WO2004029294A1 - New sequencing method for sequencing rna molecules - Google Patents
New sequencing method for sequencing rna molecules Download PDFInfo
- Publication number
- WO2004029294A1 WO2004029294A1 PCT/SE2003/001499 SE0301499W WO2004029294A1 WO 2004029294 A1 WO2004029294 A1 WO 2004029294A1 SE 0301499 W SE0301499 W SE 0301499W WO 2004029294 A1 WO2004029294 A1 WO 2004029294A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- rna
- nucleotide
- primer
- molecule
- polymerase
- Prior art date
Links
Classifications
-
- 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
- C12Q1/6869—Methods for sequencing
Definitions
- the present invention relates to methods for sequencing RNA. Furthermore, the invention relates to kits for use in the methods of the invention.
- RNA has a central role in molecular biology. For example, it is increasingly recognised that single genes can encode various proteins depending on the processing of the associated mRNAs. It appears that more than half of human genes make more than one protein based on differential splicing/modifications of precursor RNAs. In addition, the sequence of various RNA molecules can be of great value in the identification of organisms, especially micro-organisms. Furthermore there is an increasing interest in the molecular biology of RNA viruses. There is therefore a clear need for effective methods for sequencing RNA.
- RNA sequencing allows researchers to analyse the transcriptome more directly than via hybridization.
- Various methods are available for direct sequencing of RNA (described in more detail below). These are generally based on chemical or enzymatic cleavage, or a modified version of 'Sanger sequencing' as used for DNA. These methods generally employ radioactivity or fluorescence for detection in combination with a separation step, typically electrophoresis. Alterna- tively, mass-spectrometric analysis of RNA fragments or sequence ladders has also been investigated.
- the more common sequencing approaches require retro-transcription steps that generate cDNA molecules, which in turn may not accurately represent the messages (due to misincorporations, truncations etc.).
- RNA sequencing method without separation step would facilitate high throughput and integration into upstream preparation steps.
- a DNA copy of the RNA can be prepared, so-called cDNA, by annealing a DNA oligonucleotide primer to the RNA and extending the primer using a Reverse Transcriptase (RT) polymerase and deoxynucleotides. Depending on the reaction conditions the RT reaction may succeed in creating a full-length copy of the RNA.
- RT Reverse Transcriptase
- This cDNA can then be cloned into a viral or bacterial vector and can be sequenced by cycle-sequencing.
- the cDNA can be used as a template in PCR, which yields large numbers of copies of specific regions of the cDNA that can be sequenced by conventional methods of DNA sequencing.
- RNA shows much higher sensitivity to degra-
- RNA contains no thy- mine, but instead contains the closely related pyrimidine uracil.
- RNA-dependent enzyme capable of operating in the same environment as components required for detection (including nucleotide analogues) and without the risk of degrading RNA by chemical means, by intrinsic RNase activity of the polymerase, or by other, contaminating RNases.
- both MMLV and AMV RT have RNase H activity in addition to pol activity. The RNase H activity competes with the pol activity for the hybrid formed between the RNA template and the DNA primer or growing cDNA strand and degrades the RNA strand of the RNA:DNA complex.
- RNA template that is cleaved by RNase H activity is no longer an effective substrate for cDNA synthesis, decreasing both the amount and size of the cDNA. Sequencing methods, such as sequencing-by-synthesis, based on such this would suffer from reduced read-length or signal intensity.
- RNA is more prone than DNA to form complex secondary structures, which can be expected to compromise the activity of polymerase enzymes, thus demanding strategies for reduction in secondary structures or modifying the polymerase itself.
- endogenous priming a significant amount of non-specific priming (so-called endogenous priming) can occur during reverse transcription regardless of what primers are included in the reaction and that this can be avoided by development of specific reagents (Ambion Inc., USA) .
- the object of the invention is to provide a method for sequencing RNA, which is simple and avoids separation steps, and is thereby also amenable to scaling-up, automation and integration with sample preparation.
- RNA- molecule a nucleotide sequence of a RNA molecule can be analysed in a direct way by sequencing-by-synthesis.
- this aspect of the invention is a development of the PyrosequencingTM method for DNA sequences.
- kits for performing the nucleotide identification of the invention comprising in separate vials a RNA dependent polymerase, nucleotides, necessary enzymes for a sequencing-by-synthesis reaction, and optionally other necessary reagents.
- the invention relates to a method for determining the sequence of a ribo- nucleic acid molecule according to claim 33. Also, the invention refers to a kit for use in this method.
- Figure 1 Extension of a oligo (dT) ⁇ 2 -i 8 primer on a poly(rA) template with standard concentrations of dTTP. Single peaks are obtained after each dispensation corre- sponding to incorporation by the Reverse Transcriptase of one or a few nucleotides before the dTTP is consumed by apyrase.
- Figure 2 As in figure 1 but with a higher concentration of dTTP (added in the G position of the cassette). In this case one large peak is obtained presumably due to the complete extension of the primer along the template by the Reverse Transcriptase in the presence of large amounts of dTTP that apyrase does not fully consume before the end of the reaction. Note the scale is -10 - 170 relative light units.
- Figure 3 As in Figure 1 but with dCTP as added nucleotide. The incorrect nucleo- tide is not incorporated by the Reverse Transcriptase and no signal is obtained.
- Figure 4 Extension of NUSPT primer annealed to the DNA oligonucleotide E3PN19 giving the expected sequence.
- Figure 6 Extension of a oligo (dT) ⁇ 2 . ⁇ 8 primer on a poly(rA) template. Single peaks are obtained after each dispensation corresponding to incorporation by the Reverse Transcriptase of one or a few nucleotides before the dTTP is consumed by apyrase. Note that no incorporation is obtained after dispensing A, C or G.
