The 3apos is used for connecting the fluorescent label and a basic group for DNA synthesis sequencing and is provided with different cleavable connexons; -O-modified nucleotide analogs

Abstract

Disclosed herein, inter alia, are nucleotide analogs having cleavable orthogonal linkers and methods of use thereof.

Description

[0001] This application is a divisional application of a Chinese patent application filed on March 28, 2018, with Chinese patent application number 201880035369.2 and invention name “3'-O-modified nucleotide analogs with different cleavable linkers for connecting fluorescent labels to bases for DNA synthesis sequencing”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 477,947, filed on March 28, 2017, which is incorporated herein by reference in its entirety and for all purposes.

[0004] Throughout this application, reference is made to various publications and patents. Full citations of these references are found at the end of the specification immediately preceding the claims. The disclosures of these publications and patents are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art to which the present invention relates. Background Art

[0005] DNA sequencing is a fundamental tool in biological and medical research and is particularly important for personalized medicine. Various new DNA sequencing methods have been investigated with the goal of ultimately achieving the $1,000 genome goal; the main method is sequencing by synthesis (SBS), a method in which the DNA sequence is determined during a polymerase reaction (Hyman 1988; Ronagi et al. 1998; Ju et al. 2003; Li 2003; Braslavsky et al. 2003; Ruparel et al. 2005; Margulies et al. 2005; Ju et al. 2006; Wu et al. 2007; Guo et al. 2008; Bentley et al. 2008; Harris et al. 2008; Eid et al. 2009; Rothberg et al. 2011). The currently widely used high-throughput SBS technology (Bentley et al. 2008) uses a previously developed cleavable fluorescent nucleotide reversible terminator (NRT) sequencing chemistry (Ju et al. 2003; Ju et al. 2006). These cleavable fluorescent NRTs are designed based on the following principles: each of the four nucleotides (A, C, G, T) is modified by attaching a unique cleavable fluorophore to a specific position of the base and capping the 3'-OH group with a small reversible part so that it is still recognized as a substrate by the DNA polymerase. Therefore, cleavable fluorescent NRTs involve two site modifications (Ju et al. 2003; Ju et al. 2006): a fluorescent dye that acts as a reporter group on the base, and a small chemical part that is used to cap the 3'-OH to temporarily terminate the polymerase reaction after the nucleotides for sequence determination are incorporated. After incorporation and signal detection, the fluorophore is cleaved and the 3'-OH capping part is removed to resume the polymerase reaction in the next cycle. These cleavable fluorescent NRTs have been shown to be good substrates for reengineered polymerases and have been widely used in next-generation DNA sequencing systems (Ju et al. 2006; Bentley et al. 2008). In addition, it enables accurate determination of homopolymer sequences because only one base is identified in each cycle.

[0006] An SBS method that uses cleavable fluorescent nucleotide analogs as reversible terminators to sequence surface-immobilized DNA has been used (Ju et al. 2003; Li et al. 2003; Ruparel et al. 2005; Ju et al. 2006; Wu et al. 2007; Guo et al. 2008). In this method, nucleotides are modified at two specific positions so that they are still recognized as substrates by DNA polymerase: (i) different fluorophores with different fluorescence emissions are attached to specific positions of each of the four bases via cleavable linkers, and (ii) the 3'-OH group is capped with a small chemically reversible part. DNA polymerase only incorporates single nucleotide analogs that are complementary to the bases on the DNA template covalently attached to the surface. After incorporation, the unique fluorescence emission is detected to identify the incorporated nucleotide. Next, the fluorophore is removed and the 3'-OH group is chemically regenerated, which enables the next cycle of the polymerase reaction. Because the large surface on the DNA chip can have a high density of different dotted DNA templates, multiple bases can be identified in parallel per cycle, enabling simultaneous sequencing of a large number of DNA molecules. Previous work has firmly established the molecular-level strategy of SBS, which rationally modifies nucleotides by attaching cleavable fluorescent dyes to the bases and reversibly capping the 3'-OH with small moieties.

[0007] A nucleotide analog with an unprotected 3'-OH and a cleavable disulfide linker attached between the base and the fluorescent dye has been reported (Turcatti et al. 2008; Mitra et al. 2003). However, after the DNA polymerase-catalyzed extension reaction on the primer / template and imaging of the incorporated base, cleavage of the disulfide bond produces a free reactive -SH group, which must be capped with an alkylating agent (iodoacetamide as shown below) before the second extension reaction can be performed. This capping step not only adds an extra step to the process, but also limits the continuous addition of multiple nucleotides due to the long residual tail region on the nucleotide base portion. In this method, the sequencing read length is limited to 10 bases (Turcatti et al. 2008). Other disulfide-based methods require similar capping reactions to make the free SH groups unreactive (Mitra et al. 2003).

[0008] For long-read SBS strategies, it is preferred that the cleavable linkers are stable during the sequencing reaction, require less manipulation, and do not leave long tail regions on the bases after the cleavage reaction. Summary of the invention

[0009] The present invention provides a nucleotide analogue comprising: (i) deoxyribose or ribose, (ii) a base, which is linked to the 1' position of the deoxyribose or ribose, wherein the base is selected from the group consisting of: A, T, C, G and U or a derivative thereof, (iii) a blocking group, which is bound to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label, which is bound to the base via a cleavable linker.

[0010] The present invention also provides a composition comprising four different types of nucleotide analogs, wherein each type of nucleotide analogs comprises: a base selected from the group consisting of: A, T, C, G or U or a derivative thereof; deoxyribose or ribose; and a blocking group bound to the 3'-oxygen of the deoxyribose or ribose, and

[0011] (i) a first type of nucleotide analogue comprises a first type of detectable label bound to the base via a first type of linker;

[0012] (ii) the second type of nucleotide analogue comprises a second type of detectable label bound to the base via a second type of linker;

[0013] (iii) the third type of nucleotide analogue comprises a first type of detectable label bound to the base via a second type of linker; and

[0014] (iv) the fourth type of nucleotide analogue comprises a second type of detectable label bound to the base via a first type of linker;

[0015] The first type of linker is different from the second type of linker, and the first type of detectable label is different from the second type of detectable label.

[0016] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0017] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and four types of tagged nucleotide analogs under conditions that allow the nucleic acid polymerase to catalyze the incorporation of one tagged nucleotide analog into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a nucleic acid extension product,

[0018] wherein each of the at least four types of tagged nucleotide analogs comprises: a base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative thereof; a deoxyribose or ribose sugar; and a cleavable blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide, and

[0019] (i) a first type of nucleotide analogs comprises a first type of base and a first type of detectable label bound to the base via a first type of linker;

[0020] (ii) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0021] (iii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0022] (iv) a fourth type of nucleotide analogue comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0023] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0024] b) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0025] c) contacting the incorporated tagged nucleotide analog with means for cleaving a first type of linker;

[0026] d) determining whether the label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0027] e) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0028] f) cleaving the 3'-oxygen blocking group to thereby form a 3'-OH;

[0029] g) repeatedly performing steps (a)-(f) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0030] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0031] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0032] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and a first type of tagged nucleotide analogue under conditions that allow the nucleic acid polymerase to catalyze the incorporation of the tagged nucleotide analogue into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a DNA extension product,

[0033] wherein the tagged nucleotide analogs comprise a first type of base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof; a deoxyribose or ribose sugar; a cleavable blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide; and a first type of detectable label that is bound to the base via a first type of linker,

[0034] and if the tagged nucleotide is not incorporated, iteratively repeating the contacting with the second, third, and fourth types of tagged nucleotide analogs until the tagged nucleotide analog is incorporated, wherein

[0035] (i) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0036] (ii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0037] (iii) the fourth type of nucleotide analogs comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0038] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0039] b) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0040] c) contacting the incorporated nucleotide analogues with means for cleaving a first type of linker;

