WO2011034895A1 - Compositions, procédés et utilisations impliquant des analogues de nucléotides - Google Patents

Compositions, procédés et utilisations impliquant des analogues de nucléotides Download PDF

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WO2011034895A1
WO2011034895A1 PCT/US2010/048894 US2010048894W WO2011034895A1 WO 2011034895 A1 WO2011034895 A1 WO 2011034895A1 US 2010048894 W US2010048894 W US 2010048894W WO 2011034895 A1 WO2011034895 A1 WO 2011034895A1
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triphosphate
diaza
dna
nucleic acid
tctp
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PCT/US2010/048894
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Robert D. Kuchta
Gudrun Stengel
Milan Urban
Byron W. Purse
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Regents Of The University Of Colorado
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/23—Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/24—Heterocyclic radicals containing oxygen or sulfur as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • Embodiments herein report methods, compositions and uses for nucleotide analogues. Embodiments also generally report methods, compositions and uses of nucleotide analogues for generating detectible nucleic acid sequences. In certain embodiments, compositions and methods herein report synthesis of nucleic acid sequences of use for detection, amplification, and/or diagnosis. Certain embodiments report compositions and methods for amplification of target molecules. In other embodiments, compositions, methods and uses concern generating and using fluorescent cytosine analogues.
  • nucleic acids with detectible markers or agents.
  • Some applications for these labeled molecules include the visualization of PCR products, the analysis of base mutations, transcription and gene expression, in situ hybridization and four-color sequencing.
  • tagged nucleotides can serve as analytical tools for example, for diagnosis or treatment of medical conditions, nucleotide cytochemistry and RNA aptamer development.
  • compositions herein report generating highly fluorescent nucleic acids using nucleoside triphosphate analogues.
  • Embodiments included herein also generally report methods, compositions and uses of nucleotide analogues for generating detectible nucleic acid sequences.
  • compositions and methods herein concern synthesis of traceable nucleic acid sequences of use for detection, amplification, and diagnosis.
  • Certain embodiments relate to compositions and methods for amplification of target molecules.
  • compositions, methods and uses concern generating and using fluorescent cytosine analogues.
  • Some embodiments herein concern generating tagged ribonucleotide analogues.
  • ribonucleotide analogues can be used as substrates for generating R A having a detectible nucleotide.
  • Other embodiments concern uses for tagged or detectible DNA molecules using deoxyribonucleotide analogues incorporated into the molecules of interest.
  • compositions and methods disclosed herein can be used to generate detectible nucleic acid molecules using novel agents disclosed herein in combination with various polymerases and/or other reagents.
  • a large detectible probe may be generated using compositions and methods disclosed herein.
  • a large probe can be 100 bases or greater; 200 bases or greater or more.
  • Detectible probes generated herein may be used for a variety of purposes including, but not limited to, experimental purposes (e.g. isolating or tracking target molecules), medical diagnosis, disease prognosis, disease staging (e.g. cancer or other disease), labeling and detection based on specificity of the probe and/or labeling and detection based on non-specific uses of large probes.
  • Some embodiments concern applications for target molecules that include, but are not limited to, visualization of amplification products, analysis of base mutations, transcription and gene expression, in situ hybridization and four-color sequencing.
  • tagged nucleotides can serve as analytical tools including, but not limited to, diagnosis or treatment of medical conditions, nucleotide cytochemistry and RNA aptamer development. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
  • Figs. 1A-D represent comparisons between different DNA polymerases and different denaturation conditions.
  • Figs. 2 A and 2B represent electrophoretic gel analysis of PCR reactions catalyzed by a polymerase at different mixing ratios of cytosine and cytosine analogues.
  • Figs. 3A and 3B represent electrophoretic gel analysis of time courses for primer elongation past tCo-G and G-tCo base pairs.
  • FIGs. 4A and 4B illustrate dequenching of fluorescence upon enzymatic digestion of PCR products.
  • Fig. 5 illustrates a gel shift assay demonstrating modulation in molecular mass
  • Fig. 6 represents an exemplary electrophoresis gel demonstrating incorporation of a tagged ribonucleotide.
  • Fig. 7 represents an exemplary electrophoresis gel demonstrating competitive incorporation of ribonucleotides.
  • Fig. 8 represents an exemplary electrophoresis gel demonstrating competitive incorporation of ribonucleotides at various concentrations.
  • Figs. 9 A and 9B represent large fluorescent RNA generated by transcription in the presence of CTP and tCTP at different concentrations. 9A and 9B represent nucleotides generated from two different sources.
  • Tagged nucleotides can be useful analytical tools that can include, but are not limited to, diagnosis or treatment of medical conditions, cytochemistry and tagging aptamers. Certain embodiments concern fluorescent DNA of high molecular weight as a tool for studying physical properties of DNA, DNA-RNA interactions and DNA-protein interactions. Other embodiments concern generating tagged RNA molecules of use as analytical tools as well as research purposes. In addition, tagged nucleic acid molecules can play an important role in modern biotechnology for DNA sequencing, analysis and detection. While several DNA polymerases were previously found capable of incorporating large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by PCR (polymerase chain reaction) has been problematic.
  • nucleic acid molecules e.g. single-stranded, double-stranded
  • RNA/DNA hybrid nucleic acid/protein or peptide molecules of use in biological methods.
  • methods herein concern incorporating fluorescent analogues into DNA molecules.
  • methods may include PCR reactions using cytosine analogues.
  • Cytosine analogues may include, but are not limited to, 5 '-triphosphate of 1, 3-diaza-2-oxo-phenoxazine (tCo), (l,3-diaza-2-oxo-thiazine) triphosphate (d(tC)TP) and/or 1,3- diaza-2-oxophenothiazine-ribose-5 '-triphosphate (tCTP).