- Figure 7 Klenow exo ' -mediated extension of a DNA primer on a DNA template by Cy5-SS-dNTP.
- Figure 8 RT-mediated extension of a DNA primer on a RNA template by Cy5-SS- dNTP; signal over background for correct versus incorrect nucleotide.
- Figure 9 RT-mediated extension of a DNA primer on a RNA template by Cy5-SS- dNTP; real-time measurement of FRET-signal.
- Figure 10 Sequencing of the oligonucleotide E3PN19RNA using 60% Cy5-SS- dUTP, and 20% Cy5-SS-dCTP with a final nucleotide concentration of 2 ⁇ M.
- the fluorescent signals from Cy5 on the nucleotide, corrected for background, are plot- ted for each incorporation.
- Figure 11 Selectivity curve for Cy5-SS-dUTP. The fluorescent signal from Cy5 is plotted as a function of the different percentages of Cy5-SS-dUTPs in the reaction mixes.
- nucleotide By “determination of the identity of at least one nucleotide” is meant to identify the type of nucleotide, i.e A, G, C or U, that is present in the position(s) of the RNA template following directly after the 3 '-end of the oligonucleotide primer binding to the RNA template.
- One or more nucleotides in the sequence may be determined simultaneously depending on the presence of a so-called homopolymer stretch of identical bases.
- step 1) is repeated, whereupon the sequence can be deduced from positive incorporation of nucleotides.
- the activated group can be located anywhere on the dNTP molecule; in US 5,302,509, the activated group is attached to the sugar moiety at the 3 '-position, whereas in WO 93/21340, the activated group is attached to the base. Nyren discloses a third strategy in WO 98/13523 and WO98/28440 in which the activation is related to the detection of released pyrophosphate during the primer extension step.
- RNA-molecule any RNA-type, such as mRNA, tRNA, rRNA, snRNA or any other kind of RNA-molecule.
- RNA dependent polymerase any polymerase having the ability to act on a RNA-template, such as RNA dependent DNA polymerases (otherwise known as reverse transcriptases), creating a RNA:DNA duplex, and RNA dependent RNA polymerases, creating a RNA:RNA duplex.
- nucleotides is in the context of the invention meant nucleotides as well as deoxynucleotides, i.e. "building blocks” for both RNA and DNA.
- the chemistry of any of the four nucleotides making up the RNA-strand, i.e. ATP, CTP, GTP or UTP, or any analogues thereof, as well as any of the four deoxynucleotides making up the DNA-strand, i.e. dATP, dCTP, dGTP or dTTP, or any analogue thereof is readily known by a skilled person in the art.
- reaction vessel any kind of reaction vial or the like, that is suitable for a RNA sequencing analysis, such as for example a microtiter plate.
- label is meant a molecule, which is possible to detect in a suitable manner.
- label include fluorescent molecules such as fluorescein, cyanine dyes, like Cy-3, Cy-5, Cy-7, Cy-9 disclosed in U.S. 5, 268,486 (Waggoner et al.) or variants thereof, such as Cy3.5 and Cy5.5, but may also include molecules such as Rhodamine, BODIPY, ROX, TAMRA, Rl 10, R6G, Joe, HEX, TET, Alexa or Texas Red.
- labeled nucleotide or "dye-labeled nucleotide” means a nucleotide, which is connected to a label or dye-label as defined above.
- solid phase is used to define an array or a carrier.
- the term "array” refers to a heterogeneous pool of nucleic acid molecules that is distributed over a support matrix. These molecules, differing in sequence, are spaced at a distance from one another sufficient to permit the identification of discrete features of the array. It may also refer to miniaturised surfaces com- prising ordered immobilized oligonucleotides, DNA or RNA molecules.
- the term “carrier” is used to represent any support for attracting, holding or binding a polynucleotide used within the fields of biotechnology or medicine.
- a carrier can be a carrier, such as a gel, a bead (microparticles), a surface or a fiber.
- gels are acrylamide or agarose; examples of beads are solid beads, which can contain a label or a magnetic compound; beads can also be porous, such as Sepharose beads; a surface can be the surface of glass, a plastic polymer, silica or a ceramic material - these surfaces can be used to prepare so- called "arrays".
- a fiber can be a starch fiber or an optical fiber and even the end of a fiber.
- the invention provides a method for the determination of the iden- tity of at least one nucleotide in a RNA-molecule comprising the steps of:
- step (c) to (d) are repeated.
- the incorporated nucleotide(s) is (are) recorded.
- the presence or absence of incorporation is indicated by the presence of a detectable moiety.
- the detectable moiety may be removed or neutralized in step (d) after the detection.
- step (c) is performed by including a combination of sulfurylase, luciferase and apyrase enzymes in the reaction solution, which together convert the released PPi molecule to a light signal and remove excess ATP and dNTP in preparation for incorporation of the next deoxynucleotide.
- the oligonucleotide primer is a DNA or RNA oligonucleotide.
- the length of this primer is any length that is suitable for the purpose of the invention. However, in many cases a length in the interval of 10 to 30 nucleotides is suitable.
- the primer extension reaction results in the release of a residue molecule, which is detected.
- This residue molecule may for example be a PPi molecule, which is released only upon incorporation of a nucleotide.
- the detection of this PPi molecule may be performed analogous to the PyrosequencingTM reaction for DNA.
- the detection is performed by including a luciferase enzyme, as well as other necessary enzymes, such as apyrase and sulphurylase, and reagents, such as APS and luciferin, in the reaction solution, which upon release of a PPi molecule is triggered to release light.
- a luciferase enzyme as well as other necessary enzymes, such as apyrase and sulphurylase, and reagents, such as APS and luciferin
- At least one nucleotide is labelled, such as fluorescently or radioactively, thereby allowing the detection to be performed by means of detecting the presence or absence of a labelled nucleotide.
