ES2642963T3 - Labeled nucleotides - Google Patents

Description

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

Marked nucleotides Field of the invention

This invention relates to labeled nucleotides. Particularly, this invention describes nucleotides that have a removable marker and their use in polynucleotide sequencing methods.

Background

Advances in the study of molecules have been driven, in part, by the improvement of the technologies used to characterize the molecules or their biological reactions. Particularly, the study of DNA and RNA nucleic acids has benefited from the developing technologies used for sequence analysis and the study of hybridization events.

An example of the technologies that have improved the study of nucleic acids is the development of matrices made of immobilized nucleic acids. These matrices typically consist of a high density matrix of immobilized polynucleotides on a solid support material. See, for example, Fodor et al., Trends Biotech. 12: 1926, 1994, which describes the ways of assembling nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed in defined areas to allow the binding of suitably modified nucleotide phosphoramidites. Fabricated matrices can also be produced by the technique of "dotting" known polynucleotides on a solid support in predetermined positions (eg, Stimpson et al., Proc. Natl. Acad. Sci. USA 92: 6379-6383, 1995).

A further development in matrix technology is the binding of polynucleotides to solid support material to form single molecule matrices. Matrices of this type are described in the international patent application num. WO 00 / 06770.The advantage of these matrices is that reactions can be monitored at the level of a single molecule and information about a large number of unique molecules can be collected from a single reaction.

For DNA matrices to be useful, the sequences of the molecules must be determined. U.S. Patent UU. num. 5,302,509 describes a method for sequencing immobilized polynucleotides on a solid support. The method is based on the incorporation of bases A, G, C and T blocked in 3 'which have a fluorescent marker different from the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a complementary base to the target polynucleotide, but the addition by the 3 'blocking group is also prevented. The marking of the incorporated base can be determined and the blocking group is removed by chemical cleavage to allow further polymerization to occur.

Welch et al. (Chem. Eur. J. 5 (3): 951-960, 1999) describes the synthesis of modified triphosphate nucleotides with a 3'-O-blocking group that is photolabile and fluorescent. The modified nucleotides are intended to be used in DNA sequencing experiments. However, these nucleotides proved difficult to incorporate into an existing polynucleotide, due to the inability to place themselves in the active site of the enzyme polymerase.

Zhu et al. (Cytometry 28: 206-211, 1997) further describes the use of fluorescent markers attached to a nucleotide through the base group. The labeled nucleotides are intended to be used in fluorescence in situ hybridization (FlSH) experiments, where a series of embedded labeled nucleotides is required to produce a fluorescent "bar code".

WO00 / 15844, WO96 / 11937 and Prober JM et al .; Science (1987); Vol 238, no 4825 pages 336-341 describe variants in the dideoxynucleotide terminators used in the 'Sanger' electrophoretic sequencing. The nucleotides described have both 2 'and 3' positions as hydrogen.

WO99 / 57321 discloses a sequencing method in which the number of nucleotides incorporated is counted. In order to be counted, the nucleotide must have a free 3'hydroxyl portion to allow multiple incorporation events to occur.

US6136543 discloses a nucleotide with a photolabil linker between the base and the label and a free 3'-hydroxyl. D5 also discloses separately a nucleotide with a label attached to the 3 'position through a blocking portion.

Summary of the invention

In the present invention, a nucleoside or nucleotide molecule is linked to a detectable label through a cleavable linker group attached to the base, which makes the molecule useful in techniques using labeled nucleosides or nucleotides, for example, reactions sequencing, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies and other techniques

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

which use enzymes such as polymerases, reverse transcriptases, terminal transferases, or other DNA modifying enzymes. The invention is especially useful in techniques using labeled dNTPs, such as nick translation, random primer labeling, final labeling (eg, with terminal deoxynucleotidyltransferase), reverse transcription or nucleic acid amplification. The molecules of the present invention are unlike the prior art, where the label is attached to the ribose or deoxyribose sugar, or where the label is attached through a non-cleavable linker.

According to a first aspect of the invention, a nucleotide or nucleoside molecule, or an analog thereof, has a base that is attached to a detectable label through a cleavable linker.

The invention characterizes a nucleotide or nucleoside molecule, which has a base that is attached to a detectable label through a cleavable linker. The base can be a purine, or a pyrimidine. The base can be a desazapurina. The molecule may have a portion of ribose or deoxyribose sugar. Ribose or deoxyribose sugar may include a protective group attached through the 2 'or 3' oxygen atom. The protecting group can be removed to expose a 3'-OH. The molecule can be a deoxyribonucleotide triphosphate. The detectable marker can be a fluorophore. The linker may contain a disulfide bond.