  • one or more cytosine analogues may be used to generate a labeled nucleic acid molecule of interest.
  • Co is a fluorescent cytosine analogue that absorbs and emits light at about 365 and about 460 nm, respectively.
  • amplification products were fluorescent enough to visualize them in a gel by excitation with long UV light, thus eliminating the need for staining.
  • one or more DNA polymerases known in the art may be used to generate an amplified product.
  • Deep Vent polymerase can be used alone or in combination in a reaction to generate amplified products. Because tCo can substitute for cytosine, for example, as a structurally similar molecule, labeling with these modified molecules can be less invasive than labeling with dye- linked nucleotides or radio label-linked nucleotides. Some embodiments relate to generating nucleic acid molecules suited for biophysical studies.
  • compositions and methods can include one or more analogues to dCTP or CTP alone or incombination with control dCTP and/or CTP.
  • Some embodiments can include different ratios of analogues to dCTP or CTP in compositions and methods disclosed herein.
  • ratios of analogues contemplated herein may include, but are not limited to 1 : 1, 1 : 1.5, 1 : 1.67, 1 :2, 1:3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8 etc.
  • Other ratios may include 1 : 19, 1 :29, 1 :39 or other predetermined ratio for a composition contemplated herein.
  • some ratios may include more analogue than control dCTP or CTP depending on the conditions.
  • traceable agents such as fluorescent probes can serve many purposes.
  • fluorescent probes can serve as a environmentally friendly alternative to for example, radioactive markers.
  • they can offer a range of physical properties that have inspired novel technologies for nucleic acid analysis.
  • fluorescent probes can be used for real-time reaction probes in order to generate large quantities of labeled nucleic acids capable of being turned off or on due to for example, distance from a quenching agent or interruption in the sequence with an
  • fluorescent probes can serve as PCR probes that have been designed to switch between bright and dark states (detectable and undetectable states) in response to distance dependent fluorescent quenching or to intercalation of dyes into duplex DNA, R A or hybrid molecules (e.g. R A/DNA, PCA or other hybrid molecules).
  • methods can be used for detection of single nucleotide polymorphisms that exploit spectral sensitivity of certain fluorescent probes to a specific base context.
  • fluorescent probes can be used as a less invasive method for assessing a medical condition in a subject than for example, using a radioactive indicator.
  • single molecule sequencing includes replacing one, two, three or all four nucleobases up to 100 % of the molecule by a fluorescent reporter in order to create a color-coded (e.g. fluorescent) nucleic acid sequence (e.g. DNA, RNA).
  • a color-coded DNA sequence can be generated and then can be read backwards by digestion (e.g. exonuclease digestion) or forward by stepwise DNA synthesis.
  • Some embodiments include other methods in molecular biology that can take advantage of fluorescent markers for generating detectible nucleic acid molecules.
  • compositions and methods herein can include visualization of nucleic acid amplification products, analysis of base mutations, transcription and gene expression, in situ hybridization, and four-color DNA sequencing.
  • nucleic acid synthesis methods may be used where a denaturation step of about high temperatures are included which can result in no loss of enzyme activity and generation of highly fluorescent probes detectible by excitation/visualization, (e.g excitation at 365 nm). In accordance with these embodiments, no staining is required for detection of the highly fluorescent probe.
  • highly fluorescent molecules generated by compositions and methods disclosed herein may be used in disease diagnosis, progression and prognosis in a subject. In accordance with these methods, other more invasive techniques may be avoided as well as avoiding the use of potentially carcinogenic agents/dyes.
  • B family polymerases can include, but are not limited to, DNA polymerase a, DNA polymerase ⁇ , DNA polymerase ⁇ , RB 69 DNA polymerase, DNA polymerase II (e.g. E. coli and other eubacteria), herpes DNA polymerase, T4 DNA polymerase, Phi29 DNA polymerase, Tgo DNA polymerase (thermostable), Pfu DNA polymerase (thermostable), Vent DNA polymerase, Deep vent DNA polymerase and other polymerases known in the art.
  • B family DNA polymerases are defined by a series of conserved amino acid sequences. A B family DNA polymerase may contain all or a subset of these sequences. In certain embodiments,
  • compositions and methods disclosed herein can include one or more polymerases in a given nucleic acid synthesis reaction.
  • one polymerase may catalyze a reaction more readily than another.
  • Vent DNA polymerase may be used under conditions where other polymerases may not perform as efficiently or effectively.
  • Other compositions and methods disclosed herein may use two or more polymerases in order to induce synthesis of a nucleic acid sequence relative to a composition with only one polymerase.
  • a Family Polymerases A Family Polymerases
  • compositions including one or more
  • a Family polymerases can contain both replicative and repair polymerases. Replicative members from this family can include, but are not limited to, T7 DNA polymerase, as well as the eukaryotic mitochondrial DNA Polymerase ⁇ among others. Repair polymerases can include, but are not limited to, E. coli DNA pol I, Thermus aquaticus pol I (e.g. heat-resistant enzyme Taq DNA Polymerase), Bacillus
  • reagent formulations are disclosed that may be more suited for use with one or more polymerases versus other reagent formulations.
  • Other embodiments may include reagent supplementation or substitution depending, for example, on the polymerase chosen for a particular target nucleic acid synthesis.
  • betaine may be replaced in sequencing reactions under certain synthesis conditions.
  • a reagent combination used in the presence of the DNA polymerase Taq may include dtCoTP at various concentration ratios with control dCTPs and/or dtCTPs in the presence of one or more of manganese chloride (MnCl 2 ) and magnesium chloride. (MgCl 2 ).