- the label on the labelled nucleotide is cleavable.
- the detection is performed by means of detection of a change in physical properties of the RNA-molecule (i.e. the RNA:DNA duplex, or the RNA:RNA duplex) at incorporation of a nucleotide. For example, polarisation changes are detected, or an electronic detection system is used, or some optical changes due to nucleotide incorporation are recorded.
- the RNA dependent polymerase may be an RNA dependent DNA polymerase or an RNA dependent RNA polymerase.
- RNA dependent RNA polymerase it may for example originate from any RNA virus of bacteriophage, such as bacteriophage phi 6.
- the RNA dependent DNA polymerase is Reverse Transcriptase.
- the Reverse Transcriptase (RT) reaction involves extension of a DNA oligonucleotide primer on a RNA template through polymerisation of deoxynucleotides by a RT polymerase and release of pyrophosphate (PPi). It is possible to utilise this PPi in the PyrosequencingTM enzyme cascade in the same way as the PPi released during extension of a DNA oligonucleotide primer on a DNA template by a DNA polymerase.
- RNA template incorporation of a correct deoxynucleotide that is complementary to a ribonucleotide in the RNA template releases a PPi molecule that leads to light release, whereas providing the RT polymerase with an incorrect deoxynucleotide would not result in an incorporation, and thus no sig- nal.
- the signal will be proportional to the number of correct deoxynucleotides incorporated, thus making sequencing of homopolymer stretches possible.
- the RT-reaction used in the invention has been subject to a number of problems.
- the invention provides the following solutions to these problems: (1) Premature termination of primer extension leading to truncated cDNA - this is typically due to low processivity of the enzyme itself and/or secondary structure in the RNA template that causes the enzyme to pause and leave the template.
- Common solutions to this problem include the use of thermostable RT polymerases in combination with increasing the reaction temperature, which leads to a reduction in the secondary structure of the RNA template.
- Additives including glycerol, methyl mercury hy- droxide, methoxyamine-bisulfite and DMSO can be added to help destabilise nucleic acid duplexes and melt RNA secondary structure without inhibiting reverse trancriptases (Gibson et al. 1990; Mazo et al. 1979; Gerard, 1995).spermidine has also been used to improve RT activity (Aoyama, 1989). If RNA amplification methods are first used then it might also be possible to modify secondary structure by in- corporating rITP (see Sasaki et al 1998).
- T4 Gene 32 Protein has been reported to reduce secondary structure in the template (Kreader, 1996; Chandler et al 1998; Villalva et al 2001) and could be included in the RT-mediated sequencing-by-synthesis reaction.
- Other potential solutions include the ability of ret- roviral nucleocapsid protein to unwind RNA (Tanchou et al, 1995), and actinomycin D can prevent hairpin loop formation during cDNA synthesis with AMV RT (Wad- kins et al 2000). Additional oligonucleotides with 3' modifications (making them non-extendable) might be used to block interfering secondary structures at specific positions.
- Reverse trancriptases have a tendency to terminate cDNA synthesis at homo- polymer stretches of RNA (Klarman et al, 1993) that may be reduced by addition of nucleocapsid protein (DeStefano, 1995). Since the position of termination may be enzyme specific (DeStefano et al, 1991) mixes of different RT enzymes may reduce this problem.
- RNAGuard Amersham Biosciences
- RNaseOUT Invitrogen
- Reverse transcription products may be generated even without primers, so called endogenous priming.
- endogenous priming Such problems may be due to contaminating tRNA(Agranovsky 1992) and been overcome using Endo free Reverse transcriptase (Ambion).
- the RT-polymerase of the invention is for example chosen from the group comprising: HIV-1 RT, M-MuLV RT, AMV RT, RAV2 RT, Thermoscript AMV RT, Superscript II M-MuLV RT. Also included in the scope of the invention are any other RT enzymes meeting the demands of the invention as specified below in this application, including Tth DNA polymerase in the presence of Mn 2+ ions.
- RNA dependent polymerases is added to the reaction mixture of step (a).
- RT enzymes are commonly used at high temperatures (37 °C - 55 °C) and at a pH of 8,3-8,4. This is in direct contrast to the PyrosequencingTM reaction that is carried out at 28 °C and a pH of 7,6. It should be noted, however, that the optimal conditions for RT have been chosen to ensure high processivity and extension of the cDNA product over distances of several thousand bases, whereas direct RNA sequencing by RT-mediated sequencing-by-synthesis analyses would demand only extension with 10-100 bases. Thus sub-optimal conditions for RT are in some cases acceptable. Optimisation of the reaction conditions to suit all components in the cascade is possible. Also, some polymerases are thermostable and allow higher temperatures.
- the extension reaction is performed at a temperature ranging from 28 to 70 °C.
- the pH of the extension reaction solution is in the interval from 7.6 to 8.6, preferably from 8.0 to 8.4.
- Deoxynucleotide concentrations used in RT reactions are generally in the range of 0,5 - 1 mM.
- a PyrosequencingTM reaction involves low micro molar concentrations that may improve the fidelity of the reaction by reducing the risk of misincorporation.
- HIV, M-MLV and AMV RT have average processivi- ties of 50-100 nucleotides at dNTP concentrations in the range 25-150 ⁇ M (> K m dNTP ).
- M-MLV RT processivity at 25 ⁇ M dNTP is approximately 70 nt.
- 500 ⁇ M processivity for H " and H + M-MLV is 30 nt.
- An additional subject of optimisation is the balance between supplying the polymerase with deoxynucleotide at a sufficient concentration, and the activity of apyrase that is used to degrade the current deoxynucleotide in preparation for the dispensation of the next deoxynucleotide.
- the concentration of deoxynucleotides is in the interval from 1 ⁇ M to 1 mM.
- a salt is preferably added to the reaction mixture.