[0041] d) determining whether the detectable label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0042] e) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0043] f) cleaving the 3'-oxygen blocking group to thereby form 3'-OH,

[0044] g) repeatedly performing steps (a)-(f) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0045] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0046] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0047] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and four types of tagged nucleotide analogs under conditions that allow the nucleic acid polymerase to catalyze the incorporation of one tagged nucleotide analog into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a nucleic acid extension product,

[0048] wherein each of the at least four types of tagged nucleotide analogs comprises: a base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative thereof; a deoxyribose or ribose; and a blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide, and

[0049] (i) a first type of nucleotide analogs comprises a first type of base and a first type of detectable label bound to the base via a first type of linker;

[0050] (ii) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0051] (iii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0052] (iv) a fourth type of nucleotide analogue comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0053] and wherein the first type of detectable label is different from the second type of detectable label;

[0054] b) contacting the single-stranded nucleic acid with four types of nucleotide reversible terminators under conditions that allow a nucleic acid polymerase to catalyze the incorporation of one nucleotide reversible terminator into a primer, wherein each nucleotide reversible terminator comprises a blocking group for the 3'-oxygen of a deoxyribose or ribose sugar that prevents the polymerase from catalyzing the incorporation of a subsequent nucleotide under the following conditions:

[0055] (i) the polymerase fails to incorporate a tagged nucleotide analog in step a),

[0056] (ii) the nucleotide reversible terminator is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that hybridizes to the 3' terminal nucleotide residue of the primer, and

[0057] c) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0058] d) contacting the incorporated tagged nucleotide analog with means for cleaving a first type of linker;

[0059] e) determining whether the label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0060] f) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0061] g) cleaving the 3'-oxygen blocking group to thereby form a 3'-OH;

[0062] h) repeatedly performing steps (a)-(g) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0063] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0064] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0065] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and a first type of tagged nucleotide analogue under conditions that allow the nucleic acid polymerase to catalyze the incorporation of the tagged nucleotide analogue into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a DNA extension product,

[0066] wherein the tagged nucleotide analogs comprise a first type of base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof; a deoxyribose or ribose sugar; a blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide; and a first type of detectable label that is bound to the base via a first type of linker,

[0067] and if the tagged nucleotide is not incorporated, iteratively repeating the contacting with the second, third, and fourth types of tagged nucleotide analogs until the tagged nucleotide analog is incorporated, wherein

[0068] (i) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0069] (ii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0070] (iii) the fourth type of nucleotide analogs comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0071] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0072] b) contacting the single-stranded nucleic acid with four types of nucleotide reversible terminators under conditions that allow a nucleic acid polymerase to catalyze the incorporation of one nucleotide reversible terminator into a primer, wherein each nucleotide reversible terminator comprises a blocking group for the 3'-oxygen of a deoxyribose or ribose sugar that prevents the polymerase from catalyzing the incorporation of a subsequent nucleotide under the following conditions:

[0073] (i) the polymerase fails to incorporate the tagged nucleotide analog in step a), and

[0074] (ii) the nucleotide reversible terminator is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that hybridizes to the 3' terminal nucleotide residue of the primer;

[0075] c) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0076] d) contacting the incorporated nucleotide analogue with means for cleaving a first type of linker;

[0077] e) determining whether the detectable label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0078] f) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0079] g) cleaving the 3'-oxygen blocking group to thereby form 3'-OH,

[0080] h) repeatedly performing steps (a)-(g) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0081] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0082] The present invention also provides a kit for nucleic acid sequencing, comprising in separate compartments:

[0083] a) four types of tagged nucleotide analogs, wherein each type of tagged nucleotide analogs comprises a base selected from the group consisting of A, T, C, G, or U or a derivative thereof; a deoxyribose or ribose sugar; and a blocking group bound to the 3'-oxygen of the deoxyribose or ribose sugar, and

[0084] (i) a first type of nucleotide analogs comprises a first type of base and a first type of detectable label bound to the base via a first type of linker;

[0085] (ii) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0086] (iii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0087] (iv) a fourth type of nucleotide analogue comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0088] wherein the first type of linker is different from the second type of linker, and the first type of detectable label is different from the second type of detectable label. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1a and Figure 1b : For 2-color DNA SBS using the method described in Protocol 1, 3'-O-SS(DTM)-dNTP-SS-Dyes (3'-O-SS-dATP-7-SS-Rox and 3'-O-SS-dUTP-5-SS-BodipyFL) and 3'-O-SS(DTM)-dNTP-SS-Azo-Dyes (3'-O-SS-dGTP-7-Azo-Rox or 3'-O-SS-dGTP-7-SS-Azo-Rox and 3'-O-SS-dCTP-5-Azo-BodipyFL or 3'-O-SS-dCTP-5-SS-Azo-BodipyFL).

[0090] Figure 2a and Figure 2b : Synthesis of Azo linkers and general methods for synthesizing 3'-O-SS(DTM)-dNTP-SS-Azo-Dye. Amino acid derivatives of Azo linker molecules are synthesized using a recognized diazo coupling reaction. The resulting compound is coupled with DyeNHS ester to produce an acid derivative of the Azo linker labeled with a dye, which can be further converted into an NHS ester by treatment with DSC and TEA. Next, the product is coupled with the amino group of 3'-O-SS(DTM)-dNTP-SS-NH2 to produce 3'-O-SS(DTM)-dNTP-SS-Azo-Dye.

[0091] Figure 3a and Figure 3b : Synthesis examples of 3'-O-SS(DTM)-dGTP-SS-Azo-Rox and 3'-O-SS(DTM)-dTTP-SS-Azo-BodipyFL. Azo linker NHS ester labeled with Rox and BodipyFL was coupled with 3'-O-SS(DTM)-dGTP-SS-NH2 and 3'-O-SS(DTM)-dTTP-SS-NH2 to produce 3'-O-SS(DTM)-dGTP-SS-Azo-Rox and 3'-O-SS(DTM)-dTTP-SS-Azo-BodipyFL.

[0092] Figure 4 : Synthesis of 3'-O-SS(DTM)-dATP-SS-Rox.

[0093] Figure 5 : Synthesis of 3'-O-SS(DTM)-dUTP-SS-BodipyFL.

[0094] Figure 6a , Figure 6b and Figure 6c: Protocol 1: 2-color DNA SBS using 3'-O-SS(DTM)-dNTP-SS-Dyes (3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, and 3'-O-SS-dCTP-5-Azo-BodipyFL). Step 1, DNA polymerase and four nucleotide analogs (3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, and 3'-O-SS-dCTP-5-Azo-BodipyF) are added to the fixed pre-sensitized DNA template, so that the complementary nucleotide analogs can be incorporated into the growing DNA chain to terminate DNA synthesis. Step 2, tracking: DNA polymerase and four 3'-O-SS(DTM)-dNTPs (3'-O-tert-butyldisulfidemethyl (SS)-dATP, 3'-O-tert-butyldisulfidemethyl (SS)-dCTP, 3'-O-tert-butyldisulfidemethyl (SS)-dTTP and 3'-O-tert-butyldisulfidemethyl (SS)-dGTP) are added to the fixed pre-sensitized DNA template, so that complementary 3'-O-SS-nucleotide analogs can be incorporated into the subset of growing DNA chains in the pool that have not been extended with any of the dye-labeled dNTPs in step 1. The growing DNA chain is terminated with one of the four dye-labeled nucleotide analogs (A, C, G, T) or the same of the four nucleotide analogs (A, C, G, T) without dye. Step 3, after washing away the unincorporated dye-labeled nucleotides, the unique fluorescent signal from each fluorescent dye on the DNA product is detected, so that the incorporated nucleotides for sequence determination can be identified, the Rox signal indicates the incorporation of A and G, and the BodipyFL signal indicates the incorporation of T and C. Step 4, the Azo linker is cleaved by adding sodium dithionite (Na2S2O4) to the elongated DNA chain, causing the removal of Rox from the incorporated G and the removal of BodipyFL from the incorporated C. Step 5, after washing away the cleaved dye, a second round of detection of the unique fluorescent signal of each fluorescent dye on the DNA product is performed, so that the incorporated nucleotides for sequence determination can be identified. The disappearance of the Rox signal indicates the incorporation of G, and the disappearance of the BodipyFL signal indicates the incorporation of C. The retention of the Rox signal indicates the incorporation of A, and the retention of the BodipyFL signal indicates the incorporation of T. Next, in step 6, treatment of the DNA product with THP cleaves the SS linker, leading to the removal of residual fluorescent dye and regeneration of free 3'-OH groups on the DNA extension product, making the DNA extension product ready for the next cycle of DNA sequencing reaction.The presence of an additional SS bond between the Azo group and the base will result in a shorter scar on the incorporated nucleotide after THP treatment, which should result in longer reads. The structures of the modified nucleotides used in this scheme are shown in . Figure 1a and Figure 1b middle. DETAILED DESCRIPTION

