analyse sample nucleic acids obtained from formalin-fixed, paraffin-embedded (FFPE) tissue samples.
  • FFPE paraffin-embedded
  • sample nucleic acid an extended nucleic acid
  • probe and equivalent references
  • a probe “comprising” a probe sequence may consist exclusively of the probe sequence or may include something additional e.g. an additional nucleotide sequence.
  • antibody includes any of the various natural and artificial antibodies and antibody-derived proteins which are available, and their derivatives, e.g. including without limitation polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanised antibodies, human antibodies, single-domain antibodies, whole antibodies, antibody fragments such as F(ab') 2 and F(ab) fragments, Fv fragments (non-covalent heterodimers), single-chain antibodies such as single chain Fv molecules (scFv), minibodies, oligobodies, dimeric or trimeric antibody fragments or constructs, etc.
  • the term “antibody” does not imply any particular origin, and includes antibodies obtained through non-conventional processes, such as phage display.
  • Antibodies of the invention can be of any isotype (e.g. IgA, IgG, IgM i.e. an ⁇ , y or ⁇ heavy chain) and may have a K or a ⁇ light chain.
  • Sequences in the sequence listing are RNA. When the sequences are used in DNA form then the U bases will instead be present as T bases. In some embodiments, where appropriate, the reverse complement of a SEQ ID may be used for hybridisation.
  • Fig. 1A illustrates the general approach where an extended nucleic acid comprises an extension tag at its 5' end.
  • the sample nucleic acid is shown in black, and the probe is shown in grey.
  • the arrowheads indicate the 5' to 3' direction of the polynucleotides, and the dotted lines represent the extension tag.
  • Fig. 1 B illustrates the general approach where an extended nucleic acid comprises an extension tag at its 3' end.
  • Fig. 2A illustrates the general approach where a probe comprises an extension tag at its 5' end.
  • Fig. 2B illustrates the general approach where a probe comprises an extension tag at its 3' end.
  • Fig. 3A illustrates the use of an extension tag as a template for polymerase-mediated extension of a probe after hybridisation.
  • Fig. 3B illustrates the use of an extension tag as a template for polymerase-mediated extension of a sample nucleic acid after hybridisation.
  • Fig. 4A illustrates the use of an extension tag as a template for ligase-mediated extension of a probe after hybridisation.
  • Fig. 4B illustrates the use of an extension tag as a template for ligase-mediated extension of a sample nucleic acid after hybridisation.
  • Figs. 5A and 5B illustrate the use of a specific binding reagent after hybridisation.
  • Fig. 6 illustrates a possible approach to enable polymerase-mediated extension of a probe after hybridisation where an extension tag is added to the 3' end of a sample nucleic acid.
  • Fig. 7A illustrates schematically the general approach where a probe is immobilised on a support via its 5' end, and an extended nucleic acid comprises an extension tag at its 5' end.
  • Fig. 7B illustrates schematically the general approach where a probe is immobilised on a support via its 5' end and an extended nucleic acid comprises an extension tag at its 3' end.
  • Fig. 7C illustrates schematically the general approach where a probe is immobilised on a support via its 5' end and the probe comprises an extension tag at its 5' end.
  • Fig. 7D illustrates schematically the general approach where a probe is immobilised on a support via its 5' end and the probe comprises an extension tag at its 3' end.
  • Fig. 7E illustrates schematically the general approach where a probe is immobilised on a support via its 3' end and an extended nucleic acid comprises an extension tag at its 5' end.
  • Fig. 7F illustrates schematically the general approach where a probe is immobilised on a support via its 3' end and an extended nucleic acid comprises an extension tag at its 3' end.
  • Fig. 7G illustrates schematically the general approach where a probe is immobilised on a support via its 3' end and the probe comprises an extension tag at its 5' end.
  • Fig. 7H illustrates schematically the general approach where a probe is immobilised on a support via its 3' end and the probe comprises an extension tag at its 3' end.
  • Fig. 8A illustrates schematically the general approach where a sample nucleic acid is immobilised on a support via its 5' end and a probe comprises an extension tag at its 5' end.
  • Fig. 8B illustrates schematically the general approach where a sample nucleic acid is immobilised on a support via its 5' end and a probe comprises an extension tag at its 3' end.
  • Fig. 8C illustrates schematically the general approach where an extended nucleic acid is immobilised on a support via its 5' end, and wherein the extended nucleic acid comprises an extension tag at its 5' end.
  • Fig. 8D illustrates schematically the general approach where an extended nucleic acid is immobilised on a support via its 5' end, and wherein the extended nucleic acid comprises an extension tag at its 3' end.
  • Fig. 8E illustrates schematically the general approach where a sample nucleic acid is immobilised on a support via its 3' end, and a probe comprises an extension tag at its 5' end.
  • Fig. 8F illustrates schematically the general approach where a sample nucleic acid is immobilised on a support via its 3' end, and a probe comprises an extension tag at its 3' end.
  • Fig. 8G illustrates schematically the general approach where an extended nucleic acid is immobilised on a support via its 3' end, and the extended nucleic acid comprises an extension tag at its 5' end.
  • Fig. 8H illustrates schematically the general approach where an extended nucleic acid is immobilised on a support via its 3' end, and the extended nucleic acid comprises an extension tag at its 3' end.
  • Fig. 9 illustrates schematically the general approach where an extended nucleic acid comprising extension tags at both its 5' and 3' ends is used.
  • Fig. 10 illustrates schematically the general approach where a probe comprising extension tags at both its 5' and 3' ends is used.
  • Fig. 11 illustrates the general approach where an extended nucleic acid comprises an extension tag at its 5' end, and a probe comprises an extension tag at its 5' end.
  • Fig. 12 illustrates the general approach where an extended nucleic acid comprises an extension tag at its 3' end, and a probe comprises an extension tag at its 3' end.
  • Fig. 13 illustrates the concept of using an extension tag nucleotide sequence that is longer than the sample nucleic acid.
  • Fig. 14 illustrates the concept of using a longer probes or a linker to ensure that the extension tag is available for use in the analysis step.
  • Fig. 15 illustrates the concept that the probe or extended nucleic acid might be sufficiently flexible to ensure that the extension tag is available for use in the analysis step.
  • Fig. 16 illustrates an embodiment of the invention where a nucleotide sequence ligated to a sample nucleic acid or a probe (using the extension tag as a template) is longer than the extension tag nucleotide sequence.
  • Fig. 17 illustrates an embodiment of the invention where an extension tag is used as a template for polymerase-mediated extension of a primer.
  • Fig. 18 illustrates the concept of synthesising a second nucleic acid strand, using the complement of an extension tag nucleotide sequence as a template for extension of a primer.
  • Fig. 19A illustrates the probe deposition pattern used in Example 1 .
  • Fig. 19B shows the post-ligation scan image generated in the Example 1 experiments.
  • Fig. 19C shows the average signal intensities across the rectangular regions highlighted by dashed lines in Figure 19B.
  • Fig. 2OA shows the feature images from post-ligation scans in Example 2.
  • Fig. 2OB is a graph of signal intensities from post-ligation scans in Example 2.
  • Example 1 Specific detection of synthetic miRNAs using 3' ligation
  • probe sequences that were used are shown in Table 2. Each of these probes comprises a five nucleotide poly-G extension tag at its 5' end (in bold below) and a linker sequence at its 3' end for attachment to a solid support (underlined below).
  • the ligate sequence that was used is shown in Table 3 below.
  • 1 mM probe stocks were diluted to 100 ⁇ M with a 1 :1 mix of phosphate buffer (pH 9.0) and DMSO.
  • 0.5 ⁇ l of the probe and coupling buffer mix was applied to an NHS derivatised slide (Schott) in 0.5mm 2 patches.
  • the probes were deposited in the pattern shown in Figure 19A.
  • the four blocks of probe patches align with wells 1 , 3, 6 and 8 of an eight well backing plate (Agilent).
  • the coupling reaction was carried out at room temperature for approximately 1 hr. Uncoupled regions of the slide were then blocked with MiIIi-Q water.
  • Hybridisation was carried out in a hybridisation chamber using an eight well backing plate. 60 ⁇ l hybridisation mix was applied to wells 1 , 3, 6 and 8 of the backing plate. Hybridisation mix consisted of 2X Hi-RPM Hybridisation Buffer (Agilent), Nuclease Free Water (Promega) and ⁇ pmoles synthetic miRNA. miRNA-298 was added to well 1 , miRNA-665 was added to well 3, miRNA-574 was added to well 6, and a combination of all three miRNAs were added to well 8. The hybridisation chamber was then assembled according to the manufacturer's instructions. Hybridisation was carried out in a hybridisation oven at 55 0 C and 10rpm for 1 hr.
  • the hybridisation chamber was disassembled in a first wash buffer (reference 27).
  • the array was then washed for 5min in the first wash buffer (reference 27), and then for 5min in a second wash buffer (reference 27) at 37 0 C.
  • the second wash buffer was pre-warmed to 37 0 C before use (reference 27).
  • the ligation reaction was carried out in a hybridisation chamber. 60 ⁇ l of ligation mix was applied to wells 1 , 3, 6 and 8 of a backing plate. The ligation mix contained 1.5 ⁇ l 1OmM Poly A 3' Ligate (Table 3), 6 ⁇ l 10X T4 RNA Ligase 2 Reaction Buffer (NEB), 0.5 ⁇ l T4 RNA Ligase 2 (NEB) and 52 ⁇ l of Nuclease Free Water.
  • the hybridisation chamber was assembled according to the manufacturer's instructions. The reaction was incubated in a hybridisation oven at 37 0 C and 2rpm for 1 hr. The hybridisation chamber was disassembled in a first wash buffer (reference 27) and the array was washed as described above.
  • Figure 19B The array was then scanned using an Agilent scanner to generate the post-ligation image (Figure 19B). Signal intensities were analysed using the rectangular averaging line feature on GenePix Pro 4.1 software.
  • Figure 19C shows graphs of average signal intensities across the rectangular regions highlighted by dashed lines in Figure 19B. The graphs in Figure 19C align vertically with the images in Figure 19B, and the scale on the y-axis is identical for all four graphs.
  • a 1 mM stock of the MMU-MIR-574-5P.3'lig probe (Table 2) was diluted to 100 ⁇ M with a coupling buffer consisting of a 1 :1 mix of phosphate buffer (pH 9.0) and DMSO. 600 ⁇ m diameter features of probe and coupling buffer were spotted on NHS derivatised slides (Schott) using a robot. Spotting was arranged so each feature aligned to the centre of wells from an eight well backing plate (Agilent). Uncoupled regions of the slide were then blocked with MiIIi-Q water.
  • Hybridisation was carried out in hybridisation chambers (Agilent) using eight well backing plates. 50 ⁇ l hybridisation mixes were made, each consisting of 2X Hi-RPM Hybridisation Buffer (Agilent), Nuclease Free Water (Promega) and either l OOfmol, I Ofmol or 1fmol of synthetic MMU-MIR-574-5P (Table 1 ). A negative control hybridisation mix consisting only of 2X Hi-RPM Hybridisation Buffer and Nuclease Free Water was also made. 45 ⁇ l of each hybridisation mix was added to the wells of eight well backing plates. The hybridisation chambers were assembled according to the manufacturer's instructions.
  • Hybridisation was carried out in a hybridisation oven at 55 0 C and 10rpm for 16hr. Hybridisation chambers were disassembled in a first wash buffer (reference 27) at 37 0 C. Arrays were subsequently washed for 10min in the first wash buffer (reference 27) at 37 0 C, and then for 10min in the second wash buffer (reference 27) at 37 0 C. Both the first and second wash buffers were pre-warmed to 37 0 C before use (reference 27).
  • the ligation reaction was carried out in hybridisation chambers. 60 ⁇ l of ligation mix was applied to the wells of eight well backing plates. 60 ⁇ l of ligation mix contained 1.5 ⁇ l 1 OmM Poly A 3' Ligate (Table 3), 6 ⁇ l 10X T4 RNA Ligase 2 Reaction Buffer (NEB), 0.5 ⁇ l T4 RNA Ligase 2 (NEB) and 52 ⁇ l Nuclease Free Water. Hybridisation chambers were assembled according to the manufacturer's instructions. The reaction was incubated in a hybridisation oven at 37 0 C and 2rpm for 1 hr. The hybridisation chambers were disassembled in a first wash buffer (reference 27) and the arrays were washed as described above.