- the positive ion in this salt is preferably a monovalent metal ion, such as Li, K or Na.
- the negative ion of this salt is preferably an acetate ion, Ac " .
- the concentration of the salt in the reaction mixture is preferably in the interval from 10 to 100 mM.
- dATP deoxynucleotide
- PyrosequencingTM reaction a substrate for luciferase in the PyrosequencingTM reaction and will therefore give a background signal.
- the solution to this problem has been to exchange dATP for an analogue, alpha-S-dATP that the DNA polymerase can incorporate into the extended primer, but that luciferase cannot use as a substrate.
- the challenge in the RT-mediated PyrosequencingTM reaction is to identify an RT polymerase capable of incorporating such analogues. Indeed data presented here shows that RT can incorporate alpha-S-dATP.
- Alternative approaches include acceptance of the background from dATP but with software- correction of the signal, and/or the use of a mutant form of luciferase that cannot utilise dATP as a substrate.
- the deoxynucleotide dATP is exchanged for the analogue alpha-S-dATP.
- nucleotide ATP is in accordance with the discussion above exchanged for the analogue alpha-S-ATP (or alpha- S-rATP).
- the luciferase enzyme is in a mutant form, which is unable to utilise dATP as a substrate.
- the high level of secondary structure of the RNA template can cause premature truncation of the extending cDNA strand and is generally overcome through an in- crease in reaction temperature and, where possible, the use of thermostable en- zymes.
- Simple additives such as glycerol, methyl mercury hydroxide, meth- oxyamine-bisulfite, spermidine or DMSO can be added to destabilise nucleic acid duplexes and melt RNA secondary structure.
- rlTP can be incorporated when amplifying an RNA molecule to be analysed.
- T4 Gene 32 Protein has been reported to reduce secondary structure in the template and can be included in the RT-mediated sequencing-by-synthesis reaction.
- At least one RNA-secondary structure reducing reagent preferably chosen from the group comprising glycerol, methyl mercury hydroxide, methoxyamine-bisulfite, spermidine, DMSO, incorporation of rlTP (or other rNTP analogue), T4 Gene 32 Protein, retroviral nucleocapsid protein and acti- nomycin D, blocking oligonucleotide, SSB, formamide is included in the extension reaction.
- the luciferase in the PyrosequencingTM reaction is sensitive to CI " ions and this ion is generally replaced by acetate ions when preparing buffers.
- Certain RT polymerases are reportedly capable of operating in these conditions, for example ThermoScript RNase H " Reverse Transcriptase (Invitrogen Corporation, USA).
- RNA Ribonucleic Acid Sequence-Based Amplification
- TMA Transcription- Mediated Amplification
- SSR Self-Sustained Sequence Replication
- Methods such as TMA that are based on RNA transcription can also be used to prepare multiple copies of RNA from a DNA target sequence .All will, of course, yield template suitable for further analysis e.g. se- quencing. Indeed the use of such amplification methods isof great benefit in providing large quantities of high-quality template for analysis.
- the RNA molecule is subjected to a RNA amplification prior to the extension reaction.
- GTP may be exchanged for ITP in this reaction, as discussed above.
- RT polymerases have a RNase H activity and acts as a random endonuclease that digests RNA in RNA:DNA duplexes. This activity will naturally lead to a decrease in the amount of RNA template that can be used for primer extension.
- RT polymerases with low RNase H activity, and even mutants that completely lack this RNase H activity are now available (e.g. ThermoScript RNase H " Reverse Transcriptase and Superscript II RNase H " Reverse Transcriptase (Invitrogen Corporation, USA).
- Tth DNA polymerase has a very efficient intrinsic reverse transcriptase activity in the presence of Mn ions and lacks RNase H activity (Loeb et al, 1973 ; Myers and Gelfand, 1998).
- the RT-polymerase essentially lacks RNase H activity.
- essentially lacks is in the context of the invention meant a RNase H activity lower than 1.0 %, and preferably equal to or lower than 0.5 %.
- the complexity of the RNA population in an isolate leads to challenges in terms of specificity of priming.
- the DNA oligonucleotide primer will most commonly have a sequence designed to anneal only to the region of interest. This level of specificity can be enhanced by prior amplification of the RNA using various methods involving additional, region-specific primers, or by isolating and purifying the RNA of interest using oligonucleotides immobilised on a solid-phase. Indeed the oligonucleotide primer may itself be immobilised on a solid-phase, such as a biotin-streptavidin or biotin-avidin system or covalent immobilisation before or after annealing to the RNA molecule to be analysed.
- a solid-phase facilitates sequencing in complex mixtures, and also changes in buffer composition if RNA amplification is first used to prepare sufficient template for sequencing.
- the solid phase method is based on (1) immobilised oligonucleotide for capture of a specific template, and a separate sequencing primer, or (2) immobilised sequencing primer.
- the oligonucleotide primer is immobilised to a solid phase.
- the quantity of the RNA-molecule is determined by measuring the intensity of the incorporation signal and comparing it to a reference.
- the method of the invention may be used for quantitative purposes, i.e. to analyse the quantity of RNA-template in a sample.
- the invention refers to a kit for performing the nucleotide identification of the invention, comprising in separate vials a RNA dependent polymerase, nucleotides, necessary enzymes for a PyrosequencingTM reaction, and optionally other necessary reagents.
- a kit comprising necessary components and reagents for performing the method of the invention.
- the kit further comprises a RNA quantity reference sample.
- the kit may be used for quantification purposes, i.e. to analyse the quantity of RNA in a sample of interest.