  • nucleic acid synthesis compositions can include betaine without manganese chloride in a reaction having Taq polymerase and at least one fluorescent nucleotide to produce a target nucleic acid sequence.
  • embodiments may include various DMSO concentrations, alone or in combination with one or more of betaine, MnCl 2 and MgCl 2 or other reagent, in nucleic acid synthesis compositions such as those used in PCR reactions or other synthesis methods known in the art.
  • nucleic acid synthesis compositions may be void of glycerol in the presence of one or more polymerases.
  • nucleic acid synthesis compositions using Taq polymerase may contain betaine as one reagent at
  • RNA cytochemistry may be used by introducing fluorescent RNA into cells using, for example, microinjection or in vivo hybridization of fluorescent nucleic acids to endogenous RNAs.
  • a transit path of the labeled RNA can be visualized.
  • visualization may be by fluorescent microscopy or other fluorescent detecting device.
  • DNA, RNA or hybrid aptamers, short nucleic acids that bind ligands with high affinity and specificity, can be generated using tagged or labeled nucleotides disclosed herein. Aptamers are comparable to antibodies.
  • RNA aptamers have been used in diagnostic assays, including, but not limited to, ELISA, Western blotting, microarrays, capillary electrophoresis and flow cytometry.
  • fluorescent RNA aptamers can be synthesized to various lengths by direct solid-phase synthesis.
  • the fiuor can be attached to a terminal position to avoid potential negative effects of internal labeling on the aptamer activity.
  • approaches have been developed to attach an additional RNA domain to the aptamer of interest, such as malachite green, which becomes fluorescent in response to ligand binding.
  • labeling of longer RNA can be accomplished enzymatically by transcription with an RNA polymerase such as, T7 RNA polymerase, in the presence of fluorescent ribonucleotides or of initiator nucleotides, such as guanosine monophosphothioate.
  • an RNA polymerase such as, T7 RNA polymerase
  • fluorescent ribonucleotides or of initiator nucleotides, such as guanosine monophosphothioate can results in site-specific modification of the 5 '-end of the molecule that can be rendered fluorescent via subsequent coupling to a fluorescent dye.
  • methods herein concern incorporating fluorescent analogues into other RNA molecules.
  • methods may include reactions capable of incorporating a ribonucleotide analogue into a nucleic acid molecule.
  • an RNA analogue l,3-diaza-2-oxophenothiazine-ribose-5 '-triphosphate (tCTP) may be used.
  • tCTP is a fluorescent cytosine analogue that absorbs and emits light at about 365 and about 460 nm, respectively.
  • amplification products can be fluorescent enough to visualize them in a gel by excitation with long UV light, thus eliminating the need for staining.
  • RNA polymerase can be used in a reaction to generate amplified products. Because tCTP can substitute structurally for cytosine, this labeling method can be less invasive than labeling with dye-linked nucleotides and therefore produces nucleic acid molecules suited for biophysical studies.
  • RNPs RNA polymerases
  • RNPs can include, but are not limited to, T7 RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III and any other RNA polymerase known in the art.
  • isolated nucleic acids can be used for generating nucleic acid sequences.
  • Isolated nucleic acid may be derived from genomic RNA, genomic DNA, or complementary DNA (cDNA) of use in some embodiments herein.
  • isolated nucleic acids such as chemically or enzymatically synthesized DNA, may be of use for probes, primers and/or labeled detection oligonucleotides.
  • a "nucleic acid sequence” can include single-stranded and double-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogues. It is contemplated that a nucleic acid may be of 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
  • a nucleic acid sequence can be about 10 nucleotides or more, or 20 nucleotides or more, or 30 nucleotides or more, or 40 nucleotides or larger.
  • a nucleic acid sequence may be 200 bases or longer (e.g. for generating a highly fluorescent probe).
  • a large fluorescent probe may be generated using compositions and methods disclosed herein where detection may be by UV excitation/ visualization for rapid identification.
  • Nucleic acid sequences may be made by any method known in the art, for example using standard recombinant methods, synthetic techniques, or combinations thereof.
  • the nucleic acids may be cloned, amplified, or otherwise constructed, for example, incorporating cytosine analogues described herein.
  • Isolated nucleic acids may be obtained from tissue samples or other sources using any number of cloning methodologies known in the art.
  • oligonucleotide probes which selectively hybridize, under stringent conditions, to the nucleic acids of a tissue sample. Methods for construction of nucleic acid libraries are known and any such known methods may be used.
  • RNA or cDNA may be screened for the presence of an identified genetic element of interest using a probe based upon one or more sequences.
  • Various degrees of stringency of hybridization may be employed in the assay. As the conditions for hybridization become more stringent, a greater degree of complementarity between the probe and the target may be needed for duplex formation to occur.
  • the degree of stringency may be controlled by temperature, ionic strength, pH and/or the presence of a partially denaturing solvent such as formamide.
  • the stringency of hybridization can be conveniently varied by changing the
  • complementarity (sequence identity) required for detectable binding can vary in accordance with the stringency of the hybridization medium and/or wash medium.
  • the degree of complementarity can optimally be about 100 percent; but in other embodiments, sequence variations in the RNA may result in ⁇ 100% complementarity, ⁇ 90% complimentarity probes, ⁇ 80% complimentarity probes, ⁇ 70% complimentarity probes or lower depending upon the conditions.
  • primers may be compensated for by reducing the stringency of the hybridization and/or wash medium.
  • High stringency conditions for nucleic acid hybridization are well known in the art.
  • conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C.
