A method for analyzing the sequence of a target polynucleotide

Abstract

The present invention relates to a method for analyzing the sequence of a target polynucleotide by multiple polymerization of a mixture of nucleotides and a polymerase to achieve efficient and complete polymerization, thereby meeting the requirements of sequencing, improving the read length and accuracy of sequencing. Further, the present invention also relates to a kit which can be used for analyzing or sequencing a polynucleotide.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for analyzing the sequence of a target polynucleotide by multiple polymerization of a mixture of nucleotides to achieve efficient and complete polymerization to meet the needs of sequencing. Further, the present application also relates to a kit which can be used for analyzing or sequencing a polynucleotide. BACKGROUND

[0003] High-throughput sequencing, in which sequencing by synthesis (SBS) is the main commercial mainstream, mainly uses DNA polymerase and nucleotides with reversible termination and fluorescent labeling to identify DNA sequences. It can simultaneously determine the sequences of hundreds of thousands to millions of DNA molecules at a time, and has the advantages of high throughput, fast detection speed, flexibility, and low cost.

[0004] Currently, the specific process of sequencing by synthesis is to generate a large amount of DNA template by amplification or rolling circle replication, and then anchor specific sequencing primers, and add DNA polymerase and fluorescently labeled nucleotides to the reaction system at the same time; or add DNA polymerase and a mixture of fluorescently labeled nucleotides and nucleotides without fluorescent modification to the reaction system at the same time. The 3'-OH of these dNTPs is protected, so only one dNTP can be added each time. Each time a dNTP is added, the replication reaction of the DNA strand stops, followed by excitation of the fluorescent signal and collection of the signal, and then adding a chemical reagent to quench the fluorescent signal and remove the dNTP 3'-OH protecting group, so that the next round of sequencing reaction can be carried out.

[0005] However, this sequencing technology requires particularly high synthesis efficiency. In multiple copies, once incomplete synthesis occurs, it will cause signal disorder, thereby affecting the accuracy and read length of sequencing. This is also one of the key technical problems currently faced by second-generation sequencing. As people require faster sequencing time and greater throughput, the reaction efficiency of the polymerase is limited, especially the ability to polymerize nucleotides with fluorescent groups, which will affect the quality of sequencing. Although the prior art also mentions using a mixture of fluorescently labeled and non-fluorescently modified nucleotides in the reaction system to improve polymerization efficiency, the effect is still limited. Once the polymerization efficiency cannot keep up with the sequencing speed, incomplete polymerization occurs in multiple copies, which will cause signal disorder in sequencing, thereby limiting the read length and accuracy of sequencing.

[0006] Therefore, it is necessary to provide a method for sequencing a polynucleotide in order to improve the read length and accuracy of sequencing. SUMMARY

[0007] The present application increases the number of polymerization reactions and adjusts the nucleotide components involved in the polymerization reaction, so that the polymerization rate can meet the needs of sequencing, improve the read length and quality of sequencing, thereby completing the present application.

[0008] Thus, in a first aspect, the present application provides a method for analyzing the sequence of a target polynucleotide, comprising

[0009] (a) providing a target polynucleotide,

[0010] (b) contacting the target polynucleotide with a primer under conditions that allow hybridization or annealing, thereby forming a partial duplex comprising the target polynucleotide and the primer serving as a growing strand,

[0011] (c) contacting the partial duplex with a polymerase and a first nucleotide mixture under conditions that allow the polymerase to perform a nucleotide polymerization reaction, such that the growing strand is extended, wherein the first nucleotide mixture comprises at least one labeled nucleotide;

[0012] Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide; for example, comprising a nucleotide selected from the group consisting of: an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, or any combination thereof;

[0013] wherein each nucleotide in the first nucleotide mixture comprises, at its ribose or deoxyribose moiety, a protecting group capable of reversibly blocking nucleic acid chain extension (e.g., a protecting group attached through a 2' or 3' oxygen atom);

[0014] (d) contacting the product of the previous step with a polymerase and a second nucleotide mixture under conditions that allow the polymerase to perform a nucleotide polymerization reaction, such that the growing strand is extended, wherein the second nucleotide mixture comprises at least one (e.g., one, two, three, or four) (1) unlabeled nucleotide, or (2) irreversibly blocked nucleotide, or (3) a combination of the unlabeled nucleotide and irreversibly blocked nucleotide;

[0015] In certain embodiments, the second nucleotide mixture comprises at least one unlabeled nucleotide; for example, comprising a nucleotide selected from the group consisting of: an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, or any combination thereof.

[0016] wherein each unlabeled nucleotide in the second nucleotide mixture comprises, at its ribose or deoxyribose moiety, a protecting group capable of reversibly blocking nucleic acid chain extension (e.g., a protecting group attached through a 2' or 3' oxygen atom);

[0017] (e) if at least one nucleotide in the second nucleotide mixture comprises an irreversible blocking nucleotide, then this step is not performed;

[0018] if the second nucleotide mixture does not comprise an irreversible blocking nucleotide, then the product of the previous step is contacted with a polymerase and a third nucleotide mixture under conditions that allow the polymerase to perform a nucleotide polymerization reaction, such that the growing strand is extended; the third nucleotide mixture comprises at least one irreversible blocking nucleotide;

[0019] (f) detecting the presence of the label in the product of the previous step,

[0020] (g) removing the protecting group and the label contained in the product of the previous step,

[0021] (h) optionally repeating steps (c) - (g) one or more times,

[0022] thereby obtaining sequence information for the target polynucleotide.

[0023] A method for determining the sequence of a target polynucleotide can be performed by denaturing the target polynucleotide, contacting the target polynucleotide with different nucleotides, respectively, so as to form the complement of the target nucleotide, and detecting the incorporation of the nucleotide. The method utilizes polymerization, such that a polymerase extends the growing strand by incorporating the correct nucleotide complementary to the target.

[0024] For each round of polymerization, the incorporation of the nucleotide is performed by a polymerase. There are many different polymerases, and the most suitable polymerase is readily determined by one of ordinary skill in the art. Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase, or vent polymerase. Polymerases engineered to have specific properties by engineering methods can also be used.

[0025] The conditions for performing the polymerization are well known to those of skill in the art. In order to perform the polymerase reaction, it is typically first necessary to anneal a primer to the target polynucleotide, the primer being recognized by the polymerase and serving as the initiation site for subsequent extension of the growing strand. The primer can be added as a separate component relative to the target polynucleotide. Other conditions necessary for performing the polymerase reaction, including temperature, pH, buffer composition, are well known to those of skill in the art.

[0026] In certain embodiments, a first nucleotide mixture of the application is contacted with the primer and polymerase to enable a first round of polymerization. The nucleotides can be added sequentially, i.e., each type of nucleotide (A, C, G, or T / U) is added separately, or simultaneously. Depending on the rate of sequencing, a second nucleotide mixture of the application is contacted with the polymerase again to enable a second round of polymerization. The nucleotides can be added sequentially, i.e., each type of nucleotide (A, C, G, or T / U) is added separately, or simultaneously. Finally, depending on whether the second nucleotide mixture contains dideoxynucleotides or not, a third round of polymerization is selected not to be performed or to be performed. In certain embodiments, the second nucleotide mixture does not contain dideoxynucleotides, and a third nucleotide mixture of the application is contacted with the polymerase to enable a third round of polymerization. The nucleotides can be added sequentially, i.e., each type of nucleotide (A, C, G, or T / U) is added separately, or simultaneously.

[0027] In certain embodiments, unincorporated nucleotides are removed. For example, unincorporated nucleotides are removed by performing a wash step.

[0028] In certain embodiments, the label is detected. Detection can be performed by conventional methods, and the means of detecting the fluorescent label or signal are well known in the art. For example, detection can be achieved by means of a device that detects the wavelength of fluorescence. Such devices are well known in the art. For example, such a device can be a confocal scanning microscope that scans the surface of a solid support with a laser to image the fluorophores bound directly to the nucleic acid molecule being sequenced. Alternatively, each signal generated can be observed, for example, with a sensitive 2-D detector, such as a charge-coupled detector (CCD). Other techniques, such as scanning near-field optical microscopy (SNOM), can also be used.

[0029] In certain embodiments, after detection, the label can be removed with appropriate conditions.

[0030] The use of the labeled nucleotides of the application is not limited to DNA sequencing technology, but other formats including polynucleotide synthesis, DNA hybridization assays, and single nucleotide polymorphism studies can also be performed using the nucleotides of the application. Any technique involving the interaction between a nucleotide and an enzyme can utilize the molecules of the application. For example, the molecules can be used as substrates for reverse transcriptase or terminal transferase.

[0031] In certain embodiments, in step (c), the extension is template-directed extension of the target polynucleotide. In certain embodiments, the extension is extension by one nucleotide.

[0032] In certain embodiments, the first mixture of nucleotides comprises first nucleotides labeled with a first label, second nucleotides labeled with a second label, third nucleotides labeled with a third label, and fourth nucleotides labeled with a fourth label, or fourth nucleotides that are unlabeled, or first nucleotides labeled with a first label, second nucleotides labeled with a second label, third nucleotides labeled with both a first label and a second label, and fourth nucleotides that are unlabeled. Optionally, the first mixture of nucleotides further comprises at least one nucleotide that is unlabeled.

[0033] In certain embodiments, the first mixture of nucleotides comprises first nucleotides labeled with a first label, second nucleotides labeled with a second label, third nucleotides labeled with a third label, fourth nucleotides labeled with a fourth label, first nucleotides that are unlabeled, second nucleotides that are unlabeled, third nucleotides that are unlabeled, and fourth nucleotides that are unlabeled, or first nucleotides labeled with a first label, second nucleotides labeled with a second label, third nucleotides labeled with both a first label and a second label, fourth nucleotides that are unlabeled, first nucleotides that are unlabeled, second nucleotides that are unlabeled, and third nucleotides that are unlabeled.

[0034] In certain embodiments, the ratio of first nucleotides labeled with a first label to first nucleotides that are unlabeled is 20: 1 to 1 : 10 (e.g., 10: 1 to 1 : 10, 5: 1 to 1 : 5, 3: 1 to 1 : 3). In certain embodiments, the ratio of first nucleotides labeled with a first label to first nucleotides that are unlabeled is 20: 1, 1 : 10, 1 : 1, or 3: 2. In certain embodiments, the ratio of first nucleotides labeled with a first label to first nucleotides that are unlabeled is 3: 2.

[0035] In certain embodiments, the ratio of second nucleotides labeled with a second label to second nucleotides that are unlabeled is 20: 1 to 1 : 10 (e.g., 10: 1 to 1 : 10, 5: 1 to 1 : 5, 3: 1 to 1 : 3). In certain embodiments, the ratio of second nucleotides labeled with a second label to second nucleotides that are unlabeled is 20: 1, 1 : 10, 1 : 1, or 3: 2. In certain embodiments, the ratio of second nucleotides labeled with a second label to second nucleotides that are unlabeled is 3: 2.

[0036] In certain embodiments, the ratio of third nucleotides labeled with the third label to third nucleotides not labeled is 20: 1 to 1 : 10 (e.g., 10: 1 to 1 : 10, 5: 1 to 1 : 5, 3: 1 to 1 : 3). In certain embodiments, the ratio of third nucleotides labeled with the third label to third nucleotides not labeled is 20: 1, 1 : 10, 1 : 1, or 3:2. In certain embodiments, the ratio of third nucleotides labeled with the third label to third nucleotides not labeled is 3:2.

[0037] In certain embodiments, the ratio of third nucleotides labeled with the first and second labels to third nucleotides not labeled is 20: 1 to 1 : 10 (e.g., 10: 1 to 1 : 10, 5: 1 to 1 : 5, 3: 1 to 1 : 3). In certain embodiments, the ratio of third nucleotides labeled with the first and second labels to third nucleotides not labeled is 20: 1, 1 : 10, 1 : 1, or 3:2. In certain embodiments, the ratio of third nucleotides labeled with the first and second labels to third nucleotides not labeled is 3:2.

[0038] In certain embodiments, the ratio of fourth nucleotides labeled with the fourth label to fourth nucleotides not labeled is 20: 1 to 1 : 10 (e.g., 10: 1 to 1 : 10, 5: 1 to 1 : 5, 3: 1 to 1 : 3). In certain embodiments, the ratio of fourth nucleotides labeled with the fourth label to fourth nucleotides not labeled is 20: 1, 1 : 10, 1 : 1, or 3:2. In certain embodiments, the ratio of fourth nucleotides labeled with the fourth label to fourth nucleotides not labeled is 3:2.

[0039] In certain embodiments, in step (d), the second mixture of nucleotides comprises:

[0040] (1) unlabeled first nucleotides, unlabeled second nucleotides, unlabeled third nucleotides, and unlabeled fourth nucleotides; or,

[0041] (2) first irreversible block nucleotides, second irreversible block nucleotides, third irreversible block nucleotides, fourth irreversible block nucleotides; or,

[0042] (3) comprises unlabeled first nucleotides, unlabeled second nucleotides, unlabeled third nucleotides, unlabeled fourth nucleotides, and first irreversible block nucleotides, second irreversible block nucleotides, third irreversible block nucleotides, fourth irreversible block nucleotides.

[0043] In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1 to 1:100, or it may be only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1.

[0044] In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1 to 1:100, or it is only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1.

[0045] In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1 to 1:100, or it may be only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1.

[0046] In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1 to 1:100, or it may be only an irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1.

[0047] In some implementations, the first marker, the second marker, the third marker, and the fourth marker may be independently the same or different.

[0048] In some implementations, the first marker, the second marker, the third marker, and the fourth marker are different.

[0049] In some embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).

[0050] In some embodiments, the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas red, and their derivatives.

[0051] In some embodiments, the target polynucleotide comprises DNA, RNA, or any combination thereof. In some embodiments, the extended product of the nucleic acid molecule is DNA.

[0052] In some embodiments, the target polynucleotide is obtained from samples derived from eukaryotes (e.g., animals, plants, fungi), prokaryotes (e.g., bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.

[0053] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from A, T, C, G, and U.

[0054] In some implementations, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are different.

[0055] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively. In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are each independently selected from A, T, C, G, and U.

[0056] In some implementations, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are all different.

[0057] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.

[0058] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0059] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide.

[0060] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or when the first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0061] In a second aspect, this application provides a kit comprising:

[0062] (a) A first nucleotide mixture, wherein the first nucleotide mixture comprises at least one nucleotide labeled with a marker;

[0063] Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide; for example, it comprises nucleotides selected from the following: unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, unlabeled fourth nucleotide, or any combination thereof;

[0064] Each nucleotide in the first nucleotide mixture contains a protecting group (e.g., a protecting group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose portion that can reversibly block the extension of the nucleic acid chain.

[0065] (b) A second nucleotide mixture comprising: at least one (e.g., one, two, three or four) (1) an unlabeled nucleotide, or (2) an irreversible blocking nucleotide, or (3) a combination of the unlabeled nucleotide and the irreversible blocking nucleotide;

[0066] In some embodiments, the second nucleotide mixture contains at least one unlabeled nucleotide; for example, it contains a nucleotide selected from the following: an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, or any combination thereof.

[0067] Each unlabeled nucleotide in the second nucleotide mixture contains a protecting group (e.g., a protecting group attached by a 2' or 3' oxygen atom) in its ribose or deoxyribose moiety that can reversibly block the extension of the nucleic acid chain.

[0068] (c) If the second nucleotide mixture does not contain an irreversible blocking nucleotide, then the kit contains a third nucleotide mixture containing at least one irreversible blocking nucleotide;

[0069] If at least one nucleotide in the second nucleotide mixture contains an irreversible blocking nucleotide, then the kit does not contain a third nucleotide mixture.

[0070] In some embodiments, the second nucleotide mixture contains at least one unlabeled nucleotide; for example, it contains a nucleotide selected from the following: an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, or any combination thereof.

[0071] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0072] In some embodiments, the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label, or an unlabeled fourth nucleotide; or, a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide. Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide.

[0073] In some embodiments, the first nucleotide mixture comprises a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide labeled with a third marker, a fourth nucleotide labeled with a fourth marker, an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or, a first nucleotide labeled with a first marker, a second nucleotide labeled with a second marker, a third nucleotide co-labeled with the first and second markers, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0074] In some embodiments, the ratio of the first nucleotide labeled with the first marker to the unlabeled first nucleotide is 20:1 to 1:10 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3). In some embodiments, the ratio of the first nucleotide labeled with the first marker to the unlabeled first nucleotide is 20:1, 1:10, 1:1, or 3:2. In some embodiments, the ratio of the first nucleotide labeled with the first marker to the unlabeled first nucleotide is 3:2.

[0075] In some embodiments, the ratio of the second nucleotide labeled with the second marker to the unlabeled second nucleotide is 20:1 to 1:10 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3). In some embodiments, the ratio of the second nucleotide labeled with the second marker to the unlabeled second nucleotide is 20:1, 1:10, 1:1, or 3:2. In some embodiments, the ratio of the second nucleotide labeled with the second marker to the unlabeled second nucleotide is 3:2.

[0076] In some embodiments, the ratio of the third nucleotide labeled with the third marker to the unlabeled third nucleotide is 20:1 to 1:10 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3). In some embodiments, the ratio of the third nucleotide labeled with the third marker to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In some embodiments, the ratio of the third nucleotide labeled with the third marker to the unlabeled third nucleotide is 3:2.

[0077] In some embodiments, the ratio of the third nucleotide co-labeled with the first and second markers to the unlabeled third nucleotide is 20:1 to 1:10 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3). In some embodiments, the ratio of the third nucleotide co-labeled with the first and second markers to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In some embodiments, the ratio of the third nucleotide co-labeled with the first and second markers to the unlabeled third nucleotide is 3:2.

[0078] In some embodiments, the ratio of the fourth nucleotide labeled with the fourth marker to the unlabeled fourth nucleotide is 20:1 to 1:10 (e.g., 10:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3). In some embodiments, the ratio of the fourth nucleotide labeled with the fourth marker to the unlabeled fourth nucleotide is 20:1, 1:10, 1:1, or 3:2. In some embodiments, the ratio of the fourth nucleotide labeled with the fourth marker to the unlabeled fourth nucleotide is 3:2.

[0079] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide.

[0080] In some embodiments, when the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or when the first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0081] In some embodiments, the second nucleotide mixture comprises:

[0082] (1) Unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, and unlabeled fourth nucleotide; or,

[0083] (2) The first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide; or,

[0084] (3) Unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, unlabeled fourth nucleotide, and first irreversible blocking nucleotide, second irreversible blocking nucleotide, third irreversible blocking nucleotide, and fourth irreversible blocking nucleotide.

[0085] In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1 to 1:100, or it may be only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1.

[0086] In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1 to 1:100, or it is only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1.

[0087] In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1 to 1:100, or it may be only the irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1.

[0088] In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1 to 1:100, or it may be only an irreversible blocking nucleotide. In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In some embodiments, the ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1.

[0089] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0090] In some embodiments, the kit further comprises one or more selected from: nucleic acid polymerase, reagents for amplification, reagents for sequencing, or any combination thereof.

[0091] In some embodiments, the reagents used for amplification include one or more selected from: primers that are wholly or partially complementary to the polynucleotide, DNB preparation buffer, working buffer of an enzyme (e.g., nucleic acid polymerase), water, and reagents containing ions (e.g., Mg²⁺). 2+ ( ) solutions, single-stranded DNA-binding proteins, or any combination thereof.

[0092] In some embodiments, the reagents for sequencing include one or more selected from: sequencing slides, reagents for removing protecting groups and markers from nucleotides, and reagents for detecting the luminescence signal of the markers (e.g., fluorescent mixtures).

[0093] In some embodiments, the nucleic acid polymerase is a DNA polymerase, such as a thermostable DNA polymerase. In some embodiments, the thermostable DNA polymerase is obtained from *Thermus aquaticus* (Taq), *Thermus thermophiles* (Tth), *Thermus filiformis*, *Thermus flavus*, *Thermococcus literalis*, *Thermus antranildanii*, *Thermus caldophllus*, *Thermus chliarophilus*, *Thermus flavus*, *Thermus igniterrae*, *Thermus lacteus*, *Thermus oshimai*, *Thermus ruber*, *Thermus rubens*, *Thermus scotoductus*, *Thermus silvanus*, *Thermus thermophllus*, *Thermotoga maritima*, *Thermotoga neapolitana*, *Thermosipho africanus*, *Thermococcus litoralis*, *Thermococcus barossi*, *Thermococcus gorgonarius*, *Thermotoga maritima*, *Thermotoga... neapolitana, Thermosiphoafricanus, Pyrococcus woesei, Pyrococcushorikoshii, Pyrococcus abyssi, Pyrodictium occultum, Aquifexpyrophilus and Aquifexaeolieus.

[0094] In some implementations, the kit is used for the analysis of polynucleotides.

[0095] In some implementations, the kit is used for sequencing polynucleotides.

[0096] In some implementations, the presence of the marker is detected by emitting a light signal.

[0097] In some implementations, the presence of the marker is detected by one or more (e.g., two, three, four) luminescent signals.

[0098] In some implementations, the first marker, the second marker, the third marker, and the fourth marker may be independently the same or different.

[0099] In some implementations, the first marker, the second marker, the third marker, and the fourth marker are different.

[0100] In some embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).

[0101] In some embodiments, the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, EF700, Texas Red, and their derivatives.

[0102] In some embodiments, the target polynucleotide comprises DNA, RNA, or any combination thereof. In some embodiments, the extended product of the nucleic acid molecule is DNA.

[0103] In some embodiments, the target polynucleotide is obtained from samples derived from eukaryotes (e.g., animals, plants, fungi), prokaryotes (e.g., bacteria, actinomycetes), viruses, bacteriophages, or any combination thereof.

[0104] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from A, T, C, G, and U.

[0105] In some implementations, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are different.

[0106] In some embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively. In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are each independently selected from A, T, C, G, and U.

[0107] In some implementations, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are all different.

[0108] In some embodiments, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.

[0109] In some embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0110] Definitions of terms

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All patents, applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If any definition presented herein conflicts with or is inconsistent with the definitions set forth in the patents, applications, and other publications incorporated herein by reference, the definitions set forth herein shall prevail.

[0112] As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogues thereof. Polynucleotides can be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. Polynucleotide molecules can be derived from double-stranded DNA (dsDNA) (e.g., genomic DNA, PCR and amplification products, etc.), or from single-stranded DNA (ssDNA) or RNA and can be converted to dsDNA, and vice versa. The exact sequence of a polynucleotide molecule can be known or unknown. Exemplary examples of polynucleotides include: genes or gene fragments (e.g., probes, primers, EST, or SAGE tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the above sequences.

[0113] Polynucleotides can include nucleotides or nucleotide analogues. Nucleotides typically contain a sugar (such as ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides can be baseless (i.e., lacking a base). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), and deoxyadenosine monophosphate (dAMP). Deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP). Nucleotide analogs containing modified bases may also be used in the methods described herein. Exemplary modified bases that can be included in polynucleotides, whether having a natural backbone or a similar structure, include, for example, inosine, xathanine, hypoxanthine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5 -Propynouracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymidine, 5-uracil, 4-thionuracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thionated adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazoguanine, 7-deazoadenine, 3-deazoguanine, 3-deazoadenine, etc. As is known in the art, certain nucleotide analogs cannot be introduced into polynucleotides, for example, nucleotide analogs such as adenosine 5'-phosphorylsulfate.

[0114] Generally, nucleotides include nucleotides A, C, G, T, or U. As used herein, the term "nucleotide A" refers to a nucleotide containing adenine (A) or its modifications or analogues, such as ATP or dATP. "Nucleotide G" refers to a nucleotide containing guanine (G) or its modifications or analogues, such as GTP or dGTP. "Nucleotide C" refers to a nucleotide containing cytosine (C) or its modifications or analogues, such as CTP or dCTP. "Nucleotide T" refers to a nucleotide containing thymine (T) or its modifications or analogues, such as TTP or dTTP. "Nucleotide U" refers to a nucleotide containing uracil (U) or its modifications or analogues, such as UTP or dUTP. As used herein, the term "dideoxynucleotide" refers to a nucleotide deoxyribose at the 2′ and 3′ carbons of the ribose, also known as 2′,3′-dideoxynucleotide. Generally, dideoxynucleotides include dideoxynucleotides A, C, G, T, or U. The term "dideoxynucleotide A" refers to a dideoxynucleotide containing adenine (A) or an analogue, such as ddATP. "Dideoxynucleotide G" refers to a dideoxynucleotide containing guanine (G) or an analogue, such as ddGTP. "Dideoxynucleotide C" refers to a dideoxynucleotide containing cytosine (C) or an analogue, such as ddCTP. "Dideoxynucleotide T" refers to a dideoxynucleotide containing thymine (T) or an analogue, such as ddTP. "Dideoxynucleotide U" refers to a dideoxynucleotide containing uracil (U) or an analogue, such as ddUTP. As used in this article, ddNTP is one of dideoxyadenosine triphosphate (ddATP), dideoxyguanosine triphosphate (ddGTP), dideoxycytidine triphosphate (ddCTP), dideoxyuridine triphosphate (ddUTP), dideoxythymidine triphosphate (ddTTP), or a combination of two or more of them, wherein dideoxyuridine triphosphate and dideoxythymidine triphosphate do not occur simultaneously.

[0115] As used in this article, the term "marker" refers to a group that can emit a light-emitting signal under certain conditions.

[0116] As used herein, the term "luminescent label" refers to any substance that, when excited by a suitable excitation wavelength, emits fluorescence at a specific emission wavelength. Such luminescent labels can be chemiluminescent labels, for example, selected from biochemiluminescent labels that elicit different luminescence kinetics and any combination thereof, such as luciferases that elicit different luminescence kinetics and any combination thereof; such luminescent labels can be, for example, fluorophores, such as those selected from coumarin, AlexaFluor, Bodipy, luciferin, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas Red, and their derivatives.

[0117] As used herein, the term "protecting group" refers to a group that prevents polymerase (which incorporates a nucleotide containing that group into the polynucleotide chain being synthesized) from catalyzing the incorporation of another nucleotide after the incorporation of the nucleotide containing that group into the polynucleotide chain being synthesized. Such a protecting group is also referred to herein as a 3'-OH protecting group. A nucleotide containing such a protecting group is also referred to herein as a 3'-blocking nucleotide. The protecting group can be any suitable group that can be added to a nucleotide, as long as the protecting group prevents the incorporation of additional nucleotide molecules into the polynucleotide chain and is readily removable from the sugar moiety of the nucleotide without damaging the polynucleotide chain. Furthermore, the nucleotide modified with the protecting group needs to be resistant to polymerase or other suitable enzymes for incorporating the modified nucleotide into the polynucleotide chain. Therefore, an ideal protecting group exhibits long-term stability, can be efficiently incorporated by polymerase, prevents secondary or further incorporation of nucleotides, and can be removed under mild conditions, preferably aqueous conditions, without damaging the polynucleotide structure.

[0118] The prior art has described a variety of protecting groups that conform to the above descriptions. For example, WO 91 / 06678 discloses 3′-OH protecting groups including esters and ethers, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrophenyl carbonate, 2,4-sulfenyldinitro, and tetrahydrofuran ether. Metzker et al. (Nucleic Acids Research, 22(20): 4259-4267, 1994) disclosed the synthesis and application of eight 3′-modified 2-deoxyribonucleoside 5′-triphosphates (3′-modified dNTPs). WO2002 / 029003 describes the use of allyl protecting groups to cap 3′-OH groups on DNA growth chains in polymerase reactions. Preferably, various protecting groups reported in international applications WO2014139596 and WO2004 / 018497 can be used, including, for example, those in WO2014139596. Figure 1The protecting groups exemplified in A and the 3' hydroxyl protecting groups (i.e., protecting groups) defined in the claims, and, for example, those in WO2004 / 018497 Figure 3 and 4 The protecting groups exemplified in the examples and those defined in the claims. All of the above references are incorporated herein by reference in their entirety.

[0119] As used herein, the term "reversible blocking group" refers to a group that, when incorporated into a synthesizing polynucleotide chain, prevents the polymerase from proceeding to the next round of polymerization, thus terminating the polymerization reaction. In this case, only one base is incorporated into the growing nucleic acid chain in each round of polymerization. Furthermore, this group can be removed, allowing the growing nucleic acid chain to proceed to the next round of polymerization, introducing another base. Examples of reversible blocking groups include H in 3′-OH replaced by the following groups: ester, ether, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrophenyl carbonate, 2,4-sulfenyldinitro and tetrahydrofuran ether -CH2-CH=CH2, SS, or blocking the base with a large sterically hindered group and a fluorescent group, with fluorescence linked by SS.

[0120] As used herein, the term "irreversible blocking nucleotide" refers to a nucleotide that, once incorporated into a synthesizing polynucleotide chain, prevents subsequent nucleotides from being incorporated into the chain due to its blocking effect. This blocking effect is irreversible. Irreversible blocking nucleotides typically contain dideoxynucleotides, or nucleotides whose 3'-OH is replaced by groups such as methoxy (3'-OMe, i.e., 3'-OCH3), azide (3'-N3), and ethoxy (3'-OEt, i.e., 3'-OCH2CH3).

[0121] Advantages of the invention

[0122] In sequencing, this invention achieves a more efficient polymerization reaction by increasing the number of polymerization reactions, ensuring that the polymerization rate meets sequencing requirements and improving read length and quality. Furthermore, this invention achieves even higher efficiency by shortening the first round of polymerization and rapidly performing the second or third rounds, while simultaneously adjusting the nucleotide components involved in the polymerization reaction (e.g., polymerizing unlabeled nucleotide components).

[0123] The method of this invention not only increases the efficiency of the polymerization reaction and ensures a complete polymerization reaction, thereby improving the read length and quality of sequencing, but also reduces signal interference caused by incomplete fluorescence excision or incomplete fluorescence elution, improving excision efficiency and reducing sequencing error rate. Attached Figure Description

[0124] Figure 1 The flowchart of the E. coli DNA synthesis and sequencing process in Experiment 1 is shown.

[0125] Figure 2 The flowchart of the E. coli DNA synthesis and sequencing process in Experiment 2 is shown.

[0126] Figure 3 The flowchart of the simultaneous synthesis and sequencing of E. coli DNA in Experiment Group 3 is shown.

[0127] Figure 4 The flowchart of the simultaneous synthesis and sequencing of E. coli DNA in Experiment Group 4 is shown.

[0128] Figure 5 The flowcharts for sequencing E. coli DNA during synthesis are shown in experimental groups 5, 6, and 7.