- the invention in another aspect, relates to a method for determining the sequence of a ribonucleic acid molecule comprising the steps of; a) providing a single-stranded form of said ribonucleic acid molecule; b) hybridizing a primer to said single stranded form of said ribonucleic acid molecule to form a template/primer complex; c) enzymatically extending the primer by the addition of an RNA dependent polymerase and a mixture of nucleotides and a derivative of said nucleotides, wherein the derivative of said nucleotide comprises a label linked to a nucleotide via an optionally cleavable link and wherein the proportion in the mixture between the nucleotides and the derivative of said nucleotide is within the range of l-60%,l-50%, 1-40%, 1-30%, or 1-20%, preferably in the range of 5-60%, 5-50%, 5-40%, 5-30%, or 5-20%,
- steps c) to d) above are repeated at least once.
- the reason for using mixtures of nucleotides versus derivative of said nucleotides, is that two phenomena can occur in a reaction according to this aspect of the invention, which phenomena make the dilution of labelled (detectable) nucleotides with natural nucleotides preferable.
- fluorescent quenching occurs when several nucleotides are incorporated due to homopolymer stretches in the template.
- spontaneous cleavage of the S-S-bond can occur in incorporated labelled nucleotides that are in proximity to a previously incorporated and cleaved labelled nucleotide bearing a free thiol group.
- the polymerase enzymes (such as DNA polymerases and Reverse Transcriptases) exhibit a selectivity of natural nucleotides over labelled nucleotides that can differ between enzymes and between nucleotide bases. Hence, the optimum mixtures will vary between nucleotide bases and between enzymes. This explains the use of different mixes in the examples 5-6 below.
- the label is neutralized after step d) by the addition of a la- bel-interacting agent or by bleaching, preferably by photo-bleaching.
- the label can be neutralized by bleaching (photo bleaching) or by adding a compound that neutralizes the emitted fluorescence, such as another label, then reducing the emitted light by quenching.
- cleave off the label from the nucleotide it is preferable to cleave off the label from the nucleotide. This is made possible by using a linker between the nucleotide and label that is cleavable by e.g. a reducing agent.
- a linker between the nucleotide and label that is cleavable by e.g. a reducing agent.
- a method according to the above in which the link between the incorporated nucleotide and the label is cleaved after step d).
- a method according to the above is provided, in which the link between the fluorophore and nucleotide is an S-S bridge.
- the cleavage is performed by the addition of a reducing agent, thereby exposing a thiol group.
- the exposed thiol group is capped with a suitable reagent such as iodoacetamide or N-ethylmaleimide.
- the object of this aspect of the invention may be met by using a linker that is short enough to prevent interaction between adjacent labels.
- the length of the linker between the disulfide bridge and the base of the nucleotide is preferably shorter than 8 atoms.
- the linker between the disulfide bridge and the base is shorter than 8 atoms.
- step c) is performed at a pH 7.6 to 8.6, preferably from pH 8.0 to 8.4.
- the derivative of said nucleotide is a dideoxynucleotide or an acyclic nucleotide analogue.
- a mixture of natural nucleotides and a derivative of said nucleotides wherein the derivative of said nucleotides comprises a label linked to a nucleotide via an optionally cleavable link and wherein the proportion in the mixture between the nucleotides and the derivative of said nucleotides is within the range of 1-60%, 1-50%, 1-40%, 1-30%, or 1-20%, a preferred proportion is in the range of 5-20%, 5-30%, 5-40%, 5-50% or 5-60%, and even more preferred in the range of 10-20%, 10-30%, 10-40%, 10-50% or 10-60%.
- a further variant of this aspect of the invention is a kit which comprises, in separate compartments', a mixture according to previously mentioned aspects, and at least one of the following components; an RNA dependent polymerase, a reducing agent, a carrier, a capping agent, an apyrase, an alkaline phosphatase, a PP-ase, a single strand binding protein or the protein of Gene 32, for performing the method according to any of the steps in the above-mentioned methods.
- the invention also relates to a kit that contains suitable reagents for performing the method of the invention.
- kits which comprises, in separate compartments, at least two of the following components; mixture of labeled and non-labeled nucleoside triphosphates, RNA dependent polymerase, reducing agent, carrier, cap- ping agent, apyrase, single strand binding protein, for performing the method according to any of the above-mentioned embodiments.
- RNA and DNA oligonucleotides are readily commercially available and can be ordered from SGS (Sweden) and Dharmicon (USA).
- RNAguard or similar reagents can be used to protect RNA during the assay.
- ** ⁇ 50 mM reduces activity to 75% of maximum.
- HIV, M-MLV and AMV RT have average processivities of 50-100 nucleotides at dNTP concentrations in the range 25-150 ⁇ M (> K m dNTP ) .
- M-MLV RT processivity at 25 ⁇ M dNTP is approximately 70 nt.
- 500 ⁇ M processivity for H- and H+ M- MLV is 30 nt.
- the basis of the PyrosequencingTM-reaction is as follows: Themethod was developed at the Royal Institute of Technology in Sweden (Ronaghi et al.,1998, Alderborn et al.,2000), and isbased on "sequencing by synthesis" in which the deoxynucleotides are added one by one during the sequencing reaction. An automated sequencer, the PSQ96TM instrument, has recently been launched by Pyrosequencing AB (Uppsala, Sweden). The principle of the PyrosequencingTM reaction for RNA: A single stranded RNA fragment (optionally attached to a solid support), carrying an annealed DNA (optionally an RNA) sequencing primer acts as a template for the PyrosequencingTM reaction.
- RNA-dependent polymerase such as reverse transcriptase (optionally a mix of reverse transcriptases), ATP-Sulfurylase, Lucifer- ase and Apyrase.
- the nucleotide triphosphates are added sequentially according to a specified order dependent on the template and determined by the user. If the added nucleotide triphosphate matches the template, the RT polymerase will incorporate it into the growing DNA(RNA)/RNA-duplex. By this action, pyrophosphate, PP i5 will be released.