  • Other exemplary conditions are disclosed in the following Examples. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleotide content of the target sequence(s), the charge composition of the nucleic acid(s), and by the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
  • Nucleic acids may be completely complementary to a target sequence or may exhibit one or more mismatches.
  • Nucleic acid sequences of interest may also be amplified using a variety of known amplification techniques.
  • target nucleic acid sequences may be generated that are traceable using one or more traceable nucleotide.
  • PCR polymerase chain reaction
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences, to make nucleic acids to use as probes for detecting the presence of a target nucleic acid in samples, for nucleic acid sequencing, or for other purposes.
  • embodiments include, Rolling circle amplification, NEAR, developed by Ionian Technologies, Recombinase Polymerase Amplification, developed by TwistDx or other techniques known in the art.
  • Isolated nucleic acids may be prepared by direct chemical synthesis by methods such as the phosphotriester method, or using an automated synthesizer. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Nucleic acid sequences having cytosine analogues may be generated using isolated sequences as a template for generating a high density fluorescent molecule.
  • kits contemplated herein may include compositions for generating high density fluorescent nucleic acids, hybrid nucleic acid molecules, nucleic acid- protein or nucleic acid-peptide. Contemplated herein are methods for making and using a kit for analysis of or generation of such molecules.
  • the kits can include one or more containers.
  • one or more reagents may be part of a kit. Any of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container, into which compositions, may be preferably and/or suitably aliquoted.
  • Kits herein may also include an apparatus for analyzing or assessing presence of an end molecule, for example detecting or analyzing a high density fluorescent molecule produced by a kit.
  • a kit may contain one or more fluorescent cytosine or cytosine nucleotides disclosed herein. Some kits may contain one or more control samples.
  • Vent exo- and Taq pol to produce a 560 base pair long PCR fragment in the presence of various mixing ratios of dtCoTP and dCTP were compared.
  • the gene amplified codes for the beta chain of human hemoglobin and has a GC content of 53 %.
  • the template was chosen arbitrarily without consideration of the exact base sequence.
  • the PCR samples were denatured at 95°C for 45 sec. Figs.
  • FIG. 1 A and B illustrate agarose gels of the PCR products obtained with Taq and Deep Vent pol as increasing amounts of dCTP were replaced by dtCoTP.
  • the gels were exposed on a 365 nm trans-illuminator to detect the tCo emission. At this wavelength, the DNA ladder or the positive control containing only 200 ⁇ dCTP were not visible however, the PCR products obtained in the presence of moderate to high dtCoTP concentrations were clearly visible. Reactions catalyzed by Taq pol did not result in PCR product for dtCoTP concentration > 10 ⁇ , whereas Deep Vent pol tolerated dtCoTP concentrations up to 50 ⁇ .
  • Deep Vent polymerase was capable of replacing about 50 % of all cytosines with analogue, tCo, or about 60% or more assuming dCTP and dtCoTP were incorporated with equal efficiency, and that the reactions are limited by the melting temperature of the resulting PCR product rather than by the substrate specificity of this pol over a wide range of dtCoTP concentrations.
  • PCR products were not obtained when more than 10% of the total amount of dCTP was replaced by dtCoTP.
  • nucleic acid synthesis methods may be used where a denaturation step of about 99 degrees Celsius is included which can result in no loss of enzyme activity and generation of highly fluorescent probes detectible by excitation/visualization, (e.g excitation at 365 nm). In accordance with these embodiments, no staining is required for detection of the highly fluorescent probe, saving time and money.
  • highly fluorescent molecules generated by compositions and methods disclosed herein may be used in disease diagnosis and prognosis in a subject. In accordance with these methods, other more invasive techniques may be avoided as well as avoiding the use of potentially carcinogenic agents/dyes.
  • PCR reaction with Taq pol may have reduced efficiency. Although both pols insert dGTP across from tCo fairly specifically, they do it with more or less impaired efficiency. While Deep Vent pol forms the G-tCo base pair ⁇ 20-times less efficiently than the G-C pair, the difference is ⁇ 100-times for Taq pol. This suggests that the extension times lengthen significantly when DNA with high tCo density is copied.
  • inferior performance of Taq pol may be explained by the enzyme's preference for incorporating the nucleotide analogue, which is followed by subsequent pausing, and paired with the inability to translate tCo when encountered in the template.
  • DNAse I is a non-specific endonuclease that randomly cleaves DNA to release di, tri and longer oligonucleotides. Exonuclease I removes mononucleotides from single-stranded DNA in 3 '-5 ' direction. Fig.
  • FIG. 4A illustrates that substantial dequenching occurs and that it scales with increasing labeling density of the PCR products. That the labeling density really increases with increasing concentrations of free dtCoTP in the PCR reactions is confirmed by the upward shift of the PCR products in native polyacrylamide gels (Fig. 5). The mass increase may be attributed to the fact that tCo is heavier than C. In this example, the amount of dequenching peaks at 250 % for the PCR products generated in the presence of 75 ⁇ dtCoTP. Dequenching is less pronounced for the reaction with 100 ⁇ dtCoTP, suggesting, among other reasons, that the enzymes are less efficient at digesting densely tCo labeled DNA.
  • the well base stacked fluorescent base analogue 2-aminopurine is strongly quenched in duplex DNA by photoinduced electron transfer from guanine, whereas electron transfer to the poorly stacked fluorophore ethenoadenine is about 10-times worse.
  • the absence of photoinduced electron transfer from guanine to tCo despite the tight base stacking of tCo possibly indicates that tCo has a higher oxidation potential than guanine.
  • thermophilic pols show a trend for misinserting dtCoTP across a template A (Table 1). Deep Vent polymerizes dTTP 10-times more efficiently than dtCoTP across from A, whereas Taq pol prefers dTTP only by a factor of 3 (Table 1).