[0095] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided as examples only. Various changes, modifications and substitutions may be made without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0096] the term

[0097] As used herein, and unless otherwise stated, each of the following terms shall have the definitions set forth below.

[0098] A-adenine;

[0099] C-cytosine;

[0100] G-guanine;

[0101] T-thymine;

[0102] U-uracil;

[0103] DNA - deoxyribonucleic acid;

[0104] RNA-ribonucleic acid;

[0105] Unless otherwise specified, "nucleic acid" shall mean any nucleic acid molecule, including but not limited to DNA, RNA and hybrids thereof. In one embodiment, the nucleic acid bases forming the nucleic acid molecule can be bases A, C, G, T and U and derivatives thereof.

[0106] Derivatives of these bases are well known in the art and are exemplified in PCR Systems, Reagents and Consumables, Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA.

[0107] A "nucleotide residue" is a single nucleotide in the state in which it exists after being incorporated into a polynucleotide and thereby becoming a monomer of the polynucleotide. Thus, a nucleotide residue is a nucleotide monomer of a polynucleotide (e.g., DNA) that is bound to an adjacent nucleotide monomer of the polynucleotide via a phosphodiester bond at the 3' position of its sugar and to a second adjacent nucleotide monomer via its phosphate group, except that: (i) the 3' terminal nucleotide residue is bound to only one adjacent nucleotide monomer of the polynucleotide via a phosphodiester bond from its phosphate group, and (ii) the 5' terminal nucleotide residue is bound to only one adjacent nucleotide monomer of the polynucleotide via a phosphodiester bond from the 3' position of its sugar.

[0108] "Substrate" or "surface" shall mean any suitable medium in solid phase to which nucleic acids or reagents can be attached. Non-limiting examples include chips, beads, nanopore structures, and columns. In one embodiment, the solid substrate may be present in a solution (including an aqueous solution, a gel, or a fluid).

[0109] "Hybridization" shall mean that a single-stranded nucleic acid is bonded to another nucleic acid based on the well-understood principle of sequence complementarity. In one embodiment, the other nucleic acid is a single-stranded nucleic acid. The tendency of hybridization between nucleic acids depends on the temperature and ionic strength of its environment, the length of the nucleic acid, and the degree of complementarity. The influence of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch EF, Maniatis T.1989. "Molecular cloning: experimental guide (Molecular cloning: alaboratory manual)", Cold Spring Harbor Laboratory Press, New York). As used herein, the hybridization of a primer sequence or a DNA extension product with another nucleic acid shall mean sufficient bonding so that the primer or DNA extension product can be extended by generating a phosphodiester bond with an available nucleotide or nucleotide analog that can form a phosphodiester bond, respectively.

[0110] As used herein, unless otherwise specified, a base that is "unique" or "different from" another base or a recited list of bases shall mean that the base has a different structure than another base or other bases. For example, a base that is "unique" or "different from" adenine, thymine, and cytosine may include a guanine base or a uracil base.

[0111] As used herein, unless otherwise specified, a tag or label moiety that is "different from" a tag or label moiety of a reference molecule means that the tag or label moiety has a chemical structure that is different from the chemical structure of another / reference tag or label moiety.

[0112] As used herein, unless otherwise specified, "primer" means an oligonucleotide that, when forming a duplex with a polynucleotide template, is capable of serving as a polymerase incorporation point and extending from its 3' end along the template, thereby generating an extended duplex.

[0113] As used herein, "alkyl" includes branched and straight chain saturated aliphatic hydrocarbon groups with a specified number of carbon atoms, and may be unsubstituted or substituted. Thus, C1-Cn as in "C1-Cn alkyl" includes groups with 1, 2, ..., n-1 or n carbons in a straight or branched arrangement. For example, "C1-C5 alkyl" includes groups with 1, 2, 3, 4 or 5 carbon atoms in a straight or branched arrangement, and particularly includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl and pentyl.

[0114] As used herein, "alkenyl" refers to a straight or branched non-aromatic hydrocarbon group containing at least 1 carbon-carbon double bond and up to the maximum possible number of non-aromatic carbon-carbon double bonds, and may be unsubstituted or substituted. For example, "C2-C5 alkenyl" means an alkenyl group having 2, 3, 4 or 5 carbon atoms and up to 1, 2, 3 or 4 carbon-carbon double bonds, respectively. Alkenyl includes ethenyl, propenyl and butenyl.

[0115] The term "alkynyl" refers to a straight or branched chain hydrocarbon radical containing at least 1 carbon-carbon triplet and up to the maximum possible number of non-aromatic carbon-carbon triple bonds, and may be unsubstituted or substituted. Thus, "C2-C5 alkynyl" means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl.

[0116] The term "substituted" refers to a functional group as described above, such as an alkyl or hydrocarbyl group, wherein at least one bond to a hydrogen atom contained therein is replaced by a bond to a non-hydrogen or non-carbon atom, with the proviso that the normal valence is maintained and the replacement produces a stable compound. Substituted groups also include groups in which one or more bonds to a carbon or hydrogen atom are replaced by one or more bonds to a heteroatom (including double or triple bonds). Non-limiting examples of substituents include the functional groups described above, and for example N, for example to form -CN.

[0117] Nucleotide analogs are disclosed herein, which contain a 3'-O-alkyldithiomethyl (3'-O-DTM) blocking group and a fluorophore connected to a base via a DTM or other cleavable linker (Azo, allyl, 2-nitrobenzyl, azidomethyl or dimethyl ketal). Two nucleotides have a DTM linker on the base and the other two nucleotides have an alternative linker on the base. One nucleotide with a DTM linker has Dye1 connected and another nucleotide with a DTM linker has Dye2 connected. Similarly, one nucleotide with an Azo linker has Dye1 connected and another nucleotide with an Azo linker has Dye 2 connected. Therefore, the dye is connected to the linker in an orthogonal manner. After the above nucleotide set is incorporated, imaging will be performed to indicate which two possible nucleotides have been incorporated, but not specifically which one. Then, the cleavage of the Azo linker and the dye connected thereto and a second round of imaging will determine the exact nucleotide incorporated. If the dye is removed, a nucleotide with an Azo linker is incorporated. If the dye is retained, then a nucleotide with a SS linker on the base is incorporated. Ultimately, cleavage of the DTM linker will remove any remaining fluorophore on the base and restore the 3'-OH group for subsequent cycles of sequencing by synthesis (SBS).

[0118] The present invention provides a method for nucleic acid sequencing, which comprises: a) extending a sensitized DNA chain by incorporating fluorescently labeled nucleotides into the sensitized chain; and b) identifying the fluorescently labeled nucleotides to sequence the nucleic acid.

[0119] In another embodiment, the fluorescently labeled nucleotide has a label attached to the base and a cleavable blocking group at the 3'-hydroxyl group.

[0120] In another embodiment, the label is attached to the base via a cleavable linker.

[0121] In another embodiment, the 3'OH blocking group is linked to the deoxyribose via a cleavable linker. In another embodiment, the cleavable linker comprises an orthogonal chemically cleavable linker. In another embodiment, the orthogonal chemically cleavable linker comprises disulfidemethyl SS (DTM), Azo, allyl, 2-nitrobenzyl, and dimethyl ketal. In another embodiment, the 3'OH blocking group comprises SS (DTM), azidomethyl, Azo, allyl, and 2-nitrobenzyl.

[0122] In another embodiment, the nucleotide analog comprises a deaza purine base.

[0123] The present invention also provides a method for nucleic acid sequencing, which comprises: a) providing a nucleic acid template hybridized with a primer; b) extending the primer hybridized with the nucleic acid template with a fluorescently labeled nucleotide or nucleotide analog, wherein the fluorescently labeled nucleotide or nucleotide analog has a label connected to the base and a blocking group on the 3'-hydroxyl group; and c) identifying the fluorescently labeled nucleotide to sequence the nucleic acid.

[0124] In another embodiment, the fluorescently labeled nucleotide or nucleotide analog has a label on the base and a blocking group on the 3'-hydroxyl group. In another embodiment, the label is attached to the base via a cleavable linker.

[0125] In another embodiment, the nucleotide analog comprises a deaza purine base.

[0126] In another embodiment, the label is attached to the base via a cleavable linker. In another embodiment, the 3'OH blocking group is attached to the deoxyribose via a cleavable linker. In another embodiment, the cleavable linker comprises an orthogonal chemically cleavable linker. In another embodiment, the orthogonal chemically cleavable linker comprises disulfide methyl SS (DTM), Azo, allyl, and 2-nitrobenzyl. In another embodiment, the 3'OH blocking group comprises SS (DTM), azidomethyl, Azo, allyl, 2-nitrobenzyl, and dimethyl ketal.

[0127] The present invention also provides a method for simultaneously sequencing multiple different nucleic acids, which comprises: a) extending multiple sensitized DNA chains hybridized with template DNA by incorporating fluorescently labeled nucleotides, each of the template DNAs comprising one of the sensitized DNA chains; and b) identifying each fluorescently labeled nucleotide so as to simultaneously sequence multiple different nucleic acids.

[0128] In another embodiment, the fluorescently labeled nucleotide or nucleotide analog has a label on the base and a blocking group on the 3'-hydroxyl group.

[0129] In another embodiment, the label is connected to the base via a cleavable linker. In another embodiment, the nucleotide analog comprises a deazapurine base. In another embodiment, the label is connected to the base via a cleavable linker. In another embodiment, the 3'OH blocking group is connected to the deoxyribose via a cleavable linker. In another embodiment, the cleavable linker comprises an orthogonal chemically cleavable linker. In another embodiment, the orthogonal chemically cleavable linker comprises disulfide methyl SS (DTM), Azo, allyl, 2-nitrobenzyl and dimethyl ketal. In another embodiment, the 3'OH blocking group comprises SS (DTM), azidomethyl, Azo, allyl and 2-nitrobenzyl.

[0130] In another embodiment, the nucleotide analog comprises a deaza purine base.

[0131] The present invention provides a nucleotide analogue comprising: (i) deoxyribose or ribose, (ii) a base, which is linked to the 1' position of the deoxyribose or ribose, wherein the base is selected from the group consisting of: A, T, C, G and U or a derivative thereof, (iii) a blocking group, which is bound to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label, which is bound to the base via a cleavable linker.

[0132] In another embodiment, the blocking group is connected to the ribose or deoxyribose via a cleavable linker. In another embodiment, the cleavable linker connected to the blocking group comprises an orthogonal chemically cleavable linker. In another embodiment, the blocking group comprises a disulfide methyl, an azido methyl, an azo, an allyl and / or a 2-nitrobenzyl group. In another embodiment, the blocking group comprises an alkyl disulfide methyl group. In another embodiment, the base is a deazapurine base.

[0133] In another embodiment, the cleavable linker connected to the base comprises an orthogonal chemically cleavable linker. In another embodiment, the cleavable linker connected to the base is an alkyl disulfide methyl linker, an azo linker, an allyl linker, a nitrobenzyl linker, an azidomethyl linker and / or a dimethyl ketal linker. In another embodiment, the detectable label is one or more of a dye, a fluorophore, a fluorescent energy transfer label, a chemiluminescent compound, a chromophore, a mass label, electrophoresis, a mononucleotide, an oligonucleotide, or a combination thereof. In another embodiment, the detectable label is a fluorophore. In another embodiment, the detectable label is BodipyFL, R6G, ROX, Cy5 or Alexa488. In another embodiment, the nucleotide analog is 3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox or 3'-O-SS-dCTP-5-Azo-BodipyFL.

[0134] In another embodiment, the cleavable linker is an alkyldisulfidemethyl linker. In another embodiment, the nucleotide analog has the following structure:

[0135]

[0136]

[0137] In another embodiment, the cleavable linker is an azo linker. In another embodiment, the nucleotide analog has the following structure:

[0138]

[0139]

[0140] The present invention also provides a composition comprising four different types of nucleotide analogs, wherein each type of nucleotide analogs comprises: a base selected from the group consisting of: A, T, C, G or U or a derivative thereof; deoxyribose or ribose; and a blocking group bound to the 3'-oxygen of the deoxyribose or ribose, and

[0141] (i) a first type of nucleotide analogue comprises a first type of detectable label bound to the base via a first type of linker;

[0142] (ii) the second type of nucleotide analogue comprises a second type of detectable label bound to the base via a second type of linker;

[0143] (iii) the third type of nucleotide analogue comprises a first type of detectable label bound to the base via a second type of linker; and

[0144] (iv) the fourth type of nucleotide analogue comprises a second type of detectable label bound to the base via a first type of linker;

[0145] The first type of linker is different from the second type of linker, and the first type of detectable label is different from the second type of detectable label.

[0146] In another embodiment, the blocking group is connected to the ribose or deoxyribose via a cleavable linker. In another embodiment, the cleavable linker connected to the blocking group comprises an orthogonal chemically cleavable linker. In another embodiment, the blocking group comprises a disulfide methyl, an azido methyl, an azo, an allyl and / or a 2-nitrobenzyl group. In another embodiment, the blocking group comprises an alkyl disulfide methyl group.

[0147] In another embodiment, the base of one or more of the first, second, third and / or fourth type of nucleotide analogs comprises a deazapurine base. In another embodiment, the first base is A or a derivative thereof, and:

[0148] (i) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0149] (ii) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0150] (iii) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0151] (iv) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0152] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0153] (vi) The second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0154] In another embodiment, the first base is T / U or a derivative thereof, and:

[0155] (i) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0156] (ii) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0157] (iii) the second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0158] (iv) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0159] (v) the second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof, or

[0160] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof.

[0161] In another embodiment, the first base is C or a derivative thereof, and:

[0162] (i) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0163] (ii) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0164] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0165] (iv) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0166] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof,

[0167] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0168] In another embodiment, the first base is G or a derivative thereof, and:

[0169] (i) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof,

[0170] (ii) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0171] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof,

[0172] (iv) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof,

[0173] (v) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0174] (vi) The second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0175] In another embodiment, the first type of linker and / or the second type of linker comprises an orthogonal chemically cleavable linker.

[0176] In another embodiment, the first and / or second type of linker comprises one or more of an alkyldisulfidemethyl linker, an azo linker, an allyl linker, a nitrobenzyl linker, an azidomethyl linker, and / or a dimethylketal linker. In another embodiment, the first and / or second type of linker is chemically cleavable or photocleavable. In another embodiment, the first and / or second type of linker can be cleaved by a water-soluble phosphine, thereby producing a 3'-OH. In another embodiment, the water-soluble phosphine is tris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THP). In another embodiment, the first and / or second type of linker can be cleaved by sodium dithionite.