- the ATP-Sulfurylase converts the PPi into ATP
- the third enzyme, Luciferase transforms the ATP into a light signal.
- the fourth enzyme, Apyrase will degrade the excess deoxynucleotides and ATP, and the template will at that point be ready for the next reaction cycle, i.e. another nucleotide triphosphate addition.
- Luciferin and APS are substrates for the reaction. Since no PPi is released unless a deoxynucleotide is incorporated, a light signal will be produced only when the correct nucleotide is incorporated.
- the software steering the PSQ 96 system will present the results as peaks in a pyrogramTM, where the height of the peaks corresponds to the number of deoxynucleotides incorporated.
- SSB single-stranded nucleic acid binding protein
- RNA sequencing-by-synthesis of RNA is based on the use of labelled nucleotides.
- a cleavable linker arm for example a disulfide bridge
- DNA polymerase can be used by DNA polymerase to extend a DNA oligonucleotide annealed to a DNA template.
- WO 00/53812 and WO 00/50642 describe the use of a nucleotide where a disulfide-containing linker is used for coupling a dye to the nu- cleotide. This enables easy removal of the dye by redox cycling.
- RNA-dependent polymerase In the method presented here a reverse transcriptase or other RNA-dependent polymerase is used to incorporate a mixture of labelled and non-labelled nucleotides onto the DNA primer annealed to a RNA template. Unincorporated nucleotide is removed and the fluorescence of any incorporated nucleotides is measured. The fluorescent label is then cleaved from the incorporated labelled nucleotides by a re- ducing agent, such as dithiothreitol. The process can then be repeated with other nucleotides to determine the sequence of the template.
- a re- ducing agent such as dithiothreitol
- the labelled nucleotides are diluted with unlabelled nucleotides to avoid fluorescent quenching and also chemical interactions between the free thiol groups of cleaved, incorporated nucleotides, and neighbouring uncleaved labelled nucleotides, as described elsewhere in this document.
- the plate was then placed in a PSQ96 Instrument that dispensed automatically Enzyme Mix minus DNA polymerase (i.e. Sulphurylase, Luciferase and Apyrase) and Substrate (APS and luciferin) mixes followed by nucleotides.
- the nucleotides were (1) a standard concentration of dTTP giving a final concentration in the well of 2.2 ⁇ M immediately after each dispensation, (2) a 5 Ox concentrated dTTP giving a final concentration in the well of 100 ⁇ M immediately after each dispensation, and (3) a standard concentration of dCTP giving a final concentration in the well of 1.8 ⁇ M immediately after each dispensation.
- a DNA control consisting of 10 pmoles E3PN19 to which an excess of 30 pmoles NUSPT primer was annealed by incubating in 200 ⁇ L Annealing Buffer (20 mM Tris-acetate, pH 7.7, 5 mM magnesium acetate) at 65 °C for 5 minutes and then cooling to room temperature. Forty microlitres (2 pmoles) of this was used in the control well.
- Annealing Buffer (20 mM Tris-acetate, pH 7.7, 5 mM magnesium acetate
- RNA test template consisting of 100 pmoles E3PN19RNA, an RNA with the same sequence as E3PN19b, to which an excess of 300 pmol NUSPT primer was annealed by incubating in 200 ⁇ L water at 65 °C for 5 minutes and then cooling to room temperature. Twenty microlitres (10 pmoles of template) of this was used in the test well.
- E3PN19 or E3PN19RNA give a duplex with NUSPT such that the extension of the primer will give the following sequence :
- RNA/DNA duplex consisting of oligo (dT) ⁇ 2- ⁇ 8 annealed to poly (rA) (Amer- sham Biosciences). Approximately 10 pmoles of this was used in the test well.
- the wells were prepared according to the table below, made up to 40 ⁇ L with water.
- PyrosequencingTM reagents were standard products except that Klenow DNA polymerase exo- was omitted from the Enzyme Solution.
- the plate was then placed in a PSQ96 Instrument that dispensed automatically Enzyme Mix minus DNA polymerase (i.e. Sulphurylase, Luciferase and Apyrase) and Substrate (APS and luciferin) mixes followed by nucleotides.
- the nucleotides dispensed in a cyclic fashion in the order CTAG.
- NUSPT f luorescein-GT7 ⁇ 7A7AACGACGGCCAGTUCAGACGAA
- the beads were transferred to a filter plate (Multiscreen, Millipore) and washed four times with 2xAB (40 mM Tris-acetate, 10 mM MgAc 2 , pH 7.6).
- the filter plate was pre-warmed at 37 °C for 2 minutes.
- the first base was incorporated by adding 50 ⁇ L Reaction Mixture (0.5 ⁇ M Cy5-SS-dUTP, 0.5 ⁇ M dUTP, 5 U Klenow exo " , 2xAB) and incubating at 37 °C for 2 minutes.
- the beads in the wells of the filter plate were washed four times with TENT ( 40 mM Tris-HCl pH 8.8, 50 mM NaCl, 1 mM EDTA, 0.1% Tween 20) under vacuum.
- the beads were resuspended in 50 ⁇ l TENT and transferred to a fluorimeter plate to a fluorimeter plate to measure the fluorescence of the Cy 5 -labelled nucleotide (excitation 590 nm, emission 670 nm) and the fluorescence of the fluorescein-labeled primer (excitation 485nm, emission 535 nm) using a fluorimeter (Victor2, Perkin- Elmer).
- the fluorescein signal was used to normalize results for variation in transfer of beads.
- the beads were transferred back into the filter plate and the Cy 5 -label was cleaved from the incorporated dUTP by incubation with Cleavage Buffer (250 mM dithiothreitol, 50 mM NaCl, 40 mM Tris-HCl, 20 mM MgCl 2 , pH 8.4) for 3 minutes at 37°C.