  • PCR reactions rely on the use of an extremely thermostable B family DNA polymerase, Deep Vent exo-, which allows conducting the PCR reactions with a denaturation step at 99 °C without loss of enzyme activity.
  • the resulting DNA fragments are highly fluorescent and can be visualized in a gel by excitation at 365 nm, thus obviating the need for staining of the DNA bands with cancerogenic DNA intercalating dyes.
  • Figs. 1A-1D illustrates a comparison between different DNA polymerases and different denaturation conditions.
  • the PCR products obtained after 40 cycles were analyzed by agarose gel electrophoresis and the DNA bands were either visualized by excitation at 365 nm (top rows) or by staining with ethidium bromide (bottom rows).
  • Figs. 3A-3B represent gel electrophoretic analysis of time courses for primer elongation past tCo-G and G-tCo base pairs.
  • FIGs. 4 A and 4B illustrate dequenching of tCo fluorescence upon enzymatic digestion of PCR products.
  • PCR products were gel purified and treated with a mixture of DNAse I and Exonuclease I.
  • Fig. 5 illustrates a gel shift assay demonstrating the increase in molecular mass of the PCR product as tCo substitutes for cytosine.
  • Table 1 Relative catalytic efficiency for the incorporation of dtCoTP across G and A
  • Taq DNA pol was purchased from Invitrogen, Deep Vent exo- pol from New England Bio labs, DNAse I was from New England Bio labs, Exonuclease I from USB Affimetrix.
  • the PCR primers and other oligonucleotides were synthesized by Integrated DNA Technology and the plasmid coding for the globin protein is available and was a provided here.
  • the reactions contained the reaction buffers and Mg2+ concentrations recommended by the suppliers of the DNA polymerases.
  • Deep Vent exo- pol 10 mM KCl, 10 mM (NH 4 )2S0 4 , 20 mM Tris-HCl pH 8.8, 2 mM MgS0 4 , 0.1 % Triton X-100.
  • Taq pol 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5 mM MgCl 2 .
  • the reactions contained 200 ⁇ of each dNTP, 500 nM forward and reverse primer, 140 ng template and either 1 unit Deep Vent pol or 2.5 units Taq pol.
  • step 3 The sum of dtCoTP and dCTP concentration always equaled 200 ⁇ .
  • the total volume of each PCR reaction was 50 ⁇ .
  • a standard temperature protocol for reactions containing 15 % glycerol was as follows: Step 1 : 94 °C/4 min; step 2: 95 °C/45 sec; step 3: 52 °C/30 sec; step 4: 73 °C/2 min; step 5: 99 °C/45 sec; step 6: 55 °C/30 sec; step 7: 73°C/2 min; go to step 5: 40-times; final elongation: 73 °C/20 min.
  • the lower annealing temperature (step 3) was used in the first cycle to account for the lower melting temperature of tCo-free DNA.
  • the reactions were performed using an Eppendorf Mastercycler.
  • the PCR reactions were analyzed using mini 1.2 % standard agarose gels with 0.5 x TAE buffer. The gels were either exposed at 365 nm or at 254 nm after staining of the DNA bands with ethidium bromide.
  • fluorescence dequenching the excitation wavelength was set to 345 nm, the emission to 460 nm.
  • DNA primers were 5'- 32 P-labeled using T4 polynucleotide kinase (New England Biolab) and [ ⁇ - 32 ⁇ ] ⁇ .
  • the labeled primer was gel- purified and annealed to the appropriate template strands.
  • the total reaction volume was either 5 or 10 ⁇ ⁇ .
  • Polymerization was initiated by mixing equal volumes of reaction mixture and enzyme followed by incubation at 37°C.
  • the enzyme concentrations used for the determination of the Michaelis-Menten parameters Vmax and KM were 0.0125 units ⁇ L Taq pol and 0.0005 units ⁇ L Deep Vent pol, respectively.
  • primer elongation was performed with a series of dNTP concentrations and all reactions were stopped after the same amount of time, such that primer extension was below 20% for all dNTP concentrations.
  • gel loading buffer 90 % formamide with 50 mM EDTA
  • DeepVent pol were removed from the master mix after 5, 10, 20 and 30 min and quenched with two volumes of 90 % formamide with 50 mM EDTA. Analysis of the reaction products was conducted by denaturing gel electrophoresis as described above.
  • a fluorescent ribonucleotide analogue l,3-diaza-2- oxophenothiazine-ribose-5 '-triphosphate was synthesized and tested as a substrate for T7 RNA polymerase (T7 RNAP) in transcription reactions, a convenient route for generating RNA in vitro.
  • T7 RNA polymerase incorporates tCTP with about a 2- fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC.
  • T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors ⁇ 10, discrimination factors of 40 and 300 for tCTP-A base pair formation by T7 RNA polymerase operating in the elongation and initiation mode, respectively were observed. These catalytic properties make T7 RNA polymerase advantageous for synthesizing large fluorescent RNA, as demonstrated by generating an 800 nucleotide RNA, in which every cytosine was replaced with tC.
  • T7 RNA polymerase T7 RNA polymerase
  • Transcription of DNA by T7 RNA polymerase takes place in two phases, initiation and elongation.
  • T7 RNAP binds tightly to the promoter sequence via its N-terminal promoter binding domain, opens the DNA duplex and feeds the template into the active site.
  • ribonucleotide synthesis frequently results in abortive RNA products that do not exceed 8-10 bases.
  • dtCTP is a good substrate for several A and B family DNA polymerases. The analogue is incorporated with high catalytic efficiency opposite a template guanine, but also to a significant extent opposite a template adenine.
