Fluorescence-based multicycle nucleic acid sequencing reaction using imaging reagents, kits

Abstract

The present application relates to the technical field of gene sequencing, in particular to an imaging reagent and a kit for a multi-cycle nucleic acid sequencing reaction based on fluorescence. In the imaging reagent, the synergistic effect of the combination of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone and sodium ascorbate reduces the generation of free radicals, inhibits nucleic acid damage during the imaging process, reduces phasing and pre-phasing, and ultimately improves the accuracy of long read sequencing. In order to improve the sequencing read length, three functional components, namely antioxidants, fluorescence quenching inhibitors and free radical scavengers, are added to the traditional imaging reagent. Compared with the existing imaging protectants, the present application creatively selects three antioxidants, namely ethyl 3,4-dihydroxybenzoate, 4-hydroxy 3-methoxy acetophenone and sodium ascorbate, for compounding, so as to exert a synergistic effect and eliminate the need for adding fluorescence quenching inhibitors and free radical scavengers.

Description

Technical Field

[0001] This invention relates to the field of gene sequencing technology, and more specifically to imaging reagents and kits for fluorescence-based multi-cycle nucleic acid sequencing reactions. Background Technology

[0002] Continuous cycling nucleic acid sequencing methods based on fluorescently labeled nucleic acid analogs are known in the art. In such “synthetic sequencing” or “cycling sequencing” methods, multiple extension reactions are performed using DNA polymerase or DNA ligase to continuously incorporate fluorescently labeled polynucleotides complementary to the template strand. The type of polynucleotide incorporated is determined by detecting the fluorescent label signal. Detection of the fluorescence signal depends on the electromagnetic radiation emission of excited-state fluorophores, which can lead to nucleic acid damage, and the brightness of the fluorophore incorporated in each cycle of nucleotide addition decreases. Numerous analytical studies have shown that the weakening of the fluorescence signal with increasing cycle number during fluorescence detection is largely due to the fact that reactive molecules such as oxygen free radicals generated during the transition of fluorescent molecules from the ground state to the excited state can easily cause pyrimidine dimerization and nucleic acid damage, either directly or indirectly. Therefore, some studies have indicated the need to add antioxidants to imaging reagents.

[0003] For example, patent WO2006064199 discloses that the loss of fluorescence signal intensity caused by repeated fluorescence detection steps in the array configuration is partially solved by adding ascorbate to the detection solution, thereby increasing the number of available detection cycles from approximately 8 to 10 cycles without ascorbate to 25 cycles with ascorbate. Therefore, to further extend the read length of sequencing, further optimization and development of imaging reagents are needed to reduce photodamage to nucleic acids, improve fluorescence signal intensity, and enhance sequencing accuracy. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, one of the objectives of this invention is to provide an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions, in which a combination of multiple antioxidants works synergistically to extend sequencing read length and improve sequencing accuracy.

[0005] In addition, the present invention also provides a reagent kit for a fluorescence-based multi-cycle nucleic acid sequencing reaction.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An imaging reagent for a fluorescence-based multi-cycle nucleic acid sequencing reaction comprises ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate.

[0008] Optionally, the concentration ratio of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate in the above imaging reagent is 1:1:2.5. As a further preferred embodiment, the above imaging reagent does not include fluorescence quenching inhibitors and free radical scavengers.

[0009] The imaging reagent of this invention utilizes a synergistic combination of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate to reduce free radical generation, inhibit nucleic acid damage during imaging, reduce phasing and pre-phasing, and ultimately improve the accuracy of long-read sequencing. Traditional imaging reagents, in order to increase sequencing read length, typically add three functional components: antioxidants, fluorescence quenching inhibitors, and free radical scavengers. Compared to existing imaging protectants, this invention creatively selects a combination of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate to achieve a synergistic effect, eliminating the need for adding fluorescence quenching inhibitors and free radical scavengers.

[0010] Further preferably, in order to improve the stability of the imaging reagent, the imaging reagent also includes 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, which promotes the dissolution of each component in the imaging reagent and improves the overall preparation and storage stability of the imaging reagent.

[0011] Optionally, the imaging reagents described above may also include Tris buffer.

[0012] Specifically, the formulation of the above imaging reagent consists of 90–110 mM Tris, 750–850 mM NaCl, 15–25 mM ethyl 3,4-dihydroxybenzoate, 15–25 mM 4-hydroxy-3-methoxyacetophenone, 37.5–62.5 mM sodium ascorbate, 0.1–0.2 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, and pH = 8.0. As a preferred embodiment of the present invention, the imaging reagent is formulated as follows: 100mM Tris, 800mM NaCl, 20mM ethyl 3,4-dihydroxybenzoate, 20mM 4-hydroxy-3-methoxyacetophenone, 50mM sodium ascorbate, 0.1wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH=8.0.

[0013] A nucleic acid sequencing kit includes an extension reagent, a cleavage reagent, an imaging reagent, and a detergent; wherein the extension reagent includes a modified nucleotide, and the modified nucleotide molecule is attached with a reversible blocking portion; wherein the cleavage reagent includes a component that acts on the reversible blocking portion, causing the reversible blocking portion to detach from the modified nucleotide molecule; and the imaging reagent is the aforementioned imaging reagent.

[0014] Optionally, the modified nucleotide molecule is attached to a labeled molecule via a cleavable linker; the cleavable linker comprises one or more cleavable groups; the cleavable groups include disulfide bonds and / or azides; the cleavage reagent further includes a component acting on the cleavable groups to detach the labeled molecule from the modified nucleotide molecule; the reversible blocking portion is a methyl azide. As a further preferred embodiment, the labeled molecule is a fluorescent molecule.

[0015] Optionally, the detergent may include antioxidants and / or cutter removers.

[0016] Optionally, the cleavable group is an azide; the cleavage agent includes tri-(hydroxypropyl)phosphine; and the detergent includes cystine, lipoic acid, 3,3′-dithiodipropionic acid, or polyethylene glycol-modified azide.

[0017] Optionally, the detergent may also include gallic acid and / or sodium ascorbate.

[0018] In one embodiment of the present invention, the method for nucleic acid sequencing using the kit of the present invention includes: 1) providing an array of nucleic acid template molecules; 2) adding sequencing primers to hybridize with the corresponding nucleic acid template molecules; 3) adding extension reagents, where polymerase in the extension reagents incorporates modified nucleotide molecules into the sequencing primers; 4) then adding detergent to remove unreacted extension reagents; 5) then adding imaging reagents, where fluorescent molecules linked by cleavable linkers on the modified nucleotide molecules emit light under excitation light, and images of the fluorescent molecules emitting light from the array of nucleic acid template molecules are acquired, and the type of modified nucleotide incorporated into each template molecule is determined based on the image information; the imaging reagent used in the present invention includes ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate, the three components having a synergistic effect to reduce photoinduced damage to nucleic acid molecules and reduce the generation of free radicals during the process; 6) then adding... Add detergent to replace and rinse away the imaging reagent, then add cleavage reagent to remove the reversible blocking portion and labeling molecules on the modified nucleotide molecules incorporated into the sequencing primers; 7) Then add detergent to rinse and replace the unreacted cleavage reagent; the detergent in this step can eliminate the oxygen free radicals generated in the reaction system by adding antioxidants such as gallic acid and / or ascorbic acid, and at the same time add cleavage reagent scavenger, which is a compound that inhibits the interaction between reactive compounds used or generated in step 6) and polynucleotides used or generated in the sequencing process, enzymes used in the sequencing process, or other reagents or compounds used in the sequencing process, such as cystine, lipoic acid, 3,3′-dithiodipropionic acid or polyethylene glycol-modified azide, which can oxidize the residual cleavage reagent and eliminate the influence of the residual cleavage reagent on the incorporation of nucleotides in the next round; 8) Then return to step 3) for cyclic sequencing. Attached Figure Description

[0019] Figure 1 This is a graph showing the trend of fluorescence intensity changes during sequencing using the Illumina NextSeq500 reagents.

[0020] Figure 2 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Example 1 of this invention.

[0021] Figure 3 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 1 of this invention.

[0022] Figure 4 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 2 of this invention.

[0023] Figure 5 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 3 of this invention.

[0024] Figure 6 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 4 of this invention.

[0025] Figure 7 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 5 of this invention.

[0026] Figure 8 A graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 6 of this invention;

[0027] Figure 9 A graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 7 of this invention. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0029] Example 1

[0030] This embodiment provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 20 mM ethyl 3,4-dihydroxybenzoate, 20 mM 4-hydroxy-3-methoxyacetophenone, 50 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method includes:

[0031] 1) Weigh 3.0285g Tris, 11.688g NaCl, 2.4765g sodium ascorbate, 0.911g ethyl 3,4-dihydroxybenzoate, 0.8315g 4-hydroxy-3-methoxyacetophenone, and 0.25g 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate into a 250mL beaker.

[0032] 2) Add 200mL of ultrapure water to a beaker, place the rotor inside, and put the beaker on a magnetic stirrer to dissolve it;

[0033] 3) After dissolving, adjust the pH to 8.0 using concentrated hydrochloric acid;

[0034] 4) Pour the solution into a graduated cylinder and bring the volume to 250 mL. Filter the reagent after volume adjustment and store at -20℃.

[0035] Example 2

[0036] This embodiment provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 90 mM Tris, 750 mM NaCl, 15 mM ethyl 3,4-dihydroxybenzoate, 15 mM 4-hydroxy-3-methoxyacetophenone, 37.5 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0037] Example 3

[0038] This embodiment provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 110 mM Tris, 850 mM NaCl, 25 mM ethyl 3,4-dihydroxybenzoate, 25 mM 4-hydroxy-3-methoxyacetophenone, 62.5 mM sodium ascorbate, 0.2 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0039] Comparative Example 1

[0040] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 90 mM ethyl 3,4-dihydroxybenzoate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0041] Comparative Example 2

[0042] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 90 mM 4-hydroxy-3-methoxyacetophenone, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0043] Comparative Example 3

[0044] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 90 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0045] Comparative Example 4

[0046] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 50 mM ethyl 3,4-dihydroxybenzoate, 40 mM 4-hydroxy-3-methoxyacetophenone, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0047] Comparative Example 5

[0048] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 20 mM ethyl 3,4-dihydroxybenzoate, 50 mM 4-hydroxy-3-methoxyacetophenone, 20 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0049] Comparative Example 6

[0050] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 100 mM Tris, 800 mM NaCl, 50 mM ethyl 3,4-dihydroxybenzoate, 20 mM 4-hydroxy-3-methoxyacetophenone, 20 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0051] Comparative Example 7

[0052] This comparative example provides an imaging reagent for fluorescence-based multi-cycle nucleic acid sequencing reactions. Its formulation consists of 50 mM Tris, 20 mM ethyl 3,4-dihydroxybenzoate, 15 mM quinoline dimethacrylate, 50 mM M-carnitine, with the balance being water, pH 8.0. The preparation method is the same as in Example 1.

[0053] Test case

[0054] Experimental Methods: This invention uses an Illumina NextSeq 500 sequencer V2.1 for testing. Under the principle of sequencing-by-synthesis, a comparative experiment was conducted to analyze the effects of the imaging reagent from Example 1 and the comparative imaging reagent on the fluorescence signal during the sequencing reaction, as well as on sequencing read length and accuracy. Specific tests included 150 cycles and 288 microcells (titles) of the phix genome.

[0055] Experimental Groups: The experiment was conducted in 9 groups. Group 1 used the reagents supplied with the Illumina NextSeq 500 sequencer; Group 2 used the imaging reagent provided in Example 1 instead of the imaging reagent in the Illumina NextSeq 500 sequencer's reagent set, while using the same reagents as the Illumina NextSeq 500 sequencer; Group 3 used the imaging reagent provided in Comparative Example 1 instead of the imaging reagent in the Illumina NextSeq 500 sequencer's reagent set, while using the same reagents as the Illumina NextSeq 500 sequencer; Group 4 used the imaging reagent provided in Comparative Example 2 instead of the imaging reagent in the Illumina NextSeq 500 sequencer's reagent set, while using the same reagents as the Illumina NextSeq 500 sequencer; Group 5 used the imaging reagent provided in Comparative Example 3 instead of the imaging reagent in the Illumina NextSeq 500 sequencer's reagent set, while using the same reagents as the Illumina NextSeq 500 sequencer; Group 6 used the imaging reagent provided in Comparative Example 4 instead of the Illumina NextSeq 500 sequencer's reagent set. The imaging reagents in the Illumina NextSeq500 sequencer kit were used in the following groups: Group 7 used the imaging reagents provided in Comparative Example 5 instead of the imaging reagents in the Illumina NextSeq500 sequencer kit; Group 8 used the imaging reagents provided in Comparative Example 6 instead of the imaging reagents in the Illumina NextSeq500 sequencer kit; Group 9 used the imaging reagents provided in Comparative Example 7 instead of the imaging reagents in the Illumina NextSeq500 sequencer kit; and Group 9 used the imaging reagents provided in Comparative Example 7 instead of the imaging reagents in the Illumina NextSeq500 sequencer kit.

[0056] Experimental results:

[0057] 1. The sequencing results of each group after 150 cycles of single-end sequencing are shown in Table 1 below:

[0058] Table 1

[0059]

[0060] 2. The trends in fluorescence intensity during 150 single-end sequencing cycles in each group are as follows: Figures 1 to 8 As shown, where Figure 1 This graph shows the trend of fluorescence intensity changes during sequencing using commercially available reagents. Figure 2This diagram shows the trend of fluorescence intensity changes during sequencing using the imaging reagent provided by the present invention, which is composed of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone and sodium ascorbate. Figure 3 This graph shows the trend of fluorescence intensity changes when an imaging reagent composed solely of ethyl 3,4-dihydroxybenzoate is used during sequencing. Figure 4 This graph shows the trend of fluorescence intensity changes during sequencing when an imaging reagent composed solely of 4-hydroxy-3-methoxyacetophenone is used. Figure 5 This graph shows the trend of fluorescence intensity changes when an imaging reagent consisting only of sodium ascorbate is used during sequencing. Figure 6 This graph shows the trend of fluorescence intensity changes during sequencing when an imaging reagent composed of ethyl 3,4-dihydroxybenzoate and 4-hydroxy-3-methoxyacetophenone is applied. Figure 7 and Figure 8 All images show the trend of fluorescence intensity changes during sequencing when the imaging reagent, composed of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, and sodium ascorbate in a mixed ratio is applied. Figure 9 This graph shows the fluorescence intensity change trend during sequencing using an imaging reagent composed of ethyl 3,4-dihydroxybenzoate, quinoline dimethacrylate, and L-carnitine, which is disclosed in the prior art.

[0061] Depend on Figures 1-9 As can be seen from the comparative test results shown in Table 1, by Figure 1 , Figure 2 and Figure 9 The results show that the imaging reagent of this invention, composed of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate, achieves the same effect as the commercially available Illumina NextSeq500 imaging reagent in suppressing fluorescence intensity decay during sequencing reactions. It maintains overall stable fluorescence intensity throughout the sequencing process without significant attenuation. Furthermore, the data in Table 1 also show that the imaging reagent of this invention, composed of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate, achieves the same level as commercially available products in terms of phasing value, Q30, and error rate. It is superior to similar products using a combination of ethyl 3,4-dihydroxybenzoate, quinoline dimethacrylate, and L-carnitine, and can replace commercially available products.

[0062] have Figure 3 and Figure 4The results show that ethyl 3,4-dihydroxybenzoate and 4-hydroxy-3-methoxyacetophenone alone, when used as imaging reagents in sequencing reactions, cannot suppress fluorescence intensity decay, resulting in numerous phase-fixing errors during sequencing, a Q30 below 30, and a sequencing error rate exceeding 40%. Therefore, they cannot be used as imaging reagents in sequencing reactions. Figure 5 and Figure 6 The results show that, although the imaging reagent composed of ascorbic acid alone, or the combination of ethyl 3,4-dihydroxybenzoate and 4-hydroxy-3-methoxyacetophenone, exhibits a reduced fluorescence intensity decay compared to ethyl 3,4-dihydroxybenzoate and 4-hydroxy-3-methoxyacetophenone when used as an imaging reagent in sequencing reactions, it still cannot maintain overall stability of fluorescence intensity throughout the sequencing process. The data in Table 1 also show that the phasing value, Q30, and error rate of the imaging reagent composed of ascorbic acid alone, or the combination of ethyl 3,4-dihydroxybenzoate and 4-hydroxy-3-methoxyacetophenone, cannot reach the level of the imaging reagent of this invention. Therefore, it is evident that the combined use of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone, and sodium ascorbate can produce a synergistic effect, and its application as an imaging reagent in sequencing reactions can suppress fluorescence damage and improve sequencing accuracy.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An imaging reagent for a fluorescence-based multi-cycle nucleic acid sequencing reaction, characterized by, Its formulation consists of 90–110 mM Tris, 750–850 mM NaCl, 15–25 mM ethyl 3,4-dihydroxybenzoate, 15–25 mM 4-hydroxy-3-methoxyacetophenone, 37.5–62.5 mM sodium ascorbate, 0.1–0.2 wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH = 8.0; The concentration ratio of ethyl 3,4-dihydroxybenzoate, 4-hydroxy-3-methoxyacetophenone and sodium ascorbate is 1:1:2.5; excluding fluorescence quenching inhibitors and free radical scavengers.

2. The imaging reagent for a fluorescence-based multi-cycle nucleic acid sequencing reaction according to claim 1, wherein Its formula consists of: 100mM Tris, 800mM NaCl, 20mM ethyl 3,4-dihydroxybenzoate, 20mM 4-hydroxy-3-methoxyacetophenone, 50mM sodium ascorbate, 0.1wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, pH=8.

0.

3. A kit for nucleic acid sequencing, characterized by, The invention comprises an extension reagent, a cleavage reagent, an imaging reagent, and a detergent; wherein the extension reagent includes a modified nucleotide, the modified nucleotide molecule having a reversible blocking portion attached thereto; wherein the cleavage reagent includes a component that acts on the reversible blocking portion, causing the reversible blocking portion to detach from the modified nucleotide molecule; and the imaging reagent is the imaging reagent as described in any one of claims 1 to 2.

4. The kit for nucleic acid sequencing according to claim 3, wherein The modified nucleotide molecule is attached to a labeled molecule via a cleavable linker; the cleavable linker comprises one or more cleavable groups; the cleavable groups include disulfide bonds and / or azides; the cleavage agent further comprises a component acting on the cleavable groups to detach the labeled molecule from the modified nucleotide molecule; the reversible blocking portion is methyl azide; the detergent comprises an antioxidant and / or a cleavage agent scavenger.

5. The kit for nucleic acid sequencing according to claim 4, wherein The cutting agent includes tris-(hydroxypropyl)phosphine; the detergent includes cystine, lipoic acid, 3,3′-dithiodipropionic acid or polyethylene glycol-modified azide; the detergent also includes gallic acid and / or sodium ascorbate; the elongation agent also includes polymerase.

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