[0129] Figure 6 The flowcharts for sequencing E. coli DNA during synthesis are shown in experimental groups 8, 9, and 10.

[0130] Figure 7 The results show the Q30 (%) percentage for each cycle in the four experimental groups (groups 1-4). The horizontal axis represents the sequencing cycle number, and the vertical axis represents the Q30 (%) percentage for each cycle.

[0131] Figure 8 The results show the sequencing error rate (%) percentage for each cycle in the four experimental groups (groups 1-4). The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) percentage for each cycle.

[0132] Figure 9 The results show the Q30 (%) percentage for each cycle in experimental groups 2, 3, 5, 6, and 7. Using experimental group 2 as a control, the other experimental groups performed two polymerization rounds per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio. The second round used mixtures of nucleotide mixture 2 and dideoxynucleotide mixture in different ratios. The x-axis represents the sequencing cycle number, and the y-axis represents the Q30 (%) percentage for each cycle.

[0133] Figure 10The results show the sequencing error rate (%) percentage for each cycle in experimental groups 2, 3, 5, 6, and 7. Using experimental group 2 as a control, the other experimental groups performed two polymerization rounds per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio. The second round used mixtures of nucleotide mixture 2 and dideoxynucleotide mixture in different ratios. The x-axis represents the sequencing cycle number, and the y-axis represents the sequencing error rate (%) percentage for each cycle.

[0134] Figure 11 The results show the Q30 (%) percentage for each cycle in experimental groups 2, 4, 8, 9, and 10. Using experimental group 2 as a control, the other experimental groups performed three rounds of polymerization per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 with different concentrations; the second round used nucleotide mixture 2; and the third round used a dideoxynucleotide mixture. The x-axis represents the sequencing cycle number, and the y-axis represents the Q30 (%) percentage for each cycle.

[0135] Figure 12 The results show the sequencing error rate (%) percentage for each cycle in experimental groups 2, 4, 8, 9, and 10. Using experimental group 2 as a control, the other experimental groups performed three polymerization rounds per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 with different concentrations; the second round used nucleotide mixture 2; and the third round used a dideoxynucleotide mixture. The x-axis represents the sequencing cycle number, and the y-axis represents the sequencing error rate (%) percentage for each cycle. Example

[0136] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0137] Unless otherwise specified, the molecular biology experimental methods used in this invention are substantially the same as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples are described by way of illustration and are not intended to limit the scope of the invention.

[0138] 1. Key equipment used in this embodiment:

[0139] MGISEQ-2000RS sequencer, MGIDL-200H loader, MGISEQ-2000RS sequencing slides.

[0140] 2. The key reagents used in this embodiment are shown in Table 1 below:

[0141] Table 1. Reagents used

[0142] Reagent name Brand Catalogue number Cold dATP BGI 01CATP000 - 10 ml Cold dTTP BGI 01CTTP000 - 10 ml Cold dGTP BGI 01CGTP000 - 10 ml Cold dCTP BGI 01CCTP000 - 10 ml ddATP BGI 01DATP000 - 1 ml ddTTP BGI 01DTTP000 - 1 ml ddGTP BGI 01DGTP000 - 1 ml ddCTP BGI 01DCTP000 - 1 ml

[0143] Example 1

[0144] In this embodiment, various reagents are prepared in advance.

[0145] 1) Preparation of nucleotide mixture 1

[0146] As shown in Table 2, nucleotide mixture 1 contains both fluorescent dyes and reversible blocking groups, where A, T, G, and C are adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively. The fluorescent dyes attached to the nucleotides are dATP-Cy5, dGTP-Cy3, dTTP-ROX, and dCTP-EF700, all of which contain reversible blocking groups.

[0147] Table 2: Nucleotide Mixture 1

[0148] Reagent name Final concentration (nmol / L) dATP-1 200 dTTP-1 200 dGTP-1 200 dCTP-1 200

[0149] Among them, dATP-1 refers to an adenine nucleotide that has both reversible blocking group modification and Cy5 fluorescence modification, dTTP-1 refers to a thymine nucleotide that has both reversible blocking group modification and ROX fluorescence modification, dGTP-1 refers to a guanine nucleotide that has both reversible blocking group modification and Cy3 fluorescence modification, and dCTP-1 refers to a cytosine nucleotide that has both reversible blocking group modification and EF700 fluorescence modification.

[0150] 2) Preparation of nucleotide mixture 2

[0151] As shown in Table 3 below, nucleotide mixture 2 has only reversible blocking groups, where A, T, G, and C are adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively.

[0152] Table 3: Nucleotide Mixture 2

[0153] Reagent name Final concentration (nmol / L) Cold dATP 200 Cold dTTP 200 Cold dGTP 200 Cold dCTP 200

[0154] Among them, Cold dATP refers to adenine nucleotides modified only with reversible blocking groups, Cold dTTP refers to thymine nucleotides modified only with reversible blocking groups, Cold dGTP refers to guanine nucleotides modified only with reversible blocking groups, and Cold dCTP refers to cytosine nucleotides modified only with reversible blocking groups.