  • the DNA polymerases efficiently extended a d(tC-G) base pair, whereas d(tC-A) base pairs primarily resulted in chain termination.
  • Ambivalence of dtCTP incorporation may be due to the propensity of N 4 -substituted cytidine analogues for forming the imino-tautomer, which is isosteric to thymine (Chart 1).
  • PCR polymerase chain reactions
  • a lower percentage of the C's were replaced with dtC in these experiments.
  • T7 RNAP Catalytic efficiency of tCTP incorporation by T7 RNAP was assessed using synthetic DNA templates of defined sequence, using for example templates which contained a unique G or A either within the initiation region or at a remote site. Both in the elongation and initiation mode, T7 RNAP polymerized tCTP with high catalytic efficiency across from a template G. Notably, T7 RNAP appears to be more discriminative against tC-A mismatches than DNA polymerases are against d(tC-A) mismatches. To demonstrate the merit of tCTP for RNA labeling, large, 100% tC-labeled RNA were produced by transcribing an 800 base pair DNA sequence, using tCTP in place of CTP.
  • Chart 1 illustrates base pairing of the tC amino and the tC imino tautomer.
  • tCTP incorporation opposite a template guanine was used to incorporate nucleotide analogues because it possesses different catalytic properties during the initiation and elongation phases of RNA synthesis.
  • tCTP was examined during both phases by using two different synthetic DNAs. Both DNAs consisted of the 18 nucleotide (nt) T7 promotor hybridized to a complementary 37 nt DNA template (Fig. 6). Transcription usually starts at the underlined C and proceeds in 3 '-to -5' direction along a template.
  • DNA 1 was employed, which exhibited a unique guanine 12 bases away from the start site of transcription.
  • DNA 2 featured a unique guanine at position only four nucleotides from the start site, directing tCTP incorporation to the initiation region. It was necessary to start the templating region with CCT instead of CCC to avoid slippage of T7 RNAP during initiation, which can result in non-sense poly-G ribonucleotides of different lengths.
  • Fig. 6 represents a comparison of the lengths of the RNA products obtained in transcription reactions using T7 RNAP and different combinations of NTPs and labeled nucleotides.
  • T7 RNAP In the presence of only GTP and ATP (lanes 2 and 2'), transcription terminated as T7 RNAP reached the single template guanines of DNA 1 and DNA 2, respectively, and the length of the products verifies that transcription starts indeed at the underlined C.
  • T7 RNAP produces a large amount of abortive GA dinucleotide when transcribing DNA 2, as revealed by RNA labeling with [a- 32 P]ATP in the presence of only ATP and GTP (Fig. 6, lane 9).
  • RNA transcript that contains U instead of the correct C migrates distinctly different from the RNA exhibiting tC at this position. Omitting UTP from the reaction mixture in the presence of CTP or tCTP, respectively (Fig. 6, lanes 6 and 7), lead to almost complete abortion of transcription at the unique A of DNAl . In both reactions only ⁇ 7% of the corresponding abortive RNA transcripts were extended. Employing DNA2, the reaction containing ATP, GTP and CTP lead to 1 1 % extension past the unique template A (lane 6'), whereas it was 26 % for the reaction containing tCTP (lane 7') ⁇ Thus, T7 RNAP used neither ATP, GTP, CTP nor tCTP effectively to bypass the template adenine. This result suggests that T7 RNAP base pairs tCTP less
  • Fig. 6 illustrates incorporation of tCTP by T7 RNA polymerase operating either in the initiation or elongation mode. Transcription starts at the underlined C and proceeds in the 3'-to-5 ' direction. All reactions contained 0.2 units/ ⁇ T7 RNAP, 1 ⁇ DNA and 0.4 mM of each of the indicated NTPs. All RNA products are visualized based on the incorporation of [a- 32 P]GTP, except for lanes 1 and 3, where [a- 32 P]ATP was used instead, together with ATP and GTP. Lane 2 displays a poly-G ladder, which was generated as described in the experimental section.
  • Fig. 7 illustrates competitive incorporation of CTP and tCTP across from G. All reactions contained 0.2 units/ ⁇ T7 RNAP, 1 ⁇ DNA and 0.4 mM [a- 32 P]GTP and 0.4 mM ATP. The [CTP]-to-[tCTP] ratio was systematically varied as indicated below the lanes and the reactions products were analyzed after incubating for lh. The very left lane shows the no enzyme control, the poly-G ladder is located in the middle between the two reactions series.
  • tC imino tautomer is a templating effect, for example, the alignment of tC with a templating adenine induces the imino tautomer to minimize the free energy of base pairing via hydrogen bonding.
  • Such an inductive effect could be powerful if the templating and the incoming base were aligned in one plane, as in duplex DNA.
  • the X-ray structure of the open ternary T7 RNAP-DNA-NTP complex captures an interaction of the incoming nucleotide with the templating base at a putative preinsertion site that has not been observed in other open ternary polymerase -DNA-dNTP complexes so far, including BF polymerase, RB69, Taq polymerase and T7 DNA polymerase.
  • the incoming NTP binds to the fingers domain (which forms the roof of the active site in the closed ternary complex) and makes hydrogen bonding contacts with the templating base prior to entering the active site, without for example, the steric constraint of the active site and with a slight out of plane tilt.
  • T7 RNAP has a mechanism to screen for base complementarity at the preinsertion site, which may contribute to selectivity.
  • the open ternary complex of Taq polymerase however captures the incoming nucleotide bound in the active site (the insertion site), while the templating base is occluded from interactions with the incoming nucleotide.