- Cleavage Buffer 250 mM dithiothreitol, 50 mM NaCl, 40 mM Tris-HCl, 20 mM MgCl 2 , pH 8.4
- Cy5-SS-dNTPs were incorporated in the same manner as the first and cleaved as described above.
- the sequencing reaction mixes were the same for all four deoxynucleotides except for the proportion of labeled dNTPs.
- the mixes con- tained 20% Cy5-SS-dCTP, 30% Cy5-SS-dATP or 30% Cy5-SS-dGTP with the balance made up with the corresponding natural deoxynucleotide.
- RNA dependent polymerase such as a Reverse Transcriptase or more preferably Superscript II
- Example 4 Reverse transcriptase-mediated extension of a DNA primer on a RNA template by Cy5-SS-dNTP
- the sequences (5'-> 3') of the E3PN19RNA and fluorescein-labelled (FL) NUSPT oligonucleotides used in these experiments are shown below with the position of the primer site on the template underlined.
- RNA template E3PN19RNA
- 15 pmol of the complementary fluorescein-labelled DNA primer, FL-NUSPT were annealed in 5 ⁇ L water by incubating at 65 °C for 5 minutes and then cooling to room tempera- ture.
- the level of Cy5-SS-dNTP incorporated was measured by Fluorescence Resonance Energy Transfer (FRET). Measurements were performed in a fluorimeter (Victor2, Perkin-Elmer) by exciting the fluorescein on the primer at 485 nm and measuring the resonance transfer signal from any Cy5 incorporated onto the primer at 670 nm, the emission wavelength for Cy5. The results are shown in Figure 8 and clearly show that the correct nucleotide (U) gave a signal over background (absence of RT) whilst the incorrect nucleotide (C) did not.
- FRET Fluorescence Resonance Energy Transfer
- Reagents were treated with diethylpyrocarbonate, RNaseZap (Ambion) or RNAse- cure (Invitrogen) to remove RNases where necessary.
- the biotinylated oligonucleotide primer NUSPT was annealed to the RNA oligonucleotide template E3PN19RNA by incubating 40 pmole (2 pmole per replicate) NUSPT-B with 120 pmole E3PN19RNA (6 pmole per replicate) in 400 ⁇ L Anneal- ing Buffer (20 mM Tris-acetate, 5 mM MgAc 2 , pH 7.6) at 60 °C for 5 minutes and cooled to room temperature.
- the biotinylated primer annealed to the template was then captured on a solid-phase by incubating with 500 ⁇ L Binding Buffer (10 mM Tris-HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, 0.1% Tween 20) and 80 ⁇ L Streptavidin Sepharose High Performance (Amersham Biosciences) and shaking for 20 minutes.
- Binding Buffer 10 mM Tris-HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, 0.1% Tween 20
- the beads were then washed 4 times with 400 ⁇ L TE (10 mM Tris, 1 mM EDTA, pH 8.0) in filter tubes (Nanosep MF GHP 0.45 ⁇ m, Pall), resuspended in 500 ⁇ L TE and 25 ⁇ L aliquots (corresponding to 2 pmole NUSPT-B :E3PN19RNA) were transferred to the wells of a filter plate (MultiScreen; Millipore) and drained by applying vacuum.
- TE 10 mM Tris, 1 mM EDTA, pH 8.0
- reaction mixtures were as follows:
- C- and C+ 0.4 ⁇ M Cy5-SS-dCTP; 1.6 ⁇ M dCTP; 40 U RNaseOUT (Invitrogen); lx Reaction buffer as supplied with the RT enzyme (giving final concentrations of 50 mM Tris-HCl, pH 8.3 at room temperature, 75 mM KC1 and 3 mM MgCl 2 ); 100 U Superscript II RNase H " Reverse Transcriptase (Invitrogen) was included in C+.
- U- and U+ 1.2 ⁇ M Cy5-SS-dUTP; 0.8 ⁇ M dTTP; 40 U RNaseOUT (Invitrogen); lx Reaction buffer as supplied with the RT enzyme (giving final concentrations of 50 mM Tris-HCl, pH 8.3 at room temperature, 75 mM KC1 and 3 mM MgCl 2 ); 100 U Superscript II RNase H " Reverse Transcriptase (Invitrogen) was included in U+.
- a fluorescence control consisting of 200 ⁇ L TE was also included.
