Fluorescently-labeled ddNTP, synthetic method and application thereof, DNA sequencing kit and preparation method thereof

Abstract

The invention provides fluorescently-labeled ddNTP, a synthesis method and application of the fluorescently-labeled ddNTP, a DNA sequencing kit and a preparation method of the DNA sequencing kit. The fluorescently-labeled ddNTP has a structure as shown in a formula I or a formula II, the synthesis method of the fluorescently-labeled ddNTP comprises the following steps: connecting a rhodamine donor dye, a dichloro rhodamine receptor dye and ddNTP connected with a Linker group through a connecting group; wherein the Linker group is selected from any one of a propyl amino group and a propyl ethyoxyl amino group. The synthesis method disclosed by the invention is used for synthesizing the fluorescently-labeled ddNTP, the operation is simple, and the reaction conditions are mild. When the DNA sequencing kit is used for DNA sequencing, the obtained atlas has the advantages of low prepeak protrusion, slow signal intensity attenuation, good peak uniformity of the termination set, high base modulation precision, good spectral resolution and less noise interference.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA sequencing, and specifically relates to a fluorescently labeled ddNTP and a synthesis method and application thereof, and a DNA sequencing kit and a preparation method thereof. Background Art

[0002] DNA sequencing is the process of determining the sequence of nucleic acids (i.e., the order of nucleotides in DNA). It includes any method or technology used to determine the order of four base nucleotides (adenine, guanine, cytosine, and thymine). DNA sequencing technology is the most commonly used technology in molecular biology research, and its emergence has greatly promoted the development of biology. Mature DNA sequencing technology began in the mid-1970s. In 1977, Maxam and Gilbert reported a method for determining DNA sequences by chemical degradation. At the same time, Sanger invented the dideoxy chain termination method. The two methods are very different in principle, but both are based on the nucleotide starting at a fixed point and randomly terminating at a specific base to produce a series of nucleotides of four different lengths, A, T, C, and G, and then detected by electrophoresis on urea-denatured PAGE gel to obtain the DNA sequence. So far, the era of DNA sequencing has been ushered in; the fluorescent automatic sequencing technology that appeared in the early 1990s brought DNA sequencing into the era of automated sequencing. These technologies are collectively referred to as the first generation of DNA sequencing technology.

[0003] The recently developed second-generation DNA sequencing technology (Sequencing By Synthesis) has brought DNA sequencing into the era of high throughput and low cost. Sequencing By Synthesis is to add modified DNA polymerase and nucleotides with four fluorescent labels. These nucleotides with special structures are "reversible terminations". Because the 3' hydroxyl end has a chemically cleavable part, it only allows a single base to be incorporated in each cycle. At this time, the surface of the reaction plate is scanned with a laser to read the types of nucleotides polymerized in the first round of reaction for each template sequence. After that, these groups are chemically cut to restore the viscosity of the 3' end and continue to polymerize the second nucleotide. Continue in this way until each template sequence is completely polymerized into a double strand. In this way, by counting the fluorescent signal results collected in each round, the sequence of each template DNA fragment can be known.

[0004] However, at present, there are relatively few reports on the specific structure of terminators, and there are relatively few terminators and kit products prepared therefrom that can obtain better maps when used for DNA sequencing. Summary of the invention

[0005] The object of the present invention is to provide a fluorescently labeled ddNTP and a synthesis method and application thereof, and a DNA sequencing kit and a preparation method thereof. The synthesis method of the present invention is used to synthesize fluorescently labeled ddNTP, which is simple to operate and has mild reaction conditions. The DNA sequencing kit of the present invention has a low front peak protrusion and a slow signal intensity decay in the spectrum obtained during DNA sequencing, good peak uniformity of the termination set, high base calling accuracy, good spectral resolution, and less noise interference.

[0006] The above objectives of the present invention are achieved through the following technical solutions.

[0007] In a first aspect, the present invention provides a fluorescently labeled ddNTP (dideoxynucleoside triphosphate), wherein the fluorescently labeled ddNTP has a structure of Formula I or Formula II,

[0008]

[0009] In Formula I or Formula II, R1 and R2 are independently selected from hydrogen or alkyl; preferably, the alkyl group optionally forms a ring structure with the benzene ring connected to the N atom; and B is selected from any one of the four bases A, G, C, and U.

[0010] In some embodiments of the present invention, in Formula I, B is selected from any one of the three bases G, C, and U.

[0011] In some embodiments of the present invention, in Formula II, the B is A base.

[0012] In some embodiments of the present invention, the alkyl group is a C1-C10 alkyl group, preferably a C1-C6 alkyl group, and more preferably a C1-C3 alkyl group.

[0013] In some embodiments of the present invention, the two connecting bonds in Formula I or Formula II that are not connected to the six connecting sites of the benzene ring both indicate that the connecting bonds can be connected to any one of the connecting sites on either side thereof.

[0014] In some embodiments of the present invention, the fluorescently labeled ddNTP includes any one or more of fluorescently labeled ddATP having a BigDye-A structure, fluorescently labeled ddGTP having a BigDye-G structure, fluorescently labeled ddCTP having a BigDye-C structure, and fluorescently labeled ddUTP having a BigDye-U structure.

[0015]

[0016] In a second aspect, the present invention provides use of the fluorescently labeled ddNTP described in the first aspect in preparing a terminator and / or a kit for DNA sequencing.

[0017] In a third aspect, the present invention provides a method for synthesizing a fluorescently labeled ddNTP, the method comprising the following steps:

[0018] (1) reacting a dichlororhodamine acceptor dye with NHS (N-hydroxysuccinimide) to generate a dichlororhodamine acceptor dye connected with an activated ester structure;

[0019] (2) reacting a rhodamine donor dye with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and after the reaction, adding ammonia water to remove the trifluoroacetyl group to obtain a rhodamine donor dye connected with an NH2-Ph-CO- group;

[0020] (3) subjecting the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) to a substitution reaction with the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2), so that the rhodamine donor dye and the dichlororhodamine acceptor dye are linked together through -NH-Ph-CO-;

[0021] (4) allowing the dual dye product obtained in step (3) to undergo a condensation reaction with a ddNTP connected with a linker group; wherein the linker group is selected from any one of propylamino and propylethoxyamino.

[0022] In some embodiments of the present invention, the dichlororhodamine receptor dye is selected from compounds having a structure of formula ①;

[0023]

[0024] In formula ①, R1 and R2 are independently selected from hydrogen or alkyl; preferably, the alkyl group optionally forms a ring structure with the benzene ring connected to the N atom.

[0025] In some embodiments of the present invention, the alkyl group is a C1-C10 alkyl group, preferably a C1-C6 alkyl group, and more preferably a C1-C3 alkyl group.

[0026] In some embodiments of the present invention, the carboxyl group at the lower right in formula ① means that the carboxyl group can be adjacent to any one of the chlorine substituents on both sides.

[0027] In some embodiments of the present invention, the dichlororhodamine receptor dye is selected from any one of 5dR6G, 5dR110, 6dROX, and 5dTAMRA.

[0028] In some embodiments of the present invention, the rhodamine donor dye is selected from 5-carboxyfluorescein or 6-carboxyfluorescein.

[0029] In some embodiments of the present invention, the ddNTP connected with a Linker group is selected from a compound having a structure of Formula ② or Formula ③;

[0030]

[0031] In formula ② or formula ③, the B is selected from any one of the four bases A, G, C, and U; preferably, in formula ②, the B is selected from any one of the three bases G, C, and U, and in formula ③, the B is the A base.

[0032] In some embodiments of the present invention, the N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester has the structure of the following formula ④;

[0033]

[0034] In some embodiments of the present invention, the molar ratio of dichlororhodamine acceptor dye to NHS in step (1) is 1:(1-2).

[0035] In some embodiments of the present invention, the molar ratio of the rhodamine donor dye to the N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) is 1:(1-1.5).

[0036] In some embodiments of the present invention, the amount of ammonia water added in step (2) is such that the volume ratio of ammonia water to the reaction solution is 1:(8-12). In the present invention, the ammonia water refers to an aqueous solution with an ammonia concentration (mass fraction of ammonia) of 20%.

[0037] In some embodiments of the present invention, in step (3), the molar ratio of the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) to the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2) is 1:(1 to 1.5).

[0038] In some embodiments of the present invention, in step (4), the molar ratio of the double dye product obtained in step (3) to the ddNTP connected with a linker group is 1:(1-2).

[0039] In some embodiments of the present invention, in step (1), DCC (N,N'-dicyclohexylcarbodiimide) or EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) is used as a catalyst to catalyze the reaction of dichlororhodamine acceptor dye with NHS; preferably, the molar ratio of the catalyst to NHS is (0.8-1.2):1.

[0040] In some embodiments of the present invention, the reaction of the rhodamine donor dye with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) is carried out in TEA (triethylamine); preferably, the mass ratio of TEA to rhodamine donor dye is (1-3):1.

[0041] In some embodiments of the present invention, the substitution reaction in step (3) is carried out in DMF (N,N-dimethylformamide); preferably, the mass ratio of DMF to the rhodamine donor dye connected with an NH2-Ph-CO- group is (25-35):1.

[0042] In some embodiments of the present invention, the condensation reaction of step (4) is carried out in DIPEA (N,N-diisopropylethylamine); preferably, the mass ratio of DIPEA to the double dye product is (1.5-2.5):1.

[0043] In some embodiments of the present invention, the step (4) further comprises activating the double dye product obtained in step (3) with HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) before the condensation reaction occurs; preferably, the conditions of the activation treatment are: stirring at 25-45°C for 20-40 minutes under nitrogen protection, and / or the mass ratio of HBTU to the double dye product is (1.5-2.5):1.

[0044] In some embodiments of the present invention, the reaction temperature of the dichlororhodamine receptor dye and NHS in step (1) is 10-30° C., preferably 15-25° C., and the reaction time is 2-6 h, preferably 3-5 h.

[0045] In some embodiments of the present invention, the reaction temperature of the rhodamine donor dye and N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) is 10-30° C., preferably 15-25° C., and the reaction time is 3-8 h, preferably 4-6 h.

[0046] In some embodiments of the present invention, the reaction temperature of the substitution reaction in step (3) is 15 to 35° C., preferably 20 to 30° C., and the reaction time is 12 to 20 h, preferably 14 to 18 h.

[0047] In some embodiments of the present invention, the reaction temperature of the condensation reaction in step (4) is 25 to 45° C., preferably 30 to 40° C., and the reaction time is 3 to 9 hours, preferably 4 to 8 hours.

[0048] In some embodiments of the present invention, the method further comprises separating and purifying the product obtained in step (4); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; more preferably, the elution conditions for separation and purification include: mobile phase A is 1% by mass HCl, mobile phase B is MeCN (acetonitrile), and the elution time is 60 min.

[0049] In some embodiments of the present invention, the method further comprises separating and purifying the product obtained in step (3); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; more preferably, the elution conditions for separation and purification include: mobile phase A is 0.1 mol / L TEAB (pH=7.5-7.8), mobile phase B is MeCN, and the elution time is 60 min.

[0050] In some embodiments of the present invention, the method further comprises separating and purifying the product obtained in step (2); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; more preferably, the elution conditions for separation and purification include: mobile phase A is 0.1 mol / L TEAB (pH=7.5-7.8), mobile phase B is MeCN, and the elution time is 60 min.

[0051] In some embodiments of the present invention, the method further comprises separating and purifying the product obtained in step (1); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; more preferably, the elution conditions for separation and purification include: mobile phase A is 0.1 mol / L TEAB (pH=7.5-7.8), mobile phase B is MeCN, and the elution time is 60 min.

[0052] In a fourth aspect, the present invention provides a fluorescently labeled ddNTP synthesized by the synthesis method described in the third aspect.

[0053] In some embodiments of the present invention, the fluorescently labeled ddNTP has a structure of Formula I or Formula II.

[0054] In some embodiments of the present invention, the fluorescently labeled ddNTP has a structure of BigDye-A, BigDye-G, BigDye-C or BigDye-U.

[0055] In a fifth aspect, the present invention provides use of the fluorescently labeled ddNTP described in the fourth aspect in preparing a terminator and / or a kit for DNA sequencing.

[0056] In a sixth aspect, the present invention provides application of the synthesis method described in the third aspect in synthesizing the fluorescently labeled ddNTP described in the first aspect.

[0057] In some embodiments of the present invention, the synthesis method comprises the following steps:

[0058] (1) reacting a dichlororhodamine receptor dye with NHS to generate a dichlororhodamine receptor dye connected with an activated ester structure; the dichlororhodamine receptor dye is selected from a compound having a structure of formula ①;

[0059] (2) reacting a rhodamine donor dye with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and after the reaction is completed, adding ammonia water to remove the trifluoroacetyl group to obtain a rhodamine donor dye connected with an NH2-Ph-CO- group; the rhodamine donor dye is selected from 5-carboxyfluorescein or 6-carboxyfluorescein;

[0060] (3) subjecting the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) to a substitution reaction with the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2), so that the rhodamine donor dye and the dichlororhodamine acceptor dye are linked together through the -NH-Ph-CO- group;

[0061] (4) allowing the dual dye product obtained in step (3) to undergo a condensation reaction with a ddNTP connected with a linker group; wherein the ddNTP connected with a linker group is selected from a compound having a structure of formula ② or formula ③.

[0062] In some embodiments of the present invention, the synthesis method comprises the following steps:

[0063] (1) reacting a dichlororhodamine acceptor dye with NHS to generate a dichlororhodamine acceptor dye connected with an activated ester structure;

[0064] (2) reacting a rhodamine donor dye with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and after the reaction, adding ammonia water to remove the trifluoroacetyl group to obtain a rhodamine donor dye connected with an NH2-Ph-CO- group;

[0065] (3) according to the raw material combination in Table 0, the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) and the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2) are subjected to a substitution reaction, so that the rhodamine donor dye and the dichlororhodamine acceptor dye are linked together through the -NH-Ph-CO- group;

[0066] (4) The dual dye product obtained in step (3) is reacted with a ddNTP having a structure of formula ② or formula ③ and connected with a linker group by a condensation reaction according to the raw materials in Table 0.

[0067] Table 0 Raw material matching

[0068]

[0069] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddATP having a BigDye-A structure comprises the following steps:

[0070] (1) reacting 5dR6G with NHS to generate an activated ester of 5dR6G;

[0071] (2) reacting 6-FAM with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and adding ammonia water to remove the trifluoroacetyl group after the reaction to obtain 4-aminomethylbenzoic acid-6-FAM;

[0072] (3) subjecting the 5dR6G activated ester obtained in step (1) to a substitution reaction with the 4-aminomethylbenzoic acid-6-FAM obtained in step (2), so that 5dR6G and 6-FAM are linked together through the -NH-Ph-CO- group;

[0073] (4) allowing the 5dR6G-4-aminomethylbenzoic acid-6-FAM obtained in step (3) to undergo a condensation reaction with propylamino-ddATP.

[0074] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddATP having a BigDye-A structure further comprises separating and purifying the reaction product of step (1) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic conditions are: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; gradient elution is performed according to Table 1, and the product with a peak time of 30 to 38 min is collected.

[0075] Table 1

[0076] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 90 10 20 60 90 10 25 60 80 20 50 60 60 40 55 60 95 5 60 60 95 5

[0077] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddATP having a BigDye-A structure further comprises separating and purifying the reaction product of the reaction between 6-FAM and N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic column: SP ODS-AQ (50*250, 10um, 100A), the mobile phase A: 0.1mol / L TEAB (pH=7.5-7.8), B: MeCN; gradient elution is performed according to Table 2, and the product with a peak time of 38-46min is collected.

[0078] Table 2

[0079] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 90 10 20 60 90 10 25 60 80 20 50 60 60 40 55 60 95 5 60 60 95 5

[0080] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddATP having a BigDye-A structure further comprises separating and purifying the reaction product of step (3) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5-7.8), B: MeCN; gradient elution is performed according to Table 3, and the product with a peak time of 25-31min is collected.

[0081] Table 3

[0082] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 50 60 60 40 55 60 95 5 60 60 95 5

[0083] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddATP having a BigDye-A structure further comprises separating and purifying the reaction product of step (4) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 4, and the product with a peak time of 22~38min is collected.

[0084] Table 4

[0085] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 10 60 90 10 50 60 60 40 55 60 95 5 60 60 95 5

[0086] In some embodiments of the present invention, the synthesis method of fluorescently labeled ddGTP having a BigDye-G structure comprises the following steps:

[0087] (1) reacting 5dR110 with NHS to generate an activated ester of 5dR110;

[0088] (2) reacting 5-FAM with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and adding ammonia water to remove the trifluoroacetyl group after the reaction to obtain 4-aminomethylbenzoic acid-5-FAM;

[0089] (3) subjecting the activated ester 5dR110 obtained in step (1) to a substitution reaction with the 4-aminomethylbenzoic acid-5-FAM obtained in step (2), so that 5dR110 and 5-FAM are linked together via a -NH-Ph-CO- group;

[0090] (4) allowing the 5dR110-4-aminomethylbenzoic acid-5-FAM obtained in step (3) to undergo a condensation reaction with propylethoxyamino-ddGTP.

[0091] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddGTP having a BigDye-G structure further comprises separating and purifying the reaction product of step (1) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic conditions are: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; gradient elution is performed according to Table 5, and the product with a peak time of 22 to 25 min is collected.

[0092] Table 5

[0093]

[0094]

[0095] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddGTP having a BigDye-G structure further comprises separating and purifying the reaction product of the reaction of 5-FAM and N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 6, and the product with a peak time of 36~43min is collected.

[0096] Table 6

[0097] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 90 10 20 60 90 10 25 60 80 20 50 60 60 40 55 60 95 5 60 60 95 5

[0098] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddGTP having a BigDye-G structure further comprises separating and purifying the reaction product of step (3) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 7, and the product with a peak time of 22~28min is collected.

[0099] Table 7

[0100] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 50 60 60 40 55 60 95 5 60 60 95 5

[0101] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddGTP having a BigDye-G structure further comprises separating and purifying the reaction product of step (4) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 8, and the product with a peak time of 20~27min is collected.

[0102] Table 8

[0103]

[0104]

[0105] In some embodiments of the present invention, the synthesis method of fluorescently labeled ddCTP having a BigDye-C structure comprises the following steps:

[0106] (1) reacting 6dROX with NHS to generate 6dROX activated ester;

[0107] (2) reacting 6-FAM with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and adding ammonia water to remove the trifluoroacetyl group after the reaction to obtain 4-aminomethylbenzoic acid-6-FAM;

[0108] (3) subjecting the 6dROX activated ester obtained in step (1) to a substitution reaction with the 4-aminomethylbenzoic acid-6-FAM obtained in step (2), so that 6dROX and 6-FAM are linked together via a -NH-Ph-CO- group;

[0109] (4) allowing the 6dROX-4-aminomethylbenzoic acid-6-FAM obtained in step (3) to undergo a condensation reaction with propylethoxyamino-ddCTP.

[0110] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddCTP having a BigDye-C structure further comprises separating and purifying the reaction product of step (1) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic conditions are: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; gradient elution is performed according to Table 9, and the product with a peak time of 35 to 40 min is collected.

[0111] Table 9

[0112] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 65 35 25 60 65 35 30 60 55 45 45 60 45 55 55 60 95 5 60 60 95 5

[0113] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddCTP having a BigDye-C structure further comprises separating and purifying the reaction product of the reaction of 6-FAM and N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic column: SP ODS-AQ (50*250, 10um, 100A), the mobile phase A: 0.1mol / L TEAB (pH=7.5-7.8), B: MeCN; performing gradient elution according to Table 10, and collecting the product with a peak time of 38-46min.

[0114] Table 10

[0115]

[0116]

[0117] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddCTP having a BigDye-C structure further comprises separating and purifying the reaction product of step (3) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5-7.8), B: MeCN; gradient elution is performed according to Table 11, and the product with a peak time of 35-42min is collected.

[0118] Table 11

[0119] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 10 60 90 10 25 60 90 10 30 60 75 25 50 60 60 40 55 60 95 5 60 60 95 5

[0120] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddCTP having a BigDye-C structure further comprises separating and purifying the reaction product of step (4) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5-7.8), B: MeCN; gradient elution is performed according to Table 12, and the product with a peak time of 25-30min is collected.

[0121] Table 12

[0122] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 10 60 90 10 50 60 60 40 55 60 95 5 60 60 95 5

[0123] In some embodiments of the present invention, the synthesis method of fluorescently labeled ddUTP having a BigDye-U structure comprises the following steps:

[0124] (1) reacting 5dTAMRA with NHS to generate 5dTAMR activated ester;

[0125] (2) reacting 6-FAM with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and adding ammonia water to remove the trifluoroacetyl group after the reaction to obtain 4-aminomethylbenzoic acid-6-FAM;

[0126] (3) subjecting the 5dTAMR activated ester obtained in step (1) to a substitution reaction with the 4-aminomethylbenzoic acid-6-FAM obtained in step (2), so that 5dTAMRA and 6-FAM are linked together through the -NH-Ph-CO- group;

[0127] (4) The 5dTAMR-4-aminomethylbenzoic acid-6-FAM obtained in step (3) is subjected to a condensation reaction with propylethoxyamino-ddUTP.

[0128] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddUTP having a BigDye-U structure further comprises separating and purifying the reaction product of step (1) by reverse phase preparative high performance liquid chromatography; preferably, the chromatographic conditions are: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; gradient elution is performed according to Table 13, and the product with a peak time of 32 to 38 min is collected.

[0129] Table 13

[0130] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 65 35 25 60 65 35 30 60 55 45 45 60 45 55 55 60 95 5 60 60 95 5

[0131] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddUTP having a BigDye-U structure further comprises separating and purifying the reaction product of the reaction of 6-FAM and N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester in step (2) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 14, and the product with a peak time of 38~46min is collected.

[0132] Table 14

[0133] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 15 60 90 10 20 60 90 10 25 60 80 20 50 60 60 40 55 60 95 5 60 60 95 5

[0134] In some embodiments of the present invention, the method for synthesizing a fluorescently labeled ddUTP having a BigDye-U structure further comprises separating and purifying the reaction product of step (3) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 15, and the product with a peak time of 33~40min is collected.

[0135] Table 15

[0136]

[0137]

[0138] In some embodiments of the present invention, the method for synthesizing fluorescently labeled ddUTP having a BigDye-U structure further comprises separating and purifying the reaction product of step (4) by reverse phase preparative high performance liquid chromatography; preferably, chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; gradient elution is performed according to Table 16, and the product with a peak time of 24~31min is collected.

[0139] Table 16

[0140] time Flow rate (mL / min) Mobile phase A Mobile phase B 0 60 95 5 5 60 95 5 10 60 90 10 50 60 60 40 55 60 95 5 60 60 95 5

[0141] In this aspect, the raw material mass ratios, reaction temperatures, times, etc. of each reaction in the above-mentioned synthesis method that are not listed here are the same as those listed in the third aspect and are not listed again here.

[0142] In a seventh aspect, the present invention provides a terminator for DNA sequencing, wherein the terminator comprises the fluorescently labeled ddNTP described in the first aspect or the fluorescently labeled ddNTP described in the fourth aspect.

[0143] In some embodiments of the present invention, the fluorescently labeled ddNTP includes fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, and fluorescently labeled ddUTP, and the four fluorescent markers are labeled with four different dyes.

[0144] In some embodiments of the present invention, the fluorescently labeled ddATP has a structure of BigDye-A, the fluorescently labeled ddGTP has a structure of BigDye-G, the fluorescently labeled ddCTP has a structure of BigDye-C, and the fluorescently labeled ddUTP has a structure of BigDye-U.

[0145] In some embodiments of the present invention, the mass ratio of the fluorescently labeled ddATP, the fluorescently labeled ddGTP, the fluorescently labeled ddCTP, and the fluorescently labeled ddUTP is (0.09-0.13): (0.03-0.07): (0.54-0.58): (0.60-0.64).

[0146] In an eighth aspect, the present invention provides a DNA sequencing kit, which comprises the fluorescently labeled ddNTP described in the first aspect or the fluorescently labeled ddNTP described in the fourth aspect or the terminator for DNA sequencing described in the seventh aspect.

[0147] In some embodiments of the present invention, the fluorescently labeled ddNTP includes fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, and fluorescently labeled ddUTP, and the four fluorescent markers are labeled with four different dyes.

[0148] In some embodiments of the present invention, the fluorescently labeled ddATP has a structure of BigDye-A, the fluorescently labeled ddGTP has a structure of BigDye-G, the fluorescently labeled ddCTP has a structure of BigDye-C, and the fluorescently labeled ddUTP has a structure of BigDye-U.

[0149] In some embodiments of the present invention, the DNA sequencing kit further comprises a DNA polymerase and a sequencing buffer.

[0150] In a ninth aspect, the present invention provides a method for preparing the DNA sequencing kit according to the eighth aspect, the preparation method comprising the following steps:

[0151] 1) Determine the concentration of the fluorescently labeled ddNTP in the DNA sequencing kit; preferably, according to the ABIPRISMTM sequencing manual, use DNA polymerase and fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, fluorescently labeled ddUTP to perform termination cycle sequencing, and determine the concentration of the fluorescently labeled ddNTP used in the reaction by titrating each fluorescently labeled ddNTP in the single-color termination reaction and selecting the concentration that maximizes the 700th nucleotide signal.

[0152] 2) According to the concentration determined in step 1), a reagent containing a specific concentration of fluorescently labeled ddNTP is prepared.

[0153] The beneficial effects of the present invention are as follows:

[0154] The synthesis method of fluorescently labeled ddNTPs of the present invention is simple to operate and has mild reaction conditions. The DNA sequencing kit of the present invention has a low front peak protrusion and a slow signal intensity decay in the spectrum obtained during DNA sequencing, good peak uniformity of the termination set, high base calling accuracy, good spectral resolution, and less noise interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 This is the NMR spectrum of the product obtained in Example 1 (fluorescently labeled ddATP).

[0156] Figure 2 This is the NMR spectrum of the product obtained in Example 2 (fluorescently labeled ddGTP).

[0157] Figure 3 This is the NMR spectrum of the product obtained in Example 3 (fluorescently labeled ddCTP).

[0158] Figure 4 This is the NMR spectrum of the product obtained in Example 4 (fluorescently labeled ddUTP).

[0159] Figure 5 It is a signal spectrum obtained by sequencing sample 1 using the DNA sequencing kit of the present invention and the DNA sequencing kit used for comparison.

[0160] Figure 6 It is a signal spectrum obtained by sequencing sample 2 using the DNA sequencing kit of the present invention and the DNA sequencing kit used for comparison.

[0161] Figure 7 It is a sequence result diagram obtained by sequencing sample 1 using the DNA sequencing kit of the present invention and the DNA sequencing kit used for comparison.

[0162] Figure 8 It is a sequence result diagram obtained by sequencing sample 1 using the DNA sequencing kit of the present invention and the DNA sequencing kit used for comparison. DETAILED DESCRIPTION

[0163] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0164] Example 1

[0165] The synthesis method of fluorescently labeled ddATP with BigDye-A structure, the synthesis route is as follows:

[0166]

[0167] The specific synthesis steps are as follows:

[0168] (1) Synthesis of A-2

[0169] Dissolve 1g A-1 (5dR6G) in 20mL DMF, add 1.5eq NHS and 1.5eq EDCI, and react at 20°C for 24h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SPODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl, B: MeCN; perform gradient elution according to Table 1, collect the product with an elution time of 30 to 38min, and directly freeze-dry to obtain 500mg of the product for later use.

[0170] (2) Synthesis of A-5

[0171] Dissolve 1g A-3 (6-FAM) in 10mL MeCN, add 2eq TEA, then slowly add 1.2eq A-4 at 0°C, and react at 20°C for 5h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; perform gradient elution according to Table 2, and collect products with peak time of 38~46min; directly concentrate the product to 80mL, then add 10mL ammonia water and stir the reaction at 30°C for 16h, then freeze-dry to obtain 600mg of the product for use.

[0172] (3) Synthesis of A-6

[0173] 400 mg of A-2 was dissolved in 20 mL of DMF, 1.2 eq of A-5 was added, and the mixture was reacted at 25 °C for 16 h; then, the mixture was separated and purified by reverse phase preparative high performance liquid chromatography. Chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH = 7.5-7.8), B: MeCN; gradient elution was performed according to Table 3, and the product with an elution time of 25-31 min was collected and directly freeze-dried to obtain 200 mg of the product for later use.

[0174] (4) Synthesis of BigDye-A

[0175] Dissolve 200 mg of A-6 in 5 mL of DMSO, add 2 eq of HBTU, protect with N2 and stir at 35°C for 0.5 h, add 1.5 eq of A-7 (ddATP connected with propylamino group), react at 35°C for 6 h; then separate and purify by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH=7.5-7.8), B: MeCN; perform gradient elution according to Table 4, collect the product with peak time of 22-38 min and directly freeze-dry to obtain 60 mg of product. The NMR spectrum of the product is as follows Figure 1 As shown; 1 H NMR (400MHz, DMSO+D2O) δ8.00(t,J=8.5Hz,1H),7.84(t,J=10.5Hz,4H),7.58(s,1H),7.48(dd,J=18.7,8.4Hz,3H),7.00(dd,J=20.5,11.2 Hz, 5H), 6.84 (s, 2H), 6.62-6.48 (m, 3H), 3.53-3.24 (m, 8H), 3.41-3.34 (m, 4H), 2.12 (s, 6H), 1.27 (t, J = 7.1Hz, 4H), 1.22 (d, J = 6.4Hz, 6H).

[0176] Example 2

[0177] The synthesis method of fluorescently labeled ddGTP with BigDye-G structure, the synthesis route is as follows:

[0178]

[0179] The specific synthesis steps are as follows:

[0180] (1) Synthesis of G-2

[0181] Dissolve 1g G-1(5dR110) in 20mL DMF, add 1.5eq NHS and 1.5eq EDCI, and react at 20°C for 24h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SPODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl, B: MeCN; perform gradient elution according to Table 5, collect the product with a peak time of 22 to 25min, directly freeze-dry to obtain 500mg of the product for later use.

[0182] (2) Synthesis of G-5

[0183] Dissolve 1g G-3 (5-FAM) in 10mL MeCN, add 2eq TEA, then slowly add 1.2eq G-4 at 0°C, and react at 20°C for 5h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; perform gradient elution according to Table 6, and collect products with peak times of 36~43min; directly concentrate the product to 80mL, then add 10mL ammonia water and stir the reaction at 30°C for 16h, then freeze-dry to obtain 600mg of the product for use.

[0184] (3) Synthesis of G-6

[0185] Dissolve 400 mg of G-2 in 20 mL of DMF, add 1.2 eq of G-5, and react at 25 ° C for 16 hours; then use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1 mol / L TEAB (pH=7.5~7.8), B: MeCN; perform gradient elution according to Table 7, collect the product with a peak time of 22~28 min, directly freeze-dry to obtain 200 mg of the product for later use.

[0186] (4) Synthesis of BigDye-G

[0187] Dissolve 200 mg of G-6 in 5 mL of DMSO, add 2 eq of HBTU, protect with N2 and stir at 35°C for 0.5 h, add 1.5 eq of G-7 (ddGTP connected with propylethoxyamino), react at 35°C for 6 h; then separate and purify by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH = 7.5-7.8), B: MeCN; perform gradient elution according to Table 8, collect the product with an elution time of 20-27 min and directly freeze-dry to obtain 40 mg of the product. The NMR spectrum of the product is as follows: Figure 2 As shown; 1H NMR(400MHz,D2O)δ8.26(d,J=16.2Hz,1H),7.95(t,J=12.1Hz,1H),7.82(d,J=7.6Hz,2H),7.47(d,J=7.8Hz ,5H),7.20(d,J=9.2Hz,5H),7.13(t,J=10.0Hz,1H),6.98(d,J=9.2Hz,3H),6.83(d,J=9.4Hz,5H),6.74(s,5 H),6.55(d,J=9.3Hz,2H),6.45(d,J=12.2Hz,3H),4.77(s,3H),4.40(s,2H),4.21(d,J=45.0Hz,1H),4.14–3 .87 (m, 4H), 3.77 (d, J = 26.4Hz, 2H), 3.08 (d, J = 15.5Hz, 2H), 2.45 (d, J = 68.6Hz, 1H), 2.04 (d, J = 41.5Hz, 2H).

[0188] Example 3

[0189] The synthesis method of fluorescently labeled ddCTP with BigDye-C structure, the synthesis route is as follows:

[0190]

[0191]

[0192] The specific synthesis steps are as follows:

[0193] (1) Synthesis of C-2

[0194] Dissolve 1g C-1 (6dROX) in 20mL DCM, add 1.5eq NHS and 1.5eq DCC, and react at 20°C for 4h; concentrate the reaction solution to a solid, then dissolve the solid with MeCN and separate and purify it by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; perform gradient elution according to Table 9, collect the product with a peak time of 35 to 40min, then extract the obtained product with DCM, concentrate the DCM extract and dry it at 30°C to obtain 600mg of the product for use.

[0195] (2) Synthesis of C-5

[0196] Dissolve 1g C-3 (6-FAM) in 10mL MeCN, add 2eq TEA, then slowly add 1.2eq C-4 at 0°C, and react at 20°C for 5h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; perform gradient elution according to Table 10, and collect the product with a peak time of 38~46min; directly concentrate the product to 80mL, then add 10mL ammonia water and stir the reaction at 30°C for 16h, then freeze-dry to obtain 600mg of the product for use.

[0197] (3) Synthesis of C-6

[0198] Dissolve 600 mg of C-2 in 20 mL of DMF, add 1.2 eq of C-5, and react at 25 ° C for 16 h; then use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100A), mobile phase A: 0.1 mol / L TEAB (pH = 7.5 ~ 7.8), B: MeCN; perform gradient elution according to Table 11, collect the product with a peak time of 35 to 42 min, directly freeze-dry to obtain 300 mg of the product for later use.

[0199] (4) Synthesis of BigDye-C

[0200] Dissolve 300 mg of C-6 in 5 mL of DMSO, add 2 eq of HBTU, protect with N2 and stir at 35°C for 0.5 h, add 1.5 eq of C-7 (ddCTP connected with propylethoxyamino), react at 35°C for 6 h; then separate and purify by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH = 7.5-7.8), B: MeCN; perform gradient elution according to Table 12, collect the product with a peak time of 25 to 30 min and directly freeze-dry to obtain 100 mg of the product. The NMR spectrum of the product is as follows Figure 3 As shown; 1H NMR(400MHz, DMSO+D2O)δ8.03(d,J=7.0Hz,2H),7.96–7.78(m,4H),7.62–7.46(m,4H), 7.00(d,J=8.9Hz,2H),6.76(s,1H),6.56(d,J=39.7Hz,3H),4.89(s,2H),4.75-4.57(m ,6H),4.25-4.01(m,2H),3.82-3.59(m,1H),3.51-3.39(m,7H),3.8-3.07(m,2H),3.02 -2.88(m,3H),2.64(d,J=22.7Hz,4H),2.41(d,J=55.6Hz,4H),1.90(t,J=37.9Hz,9H).

[0201] Example 4

[0202] The synthesis method of fluorescently labeled ddUTP with BigDye-U structure, the synthesis route is as follows:

[0203]

[0204] The specific synthesis steps are as follows:

[0205] (1) Synthesis of U-2

[0206] Dissolve 1g U-1 (5dTAMRA) in 20mL DCM, add 1.5eq NHS and 1.5eq DCC, and react at 20°C for 4h; concentrate the reaction solution to a solid, then dissolve the solid with MeCN and separate and purify it by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 1% HCl by mass, B: MeCN; perform gradient elution according to Table 13, collect the product with a peak time of 32 to 38min, then extract the obtained product with DCM, concentrate the DCM extract and dry it at 40°C to obtain 600mg of the product for later use.

[0207] (2) Synthesis of U-5

[0208] Dissolve 1g U-3 (6-FAM) in 10mL MeCN, add 2eq TEA, then slowly add 1.2eq C-4 at 0°C, and react at 20°C for 5h; then directly use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10um, 100A), mobile phase A: 0.1mol / L TEAB (pH=7.5~7.8), B: MeCN; perform gradient elution according to Table 14, and collect the product with a peak time of 38~46min; directly concentrate the product to 80mL, then add 10mL ammonia water and stir the reaction at 30°C for 16h, then freeze-dry to obtain 600mg of the product for use.

[0209] (3) Synthesis of U-6

[0210] Dissolve 600 mg of U-2 in 20 mL of DMF, add 1.2 eq of U-5, and react at 25° C. for 16 h; then use reverse phase preparative high performance liquid chromatography for separation and purification, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH=7.5-7.8), B: MeCN; perform gradient elution according to Table 15, collect the product with a peak time of 33-40 min, directly freeze-dry to obtain 300 mg of the product for later use.

[0211] (4)Synthesize BigDye-U

[0212] Dissolve 300 mg of U-6 in 5 mL of DMSO, add 2 eq of HBTU, protect with N2 and stir at 35°C for 0.5 h, add 1.5 eq of U-7 (ddUTP connected with propylethoxyamino), react at 35°C for 6 h; then separate and purify by reverse phase preparative high performance liquid chromatography, chromatographic conditions: chromatographic column: SP ODS-AQ (50*250, 10 um, 100 A), mobile phase A: 0.1 mol / L TEAB (pH = 7.5-7.8), B: MeCN; perform gradient elution according to Table 16, collect the product with an elution time of 24 to 31 min and directly freeze-dry to obtain 85 mg of the product. The NMR spectrum of the product is as follows Figure 4 As shown; 1H NMR (400MHz, DMSO+D2O) δ8.00(t,J=8.5Hz,1H),7.84(t,J=10.5Hz,3H),7.58(s,1H),7.48(dd,J=18.7,8.4Hz,4H),7.00(dd,J=20.5,11.2H z,3H),6.95-6.78(m,4H),6.62-6.48(m,4H),4.77-4.68(m,4H),4.08-3.89(m,1H),3.17(s,9H),3.10-2.88(m,5H),1.22(d,J=6.4Hz,6H).

[0213] Application Example (Preparation of DNA Sequencing Kit)

[0214] According to ABIPRISMTM sequencing manual (PE Applied Biosystems, Foster City, CA), energy transfer dyes are used, and Taq DNA polymerase and fluorescently labeled ddNTP (fluorescently labeled ddATP synthesized in Example 1, fluorescently labeled ddGTP synthesized in Example 2, fluorescently labeled ddCTP synthesized in Example 3, fluorescently labeled ddUTP synthesized in Example 4) are used to terminate cycle sequencing. The concentration of dNTP used in the sequencing reaction solution is: dATP, dCTP and dTTP (or dUTP) concentration is 100 μM, and dITP concentration is 500 μM. The concentration of the terminator used in the reaction is determined by titrating each terminator in the monochromatic termination reaction and selecting the concentration that maximizes the 700th nucleotide signal. The concentration of four fluorescently labeled ddNTPs is adjusted according to the relative brightness of each dye, so as to obtain the approximately equivalent signal of each color in the four-color reaction. The dye and terminator set used a 10 nm virtual filter on the ABI 3730xL sequencer CCD camera, centered at 545, 570, 595, and 625 nm. The final concentration setting results of the fluorescently labeled ddNTPs are shown in Table 17.

[0215] Table 17 Concentrations of fluorescently labeled ddNTPs used in the reaction

[0216]

[0217] Prepare the fluorescent-labeled ddNTP mixed solution according to the final concentration in Table 17, and prepare the mixed reagent for BAC DNA sample sequencing according to the reagent dosage in Table 18.

[0218] Table 18 Mixed reagents for sequencing BAC DNA samples

[0219]

[0220] Computer test

[0221] The DNA sequencing kit prepared by the above application examples was compared with the DNA sequencing kit prepared by using the terminator raw materials produced by Haining Boshang Biotechnology Co., Ltd. according to the reagent dosage in Table 18, and the same method was used to detect the same BAC DNA sample. The specific operation is: use the BAC DNA sample provided by Suzhou Zixi Biotechnology Co., Ltd. in a Perkin-Elmer 9600 thermal cycler and cycle according to the following scheme: initial denaturation at 95°C for 5 minutes, and then perform 30 cycles of denaturation at 95°C for 30 seconds, 55°C for 20 seconds, and 60°C for 4 minutes. Use a Centric-sep spin column to remove excess terminators, vacuum dry the sample, and then resuspend it with 2μL or 4μL formamide, heat to 95°C and keep warm for 2 minutes, take 2μL of the sample and load it into the ABI 3730xL sequencer for sequencing. According to the above method, two samples were measured in sequence; among them, the signal spectra of sample 1 and sample 2 are as shown respectively. Figure 5 and Figure 6 The sequence results of sample 1 and sample 2 are shown in Figure 7 and Figure 8 shown.

[0222] like Figure 5 and 6 As shown, the pre-peak protrusion in the signal spectrum measured by the DNA sequencing kit of the present invention is significantly smaller than that of the DNA sequencing kit used for comparison, so the DNA sequencing kit of the present invention is better than the DNA sequencing kit used for comparison in controlling the pre-peak protrusion; and as the detection process proceeds, the signal intensity attenuation of the DNA sequencing kit of the present invention is significantly less than that of the DNA sequencing kit used for comparison, indicating that the DNA sequencing kit of the present invention is superior to the DNA sequencing kit used for comparison in terms of intensity attenuation.

[0223] like Figure 7 and 8 As shown, the DNA sequencing kit of the present invention has better peak uniformity of the termination set than the DNA sequencing kit used for comparison, the base calling accuracy of the DNA sequencing kit of the present invention is higher than that of the DNA sequencing kit used for comparison, the DNA sequencing kit of the present invention has better spectral resolution, and also reduces noise interference compared with the DNA sequencing kit used for comparison.

[0224] In summary, compared with the DNA sequencing kit used as a comparison, the DNA sequencing kit of the present invention reduces the occurrence of pre-peak protrusions, reduces the signal intensity decay rate, improves the peak uniformity, increases the base calling accuracy, has better spectral resolution, and reduces noise interference.

[0225] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A fluorescently labeled ddNTP, characterized in that The fluorescently labeled ddNTP has a structure of Formula I or Formula II, In Formula I or Formula II, R1 and R2 are independently selected from hydrogen or alkyl; preferably, the alkyl group optionally forms a ring structure with the benzene ring connected to the N atom; and B is selected from any one of the four bases A, G, C, and U.

2. The fluorescent-labeled ddNTP according to claim 1, characterized in that The alkyl group is a C1-C10 alkyl group, preferably a C1-C6 alkyl group, and more preferably a C1-C3 alkyl group; preferably, the fluorescently labeled ddNTP includes any one or more of a fluorescently labeled ddATP having a BigDye-A structure, a fluorescently labeled ddGTP having a BigDye-G structure, a fluorescently labeled ddCTP having a BigDye-C structure, and a fluorescently labeled ddUTP having a BigDye-U structure, 3. A method for synthesizing fluorescently labeled ddNTPs, characterized in that: The synthesis method comprises the following steps: (1) reacting a dichlororhodamine acceptor dye with NHS to generate a dichlororhodamine acceptor dye connected with an activated ester structure; (2) reacting a rhodamine donor dye with N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester, and after the reaction, adding ammonia water to remove the trifluoroacetyl group to obtain a rhodamine donor dye connected with an NH2-Ph-CO- group; (3) subjecting the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) to a substitution reaction with the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2), so that the rhodamine donor dye and the dichlororhodamine acceptor dye are linked together through the -NH-Ph-CO- group; (4) allowing the dual dye product obtained in step (3) to undergo a condensation reaction with a ddNTP connected with a linker group; wherein the linker group is selected from any one of propylamino and propylethoxyamino.