The invention further features a method of labeling a nucleic acid molecule, where the method includes incorporating a nucleotide or nucleoside molecule into the nucleic acid molecule, where the nucleotide or nucleoside molecule has a base that is attached to a detectable label. through a cleavable linker. The incorporation step can be achieved through a terminal transferase, a polymerase or a reverse transcriptase. The base can be a purine, or a pyrimidine. The base can be a desazapurina. The nucleotide or nucleoside molecule may have a portion of ribose or deoxyribose sugar. Ribose or deoxyribose sugar may include a protective group attached through the 2 'or 3' oxygen atom. The protecting group can be removed to expose a 3'-OH group. The molecule can be a deoxyribonucleotide triphosphate. The detectable marker can be a fluorophore. The linker may contain a disulfide bond. The detectable label and / or the cleavable linker may be of sufficient size to prevent the incorporation of a second nucleotide or nucleoside in the nucleic acid molecule.

In another aspect, the invention characterizes a method for determining the sequence of a single stranded target polynucleotide, where the method includes monitoring the sequential incorporation of complementary nucleotides, where each nucleotide has a base that is attached to a detectable marker through of a cleavable linker, and where the identity of each incorporated nucleotide is determined by detecting the marker attached to the base, and subsequent removal of the marker.

The invention further characterizes a method for determining the sequence of a single stranded target polynucleotide, where the method includes: (a) providing nucleotides, where the nucleotides have a base that is attached to a detectable label through a cleavable linker, and where the detectable marker bound to each type of nucleotide can be distinguished by the detection of the detectable marker used for other types of nucleotides; (b) incorporate a nucleotide into the complement of the single stranded target polynucleotide; (c) detect the nucleotide marker of (b), thereby determining the type of nucleotide incorporated; (d) remove the nucleotide marker from (b); and (e) optionally repeat steps (b) - (d) one or more times, and thereby determine the sequence of the single stranded target polynucleotide.

In the methods described in the present description, each of the nucleotides can be contacted sequentially with the objective, with the elimination of the nucleotides not incorporated before the addition of the next nucleotide, where the detection and elimination of the marker and the group of Blocking is carried out after the addition of each nucleotide or after the addition of the four nucleotides.

In the methods, all nucleotides can contact the target simultaneously, that is, a composition comprising all the different nucleotides that contact the target, and unincorporated nucleotides are removed before detection and after detection. Marker removal.

The methods may comprise a first stage and a second stage, where in the first stage, a first composition comprising two of the four types of nucleotides is contacted with the target, and unincorporated nucleotides are removed prior to detection and after removal of the marker, and where in the second stage, a second composition comprising the two nucleotides not included in the first composition is contacted with the target, and the unincorporated nucleotides are removed before detection and after the elimination of the marker, and where the first stage and the second stage can optionally be repeated one or more times.

The methods described in the present description may further comprise a first stage and a second stage, where in the first stage, a composition comprising one of the four nucleotides is contacted with the target, and the unincorporated nucleotides are removed before the detection and subsequent to the removal of the marker, and where in the second stage, a second composition comprising the three nucleotides not included in the first composition is contacted with the target, and the unincorporated nucleotides are removed before detection Y

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

after the removal of the marker, and where the first stage and the second stage can optionally be repeated one or more times.

The methods described in the present description may further comprise a first stage and a second stage, where in the first stage, a first composition comprising three of the four nucleotides is contacted with the target, and the unincorporated nucleotides are removed before of the detection and subsequent to the removal of the marker, and where in the second stage, a composition comprising the nucleotide not included in the first composition is contacted with the target, and the unincorporated nucleotides are removed before detection and after the removal of the marker, and where the first stage and the second stage can optionally be repeated one or more times.

In a further aspect, the invention characterizes a case, where the case includes: (a) individual nucleotides, where each nucleotide has a base that is attached to a detectable label through a cleavable linker, and where the detectable label attached to each nucleotide can be distinguished with the detection of the detectable marker used for the other three nucleotides, and (b) packaging materials for these. The kit may also include an enzyme and appropriate buffers for the action of the enzyme.

Nucleotides / nucleosides are suitable for use in many different DNA-based methodologies, which include DNA synthesis and DNA sequencing protocols.

According to another aspect of the invention, a method for determining the sequence of an objective polynucleotide comprises monitoring the sequential incorporation of complementary nucleotides, wherein the nucleotides comprise a detectable label attached to the nucleotide base portion through a linker. cleavable, detect incorporation by monitoring the marker and remove the marker to allow the incorporation of the additional nucleotide.

Description of the figures

Figure 1 shows the structure of an exemplary nucleotide useful in the invention. For each structure, X can be H, phosphate, diphosphate or triphosphate. Ri and R2 can be the same or different, and can be selected from H, OH, or any group that can be transformed into an OH.

Figure 2 shows linker structures useful in the invention, including (1) disulfide linkers in addition to a general definition of linkers that can be used.

Figure 3 shows some functional molecules useful in the invention, including some cleavable linkers. In these structures, R1 and R2 can be the same or different, and can be H, OH, or any group that can be transformed into an OH group, including a carbonyl group. R3 represents one or more substituents independently selected from alkyl, alkoxy, amino or halogen groups.

Figure 4 shows a denaturing gel showing the incorporation of the triphosphate of Example 1 using Klenow polymerase.

Detailed description of the invention

The present invention relates to nucleotides and nucleosides that are modified by binding a label through a cleavable linker, which makes the molecule useful in techniques where the labeled molecule interacts with an enzyme, such as sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, techniques that use enzymes such as polymerase, reverse transcriptase, terminal transferase, techniques that use labeled dNTPs (eg, nick translation, random primer labeling, final labeling (eg, with terminal deoxynucleotidyltransferase), reverse transcription or nucleic acid amplification).

As is known in the art, a "nucleotide" consists of a nitrogen base, a sugar, and one or more phosphate groups. In RNA, sugar is a ribose, and in DNA a deoxyribose, that is, a sugar that lacks a hydroxyl group that is present in ribose. The nitrogen base is a derivative of purine or pyrimidine. The purines are adenosine (A) and guanidine (G), and the pyrimidines are citidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is bound to the N-1 of a pyrimidine or to the N-9 of a purine. A nucleotide is also a phosphate ester of a nucleoside, where esterification occurs in the hydroxyl group attached to the C-5 of the sugar. Nucleotides are usually mono, di or triphosphates.

A "nucleoside" is structurally similar to a nucleotide, but lacks the phosphate portions. An example of a nucleoside analog would be one where the label is attached to the base and there is no phosphate group attached to the sugar molecule.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

Although the base is usually mentioned as a purine or pyrimidine, the expert will appreciate that derivatives and analogs are available which do not alter the ability to experience the pairing of Watson-Crick nucleotide or nucleoside bases. "Derivative" or "analog" means a compound or molecule whose central structure is the same as, or closely resembles that of a parental compound, but which has a chemical or physical modification, such as a different or additional side group, which It allows the nucleotide derivative or nucleoside to bind to another molecule. For example, the base can be a desazapurina. Derivatives must be able to experience Watson-Crick mating. "Derivative" and "analogous" also mean a synthetic derivative of nucleotide or nucleoside having modified base portions and / or modified sugar portions. Such derivatives and analogs are discussed in, for example, Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs may further comprise modified phosphodiester bonds, which include phosphorothioate, phosphorodithioate, alkyl phosphonate, phosphoranilidate and phosphoramidate linkages. Analogs must be able to experience Watson-Crick base pairing. "Derivative" and "analog", as used herein, may be used interchangeably, and are encompassed by the terms "nucleotide" and "nucleoside" as defined in the present description.

The present invention can make use of conventional detectable markers. Detection can be carried out by any suitable method, including fluorescence spectroscopy or by other optical means. The preferred marker is a fluorophore, which, after energy absorption, emits radiation at a defined wavelength. Many suitable fluorescent markers are known. For example, Welch et al. (Chem. Eur. J. 5 (3): 951-960, 1999) describes functionalized dansyl fluorescent portions that can be used in the present invention. Zhu et al. (Cytometry 28: 206-211, 1997) describe the use of the fluorescent markers Cy3 and Cy5, which can also be used in the present invention. Suitable markers for use are also described in Prober et al. (Science 238: 336-341,1987); Connell et al. (BioTechniques 5 (4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15 (11): 4593-4602, 1987) and Smith et al. (Nature 321: 674, 1986). Other commercially available fluorescent markers include, but are not limited to, fluorescefna, rhodamine (which includes TMR, texas red and Rox), alexa, bodipy, acridine, coumarin, pyrene, benzantracene and cyanines.

Multiple markers can also be used in the invention. For example, bifluoroforo FRET cassettes (Tet. Letts. 46: 8867-8871, 2000) are well known in the art and can be used in the present invention. Multi fluor dendrimeric systems (J. Amer. Chem. Soc.123: 8101-8108, 2001) can also be used.

Although fluorescent markers are preferred, other forms of detectable markers will be useful for those skilled in the art. For example, microparticles, which include quantum dots (Empodocles, and others, Nature 399: 126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72: 6025-6029, 2000), microbeads (Lacoste and others, Proc. Natl. Acad. Sci USA 97 (17): 9461-9466, 2000), and markers detectable by mass spectrometry, can be used.

Multiple component markers can also be used in the invention. A multi-component marker is one that is dependent on the interaction with an additional compound for detection. The most common multi-component marker used in biology is the streptavidin biotin system. Biotin is used as the marker attached to the nucleotide base. Streptavidin is then added separately to allow detection to occur. Other multiple component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection.

The marker (or the marker and the construction of the linker) may be of sufficient size or structure to act as a block for the incorporation of another nucleotide on the nucleotide of the invention. This allows controlled polymerization to take place. The block may be due to steric hindrance, or it may be due to a combination of size, load and structure.

The invention will be further described with reference to nucleotides. However, unless otherwise indicated, it is intended that the reference to nucleotides be applied in addition to nucleosides. The invention will be further described with reference to DNA, although the description will also be applicable to RNA, PNA and other nucleic acids, unless otherwise indicated.

Modified nucleotides of the invention use a cleavable linker to bind the label to the nucleotide. The use of a cleavable linker ensures that the label can, if required, be removed after detection, avoiding any interference signal with any labeled nucleotide subsequently incorporated.

The cleavable linkers are known in the art, and conventional chemistry can be applied to bind a linker to a nucleotide base and a label. The linker can be cleaved by exposure to radicals, metals, reducing agents or oxidation agents. Suitable linkers can be adapted from standard chemical blocking groups, as described in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Suitable additional cleavage linkers that are used in solid phase synthesis are described in Guillier et al. (Chem. Rev. 100: 2092-2157, 2000).

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

The use of the term "cleavable linker" is not intended to imply that the complete linker is required to be removed from the nucleotide base. The cleavage site can be placed in a position in the linker that ensures that part of the linker remains attached to the nucleotide base after cleavage.

The linker can be attached at any position at the nucleotide base as long as the Watson-Crick base pairing can still be performed. In the context of the purine bases, it is preferred that the linker is attached through the purine position 7 or the preferred desazapurine analog, through an 8-modified purine, through an N-6 modified adenosine or a modified guanine in N-2. For pyrimidines, the binding is preferably through position 5 in the cytosine, thymidine or uracil and position N-4 in the cytosine. Suitable nucleotide structures are shown in Figure 1. For each structure in Figure 1, X may be H, phosphate, diphosphate or triphosphate. R1 and R2 may be the same or different, and may be selected from H, OH, or any group that can be transformed into an OH, which includes, but is not limited to, carbonyl.

Suitable linkers are generally shown in Figure 2 and include, but are not limited to, disulfide linkers (1), cleavage under reducing conditions, oxidative conditions, cleavage through the use of safety capture linkers and cleavage by mechanisms of elimination.

D. Excision under reducing conditions

There are many known linkers that are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts have been used to cleave benzyl and benzyloxycarbonyl groups. The reduction of disulfide bonds is also known in the art.

E. Excision under oxidative conditions

Oxidation-based approaches are well known in the art. These include the oxidation of p-alkoxybenzyl groups and the oxidation of the sulfur and selenium linkers. The use of aqueous iodine to cleave disulfides and other linkers based on selenium or sulfur are also within the scope of the invention.

F. Safety capture linkers

Security capture linkers are those that are cleaved in two stages. In a preferred system, the first stage is the generation of a reactive nucleophilic center, following a second stage that involves an intramolecular cyclization resulting in excision. For example, the levulfnic ester bonds can be treated with hydrazine or photochemistry to release an active amine, which can then be cycled to cleave an ester in another part of the molecule (Burgess et al., J. Org. Chem. 62: 5165-5168, 1997).

G. Excision by elimination mechanisms

Elimination reactions can also be used. For example, base catalyzed elimination of groups such as Fmoc and cyanoethyl, and palladium catalyzed reductive elimination of allylic systems can be used.

As well as the cleavage site, the linker may comprise a spacer unit. The spacer separates the nucleotide base from the cleavage site or marker. The length of the linker is not important as long as the marker is kept at a sufficient distance from the nucleotide so as not to interfere with any interaction between the nucleotide and an enzyme.

The modified nucleotides may further comprise additional groups or modifications in the sugar group. For example, a dideoxyribose derivative, which lacks two oxygen in the ribose ring structure (in positions 2 'and 3'), can be prepared, and can be used as a block for further incorporation of nucleotides into a chain of growing oligonucleotide. The protecting group is intended to prevent the incorporation of nucleotides into a nascent polynucleotide chain, and can be removed under defined conditions to allow polymerization to occur. Unlike the prior art, there is no detectable marker attached at the 3 'position of the ribose. This ensures that the steric hindrance with the polymerase enzyme is reduced, although it still allows the control of incorporation using the protective group.

The expert will appreciate how to bind a suitable protecting group to the ribose ring to block interactions with 3'-OH. The protective group can be joined directly in the 3 'position, or it can be joined in the 2' position (the protective group is of sufficient size or load to block the interactions in the 3 'position). Alternatively, the protecting group can be joined in both 3 'and 2' positions, and can be cleaved to expose the 3'OH group.

Suitable protecting groups will be apparent to the expert, and can be formed from any suitable protective group described in Green and Wuts, supra. The protecting group must be removed (or modified) to produce a 3 'OH group. The process used to obtain the 3'OH group can be any suitable chemical or enzymatic reaction.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

60

65

The labile linker may consist of cleavable functionality under identical conditions for blocking. This will make the check-out process more efficient since only one treatment will be needed to cleave both the marker and the block. Thus, the linker may contain functional groups as described in Figure 3, which could be cleaved with hydroxyl functionality either in the residual nucleoside or in the deleted label. The linker may also consist of a completely different chemical functionality that turns out to be labile to the conditions used to cleave the block.

The term "alkyl" covers the alkyl groups of both the straight chain and the branched chain. Unless the context indicates otherwise, the term "alkyl" refers to groups having 1 to 8 carbon atoms, and typically 1 to 6 carbon atoms, for example 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl butyl, 3-methyl butyl, and n-hexyl and its isomers.

Examples of cycloalkyl groups are those that have 3 to 10 ring atoms, particular examples include those derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane, bicycloheptane and decalin.

Examples of alkenyl groups include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, butenyl, buta-1,4-dienyl, pentenyl, and hexenyl.

Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl and cyclohexenyl.

The term "alkoxy" refers to a C1-6 alkoxy unless otherwise indicated: -OR, where R is a C1-6 alkyl group. Examples of C1-6 alkoxy groups include, but are not limited to. a, -OMe (methoxy), -OEt (ethoxy), -O (nPr) (n-propoxy), -O (iPr) (isopropoxy), -O (nBu) (n-butoxy), -O (sBu) (sec-butoxy), -O (iBu) (isobutoxy), and -O (tBu) (tert-butoxy).

The term "amino" refers to groups of the NR1R2 type, wherein R1 and R2 are independently selected from hydrogen, a C1-6 alkyl group (also referred to as C1-6 alkylamino or C1-6 alkylamino).

The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.

The nucleotide molecules of the present invention are suitable for use in many different methods where nucleotide detection is required.

DNA sequencing methods, such as those described in US Patent No. 5,302,509 can be carried out using nucleotides.

A method for determining the sequence of a target polynucleotide can be carried out by contacting the target polynucleotide separately with the different nucleotides to form the complement to that of the target polynucleotide and detect the incorporation of the nucleotides. Such a method uses polymerization, in which a polymerase enzyme extends the complementary chain by incorporating the correct nucleotide complementary to that of the target. The polymerization reaction also requires a specific primer to initiate polymerization.

For each cycle, the incorporation of the labeled nucleotide is carried out by the enzyme polymerase, and then the incorporation event is determined. There are many different polymerase enzymes, and it will be evident to the person with experience in the art, which is the most appropriate to use. Preferred enzymes include aDn polymerase I, the Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or vent polymerase. In addition, a polymerase designed by engineering can be used to have specific properties.

Sequencing methods are preferably carried out with the target polynucleotide disposed on a solid support. Multiple target polynucleotides can be immobilized on the solid support through linker molecules, or they can be attached to particles, for example, microspheres, which can also be attached to a solid support material.

The polynucleotides can be attached to the solid support by a number of means, including the use of biotin-avidin interactions. Methods for immobilization of polynucleotides on a solid support are well known in the art, and include lithographic techniques and "tapping" of individual polynucleotides at defined positions on a solid support. Suitable solid supports are known in the art, and include glass and pearl slides, silicon and ceramic surfaces and plastic materials. The support is normally a flat surface although microscopic beads (microspheres) can also be used and in turn, can be attached to another solid support by known means. The microspheres can be of any suitable size, typically in the range of 10 nm to 100 nm in diameter. In a preferred embodiment, the polynucleotides adhere directly to a flat surface, preferably a flat glass surface. The union will preferably be by means of a covalent bond. Preferably, the matrices that are used are single-molecule matrices comprising polynucleotides in different optically resolved areas, for example, as described in international application no. WO 00/06770.

5

10

fifteen

twenty

25

30

35

40

Four. Five

fifty

55

The sequencing method can be carried out in both matrices of a single polynucleotide molecule and molecules of multiple polynucleotides, that is, matrices of different individual polynucleotide molecules and matrices of different regions comprising multiple copies of a single polynucleotide molecule. Single molecule matrices allow each individual polynucleotide to be solved separately. The use of single molecule matrices is preferred. Sequencing of single-molecule matrices allows a spatially non-destructive spatial matrix to be formed.

The method makes use of the polymerization reaction to generate the complementary sequence of the objective. The conditions necessary for the polymerization to occur will be apparent to the expert.

To carry out the polymerase reaction it will normally be necessary to first hybridize a primer sequence with the target polynucleotide, the primer sequence is recognized by the enzyme polymerase and acts as an initiation site for subsequent extension of the complementary chain. The primer sequence can be added as a separate component with respect to the target polynucleotide. Alternatively, the primer and the target polynucleotide can each be part of a single chain molecule, where the primer portion forms an intramolecular duplex with a portion of the target, that is, a hairpin loop structure. This structure can be immobilized on the solid support at any point of the molecule. Other conditions necessary to carry out the polymerase reaction, which include temperature, pH, buffer compositions, etc., will be apparent to those skilled in the art.

The modified nucleotides of the invention are then contacted with the target polynucleotide, to allow polymerization to occur. Nucleotides can be added sequentially, that is, the separate addition of each type of nucleotide (A, T, G or C) or added together. If added together, it is preferred that each type of nucleotide be labeled with a different marker.

This polymerization step is allowed to proceed for a sufficient time to allow the incorporation of a nucleotide.

Nucleotides that are not incorporated are then removed, for example, by subjecting the matrix to a washing step, and then the detection of the incorporated markers can be carried out.

The detection can be by conventional means, for example, if the marker is a fluorescent portion, the detection of a built-in base can be carried out by using a confocal scanning microscope to explore the surface of the matrix with a laser, to represent the image of a fluorophore directly attached to the incorporated base. Alternatively, a sensitive 2-D detector, such as a coupled load detector (CCD), can be used to display the generated individual signals. However, other techniques such as near field scanning optical microscopy (SNOM) are available, and can be used when images of dense matrices are formed. For example, using SNOM, individual polynucleotides can be distinguished when a distance of less than 100 nm is separated, for example, 10 nm to 10pm. For a description of near-field scanning optical microscopy, see Moyer et al., Laser Focus World 29:10, 1993. A suitable apparatus used to form the images of polynucleotide matrices is known and the establishment of the technique will be apparent. for the expert

After detection, the marker can be removed using suitable conditions that cleave the linker.

The use of modified nucleotides is not limited to DNA sequencing techniques, and other techniques, which include polynucleotide synthesis, DNA hybridization assays and single nucleotide polymorphism studies can be carried out using the nucleotides of the invention. Any technique that involves the interaction between a nucleotide and an enzyme can make use of the molecules of the invention. For example, the molecule can be used as a substrate for a reverse transcriptase or a terminal transferase enzyme.

Suitable structures are described in the following Examples and are shown in the attached figures.

Examples

Example 1. Synthesis of a Disulfide Bond.

5

10

fifteen

twenty

25

image 1

t-Butyl-N- (2-mercaptoethyl) carbamate (3 mmol, 0.5 mL) was added dropwise to a solution of 1.32g (6.0 mmol) of aldritiol in 15 mL of MeOH. After 1.5 h the reaction was complete and the solvent evaporated. The crude product was purified by chromatography on silica with ethyl acetate: petroleum ether (1: 4). Product 1a was obtained as a slightly yellow oil (0.76 g, 2.67 mmol, 89%). 1H NMR (500 Mhz, D6-DMSO): d = 1.38 (s, 9 H, tBu), 2.88 (t, J = 6.6 Hz, 2 H, SCH2) 3.20 (q, J = 6.6 Hz, 2 H, CH2NH ), 7.02 (bs, 1 H, NH), 7.24 (ddd, J = 7.3 Hz, J = 4.9 Hz, J = 1.0 Hz, 1 H, H-5), 7.77 (dt, J = 8.1 Hz, J = 1.0 Hz, 1 H, H-3), 7.82 (ddd, J = 8.1 Hz, J = 7.4 Hz, J = 1.8 Hz, 1 H, H-4), 8.46 (ddd, J = 4.9 Hz, J = 1.8 Hz, J = 1.0 Hz, 1 H, H-6).

image2

To deprotect the 1a amine, 17 mg of 1a (60 pmol) was dissolved in a mixture of 0.5 mL of DCM and 0.5 mL of trifluoroacetic acid. This mixture was stirred for 2.5 h at room temperature and then the solvents were removed under reduced pressure. The residue was dissolved three times again in 2 mL of DCM and evaporated to dryness. The unprotected product was dried under high vacuum for 3 h and then dissolved in 1 mL of dry DMF. It was assumed that the check out was complete.

To a solution of 15 mg of 5-carboxy tetramethyl rhodamine (35 pmol) in 2 mL of DMF, 8.0 mg of N-hydroxy succinimide (70 pmol) and 7.8 mg DCC (38 pmol) were added. The mixture was stirred for 6 h in the dark. Then 22 pL of DIPEA (126 pmol) and the deprotection solution 1a in 1 mL of DMF were added. After stirring the reaction mixture overnight in the dark, the solvent was removed under reduced pressure. The residue was dissolved in DCM and washed with saturated NaCl solution. After drying over MgSO4 the crude mixture was purified on silica with CHCl3: MeOH (3: 1) as solvent. 1b was isolated as a dark red solid with a yield of 90% (19.2 mg, 31.4 pmol). 1H NMR (500 MHz, D6-DMSO): 5 = 3.09 (t, J = 6.7 Hz, 2 H, SCH2), 3.63 (q, J = 6.2 Hz, 2 H, CH2NH), 6.48 - 6.53 (m, 6 H, H-Anthracene), 7.23-7.26 [m, 1 H, H-5 (pyridine)], 7.32 (d, J = 7.9 Hz, 1 Hz, H-3), 7.81 - 7.82 [m, 2 H,

5

10

fifteen

twenty

25

30

35

40

H-3 + H-4 (pyridine)], 8.21 (d, J = 7.9 Hz, 1 H, H-4), 8.43 (s, 1 H, H-6), 8.47 [dt, J = 4.7 Hz, J = 1.3 Hz, 1 H, H-6 (pyridine)], 9.03 (t, J = 5.2 Hz, 1 H, NH).

image3

Mercaptopropionic acid (20.6pmol, 1.8 mL) was added to a solution of 19.6 mg of 1b (32.7 pmol) in 2 mL of MeOH. The mixture was stirred for 2.5 h in the dark. The solvent was removed under reduced pressure. The crude product was purified by silica chromatography with CHCl3: MeOH: AcOH 15: 1: 0.5 as the solvent mixture. 15.5 mg (26 pmol, 80%) of 1c dark red crystals could be isolated. 1H NMR (500 MHz, D2O): 5 = 2.53 (t, J = 7.0 Hz, 2 H, CH2COOH), 2.88 (t, J = 7.0 Hz, 2 H, CH2CH2COOH), 2.96 - 2.99 (m, 2 H, CH2CH2NH), 3.73 (t, J = 6.3 Hz, 2 H, CH2NH), 6.53 (d, J = 2.4 Hz, 2 H, H-Anthracene), 6.81 (dd, J = 9.5 Hz, J = 4.5 Hz, 2 H, H-Anthracene), 7.12 (d, J = 9.5 Hz, 2 H, H- Anthracene), 7.48 (d, J = 7.9 Hz, 1 H, H-3), 7.95 (dd, J = 8.1 Hz, J = 1.9 Hz, 1 H, H-2) 8.13 (d, J = 1.9 Hz, 1 H, H-1). + See electro pulverization (C30H31N3O6S2): expected 593.17; found 594.3 [M + H], 616.2 [M + Na].

image4

To a solution of 25.8 mg of 1c (43.4 pmol) in 3 mL of DMF (dry) was added 9.9 mg N-hydroxy succinimide (86.8 pmol) and 9.7 mg of DCC (47.1 pmol). The mixture was stirred for 5 h in the dark at room temperature and then put in the refrigerator overnight. The mixture was filtered through a cotton plug in a new flask and to this was added a solution of 865 pL of propargilamino dUTP (14.7 pmol, 17 pmol in 1 mL of H2O) and 3 mL of sodium borate buffer (0.1 M solution , pH 9). The mixture was stirred overnight. After the elimination of the solvents the residue was dissolved in as little water as possible and purified by HPLC. A Zorbax C18 column with 0.1 M triethylammonium bicarbonate (TEAB) and acetonitrile was used as buffers. 31P NMR (400 MHz, D2O): 5 = -4.73 (d), -9.93 (d), 19.03 (t) .- see electro spray (C42H47N6O19P3S2 assuming 4 H + against ions): expected 1096.16; found 1092.9.UV in Water: A (max) = 555 nm A (555) = 0.885 (c = 0.036 pmol).

Triphosphate (1) was successfully incorporated using Klenow DNA polymerase. The reaction was carried out under the following conditions: 50 mM Tris. HCl (pH 7.5), 10 mM NaCl, 2 mM DTT, 0.1 mM EDTA, 5 mM MgCh, 2pM compound 3, 100 nM DNA template (previously labeled with P32 and T4 polynucleotide kinase) and 10 units of commercial exo-Klenow (Amersham Corp., Arlington Heights, Illinois, uSa). The DNA templates were self-complementary hairpins (5'-TACCgTCgACgTCgACgCT ggCg-AgCgT gCT gCggTTTTT (C6-

amino) TTACCgCAgCACgCTCgCCAgCg; Sec. With num. of ident: 1). The reaction was carried out in a volume of 100 pL at 37 ° C with time points taken at 0, 1, 3, 5 and 10 min. The reaction products were electrophoresed in a denaturing polyacrylamide gel (8 M urea) at 20% and images were formed in a Phosphorlmager for Typhoon. An extension of a single complete base in 1 minute was observed indicating an efficient incorporation of the polymerase (disulfide bond gel Figure. 4). A second set of rails is shown where the material is exposed to the DTT after incorporation. A different band change can be observed which shows the removal of the dye from the DNA construct, thus showing a polymerase incorporation and cleavage cycle using this disulfide compound.

Claims (12)

5 10 fifteen twenty 25 30 35 1. A method for detecting the extension of a primer hybridized to a target polynucleotide, comprising: (a) provide at least one nucleotide comprising (1) a fluorescent marker bound by a nucleotide base linker, and (2) a 2 'or 3' oxygen-linked protective group of the nucleotide sugar, wherein the linker can be cleaved by exposure to radicals, metals, reducing agents or oxidizing agents; (b) extending the primer hybridized with the nucleotide only if the nucleotide base is complementary to the base at the corresponding position of the target polynucleotide; Y (c) detect the presence of the marker in the extended primer; thus detecting the extension of the primer. 2. The method of claim 1, wherein step (a) provides four different nucleotides. 3. The method of claim 1 or 2, further comprising: (d) exposing the extended primer to radicals, metals, reducing agents or oxidizing agents to cleave the fluorescent label. 4. The method of claim 1, wherein the linker and the protecting group are cleavable under identical conditions. 5. The method of claim 4, further comprising: (d) exposing the extended primer to a single set of cleavage conditions to remove the linker and the protecting group. 6. The method of claims 1-5, wherein the base is a purine, pyrimidine or deazapurine. 7. The method of claims 1-6, wherein the cleavable linker comprises a protected hydroxyl functionality. 8. The method of claims 1-7 wherein the linker comprises a cleavable alflic system. 9. The method of claims 1-8 wherein the polynucleotide is attached to a solid support. 10. An extended primer comprising a nucleotide having a base that is linked to a fluorescent label through a cleavable linker, wherein the molecule has a portion of ribose or deoxyribose sugar comprising a protective group attached through the atom of 2 'or 3' oxygen wherein the linker comprises a cleavable alflic system. 11. An array of extended primers each comprising a nucleotide that has a base that is linked to a fluorescent label through a cleavable linker, wherein the molecule has a portion of ribose or deoxyribose sugar comprising a protective group attached to through the 2 'or 3' oxygen atom where the linker comprises a cleavable alflic system, the matrix containing four different nucleotides. 12. The extended primer of claim 10 or the matrix of claim 11, wherein the linker and the protecting group are cleavable under identical conditions.