[0177] In another embodiment, the detectable label of the first and / or second type is one or more of a dye, a fluorophore, a fluorescent energy transfer label, a chemiluminescent compound, a chromophore, a mass label, electrophoresis, a mononucleotide, an oligonucleotide, or a combination thereof. In another embodiment, the detectable label of the first and / or second type is a fluorophore. In another embodiment, the detectable label of the first and / or second type is BodipyFL, R6G, ROX, Cy5, or Alexa488.

[0178] In another embodiment, the composition comprises one or more of 3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, or 3'-O-SS-dCTP-5-Azo-BodipyFL.

[0179] In another embodiment, the first type of linker is an alkyldisulfidemethyl linker and the second type of linker is an azo linker. In another embodiment, the nucleotide analog is selected from the group comprising:

[0180]

[0181]

[0182] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0183] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and four types of tagged nucleotide analogs under conditions that allow the nucleic acid polymerase to catalyze the incorporation of one tagged nucleotide analog into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a nucleic acid extension product,

[0184] wherein each of the at least four types of tagged nucleotide analogs comprises: a base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative thereof; a deoxyribose or ribose sugar; and a cleavable blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide, and

[0185] (i) a first type of nucleotide analogs comprises a first type of base and a first type of detectable label bound to the base via a first type of linker;

[0186] (ii) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0187] (iii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0188] (iv) a fourth type of nucleotide analogue comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0189] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0190] b) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0191] c) contacting the incorporated tagged nucleotide analog with means for cleaving a first type of linker;

[0192] d) determining whether the label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0193] e) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0194] f) cleaving the 3'-oxygen blocking group to thereby form a 3'-OH;

[0195] g) repeatedly performing steps (a)-(f) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0196] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0197] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0198] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and a first type of tagged nucleotide analogue under conditions that allow the nucleic acid polymerase to catalyze the incorporation of the tagged nucleotide analogue into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a DNA extension product,

[0199] wherein the tagged nucleotide analogs comprise a first type of base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof; a deoxyribose or ribose sugar; a cleavable blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide; and a first type of detectable label that is bound to the base via a first type of linker,

[0200] and if the tagged nucleotide is not incorporated, iteratively repeating the contacting with the second, third, and fourth types of tagged nucleotide analogs until the tagged nucleotide analog is incorporated, wherein

[0201] (i) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0202] (ii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0203] (iii) the fourth type of nucleotide analogs comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0204] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0205] b) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0206] c) contacting the incorporated nucleotide analogues with means for cleaving a first type of linker;

[0207] d) determining whether the detectable label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0208] e) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0209] f) cleaving the 3'-oxygen blocking group to thereby form 3'-OH,

[0210] g) repeatedly performing steps (a)-(f) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0211] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0212] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0213] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and four types of tagged nucleotide analogs under conditions that allow the nucleic acid polymerase to catalyze the incorporation of one tagged nucleotide analog into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a nucleic acid extension product,

[0214] wherein each of the at least four types of tagged nucleotide analogs comprises: a base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative thereof; a deoxyribose or ribose; and a blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide, and

[0215] (i) a first type of nucleotide analogs comprises a first type of base and a first type of detectable label bound to the base via a first type of linker;

[0216] (ii) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0217] (iii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0218] (iv) a fourth type of nucleotide analogue comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0219] and wherein the first type of detectable label is different from the second type of detectable label;

[0220] b) contacting the single-stranded nucleic acid with four types of nucleotide reversible terminators under conditions that allow a nucleic acid polymerase to catalyze the incorporation of one nucleotide reversible terminator into a primer, wherein each nucleotide reversible terminator comprises a blocking group for the 3'-oxygen of a deoxyribose or ribose sugar that prevents the polymerase from catalyzing the incorporation of a subsequent nucleotide under the following conditions:

[0221] (i) the polymerase fails to incorporate a tagged nucleotide analog in step a),

[0222] (ii) the nucleotide reversible terminator is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that hybridizes to the 3' terminal nucleotide residue of the primer, and

[0223] c) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0224] d) contacting the incorporated tagged nucleotide analog with means for cleaving a first type of linker;

[0225] e) determining whether the label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0226] f) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0227] g) cleaving the 3'-oxygen blocking group to thereby form a 3'-OH;

[0228] h) repeatedly performing steps (a)-(g) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0229] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0230] The present invention provides a method for determining the nucleotide sequence of a single-stranded nucleic acid, comprising:

[0231] a) contacting the single-stranded nucleic acid with a nucleic acid polymerase and a first type of tagged nucleotide analogue under conditions that allow the nucleic acid polymerase to catalyze the incorporation of the tagged nucleotide analogue into the primer when it is complementary to a nucleotide residue of the single-stranded nucleic acid that is immediately 5′ to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3′-terminal nucleotide residue of the primer, so as to form a DNA extension product,

[0232] wherein the tagged nucleotide analogs comprise a first type of base that is adenine, guanine, cytosine, thymine, or uracil, or a derivative of each thereof; a deoxyribose or ribose sugar; a blocking group that is bound to the 3'-oxygen of the deoxyribose or ribose sugar, preventing a polymerase from catalyzing the incorporation of a subsequent nucleotide; and a first type of detectable label that is bound to the base via a first type of linker,

[0233] and if the tagged nucleotide is not incorporated, iteratively repeating the contacting with the second, third, and fourth types of tagged nucleotide analogs until the tagged nucleotide analog is incorporated, wherein

[0234] (i) the second type of nucleotide analogs comprises a second type of base and a second type of detectable label bound to the base via a second type of linker;

[0235] (ii) the third type of nucleotide analogs comprises a third type of base and a first type of detectable label bound to the base via a second type of linker; and

[0236] (iii) the fourth type of nucleotide analogs comprises a fourth type of base and a second type of detectable label bound to the base via a first type of linker;

[0237] wherein the first type of linker is different from the second type of linker, and wherein the first type of detectable label is different from the second type of detectable label;

[0238] b) contacting the single-stranded nucleic acid with four types of nucleotide reversible terminators under conditions that allow a nucleic acid polymerase to catalyze the incorporation of one nucleotide reversible terminator into a primer, wherein each nucleotide reversible terminator comprises a blocking group for the 3'-oxygen of a deoxyribose or ribose sugar that prevents the polymerase from catalyzing the incorporation of a subsequent nucleotide under the following conditions:

[0239] (i) the polymerase fails to incorporate the tagged nucleotide analog in step a), and

[0240] (ii) the nucleotide reversible terminator is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that hybridizes to the 3' terminal nucleotide residue of the primer;

[0241] c) identifying whether a nucleotide analog comprising a first type or a second type of detectable label was incorporated in step (a);

[0242] d) contacting the incorporated nucleotide analogue with means for cleaving a first type of linker;

[0243] e) determining whether the detectable label was removed by the means for cleavage in step (c) to thereby determine the identity of the incorporated nucleotide analogue;

[0244] f) contacting the incorporated tagged nucleotide analog with means for cleaving a second type of linker;

[0245] g) cleaving the 3'-oxygen blocking group to thereby form 3'-OH,

[0246] h) repeatedly performing steps (a)-(g) for each nucleotide residue of the single-stranded nucleic acid being sequenced, wherein in each repetition of step (a), the tagged nucleotide is incorporated into the nucleic acid extension product generated by the previous repetition of step (a) if it is complementary to a nucleotide residue of the single-stranded nucleic acid that is located 5' to the nucleotide residue of the single-stranded nucleic acid that is hybridized to the 3'-terminal nucleotide residue of the nucleic acid extension product,

[0247] In order to thereby determine the nucleotide sequence of the single-stranded nucleic acid.

[0248] In another embodiment, the 3'-oxygen blocking group of the nucleotide reversible terminator is bound to the 3'-oxygen through at least one orthogonal chemically cleavable linker. In another embodiment, the blocking group of the nucleotide reversible terminator comprises disulfide methyl, azido methyl, azo, allyl and / or 2-nitrobenzyl. In another embodiment, the blocking group of the nucleotide reversible terminator comprises alkyl disulfide methyl. In another embodiment, the blocking group of the nucleotide reversible terminator is chemically cleaved or photocleaved. In another embodiment, the blocking group of the nucleotide reversible terminator is cleaved by a water-soluble phosphine, thereby producing 3'-OH. In another embodiment, the water-soluble phosphine is tris-(2-carboxyethyl) phosphine (TCEP) or tris(hydroxypropyl) phosphine (THP).

[0249] In another embodiment, the nucleotide reversible terminator is 3'-O-SS(DTM)-dNTP (3'-O-tert-butyldisulfidemethyl (SS)-dATP, 3'-O-tert-butyldisulfidemethyl (SS)-dCTP, 3'-O-tert-butyldisulfidemethyl (SS)-dTTP, and 3'-O-tert-butyldisulfidemethyl (SS)-dGTP).

[0250] In another embodiment, the blocking group of the nucleotide reversible terminator is cleaved by sodium dithionite.

[0251] In another embodiment, the 3'-oxygen blocking group of the tagged nucleotide analog is bound to the 3'-oxygen through at least one orthogonal chemically cleavable linker. In another embodiment, the blocking group of the tagged nucleotide analog comprises a disulfidemethyl, an azidomethyl, an azo, an allyl, and / or a 2-nitrobenzyl group. In another embodiment, the blocking group of the tagged nucleotide analog comprises an alkyldisulfidemethyl group.

[0252] In another embodiment, the base of one or more of the first, second, third and / or fourth type of nucleotide analogs comprises a deaza purine base.

[0253] In another embodiment, the first base is A or a derivative thereof, and:

[0254] (i) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0255] (ii) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0256] (iii) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0257] (iv) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0258] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0259] (vi) The second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0260] In another embodiment, the first base is T / U or a derivative thereof, and:

[0261] (i) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0262] (ii) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0263] (iii) the second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0264] (iv) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0265] (v) the second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof, or

[0266] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof.

[0267] In another embodiment, the first base is C or a derivative thereof, and:

[0268] (i) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0269] (ii) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0270] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0271] (iv) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0272] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof, or

[0273] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0274] In another embodiment, the first base is G or a derivative thereof, and:

[0275] (i) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof,

[0276] (ii) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0277] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof,

[0278] (iv) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof,

[0279] (v) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0280] (vi) The second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0281] In another embodiment, the first type of linker and / or the second type of linker comprises an orthogonal chemically cleavable linker.

[0282] In another embodiment, the first and / or second type of linker comprises one or more of an alkyldisulfidemethyl linker, an azo linker, an allyl linker, a nitrobenzyl linker, an azidomethyl linker, and / or a dimethylketal linker.

[0283] In another embodiment, the first and / or second type of linker is chemically cleavable or photocleavable. In another embodiment, the first and / or second type of linker can be cleaved by a water-soluble phosphine, thereby generating a 3'-OH. In another embodiment, the water-soluble phosphine is tris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THP). In another embodiment, the first and / or second type of linker can be cleaved by sodium dithionite.

[0284] In another embodiment, the detectable label of the first and / or second type is one or more of a dye, a fluorophore, a fluorescent energy transfer label, a chemiluminescent compound, a chromophore, a mass tag, electrophoresis, a mononucleotide, an oligonucleotide, or a combination thereof. In another embodiment, the detectable label of the first and / or second type is a fluorophore. In another embodiment, the detectable label of the first and / or second type is BodipyFL, R6G, ROX, Cy5, or Alexa488. In another embodiment, the tagged nucleotide is selected from the group comprising one or more of the following: 3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, or 3'-O-SS-dCTP-5-Azo-BodipyFL.

[0285] In another embodiment, the first type of linker is an alkyldisulfidemethyl linker and the second type of linker is an azo linker.

[0286] In another embodiment, the tagged nucleotide analogs are selected from the group comprising:

[0287]

[0288]

[0289]

[0290] as well as

[0291]

[0292] The present invention also provides a kit for nucleic acid sequencing, comprising in separate compartments:

[0293] a) four types of tagged nucleotide analogs, wherein each type of tagged nucleotide analogs comprises a base selected from the group consisting of A, T, C, G, or U or a derivative thereof; a deoxyribose or ribose sugar; and a blocking group bound to the 3'-oxygen of the deoxyribose or ribose sugar, and

[0294] (i) a first type of nucleotide analogue comprises a first type of detectable label bound to the base via a first type of linker;

[0295] (ii) the second type of nucleotide analogue comprises a second type of detectable label bound to the base via a second type of linker;

[0296] (iii) the third type of nucleotide analogue comprises a first type of detectable label bound to the base via a second type of linker; and

[0297] (iv) the fourth type of nucleotide analogue comprises a second type of detectable label bound to the base via a first type of linker;

[0298] Each type of nucleotide has a different base.

[0299] b) the first type of linker is different from the second type of linker, and the first type and the second type of detectable labels are different reagents suitable for nucleic acid polymerization; and

[0300] c) Instructions for use.

[0301] In another embodiment, the first type of linker is a cleavable allyl linker and the second type of linker is a cleavable azo linker or a disulfidemethyl linker.

[0302] In another embodiment, the kit further comprises four types of nucleotide reversible terminators, wherein each nucleotide reversible terminator comprises a blocking group for the 3'-oxygen of deoxyribose or ribose, which prevents the polymerase from catalyzing the incorporation of subsequent nucleotides.

[0303] In another embodiment, the first base is A or a derivative thereof, and:

[0304] (i) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0305] (ii) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0306] (iii) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0307] (iv) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0308] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0309] (vi) The second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0310] In another embodiment, the first base is T / U or a derivative thereof, and:

[0311] (i) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0312] (ii) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0313] (iii) the second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof,

[0314] (iv) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof,

[0315] (v) the second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof, or

[0316] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof.

[0317] In another embodiment, the first base is C or a derivative thereof, and:

[0318] (i) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof,

[0319] (ii) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0320] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof,

[0321] (iv) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof,

[0322] (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof, or

[0323] (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0324] In another embodiment, the first base is G or a derivative thereof, and:

[0325] (i) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof,

[0326] (ii) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof,

[0327] (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof,

[0328] (iv) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof,

[0329] (v) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or

[0330] (vi) The second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

[0331] The present invention provides a nucleotide analog comprising (i) a base, (ii) a deoxyribose or ribose sugar, (iii) an alkyldisulfidemethyl moiety bound to the 3'-oxygen of the deoxyribose or ribose sugar, and (iv) a detectable label bound to the base via a disulfidemethyl linker or an alternative cleavable linker such as Azo.

[0332] The present invention also provides a composition comprising four different types of the above-mentioned nucleotide analogs having an alkyldithiomethyl group bound to the 3'-oxygen of deoxyribose or ribose: (1) a nucleotide comprising a cleavable Azo linker between a base and Dye1; (2) a nucleotide comprising a cleavable Azo linker between a base and Dye2; (3) a nucleotide comprising a cleavable DTM linker between a base and Dye1; and (4) a nucleotide comprising a cleavable DTM linker between a base and Dye2.

[0333] The present invention also provides a nucleic acid sequencing method, which comprises:

[0334] a) Provide

[0335] 1) Nucleic acids,

[0336] 2) Nucleic acid polymerase,

[0337] 3) a primer capable of hybridizing to the nucleic acid, and

[0338] 4) four different labeled nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety attached to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label attached to the base via a disulfidemethyl linker or an Azo linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable Azo linker and Dye1; (2) a cleavable Azo linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0339] b) incorporating the nucleic acid polymerase into the primer together with one or more of the nucleotide analogs to generate an extended strand;

[0340] c) detecting said unique detectable label of each incorporated nucleotide analogue, so as to thereby identify each incorporated nucleotide in said extended strand in two steps,

[0341] 1) Imaging after incorporation to provide two possible nucleotides,

[0342] 2) Imaging after cleavage of the Azo linker with sodium dithionite to determine the exact nucleotide incorporated;

[0343] d) cleaving the DTM cleavable linker of any residual Dye and DTM blocking group attached to the 3'-OH in preparation for the next cycle; and

[0344] e) repeating steps a) to d);

[0345] The nucleic acid is thereby sequenced.