  • Figure 2OA shows the feature images from post-ligation scans at identical image settings.
  • the signal intensities of each feature were extracted using the Feature Extraction software version 10.1.1.1 ( Figure 20B).
  • Figure 2OB is a graph of feature extracted F532 Median - BF534 signal intensities from post-ligation scans. Replicates were carried out on different NHS derivatised slides and processed on the same day.

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Abstract

L’invention concerne des procédés, des dispositifs et des produits associés pour analyser des acides nucléiques, y compris des acides nucléiques courts. Les procédés de l’invention utilisent une étape d’hybridation pour faciliter la discrimination d’acides nucléiques d’intérêt parmi d’autres acides nucléiques. Les procédés de l’invention utilisent un marqueur d’extension pour l’analyse des acides nucléiques d’intérêt. Cette combinaison d’une hybridation et d’une analyse utilisant un marqueur d’extension facilite l’analyse des acides nucléiques, et est particulièrement avantageuse pour l’analyse d’acides nucléiques courts. Elle permet de détecter et de manipuler facilement les acides nucléiques.
PCT/GB2009/001057 2008-04-22 2009-04-22 Analyse d’acides nucléiques WO2009130480A1 (fr)

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WO2011157617A1 (fr) * 2010-06-17 2011-12-22 Febit Holding Gmbh Ensemble complexe de banques de miarn
CN102719433A (zh) * 2011-03-30 2012-10-10 华中农业大学 osa-MIR167a基因在调控水稻株型中的应用
CN103361348A (zh) * 2012-03-29 2013-10-23 中国科学院遗传与发育生物学研究所 与水稻叶片宽度调控相关microRNA及其编码核酸分子与应用
CN105018494A (zh) * 2014-07-10 2015-11-04 上海益诺思生物技术有限公司 mo-miR-374在制备肾毒性生物标志物中的用途
US20170088854A1 (en) * 2009-12-06 2017-03-30 A.B. Seeds Ltd. MicroRNA Compositions and Methods for Enhancing Plant Resistance to Abiotic Stress
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Cited By (11)

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Publication number Priority date Publication date Assignee Title
WO2010107397A1 (fr) * 2009-03-19 2010-09-23 Agency For Science, Technology And Research Modulateurs de l'apoptose et leurs utilisations
US20170088854A1 (en) * 2009-12-06 2017-03-30 A.B. Seeds Ltd. MicroRNA Compositions and Methods for Enhancing Plant Resistance to Abiotic Stress
WO2011157617A1 (fr) * 2010-06-17 2011-12-22 Febit Holding Gmbh Ensemble complexe de banques de miarn
CN102719433A (zh) * 2011-03-30 2012-10-10 华中农业大学 osa-MIR167a基因在调控水稻株型中的应用
CN103361348A (zh) * 2012-03-29 2013-10-23 中国科学院遗传与发育生物学研究所 与水稻叶片宽度调控相关microRNA及其编码核酸分子与应用
CN103361348B (zh) * 2012-03-29 2015-10-14 中国科学院遗传与发育生物学研究所 与水稻叶片宽度调控相关microRNA及其编码核酸分子与应用
CN105018494A (zh) * 2014-07-10 2015-11-04 上海益诺思生物技术有限公司 mo-miR-374在制备肾毒性生物标志物中的用途
CN105018494B (zh) * 2014-07-10 2018-02-02 上海益诺思生物技术股份有限公司 rno‑miR‑374在制备肾毒性生物标志物中的用途
CN107446928A (zh) * 2017-09-25 2017-12-08 南开大学 一个花椰菜器官发育调控miRNA序列及其应用
CN107446928B (zh) * 2017-09-25 2021-02-05 南开大学 一个花椰菜器官发育调控miRNA序列及其应用
WO2019173799A1 (fr) * 2018-03-08 2019-09-12 Caris Science, Inc. Sondes oligonucléotidiques et leurs utilisations

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