  • Two studies confirmed that the closure of the fingers domain and the alignment of the templating base with the incoming base are both fast steps on the reaction coordinate of nucleotide polymerization by Klenow fragment. This suggests that Klenow is in fast equilibrium between the open and closed conformation and that it screens for base complementarity at the insertion site after formation of Watson-Crick hydrogen bonds between incoming dNTP and a templating base.
  • T7 RNAP has a mechanism to reject the incoming tCTP at the preinsertion site, whereas Klenow allows dtCTP access to the insertion site where electronic constraints and planar base stacking facilitate the formation of the imino tautomer, which is then accepted due to its resemblance to a T-A base pair.
  • tC labeling of large RNA The generation of full length products on DNA1-4 in assays containing ATP, GTP, UTP, and tCTP suggested that it should be possible to use tCTP and T7 RNAP to generate highly fluorescent RNA.
  • T7 RNAP To present T7 RNAP with another challenge and to demonstrate the merit of tCTP incorporation for fluorescent labeling of large RNA transcripts, about 800 nucleotides of a Borellia miyamotoi flaggellin protein gene were transcribed in the presence of tCTP. Formation of full length RNA requires T7 RNAP to incorporate 3 consecutive tCTPs at 6 different positions.
  • RNA transcripts obtained at different mixing ratios of tCTP and CTP and separated by agarose gel electrophoresis As described recently for tCo labeled PCR products, tCo being a fluorescent oxo-ortholog of tC, it is possible to visualize the nucleic acids based on the tC fluorescence only, thus obviating the need for ethidium bromide staining.
  • Fluorescent 890 nucleotide RNA was obtained at all tCTP/CTP ratios tested, from a ratio of 1/15 to the point of full substitution of CTP with tCTP.
  • the same reaction conditions were used to transcribe a catalytic RNA, the 207 nucleotide long E. coli riboswitch for vitamin B12 (Fig. 9B).
  • RNA transcripts For both RNA transcripts, increasing the tC content led to slightly faster migration of the product RNA, and the overall yield was slightly diminished. The slightly faster migration could indicate premature termination at a specific site, or more likely, the RNA adopts a folded structure due to the increased hydrophobicity of the tC as compared to C. Despite the lower RNA yield at high tC labeling density, T7 RNAP transcription in the presence of tCTP is unexpectedly efficient.
  • Fig. 8 represents incorporation of UTP and tCTP across from a template A. All reactions contained 0.2 units/ ⁇ T7 RNAP, 1 ⁇ DNA4, 0.4 mM GTP, 0.4 mM ATP, some ⁇ x- [ 32 P]-GTP and increasing concentrations of UTP (left side) or tCTP (right side). Each reaction was stopped after 1 h. The UTP and tCTP concentrations, respectively, were as follows: 1, 5, 10, 25, 50, 100, 200 ⁇ .
  • Figs. 9A and 9B represent large fluorescent RNA generated by transcription in the presence of CTP and tCTP at different concentrations.
  • the top row shows the agarose gel exposed by UV light prior to staining with ethidium bromide (EtBr).
  • the CTP and tCTP concentrations are provided below the imageimages.
  • the reaction in lane 1 reactions next to the marker contained DNA template, pimers, enzyme but no CTP or tCTP, the control in lane 2 contained all four natural NTPs but no template.
  • Table 4 Kinetic parameters for the insertion of tCTP and UTP opposite adenine.
  • V max /K M for the incorporation of UTP into DNA X, divided by V max /K M for the incorporation of tCTP into DNA X.
  • the base analogue tC combines several properties that make it an attractive candidate for the design of regulatory and catalytic RNAs: as with other N 4 -substituted cytosine analogues it likely engages in variable hydrogen bonding patterns depending on its tautomerization state, it stabilizes DNA-RNA and DNA-DNA duplexes, and it is fluorescent and traceable within the limits of its fluorescence quantum yield. It is possible that this base analogue may be used for producing functionally expanded nucleic acid libraries (e.g. R A or DNA libraries) using T7 transcription combined with selection processes such as SELEX or other selection process.
  • functionally expanded nucleic acid libraries e.g. R A or DNA libraries
  • RNA polymerase and RNAse OutTM were from Invitrogen.
  • Synthetic oligonucleotides were purchased from Integrated DNA Technology and the DNA sample used for transcription of the Borrelia miyamotoi gene (locus D3777, region 354-1241; Gen Bank 43777) was provided and PCR amplified to introduce the T7 promotor.
  • Synthesis of tCTP was performed using the known synthesis of the l,3-diaza-2-oxophenothiazine nucleobase, Vorbruggen's silyl-Hilbert- Johnson
  • reaction mixture was heated at reflux for 2.5 hours, the allowed to cool to room temperature.
  • the reaction mixture was then poured into 5% NaHC0 3 solution (100 ml), and extracted with CH 2 C1 2 (2 x 100 ml). After drying over anhydrous Na 2 S0 4 , the solvent was removed by rotary evaporation and the product was purified by flash chromatography (5% hexanes in EtOAc), yielding the product as a yellow oil (395 mg, 91%), which was found to consist of only the desired ⁇ anomer.
  • tC-ribonucleoside triphosphate [00093] tC-ribonucleoside triphosphate.
  • tC (30 mg, 0.09 mmol) was dissolved in trimethyl phosphate (0.5 mL) under argon and cooled on ice.
  • POCl 3 (9 uL, 1.1 equivalent) in trimethyl phosphate (0.05 mL) was added dropwise and the mixture was stirred for 2 h while warming up to room temperature.
  • Tributylammonium pyrophosphate (0.6 g, 1.1 mmol, 12 equivalents) in DMF (1 mL) was added followed by several droplets of tributyl amine.