- NASBA isothermal enzymatic in vitro nucelic acid amplification optimised for the diagnosis of HIV- 1 infection. J.Virol. Methods (35) 273-286.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/529,352 US20060166203A1 (en) | 2002-09-27 | 2003-09-26 | New sequencing method for sequencing rna molecules |
AU2003265185A AU2003265185A1 (en) | 2002-09-27 | 2003-09-26 | New sequencing method for sequencing rna molecules |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US41375202P | 2002-09-27 | 2002-09-27 | |
US60/413,752 | 2002-09-27 | ||
SE0202867-8 | 2002-09-27 | ||
SE0202867A SE0202867D0 (en) | 2002-09-27 | 2002-09-27 | New sequencing method |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2004029294A1 true WO2004029294A1 (en) | 2004-04-08 |
Family
ID=32044763
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE2003/001499 WO2004029294A1 (en) | 2002-09-27 | 2003-09-26 | New sequencing method for sequencing rna molecules |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2003265185A1 (en) |
WO (1) | WO2004029294A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008073890A2 (en) * | 2006-12-12 | 2008-06-19 | Helicos Biosciences Corporation | Buffer composition |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4862849A (en) * | 1986-08-07 | 1989-09-05 | Wilson Dallas W | RPM activated, powered limiter for pressure time vehicle engine fuel systems |
WO1990013666A1 (en) * | 1989-05-11 | 1990-11-15 | Amersham International Plc | Sequencing method |
WO1998044152A1 (en) * | 1997-04-01 | 1998-10-08 | Glaxo Group Limited | Method of nucleic acid sequencing |
WO2000043540A1 (en) * | 1999-01-22 | 2000-07-27 | Pyrosequencing Ab | A method of dna sequencing |
WO2000050642A1 (en) * | 1999-02-23 | 2000-08-31 | Caliper Technologies Corp. | Sequencing by incorporation |
WO2000053812A2 (en) * | 1999-03-12 | 2000-09-14 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
WO2001094546A2 (en) * | 2000-06-02 | 2001-12-13 | Dna Sciences, Inc. | Primer extension using a mixture of labelled and unlabelled nucleotides |
WO2002020836A2 (en) * | 2000-09-07 | 2002-03-14 | Pyrosequencing Ab | Method of sequencing dna |
WO2002020837A2 (en) * | 2000-09-08 | 2002-03-14 | Pyrosequencing Ab | Method of nucleic acid typing or sequencing |
-
2003
- 2003-09-26 WO PCT/SE2003/001499 patent/WO2004029294A1/en not_active Application Discontinuation
- 2003-09-26 AU AU2003265185A patent/AU2003265185A1/en not_active Abandoned
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4862849A (en) * | 1986-08-07 | 1989-09-05 | Wilson Dallas W | RPM activated, powered limiter for pressure time vehicle engine fuel systems |
WO1990013666A1 (en) * | 1989-05-11 | 1990-11-15 | Amersham International Plc | Sequencing method |
WO1998044152A1 (en) * | 1997-04-01 | 1998-10-08 | Glaxo Group Limited | Method of nucleic acid sequencing |
WO2000043540A1 (en) * | 1999-01-22 | 2000-07-27 | Pyrosequencing Ab | A method of dna sequencing |
WO2000050642A1 (en) * | 1999-02-23 | 2000-08-31 | Caliper Technologies Corp. | Sequencing by incorporation |
WO2000053812A2 (en) * | 1999-03-12 | 2000-09-14 | President And Fellows Of Harvard College | Replica amplification of nucleic acid arrays |
WO2001094546A2 (en) * | 2000-06-02 | 2001-12-13 | Dna Sciences, Inc. | Primer extension using a mixture of labelled and unlabelled nucleotides |
WO2002020836A2 (en) * | 2000-09-07 | 2002-03-14 | Pyrosequencing Ab | Method of sequencing dna |
WO2002020837A2 (en) * | 2000-09-08 | 2002-03-14 | Pyrosequencing Ab | Method of nucleic acid typing or sequencing |
Non-Patent Citations (3)
Title |
---|
BLANCHARD C.L. ET AL.: "Cucumber mosaic Virus RNA 5 Is a Mixed Population derived from the Conserved 3'-terminal regions of Genomic RNAs 2 and 3", VIROLOGY, vol. 217, no. 2, 1996, pages 598 - 601, XP002976879 * |
PIRRUNG M.C. ET AL.: "Solid-Phase, single Nucleotide primer extension of DNA/RNA Hybrids by reverse Transcriptases", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 11, no. 8, 2001, pages 2437 - 2440, XP002976878 * |
SASAKI N. ET AL.: "Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate", GENE, vol. 222, no. 1, 1998, pages 17 - 24, XP004151160 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008073890A2 (en) * | 2006-12-12 | 2008-06-19 | Helicos Biosciences Corporation | Buffer composition |
WO2008073890A3 (en) * | 2006-12-12 | 2008-12-11 | Helicos Biosciences Corp | Buffer composition |
Also Published As
Publication number | Publication date |
---|---|
AU2003265185A1 (en) | 2004-04-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060166203A1 (en) | New sequencing method for sequencing rna molecules | |
JP4638388B2 (en) | Probe and method for hepatitis C virus typing using single probe analysis | |
EP1159454B1 (en) | Polynucleotide sequencing method | |
JP3903059B2 (en) | Oligonucleotide primers for HCV nucleic acid amplification | |
EP2722403B1 (en) | Method for preventing high molecular weight products during amplification | |
JP6316185B2 (en) | Methods for sequencing, amplification and detection of nucleic acids containing internally labeled primers | |
CN113748216A (en) | Single-channel sequencing method based on self-luminescence | |
KR20120020067A (en) | Kit for detecting hepatitis c virus and method for detecting hepatitis c virus using the same | |
JP5692954B2 (en) | Assay for detecting and quantifying HIV-1 | |
JP2024032995A (en) | Compositions and methods for amplifying or detecting varicella zoster virus | |
US8222389B2 (en) | Method for lowering both sequence variations and increase of base line effects in a diagnostic hybridisation assay, assay for performing such a method and probe for use in the assay | |
WO2021031109A1 (en) | Method for sequencing polynucleotides on basis of optical signal dynamics of luminescent label and secondary luminescent signal | |
JP2022079726A (en) | Compositions and Methods for Detecting or Quantifying Hepatitis B Virus | |
US20050244813A1 (en) | Detection of human papillomavirus e6 mrna | |
WO2004029294A1 (en) | New sequencing method for sequencing rna molecules | |
CN108291252B (en) | General method for stabilizing specific RNA | |
CN114286867B (en) | Method for sequencing polynucleotide based on luminous marker optical signal dynamics and secondary luminous signal | |
KR102226356B1 (en) | Primers for detecting Dengue virus by LAMP | |
US20160273036A1 (en) | Photoinduced electron transfer (pet) primer for nucleic acid amplification | |
US20220064726A1 (en) | Method for detecting polymerase incorporation of nucleotides | |
EP1253206A2 (en) | Method of amplifying or detecting hiv-1 rna | |
KR20130092359A (en) | Oligonucleotide sets for detection of human papillomavirus |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase | ||
ENP | Entry into the national phase |
Ref document number: 2006166203 Country of ref document: US Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10529352 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 10529352 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |