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WO2008037568A2 - Terminateurs réversibles pour un séquençage efficace par synthèse - Google Patents

Terminateurs réversibles pour un séquençage efficace par synthèse Download PDF

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WO2008037568A2
WO2008037568A2 PCT/EP2007/059207 EP2007059207W WO2008037568A2 WO 2008037568 A2 WO2008037568 A2 WO 2008037568A2 EP 2007059207 W EP2007059207 W EP 2007059207W WO 2008037568 A2 WO2008037568 A2 WO 2008037568A2
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group
cleavable
compound according
hydrogen
detectable
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PCT/EP2007/059207
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WO2008037568A3 (fr
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Diana Katerina Knapp
Joachim W. Engels
Angelica Keller
Yangzhou Li
Julius Gagilas
Saulius Serva
Alina Stura
Andras Foldesi
Camilla Estmer Nilsson
Marek Kwiatkowski
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Quiatech Ab
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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
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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

Definitions

  • the present invention relates to novel nucleic acid chain extension terminators, their use in nucleic acid sequencing as well as a method for preparing such compounds.
  • Hyman E.D., Anal. Biochem., 174:423 (1988) discloses the addition of a nucleotide to a an immobilised DNA template/primer complex in the presence of a polymerase and determination of polymerisation reaction by detecting the pyrophosphate liberated as a result of the polymerisation.
  • Jett et al. J. Biomol. Struct. Dyn., I, p. 301, 1989 discloses a method wherein a single stranded DNA or RNA molecule of labelled nucleotides, complementary to the sequence to be determined, is suspended in a moving flow stream. Individual bases are then cleaved sequentially from the end of the suspended sequence and determined by a detector passed by the flow stream.
  • EP-A-223 618 discloses the use of an immobilized DNA template, primer and polymerase exposed to a flow containing only one species of deoxynucleotide at a time. A downstream detection system then determines whether deoxynucleotide is incorporated into the copy or not by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the complex of DNA template and polymerase.
  • WO 90/13666 proposes a method directly measuring the growth of the template copy rather than determining it indirectly from compositions in the flow medium. Only one of the four nucleotides is present at the same time, and the polymerization events reflecting the incorporation of a nucleotide or not are detected by spectroscopic means (evanescent wave spectroscopy, fluorescence detection, absorption spectroscopy) or by the individual nucleotides being labelled.
  • spectroscopic means evanescent wave spectroscopy, fluorescence detection, absorption spectroscopy
  • the Cm group can be easily introduced at a selected position in nucleosides upon introduction of the methylthiomethyl group at this position, its activation (eg by means of sulfuryl chloride) and reacting of obtained chloromethyl derivative with potassium cyanide in DMF, similarly to the method described by us in US 6309836. It is important to remember that the cyano group mentioned here acts only as a one of many possible electron withdrawing groups, thus other groups characterised by similar ability can be used instead in all of the above listed compounds. It is, however, natural that due to the size restriction enforced by most of polymerases the size of the alternative electron withdrawing groups should be kept to the necessary minimum.
  • EP 1291354 discloses a linker between the nucleobase and a detectable group that can be cleaved by a reducing reagent.
  • the similar type of linkers can be found in WO 2004/018497. This linker containing a dithio- unit could be cleaved by means of phosphines or dithiotreitol.
  • US6664079 uses photolabile linkers or linkers cleavable by means of palladium ions. Any of these linkers could be used together with the present method of 3'-0 protection, and would be equivalent to two steps procedure for complete deprotection of incorporated nucleotide. It would be desirable, however, to perform these cleavages in one single step.
  • the function connecting the detectable moiety should contain a unit cleavable by means of the same reagent as is necessary for the cleavage of the 3'-O- protecting group.
  • a silyl group or a disyloxyl linker (US 6291669) could be a working alternative as both are fluoride-labile.
  • cleavable functions, analogues to the presented 3'- blocking units can be constructed, used for connection of detectable group to the nucleobase, and conveniently removed under the same conditions as are used for the removal of the 3-O-protection.
  • One object of the present invention is to provide nucleotide derivatives which are useful as chain terminators and which upon removal of the cleavable 3 '-protecting group may readily be converted into nucleotides or nucleotide analogues that may be further extended.
  • Another object of the present invention is to provide a method for nucleotide sequence determination using the novel chain terminators.
  • Another object of the present invention is to provide a process of preparing novel chain terminators according to the invention.
  • a still further object of the invention is to provide a kit comprising, in separate containers, the novel chain terminators of the invention, and necessary enzymes and/or nucleotides and/or reagents for preparing oligo- or polynucleotides.
  • Yet another object of the invention is to provide a kit comprising, in separate containers, the novel chain terminators of the invention and necessary enzymes and/or nucleotides and/or reagents, for determining the sequence of a nucleic acid.
  • the invention provides a method for sequencing a nucleic acid including the steps of : a) providing a target nucleic acid array including a plurality of target nucleic acids; b) contacting a sequencing primer with the target nucleic acids thereby forming target-primer complexes between complementary portions of the sequencing primers and the target nucleic acids; c) incorporating a first 3'-O-modif ⁇ ed nucleotide into at least one sequencing primer portion of the target-primer complexes, the first 3'-O-modified nucleotide being complementary to the target nucleic acid; and d) detecting the incorporation of the first 3'-0 -modified nucleotide, wherein the first 3'-0 -modified nucleotide is complementary to the target sequence at the first 3'-0 - modified nucleotide's site of incorporation.
  • the detecting step can be performed before or after removing a 3'-0 moiety.
  • This sequencing method is effective for producing a plurality of nucleotide sequences wherein the nucleotide sequences correspond to overlapping nucleotide sequences of the target nucleic acid.
  • the array may be composed with the plurality of sequencing primers forming primer-target complex upon contacting with a mixture containing plurality of target nucleic acids.
  • label or “detectable group” means a molecule, which is possible to detect in a suitable manner.
  • label or “dye- label” include fluorescent molecules such as fluorescein, cyanine dyes, like Cy3, Cy-5, Cy-7, Cy-9 disclosed in U.S. 5268486 (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 detectable group as defined above.
  • 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 miniaturized surfaces comprising ordered immobilized oligonucleotides, DNA or RNA molecules.
  • the nucleobase B may be natural or synthetic. Natural nucleobases include common nucleobases, such as adenine, guanine, cytosine, thymine and uracil, as well as less common nucleobases, such as xanthine, hypoxanthine or 2-aminopurine. Synthetic nucleobases B are analogues to the natural nucleobases and capable of interacting with other nucleobases in a specific, hydrogen bond determined way.
  • X and Y can independently be oxygen or sulphur atom, although the most preferred for both positions is oxygen.
  • the ability of the 3'-O- protecting group to be cleaved is to large extent depending on the nature of function Z.
  • Z is a chemical group characterized by strong electron withdrawing properties. This prerequisite is valid no matter which chemical mechanism is considered to be operating at a particular case, since both beta-elimination mechanism and nucleophilic substitution demands the presence of the mentioned electron withdrawing function.
  • Z maid be chosen from a large group of functions known to those skilled in the art, and can be selected among others from the following groups: nitro, cyano, sulfono, sulfoxide, trihalogenomethyl, aldehydo, ketone, ester, phenyl or substituted phenyl.
  • the substituent Rl denotes hydrogen, hydroxyl or a protected hydroxyl. Any known permanent or transient protecting group can be applied for this purpose, however the most preferred function for this protection is methyl group.
  • R2 and R3 are together or separately hydrogen, an alkyl or a substituted alkyl group.
  • R5 can be hydrogen or an additional electron withdrawing group.
  • R5 can be identical with Z or different.
  • nucleotide triphosphate carrying the detectable label located right on the 3'-O-protecting group.
  • nucleotide if accepted by the polymerase and incorporated into the primer, could subsequently be 3'-0 deprotected by a single reaction with simultaneous removal of the detectable function.
  • R4 is hydrogen or a chemical moiety consisting of a linker molecule L, linking a detectable group to the rest of the structure (I).
  • linker molecule L linking a detectable group to the rest of the structure (I).
  • linkers can be of different length, consist of other atoms than just carbon and contain different proportions of the heteroatoms.
  • linkers importance is only in connecting a detectable label to the rest of the structure without substantial increase of the molecules size or its solubility parameters.
  • Detectable moiety can be chosen from a vast number of such moieties known to those skilled in the art.
  • exemplary such moieties are radioactively labelled functions, luminescent, electroluminescent or fluorescent labels, and labels that absorb characteristic visible or infrared light.
  • the detectable group is a fluorescent label.
  • a way of keeping the 3 '-protecting structure at the minimum size is to place the detectable group at the nucleobase. The most favoured position for location of any functional group at a particular base has been elaborated in details during the last decade.
  • 5-C atom for cytosine, 5-C-methyl thymine, 7-deazaguanine and 7-deazaadenine are the optimal positions for attachment a linker to the base to assure that the nucleotide will be recognized by the polymerase and the incorporated base will perform well in a base-pairing with a nucleotide from the opposite strand.
  • Placing a detectable group on the nucleobase renders the presence of the R4 unnecessary, so in such case the R4 group can be downsized to hydrogen.
  • Cyanoethoxymethyl group is known from the literature to be labile upon treatment with fluoride anions, especially if the reagent is tetrabutylamminium fluoride (TBAF) and dissolved in an aprotic colvent like tetrahydrofuran (THF). Cyanoethyl group, when exists as an ether at the 2'-O- position, is regarded as stable even in the presence of fluoride anion as was shown before by Sekine et al. ( J. Org. Chem. 2005, 70, 10453-10460).
  • TBAF tetrabutylamminium fluoride
  • THF tetrahydrofuran
  • the detectable group is placed either on R4 or R6 or is absent, thus either R4 is hydrogen and the detectable group is placed on R6, or R6 is hydrogen and the detectable group is placed on R4, or both R4 and R6 are hydrogens.
  • Numbers m and n are independently 0 or 1.
  • the compounds of Formula I may be used as pure chain extension inhibitors, or chain terminators, for example, in DNA sequencing according to the chain termination method, as is per se known in the art, the advantages of the compounds are, of course, better benefited from when the convenient deprotection capabilities of the compounds are utilized. This is, for example, the case when the compounds I are used in nucleic acid sequencing methods based on the sequential incorporation and determination of individual nucleotides in a growing nucleic acid copy strand as described in, for example, the aforementioned WO 91/06678, US-A- 5,302,509, DE-A-414 1178 and WO 93/21340.
  • SBS Sequencing by Synthesis
  • Another aspect of the invention therefore provides a method for determining the sequence of a nucleic acid, which method comprises providing a single- stranded template comprising the nucleic acid to be determined, and at least partially synthesizing a complementary nucleic acid molecule in a stepwise serial manner by the addition of nucleotides in which the identity of each nucleotide incorporated into the complementary nucleic acid molecule is determined subsequent to its incorporation, wherein said nucleotides are compounds of Formula I as defined above, and wherein the 3 '-blocking group is removed from the nucleotide after its incorporation to permit further extension of the nucleic acid molecule.
  • a method for determining the sequence of a nucleic acid comprises the following steps: (i) providing a single-stranded template comprising the nucleic acid to be sequenced, (ii) hybridising a primer to the template to form a template/primer complex, (iii) subjecting the primer to an extension reaction by the addition of compounds of Formula I with different nucleobases B corresponding the four bases A, C, T and G or analogues thereof, (iv) determining the type of the compound of Formula I added to the primer, (v) selectively hydro lysing the 3'-O- protective group, and (vi) repeating steps (iii) to (v) sequentially and recording the order of incorporation of compounds of Formula I.
  • the different compounds of Formula I in step (iii) may be added in sequence, in which case the four different compounds I may carry the same detectable group (label). Alternatively, the different compounds I may have different labels and may added at the same time.
  • the template/primer complex is bound to a solid-phase support, such as a sequencing chip, or porous and non-porous beads for example.
  • the template may be attached to the solid support via a binding linker, which, for instance, is ligated to the 5 '-end of the template or incorporated in one of the ends of the template by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the binding linker may then be attached to the solid support for instance by use of chemical covalent bond or using non-covalent interactions exemplified by a streptavidin coupling system.
  • the primer may be attached to the solid support.
  • the compounds of Formula I may, of course, also conveniently be used in so-called mini- sequencing (Syvanen A-C et al., Genomics 8:684-692 (1990).
  • 5'-O-Dimethoxytrityl-3'-O-t-butyldimethylsilyl-thymidine (2) 5'-O-DMTr-thymidine (1) (5.34 g, ⁇ 10 mmol) was co-evaporated with dry pyridine (2x) then re-dissolved in the same solvent ( ⁇ 80 ml). Imidazole (2.72 g, 40 mmol) was added followed by tert-butyldimethylsilyl chloride (3.01g, 20 mmol). Stirring at room temperature was maintained for -3.75 h, then the reaction mixture was poured into saturated aqueous NaHCO3 and extracted with dichloromethane (3x).
  • Thymidine derivative 5 (1.71 g, 3.8 mmol) was dissolved in dry t-butanol. To the stirred solution acrylonitrile (1.31 ml, ) was added followed by cesium carbonate (350 mg) and the resulting inhomogeneous mixture was stirred under argon for 1 h. The reaction was filtered through a Celite pad, the pad was washed with dichloromethane. The organic phase was evaporated and the residue was separated by short column chromatography (cyclohexane-ethyl acetate) to give the fully protected nucleoside 6 (1.24 g, 2.46 mmo 1, 65%).
  • Thymidine derivative 6 (1.54 g, 3.06 mmol) was dissolved in methanol and aqueous ammonia (32%, 20 ml) was added followed by dioxane ( ⁇ 5 ml). The mixture was stirred at room temperature for 6.5 h. Then the solvents were evaporated and the residue co-evaporated with methanol (3x) and after that with dichloromethane (2x). The resulted glass was separated by short column chromatography (dichloromethane-methanol) to give the fully protected nucleoside 7 (828 mg, 2.8 mmol, 92%).
  • 3'-O-(2-cyanoethyl)thymidine (7) (55 mg, 0.19 mmol, 1.0 eq.) was co-evaporated two times with dry pyridine (3 ml) and dried overnight in vacuo at the rotary vane
  • the flask was filled with argon and closed with a septum. The following steps were carried out under a slight positive argon pressure.
  • the nucleoside was dissolved in anhydrous pyridine (190 ⁇ l) and anhydrous dioxane (570 ⁇ l). Afterwards 190 ⁇ l (0.19 mmol, 1.0 eq.) of a freshly prepared 1 M solution of the phosphitylating agent 2-chloro-4H-l,2,3-benzo-dioxaphosphorin-4-one in anhydrous dioxane were added to the well stirred solution of the nucleoside.
  • Thymidine (1) (2.42 g, 10 mmol) was co-evaporated with dry pyridine (3x) and dissolved in the same solvent (100 ml). The solution was placed in an ice-bath and benzoyl chloride (1.28 ml, 11 mmol) dissolved in dry dichloromethane (10 ml) was added drop wise (ca 10 min). Stirring at ice-bath temperature was maintained for ⁇ 3.5 h, and then the reaction mixture was poured into saturated aqueous NaHCO3 and extracted with dichloromethane (3x). The pooled organic phase was dried over MgSO4, filtered and evaporated.
  • thymidine derivative 3 (2.45 g, 6.03 mmol) was dissolved in dry dichloromethane (50 ml). Triethylamine (0.84 ml) was added followed by molecular sieves (3 A, ca 40 ml, pre- activated in an oven at ca 95 0 C), and the heterogeneous mixture was gently stirred for 2h at room temperature under argon.
  • 3'-O-(2-Cyanoethoxy)methyl-thymidine triphosphate (6) 3'-O-Cyanoethoxymethyl-thymidine (5) (90mg, 0.27mmol, leq) was co-evaporated three times with dry pyridine and dried in vacuo overnight. The starting material was then transferred into a reaction vessel equipped with a magnetic stirring bar and a septum and dissolved in anhydrous dimethylformamide (4 ml) and anhydrous pyridine (1 ml).
  • N-Benzoyl-2',3'-O-trimethylsilanyl-5-iodo-2'-deoxyuridine (2) was prepared.
  • 5-iodo-2'- deoxyuridine (1) 700 mg, 2 mmol
  • dry pyridine (20 ml) were added ethyldiisopropylamine (1.74 ml, 10 mmol) and chlorotrimethylsilane (0.63 ml, 5 mmol). After the mixture was stirred at room temperature for 30 min, benzoyl chloride (0.35 ml, 3 mmol) was added.
  • N-Benzoyl-5-iodo-2'-deoxyuridine (3) N-Benzoyl-2',3'-O-trimethylsilanyl-5-iodo-2'- deoxyuridine (2) (1.20 g, 2 mmol) was dissolved in 20 ml of CHC13 / MeOH (1 : 1) and CF3COOH was added. The mixture was stirred for 30 min at room temperature. The solvent was removed in vacuum and the residue was crystallized from MeOH to give N-benzoyl-5-iodo-2'- deoxyuridine as a white solid. The mother liquid was repeated to crystallize for two times to give the combined solid (0.78 g, 86%).
  • N-Benzoyl-5-iodo-2'-deoxyuridine (3) (4.57 g, 10 mmol) was dissolved in 20 ml of dry pyridine and 40 ml of DMF, and then Et3N (2.3 ml, 15 mmol) and MMTrCl (4.0 g, 13 mmol) were added. The resulting mixture was stirred for 19 h. Another part of MMTrCl (0.3 g, 1 mmol) was added and the mixture was stirred for 3 h. The reaction was quenched by addition of 20 ml of EtOH.
  • N-Benzoyl-3 '-O-cyanoethyl-5 '-O-MMTr-5-iodo-2'-deoxyuridine (5) N-Benzoyl-5 '-0-MMTr- 5-iodo-2'-deoxyuridine (4) (4.5 g, 6.2 mmol) was dissolved in 31 ml of t-BuOH, and then acrylonitrile (8.25 ml, 126 mmol) and Cs2CO3 (2.21 g, 6.3 mmol) were added. The mixture was stirred vigorously at room temperature for 3 h.
  • N-Benzoyl-3 '-O-cyanoethyl-5 '-O- MMTr-5-iodo-2'-deoxyuridine (5) 120 mg, 0.13 mmol was dissolved in 6 ml of MeOH and aqueous ammonia (2 ml) was added at O 0 C. The resulting mixture was stirred at room temperature for 2 h. After removal of the solvent, the residue was purified by flash column chromatography (MeOH in CH2C12 0 - 1%) to give 3'-O-cyanoethyl-5'-O-MMTr-5-iodo-2'- deoxyuridine (66 mg, 75%).
  • 3 '-O-Canoethyl-5 '-O-MMTr-5-iodo-2'- deoxyuridine (6) (680 mg, 1 mmol) was dissolved 10 ml of CHC13 and 80% AcOH (10 ml) was added at O 0 C. The resulting mixture was stirred at room temperature for 30 min. After removal of the solvent, the residue was purified by flash column chromatography (MeOH in CH2C12 0 - 5%) to give the title compound (370 mg, 91%).
  • 2-(2-(2-Azidoethoxy)ethoxy)ethanol (2) 2-(2-(2-Chloroethoxy)ethoxy)ethanol (1). (10.0 g, 56.9 mmol, 1.0 eq.) were dissolved in 50 ml ethanol. To this solution sodium iodide (1.7 g, 11.9 mmol, 0.2 eq.) and sodium azide (11.1 g, 170.8 mmol, 3.0 eq.) were added and the resulting mixture refluxed for 6 days. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in methylene chloride and stored overnight at 4°C. After filtration and concentration the slightly yellow oil was purified by distillation.
  • reaction mixture was then concentrated to about half the volume under reduced pressure, extracted two times with ethyl acetate (20 ml), and one time with methylene chloride (20 ml), dried over Na2SO4 and concentrated.
  • nucleoside containing a methylthiomethyl substituents can react upon activation with appropriate electrophile like sulfuryl chloride, N-bromosuccinimide or CuBr2 with a spectrum of nucleophilic reagents.
  • the secondary alcohol (2) was used as the nucleophile and was found to react smoothly with the activated compound (1) to form derivative (3), conforming the results of Sawada and Ito (Tet. Lett. 2001, 42, 2501-2504) who found that primary, secondary and even tertiary alcohols react efficiently to form appropriate formacetal derivatives.
  • the following removal of MMTr group and introduction of the triphosphate residue was described in the earlier Examples.
  • the alcohol (1) can react with carbonyl diimidazole (CDI), disuccinamidyl carbonate (DSC) or phosgene to form a reactive derivative (2).
  • Such derivatives can react with primary and secondary amines like the amine group present in the compound (3) to form the carbamido derivative (4).
  • All reactions represent standard procedures, known from the textbooks of organic chemistry. A following process consisting of four consecutive reactions is introducing the triphosphate moiety on the 5 '-position, and a labeling function on the base. These steps were listed in the Example above.
  • Example 8 Cleavage of the 3'-O- CEM from a primer containing this modification.
  • 0.1M TEAAc and buffer B 80% ACN in 0.1M TEAAc, gradient: 0-40% B in 12 min.
  • 2,7 ⁇ M RevertAid M-MuLV RT was incubated for the indicated time at 37°C (buffer Tango) with 50 ⁇ M TTP-CNE and 10OnM of labeled DNA duplex.
  • Polymerases was incubated for a 30 min at 37°C (buffer Tango) with 50 ⁇ M TTP-CEM and 1OnM of labeled DNA duplex.
  • reaction samples were taken, enriched with lOO ⁇ M of TTP and further incubated for an additional 15 minutes in order to test if reaction products contain the blocking 3' group (and thus are resistant to the extension).
  • Polymerases were incubated for a 60 min at 37°C (buffer Tango) with 50 ⁇ M TTP-CE and 10 nM of labeled DNA duplex.
  • reaction samples were taken, enriched with lOO ⁇ M of TTP and further incubated for an additional 15 minutes in order to test if reaction products contain the blocking 3' group (and thus are resistant to the extension).

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Abstract

La présente invention concerne des composés de structure générale (I) ou des sels de ceux-ci, où B représente une base nucléotidique, X et Y représentent indépendamment oxygène ou soufre, Z représente un groupe chimique caractérisé par des propriétés de puissant électro-attracteur, R1 représente hydrogène, hydroxyle ou un hydroxyle protégé, R2 et R3 représentent conjointement ou séparément hydrogène ou un hydrocarbyle, R4 représente hydrogène ou un groupe chimique constitué d'une molécule de liaison L, qui relie un groupe détectable au reste de la structure (I), R5 représente hydrogène ou un groupe électro-attracteur supplémentaire et peut être identique à Z ou différent de celui-ci, R6 représente hydrogène ou un groupe chimique constitué d'une unité de liaison L, d'un groupe clivable et d'un groupe détectable réunis dans l'ordre suivant R6 = -L- groupe clivable-groupe détectable. Le groupe détectable est placé sur R4 ou sur R6 ou est absent et R4 représente alors hydrogène et le groupe détectable est placé sur R6 ou R6 représente hydrogène et le groupe détectable est placé sur R4 ou alors R4 et R6 représentent tous les deux hydrogène. Les nombres m et n représentent indépendamment 0 ou 1. Les composés de formule (I) sont utilisés comme terminateurs d'extension de chaîne réversibles. Cette invention concerne également l'utilisation des composés de formule (I) dans un séquençage d'acides nucléiques, ainsi qu'un procédé de préparation de composés de formule (I).
PCT/EP2007/059207 2006-09-04 2007-09-04 Terminateurs réversibles pour un séquençage efficace par synthèse WO2008037568A2 (fr)

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US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
US9150896B2 (en) 2012-09-06 2015-10-06 Illumina, Inc. Nucleotides and primers with removable blocking groups
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
US9868947B2 (en) 2015-05-04 2018-01-16 Washington University Compositions and methods for the construction of a random allelic series
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US10738072B1 (en) 2018-10-25 2020-08-11 Singular Genomics Systems, Inc. Nucleotide analogues
EP3699283A1 (fr) 2014-10-20 2020-08-26 Molecular Assemblies Inc. Enzymes indépendantes de la matrice modifiées pour la synthèse de polydésoxynucléotides
US10822653B1 (en) 2019-01-08 2020-11-03 Singular Genomics Systems, Inc. Nucleotide cleavable linkers and uses thereof
WO2021123074A1 (fr) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Procédés de séquençage par synthèse au moyen d'un schéma de marquage consécutif
US11085076B2 (en) 2015-09-28 2021-08-10 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
WO2022083686A1 (fr) * 2020-10-21 2022-04-28 深圳华大生命科学研究院 Nucléoside ou nucléotide modifié
WO2022103499A1 (fr) 2020-11-11 2022-05-19 Microsoft Technology Licensing, Llc Contrôle spatial de synthèse de polynucléotides par coiffage de brin
US11591647B2 (en) 2017-03-06 2023-02-28 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US12018325B2 (en) 2017-03-28 2024-06-25 The Trustees Of Columbia University In The City Of New York 3′-O-modified nucleotide analogues with different cleavable linkers for attaching fluorescent labels to the base for DNA sequencing by synthesis
US12188086B2 (en) 2018-03-15 2025-01-07 The Trustees Of Columbia University In The City Of New York Nucleotide analogues and use thereof for nucleic acid sequencing and analysis preliminary class
US12215124B2 (en) 2018-02-21 2025-02-04 Singular Genomics Systems, Inc. Fluorinated nucleotides and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