4. The synthesis method according to claim 3, characterized in that The dichlororhodamine receptor dye is selected from a compound having a structure of formula ①; preferably, the dichlororhodamine receptor dye is selected from any one of 5dR6G, 5dR110, 6dROX, and 5dTAMRA; In formula ①, R1 and R2 are independently selected from hydrogen or alkyl; preferably, the alkyl optionally forms a ring structure with the benzene ring connected to the N atom; more preferably, the alkyl is a C1-C10 alkyl, preferably a C1-C6 alkyl, more preferably a C1-C3 alkyl; and / or, the rhodamine donor dye is selected from 5-carboxyfluorescein or 6-carboxyfluorescein; And / or, the ddNTP connected with a Linker group is selected from a compound having a structure of Formula ② or Formula ③; In formula ② or formula ③, the B is selected from any one of the four bases A, G, C, and U; preferably, in formula ②, the B is selected from any one of the three bases G, C, and U, and in formula ③, the B is the A base; and / or, in said step (1), the molar ratio of dichlororhodamine acceptor dye to NHS is 1:(1-2); And / or, in step (2), the molar ratio of the rhodamine donor dye to the N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester is 1:(1-1.5); And / or, the amount of ammonia water added in step (2) is such that the volume ratio of ammonia water to the reaction solution is 1:(8-12); And / or, in the step (3), the molar ratio of the dichlororhodamine acceptor dye connected with an activated ester structure obtained in step (1) to the rhodamine donor dye connected with an NH2-Ph-CO- group obtained in step (2) is 1:(1-1.5); And / or, in the step (4), the molar ratio of the double dye product obtained in step (3) to the ddNTP connected with a linker group is 1:(1-2).

5. The synthesis method according to claim 3 or 4, characterized in that: In the step (1), DCC or EDCI is used as a catalyst to catalyze the reaction of dichlororhodamine acceptor dye and NHS; preferably, the molar ratio of the catalyst to NHS is (0.8-1.2):1; And / or, the reaction of the rhodamine donor dye and in step (2) is carried out in TEA; preferably, the mass ratio of TEA to the rhodamine donor dye is (1-3):1; And / or, the substitution reaction in step (3) is carried out in DMF; preferably, the mass ratio of DMF to the rhodamine donor dye connected with NH2-Ph-CO- group is (25-35):1; And / or, the condensation reaction of step (4) is carried out in DIPEA; preferably, the mass ratio of DIPEA to the double dye product is (1.5-2.5):1; And / or, the step (4) further comprises activating the double dye product obtained in step (3) with HBTU before the condensation reaction occurs; preferably, the activation treatment conditions are: stirring at 25-45°C for 20-40 minutes under nitrogen protection, and / or, the molar ratio of HBTU to the double dye product is (1.5-2.5):

1.

6. The synthesis method according to any one of claims 3 to 5, characterized in that In the step (1), the reaction temperature of the dichlororhodamine receptor dye and NHS is 10 to 30° C., preferably 15 to 25° C., and the reaction time is 2 to 6 hours, preferably 3 to 5 hours; And / or, in step (2), the reaction temperature of the rhodamine donor dye and the N-trifluoroacetyl-4-aminomethylbenzoic acid activated ester is 10 to 30° C., preferably 15 to 25° C., and the reaction time is 3 to 8 hours, preferably 4 to 6 hours; And / or, the reaction temperature of the substitution reaction in step (3) is 15 to 35° C., preferably 20 to 30° C., and the reaction time is 12 to 20 hours, preferably 14 to 18 hours; And / or, the reaction temperature of the condensation reaction in step (4) is 25 to 45° C., preferably 30 to 40° C., and the reaction time is 3 to 9 hours, preferably 4 to 8 hours; And / or, the method further comprises separating and purifying the product obtained in step (4); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; And / or, the method further comprises separating and purifying the product obtained in step (3); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; And / or, the method further comprises separating and purifying the product obtained in step (2); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography; And / or, the method further comprises separating and purifying the product obtained in step (1); preferably, the separation and purification is carried out by reverse phase preparative high performance liquid chromatography.

7. Use of the fluorescently labeled ddNTP according to claim 1 or 2 or the fluorescently labeled ddNTP synthesized by the synthesis method according to any one of claims 3 to 6 in preparing terminators and / or kits for DNA sequencing.

8. A terminator for DNA sequencing, characterized in that The terminator comprises the fluorescently labeled ddNTP according to claim 1 or 2 or the fluorescently labeled ddNTP synthesized by the synthesis method of any one of claims 3 to 6; preferably, the fluorescently labeled ddNTP comprises fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, and fluorescently labeled ddUTP, and the four fluorescent markers are labeled with 4 different dyes; more preferably, the fluorescently labeled ddATP has a structure of BigDye-A, the fluorescently labeled ddGTP has a structure of BigDye-G, the fluorescently labeled ddCTP has a structure of BigDye-C, and the fluorescently labeled ddUTP has a structure of BigDye-U; and / or, the mass ratio of the fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, and fluorescently labeled ddUTP is (0.09 to 0.13): (0.03 to 0.07): (0.54 to 0.58): (0.60 to 0.64).

9. A DNA sequencing kit, characterized in that: The kit comprises the fluorescently labeled ddNTP according to claim 1 or 2, or the fluorescently labeled ddNTP synthesized by the synthesis method according to any one of claims 3 to 6, or the terminator for DNA sequencing according to claim 8; preferably, the kit also comprises a DNA polymerase and a sequencing buffer.

10. The method for preparing the DNA sequencing kit according to claim 9, characterized in that: The preparation method comprises the following steps: 1) Determine the concentration of the fluorescently labeled ddNTP in the DNA sequencing kit; preferably, according to the ABIPRISMTM sequencing manual, use DNA polymerase and fluorescently labeled ddATP, fluorescently labeled ddGTP, fluorescently labeled ddCTP, fluorescently labeled ddUTP to perform termination cycle sequencing, and determine the concentration of the fluorescently labeled ddNTP used in the reaction by titrating each fluorescently labeled ddNTP in the single-color termination reaction and selecting the concentration that maximizes the 700th nucleotide signal; 2) According to the concentration determined in step 1), a reagent containing a specific concentration of fluorescently labeled ddNTP is prepared.

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