[0346] The present invention also provides a kit for nucleic acid sequencing, comprising in separate compartments:

[0347] a) a plurality of nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety attached to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label attached to the base via a disulfidemethyl linker or an azo linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable azo linker and Dye1; (2) a cleavable Azo linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0348] b) reagents suitable for nucleic acid polymerization; and

[0349] c) Instructions for use.

[0350] The present invention provides a nucleotide analog comprising (i) a base, (ii) a deoxyribose or ribose sugar, (iii) an alkyldisulfidemethyl moiety bound to the 3'-oxygen of the deoxyribose or ribose sugar, and (iv) a detectable label bound to the base via a disulfidemethyl linker or an alternative cleavable linker such as Azo.

[0351] The present invention also provides a composition comprising four different types of the above-mentioned nucleotide analogs having an alkyldithiomethyl group bound to the 3'-oxygen of deoxyribose or ribose: (1) a nucleotide comprising a cleavable Azo linker between a base and Dye1; (2) a nucleotide comprising a cleavable Azo linker between a base and Dye2; (3) a nucleotide comprising a cleavable DTM linker between a base and Dye1; and (4) a nucleotide comprising a cleavable DTM linker between a base and Dye2.

[0352] The present invention also provides a nucleic acid sequencing method, which comprises:

[0353] a) Provide

[0354] 1) Nucleic acids,

[0355] 2) Nucleic acid polymerase,

[0356] 3) a primer capable of hybridizing to the nucleic acid, and

[0357] 4) four different labeled nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety attached to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label attached to the base via a disulfidemethyl linker or an Azo linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable 2-nitrobenzyl (2NB) linker and Dye1; (2) a cleavable 2NB linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0358] b) incorporating the nucleic acid polymerase into the primer together with one or more of the nucleotide analogs to generate an extended strand;

[0359] c) detecting said unique detectable label of each incorporated nucleotide analogue, so as to thereby identify each incorporated nucleotide in said extended strand in two steps,

[0360] 1) Imaging after incorporation to provide two possible nucleotides,

[0361] 2) Imaging after photocleavage of the 2-nitrophenyl linker with 340 nm light to determine the exact nucleotide incorporated;

[0362] d) cleaving the DTM cleavable linker of any residual Dye and DTM blocking group attached to the 3'-OH in preparation for the next cycle; and

[0363] e) repeating steps a) to d);

[0364] The nucleic acid is thereby sequenced.

[0365] The present invention also provides a kit for nucleic acid sequencing, comprising in separate compartments:

[0366] a) a plurality of nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety attached to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label attached to the base via a disulfidemethyl linker or a 2-nitrobenzyl (2NB) linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable 2NB linker and Dye1; (2) a cleavable 2NB linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0367] b) reagents suitable for nucleic acid polymerization; and

[0368] c) Instructions for use.

[0369] The present invention provides a nucleotide analog comprising (i) a base, (ii) a deoxyribose or ribose sugar, (iii) an alkyldisulfidemethyl moiety bound to the 3'-oxygen of the deoxyribose or ribose sugar, and (iv) a detectable label bound to the base via a disulfidemethyl linker or an alternative cleavable linker such as Azo.

[0370] The present invention also provides a composition comprising four different types of the above-mentioned nucleotide analogs having an alkyldithiomethyl group bound to the 3'-oxygen of deoxyribose or ribose: (1) a nucleotide comprising a cleavable 2-nitrobenzyl (2NB) linker between the base and Dye1; (2) a nucleotide comprising a cleavable 2NB linker between the base and Dye2; (3) a nucleotide comprising a cleavable DTM linker between the base and Dye1; and (4) a nucleotide comprising a cleavable DTM linker between the base and Dye2.

[0371] The present invention also provides a nucleic acid sequencing method, which comprises:

[0372] a) Provide

[0373] 1) Nucleic acids,

[0374] 2) Nucleic acid polymerase,

[0375] 3) a primer capable of hybridizing to the nucleic acid, and

[0376] 4) four different labeled nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety attached to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label attached to the base via a disulfidemethyl linker or an Azo linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable allyl linker and Dye1; (2) a cleavable allyl linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0377] b) incorporating the nucleic acid polymerase into the primer together with one or more of the nucleotide analogs to generate an extended strand;

[0378] c) detecting said unique detectable label of each incorporated nucleotide analogue, so as to thereby identify each incorporated nucleotide in said extended strand in two steps,

[0379] (i) imaging after incorporation to provide two possible nucleotides,

[0380] (ii) imaging after cleavage of the allylic linker with Pd(0) to determine the exact nucleotide incorporated;

[0381] d) cleaving the DTM cleavable linker of any residual Dye and DTM blocking group attached to the 3'-OH in preparation for the next cycle; and

[0382] e) repeating steps a) to d);

[0383] The nucleic acid is thereby sequenced.

[0384] The present invention also provides a kit for nucleic acid sequencing, comprising in separate compartments:

[0385] a) a plurality of nucleotide analogs, each comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an alkyldisulfidemethyl moiety bound to the 3'-oxygen of the deoxyribose or ribose, and (iv) a detectable label bound to the base via a disulfidemethyl linker or an Azo linker, and four orthologous combinations of a dye and a cleavable linker: (1) a cleavable allyl linker and Dye1; (2) a cleavable allyl linker and Dye2; (3) a cleavable DTM linker and Dye1; and (4) a cleavable DTM linker and Dye2;

[0386] b) reagents suitable for nucleic acid polymerization; and

[0387] c) Instructions for use.

[0388] Experimental Discussion

[0389] Scenario 1:

[0390] Two-color DNA SBS was performed using 3'-O-SS(DTM)-dNTP-SS-Dye (3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, and 3'-O-SS-dCTP-5-Azo-BodipyFL).

[0391] Step 1, DNA polymerase and four nucleotide analogs (3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox and 3'-O-SS-dCTP-5-Azo-BodipyF) are added to the fixed pre-sensitized DNA template, so that the complementary nucleotide analogs can be incorporated into the growing DNA chain to terminate DNA synthesis.

[0392] Step 2, tracking: DNA polymerase and four 3'-O-SS(DTM)-dNTPs (3'-O-tert-butyldisulfidemethyl (SS)-dATP, 3'-O-tert-butyldisulfidemethyl (SS)-dCTP, 3'-O-tert-butyldisulfidemethyl (SS)-dTTP and 3'-O-tert-butyldisulfidemethyl (SS)-dGTP) are added to the fixed pre-sensitized DNA template, so that complementary 3'-O-SS-nucleotide analogs can be incorporated into the subset of growing DNA chains in the pool that have not been extended with any of the dye-labeled dNTPs in step 1. The growing DNA chain is capped with one of the four dye-labeled nucleotide analogs (A, C, G, T) or the same of the four nucleotide analogs (A, C, G, T) without a dye.

[0393] Step 3, after washing away the unincorporated dye-labeled nucleotides, the unique fluorescent signal from each fluorescent dye on the DNA product is detected, enabling identification of the incorporated nucleotides for sequence determination, with the Rox signal indicating the incorporation of A and G, and the BodipyFL signal indicating the incorporation of T and C.

[0394] Step 4, the Azo linker is cleaved by adding sodium dithionite (Na2S2O4) to the elongated DNA strand, resulting in the removal of Rox from the incorporated G and BodipyFL from the incorporated C.

[0395] Step 5, after washing away the cleaved dye, a second round of detection of the unique fluorescent signal of each fluorescent dye on the DNA product is performed, enabling identification of the incorporated nucleotides for sequence determination. The disappearance of the Rox signal indicates the incorporation of G, and the disappearance of the BodipyFL signal indicates the incorporation of C. The retention of the Rox signal indicates the incorporation of A, and the retention of the BodipyFL signal indicates the incorporation of T.

[0396] Next, in step 6, treatment of the DNA product with THP cleaves the SS linker, resulting in removal of residual fluorescent dye and regeneration of free 3'-OH groups on the DNA extension product, making the DNA extension product ready for the next cycle of DNA sequencing reaction. The presence of another SS bond between the Azo group and the base will result in a shorter scar on the incorporated nucleotide after THP treatment, which should result in longer reads. The structures of the modified nucleotides used in this protocol are shown in Figure 1a and 1b middle.

[0397] References:

[0398] 1. Hyman ED (1988) A new method of sequencing DNA. Anal Biochem, 174: 423-436.

[0399] 2. Ronaghi M et al. (1998) A sequencing method based on real-time pyrophosphate. Science 281: 363-365.

[0400] 3. Ju J et al. (2003) Massive Parallel Method for Decoding DNA and RNA, U.S. Patent No. 6,664,079.

[0401] 4. Li Z, Bai X, Ruparel H, Kim S, Turro NJ, Ju J (2003) A photocleavable fluorescent nucleotide for DNA sequencing and analysis. Proc Natl Acad Sci USA 100: 414-419.

[0402] 5. Braslavsky I et al. (2003) Sequence information can be obtained from single DNA molecules. Proceedings of the National Academy of Sciences of the United States of America, 100: 3960-3964.

[0403] 6. Ruparel H et al. (2005) Design and synthesis of a 3'-O-allylphotocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis. Proceedings of the National Academy of Sciences of the United States of America, 102: 5932-5937.

[0404] 7. Margulies M et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376-380.

[0405] 8. Ju J et al. (2006) Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proceedings of the National Academy of Sciences of the United States of America, 103: 19635-19640.

[0406] 9. Wu J et al. (2007) 3'-O-modified nucleotides as reversible terminators for pyrosequencing. Proc Natl Acad Sci USA 104: 16462-16467.

[0407] 10. Guo J et al. (2008) Four-color DNA sequencing with 3'-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc Natl Acad Sci U S A 105:9145-9150.

[0408] 11. Bentley DR et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456: 53-59.

[0409] 12. Harris TD et al. (2008) Single-molecule DNA sequencing of a viral genome. Science, 320: 106-109.

[0410] 13. Eid, J. et al. (2009) Real-time DNA sequencing from single polymerase molecules. Science, 323: 133-138.

[0411] 14. Rothberg JM et al. (2011) An integrated semiconductor device enabling non-optical genome sequencing. Nature, 475: 348-352.

[0412] 15. Turcatti G, Romieu A, Fedurco M, Tairi AP (2008) A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acids Res 36: e25.

[0413] 16. Mitra RD et al. (2003) Fluorescent in situ sequencing on polymerase colonies. Analytical Biochemistry, 320: 55-65.

Claims

1. A composition comprising four different types of nucleotide analogs, wherein each type of nucleotide analogs comprises: a base selected from the group consisting of: A, T, C, G or U or a derivative thereof; a deoxyribose or ribose; and a blocking group bound to the 3′-oxygen of the deoxyribose or ribose, and (i) a first type of nucleotide analogs comprises a first base and a first type of detectable label bound to the base via a first type of linker; (ii) the second type of nucleotide analogs comprises a second base and a second type of detectable label bound to the base via a second type of linker; (iii) a third type of nucleotide analogue comprises a third base and the first type of detectable label bound to the base via the second type of linker; and (iv) a fourth type of nucleotide analogue comprises a fourth base and the second type of detectable label bound to the base via the first type of linker; wherein the first type of linker is different from the second type of linker, and the first type of detectable label is different from the second type of detectable label.

2. The composition of claim 1, wherein the blocking group is linked to the ribose or deoxyribose via a cleavable linker.

3. The composition of claim 1, wherein the cleavable linker attached to the blocking group comprises an orthogonal chemically cleavable linker.

4. The composition of claim 1, wherein the blocking group comprises a disulfide methyl group, an azido methyl group, an azo group, an allyl group and / or a 2-nitrobenzyl group.

5. The composition of claim 1, wherein the blocking group comprises an alkyldisulfidemethyl group.

6. The composition of claim 1, wherein the base of one or more of the first, second, third and / or fourth types of nucleotide analogs comprises a deaza purine base.

7. The composition of claim 1, wherein the first base is A or a derivative thereof, and: (i) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof, (ii) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof, (iii) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof, (iv) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof, (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or (vi) The second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

8. The composition of claim 1, wherein the first base is T / U or a derivative thereof, and: (i) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof, (ii) the second base is C or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof, (iii) the second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is G or a derivative thereof, (iv) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is C or a derivative thereof, (v) the second base is G or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof, or (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof.

9. The composition of claim 1, wherein the first base is C or a derivative thereof, and: (i) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is G or a derivative thereof, (ii) the second base is A or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is T / U or a derivative thereof, (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is G or a derivative thereof, (iv) the second base is T / U or a derivative thereof, the third base is G or a derivative thereof, and the fourth base is A or a derivative thereof, (v) the second base is G or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof, or (vi) The second base is G or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof.

10. The composition of claim 1, wherein the first base is G or a derivative thereof, and: (i) the second base is C or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is A or a derivative thereof, (ii) the second base is C or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is T / U or a derivative thereof, (iii) the second base is T / U or a derivative thereof, the third base is A or a derivative thereof, and the fourth base is C or a derivative thereof, (iv) the second base is T / U or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is A or a derivative thereof, (v) the second base is A or a derivative thereof, the third base is T / U or a derivative thereof, and the fourth base is C or a derivative thereof, or (vi) The second base is A or a derivative thereof, the third base is C or a derivative thereof, and the fourth base is T / U or a derivative thereof.

11. The composition of claim 1, wherein the first type of linker and / or the second type of linker comprises an orthogonal chemically cleavable linker.

12. The composition of claim 1, wherein the first and / or second type of linker comprises one or more of an alkyldisulfidemethyl linker, an azo linker, an allyl linker, a nitrobenzyl linker, an azidomethyl linker, and / or a dimethylketal linker.

13. The composition of claim 1, wherein the first and / or second type of linker is chemically cleavable or photocleavable.

14. The composition of claim 1, wherein the first and / or second type of linker is cleavable by a water-soluble phosphine, thereby generating a 3'-OH.

15. The composition of claim 14, wherein the water-soluble phosphine is tris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine (THP).

16. The composition of claim 1, wherein the first and / or second type of linker is cleavable by sodium dithionite.

17. The composition of claim 1, wherein the first and / or second type of detectable label is one or more of a dye, a fluorophore, a fluorescent energy transfer tag, a chemiluminescent compound, a chromophore, a mass tag, an electrophoresis, a mononucleotide, an oligonucleotide, or a combination thereof.

18. The composition of claim 1, wherein the first and / or second type of detectable label is a fluorophore.

19. The composition of claim 1, wherein the first and / or second type of detectable label is BodipyFL, R6G, ROX, Cy5 or Alexa488.

20. The composition of claim 1, comprising one or more of 3'-O-SS-dATP-7-SS-Rox, 3'-O-SS-dTTP-5-SS-BodipyFL, 3'-O-SS-dGTP-7-Azo-Rox, or 3'-O-SS-dCTP-5-Azo-BodipyFL.

21. The composition of claim 1, wherein the first type of linker is an alkyldisulfidemethyl linker and the second type of linker is an azo linker.

22. The composition of claim 1, wherein the nucleotide analog is selected from the group consisting of:

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