WO2001025247A1 (fr) * 1999-10-05 2001-04-12 Quiatech Ab Composes de protection des hydroxyles et procedes d'utilisation
WO2002029003A2 (fr) * 2000-10-06 2002-04-11 The Trustees Of Columbia University In The City Of New York Methode d'analyse massivement parallele destinee a decoder l'adn et l'arn
WO2004018497A2 (fr) * 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5302509A (en) * 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
WO2001025247A1 (fr) * 1999-10-05 2001-04-12 Quiatech Ab Composes de protection des hydroxyles et procedes d'utilisation
WO2002029003A2 (fr) * 2000-10-06 2002-04-11 The Trustees Of Columbia University In The City Of New York Methode d'analyse massivement parallele destinee a decoder l'adn et l'arn
WO2004018497A2 (fr) * 2002-08-23 2004-03-04 Solexa Limited Nucleotides modifies

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OHGI T ET AL: "A New RNA Synthetic Method with a 2'-O-(2-Cyanoethoxymethyl) Protecting Group" ORGANIC LETTERS, AMERICAN CHEMICAL SOCIETY, COLUMBUS, OH; US, vol. 7, no. 16, 1 January 2005 (2005-01-01), pages 3477-3480, XP003009345 ISSN: 1523-7060 cited in the application *
SANEYOSHI H ET AL: "A General Method for the Synthesis of 2'-O-Cyanoethylated Oligoribonucleotides Having Promising Hybridization Affinity for DNA and RNA and Enhanced Nuclease Resistance" JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, vol. 70, no. 25, 1 January 2005 (2005-01-01), pages 10453-10460, XP003017305 ISSN: 0022-3263 cited in the application *
UMEMOTO T ET AL: "Oligoribonucleotide synthesis by the use of 1-(2-cyanoethoxy)ethyl (CEE) as a 2'-hydroxy protecting group" TETRAHEDRON LETTERS, ELSEVIER, AMSTERDAM, vol. 45, no. 52, 20 December 2004 (2004-12-20), pages 9529-9531, XP004727393 ISSN: 0040-4039 cited in the application *

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US9150896B2 (en) 2012-09-06 2015-10-06 Illumina, Inc. Nucleotides and primers with removable blocking groups
US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
US10249038B2 (en) 2013-03-15 2019-04-02 Qiagen Sciences, Llc Flow cell alignment methods and systems
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US9868947B2 (en) 2015-05-04 2018-01-16 Washington University Compositions and methods for the construction of a random allelic series
US12006540B2 (en) 2015-09-28 2024-06-11 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
US11999999B2 (en) 2015-09-28 2024-06-04 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
US11085076B2 (en) 2015-09-28 2021-08-10 The Trustees Of Columbia University In The City Of New York Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis
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US11591647B2 (en) 2017-03-06 2023-02-28 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US11773439B2 (en) 2017-03-06 2023-10-03 Singular Genomics Systems, Inc. Nucleic acid sequencing-by-synthesis (SBS) methods that combine SBS cycle steps
US12018325B2 (en) 2017-03-28 2024-06-25 The Trustees Of Columbia University In The City Of New York 3′-O-modified nucleotide analogues with different cleavable linkers for attaching fluorescent labels to the base for DNA sequencing by synthesis
US12215124B2 (en) 2018-02-21 2025-02-04 Singular Genomics Systems, Inc. Fluorinated nucleotides and uses thereof
US12188086B2 (en) 2018-03-15 2025-01-07 The Trustees Of Columbia University In The City Of New York Nucleotide analogues and use thereof for nucleic acid sequencing and analysis preliminary class
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US12145960B2 (en) 2018-10-25 2024-11-19 Singular Genomics Systems, Inc. Nucleotide analogues
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US10738072B1 (en) 2018-10-25 2020-08-11 Singular Genomics Systems, Inc. Nucleotide analogues
US11970735B2 (en) 2019-01-08 2024-04-30 Singular Genomics Systems, Inc. Nucleotide cleavable linkers and uses thereof
US10822653B1 (en) 2019-01-08 2020-11-03 Singular Genomics Systems, Inc. Nucleotide cleavable linkers and uses thereof
WO2021123074A1 (fr) 2019-12-18 2021-06-24 F. Hoffmann-La Roche Ag Procédés de séquençage par synthèse au moyen d'un schéma de marquage consécutif
WO2022083686A1 (fr) * 2020-10-21 2022-04-28 深圳华大生命科学研究院 Nucléoside ou nucléotide modifié
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