  • T7 transcription of synthetic oligonucleotides 20 transcription reactions contained 1 ⁇ DNA construct, 0.4 mM of each NTP, 5 mM DTT, [a- 32 P]GTP, commercial reaction buffer (40 mM Tris-HCl pH 8, 8 mM MgCl 2 , 2 mM spermidine, 25 mM NaCl) and 0.2 units/ ⁇ ⁇ T7 RNA polymerase.
  • the DNA constructs were prepared by hybridizing the T7 promotor (5'-TAA TAC GAC TCA CTA TAG-3' SEQ ID NO.
  • RNA products were separated by denaturing gel electrophoresis (20 % polyacrylamide, 8 M urea gels) and visualized using phosphorimagery.
  • fractioncTP being the amount of RNA extended by CTP
  • (k ca t/K M )tcTP the catalytic efficiency for tCTP incorporation
  • (k cat /KM)cTP the catalytic efficiency for CTP incorporation.
  • the kinetic parameters for tCTP incorporation across from A were measured under standard conditions. Assays contained 1 ⁇ DNA 3 or DNA 4, 0.4 mM ATP, 0.4 mM GTP, some a-[ 32 P]-GTP and either: 1, 5, 10, 25, 50, 100, 200 ⁇ UTP or 1, 5, 10, 25, 50, 100, 200 ⁇ tCTP. Reaction time was 1 hour.
  • RNA extended beyond 11 nucleotides was determined, in case of DNA 4, the percentage of RNA extended beyond 3 nucleotides was quantified.
  • the percentage of extended RNA was plotted versus the UTP or tCTP concentration and the Michaelis-Menten parameters were derived by non-linear curve fitting.
  • RNAse out 20 transcription reactions contained 1 ⁇ , PCR amplified DNA template, 0.4 mM ATP, 0.4 mM GTP, 0.4 mM UTP, 5 mM DTT, 0.6 units/ ⁇ _, RNAse out, commercial reaction buffer (40 mM Tris-HCl pH 8, 8 mM MgCl 2 , 2 mM spermidine, 25 mM NaCl) and 2 units/ ⁇ _, T7 RNA polymerase.
  • commercial reaction buffer 40 mM Tris-HCl pH 8, 8 mM MgCl 2 , 2 mM spermidine, 25 mM NaCl
  • CTP and tCTP were added to match the following concentrations: 400 ⁇ CTP, no tCTP; 375 ⁇ CTP, 25 ⁇ tCTP; 350 ⁇ CTP, 50 ⁇ tCTP; 300 ⁇ CTP, 100 ⁇ tCTP; 200 ⁇ CTP, 200 ⁇ tCTP; 100 ⁇ CTP, 300 ⁇ tCTP; no CTP, 400 ⁇ tCTP.
  • the samples were incubated for 1 hour, mixed with gel loading buffer (10 % ficoll 400, 10 % glycerol, 1 x TBE) and the RNA was separated using an 1.2 % agarose gel. The gels were exposed on an UV transilluminator prior to and after staining with ethidium bromide.
  • nucleic acid sequences may be synthesized using Taq polymerase for the PCR by modifying reaction conditions. Two sets of conditions were developed that allow Taq polymerase to work well in PCR. In one example, betaine was not present in the reaction mix. It was discovered that Taq works in the presence of 25uM dtCoTP, 175uM dCTP, 200uM of the other three dNTPs, 3.5mM MgC12 and 0.5mM MnC12. Standard PCR cycles may be used, or even better do the denaturation step at 97C for 20 sec. One key ingredient was to include the MnCl 2 .
  • dtCoTP and dNTPs can vary the ratio of dtCoTP and dNTPs or use dtCTP in various ratios.
  • other PCR reagent compositions can include Co 2 in the metals, but in certain cases Mn 2 worked more efficiently as a catalyst in the reaction.
  • Other exemplary methods may include using 1 M betaine in a nucleic acid sequence synthesizing reaction.
  • Mn+2 was not needed when betaine was present in the PCR reaction.

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Abstract

La présente invention concerne des compositions, des procédés et des utilisations impliquant des analogues de la cytosine triphosphate. Dans certains modes de réalisation, lesdits analogues de la cytosine triphosphate peuvent être utilisés pour générer des séquences d'acides nucléiques à haute densité de fluorescence pour diverses utilisations.
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WO2021260107A1 (fr) 2020-06-25 2021-12-30 Stealth Labels Biotech Ab Analogues fluorescents de la cytosine et leur application en transcription et traduction

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US20020119455A1 (en) * 1997-02-12 2002-08-29 Chan Eugene Y. Methods and products for analyzing polymers
US20040054162A1 (en) * 2001-10-30 2004-03-18 Hanna Michelle M. Molecular detection systems utilizing reiterative oligonucleotide synthesis
US20090155856A1 (en) * 2007-08-15 2009-06-18 Hayato Miyoshi Nucleic acid amplification method

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US20020119455A1 (en) * 1997-02-12 2002-08-29 Chan Eugene Y. Methods and products for analyzing polymers
US20040054162A1 (en) * 2001-10-30 2004-03-18 Hanna Michelle M. Molecular detection systems utilizing reiterative oligonucleotide synthesis
US20090155856A1 (en) * 2007-08-15 2009-06-18 Hayato Miyoshi Nucleic acid amplification method

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Title
STENGEL G. ET AL: "Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase alpha and Klenow Fragment", BIOCHEMISTRY, vol. 48, no. 31, 6 July 2009 (2009-07-06), pages 7547 - 7555 *

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WO2021260107A1 (fr) 2020-06-25 2021-12-30 Stealth Labels Biotech Ab Analogues fluorescents de la cytosine et leur application en transcription et traduction

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