[0155] 3) Preparation of nucleotide mixture 3

[0156] As shown in Table 4 below, all nucleotide mixtures 3 are dideoxynucleotides, where ddATP refers to adenosine triphosphate dideoxynucleotide, ddTTP refers to thymine triphosphate dideoxynucleotide, ddGTP refers to guanine triphosphate dideoxynucleotide, and ddCTP refers to cytosine triphosphate dideoxynucleotide.

[0157] Table 4: Nucleotide Mixture 3

[0158] Reagent name Final concentration (nmol / L) ddATP 200 ddTTP 200 ddGTP 200 ddTTP 200

[0159] 4) Preparation of Synthetic Reagent 1

[0160] Prepare and mix the synthetic reagent 1 as shown in Table 5 below for later use. The nucleotide in synthetic reagent 1 is a nucleotide that is modified with both a fluorescent dye and a reversible blocking group.

[0161] Table 5: Synthetic Reagent 1

[0162] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 1 1.2 DNA polymerase 1.2

[0163] 5) Preparation of Synthetic Reagent 2

[0164] Prepare and mix the synthetic reagent 2 as shown in Table 6 below for later use. The nucleotides in this synthetic reagent 2 are nucleotides modified only with reversible blocking groups.

[0165] Table 6: Synthetic Reagent 2

[0166] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 2 1.2 DNA polymerase 1.2

[0167] 6) Preparation of synthetic reagents 3-1, 3-2, 3-3 and 3-4. These nucleotide mixtures are prepared by mixing nucleotide mixture 1 and nucleotide mixture 2 with different concentration ratios. Refer to Table 7-10 for the specific concentration ratios.

[0168] Prepare the synthetic reagent 3-1 as shown in Table 7 below and mix well for later use.

[0169] Table 7: Synthetic Reagents 3-1

[0170] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 1 0.6 Nucleotide mix 2 0.6 DNA polymerase 1.2

[0171] 7) Preparation of synthetic reagent 3-2

[0172] Prepare the synthetic reagent 3-2 as shown in Table 8 below and mix well for later use.

[0173] Table 8: Synthetic Reagents 3-2

[0174] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 1 0.72 Nucleotide mix 2 0.48 DNA polymerase 1.2

[0175] 8) Preparation of synthetic reagent 3-3

[0176] Prepare the synthetic reagent 3-3 as shown in Table 9 below and mix well for later use.

[0177] Table 9: Synthetic Reagents 3-3

[0178]

[0179]

[0180] 9) Preparation of synthetic reagents 3-4

[0181] Prepare and mix the synthetic reagents 3-4 as shown in Table 10 below for later use.

[0182] Table 10: Synthetic Reagents 3-4

[0183] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 1 1.14 Nucleotide mix 2 0.06 DNA polymerase 1.2

[0184] 10) Preparation of Synthetic Reagent 4

[0185] Prepare and mix the synthetic reagent 4 as shown in Table 11 below for later use.

[0186] Table 11: Synthetic Reagent 4

[0187] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 3 1.2 DNA polymerase 1.2

[0188] 11) Preparation of synthetic reagents 5-1, 5-2 and 5-3: These nucleotide mixtures were prepared by mixing nucleotide mixture 2 and nucleotide mixture 3 with different concentration ratios. The specific concentration ratios are shown in Table 12-14.

[0189] Prepare the synthetic reagent 5-1 as shown in Table 12 below and mix well for later use.

[0190] Table 12: Synthetic Reagents 5-1

[0191]

[0192]

[0193] 12) Preparation of synthetic reagent 5-2

[0194] Prepare the synthetic reagent 5-2 as shown in Table 13 below and mix well for later use.

[0195] Table 13: Synthetic Reagents 5-2

[0196] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 2 12 Nucleotide mix 3 0.12 DNA polymerase 1.2

[0197] 13) Preparation of synthetic reagent 5-3

[0198] Prepare the synthetic reagent 5-3 as shown in Table 14 below and mix well for later use.

[0199] Table 14: Synthetic Reagents 5-3

[0200] Reagent name Amount (ml) Reaction buffer 57.6 Nucleotide mix 2 12 Nucleotide mix 3 0.24 DNA polymerase 1.2

[0201] Example 2

[0202] All experiments below used E. coli single-stranded circular DNA (i.e., MGI standard library reagent V3.0) as a template and used the MGISEQ-2000RS high-throughput sequencing kit (MGI) to prepare DNA nanospheres and load them onto the chip for subsequent sequencing.

[0203] Experimental Group 1: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagent in well #1 of the kit was removed and replaced with the prepared synthesis reagent 1, i.e., the reaction solution of a nucleotide mixture containing both fluorescent and reversibly blocking modification groups. Only one polymerization round was performed per cycle, and the polymerized nucleotides were a mixture of nucleotides modified with both fluorescent and reversibly blocking modification groups. The sequencing was performed using the MGISEQ-2000RS sequencing platform according to... Figure 1 The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0204] Experimental Group 2: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagent in well #1 was removed and replaced with the synthesis reagent 3-2 from the test group; that is, the reaction solution containing a nucleotide mixture 1 with both fluorescent and reversibly blocking modified groups prepared in a 3:2 ratio, and a nucleotide mixture 2 containing purely reversibly blocking nucleotides mixed in this nucleotide mixture. Each reaction cycle consisted of only one polymerization round, and the polymerized nucleotides were a mixture of nucleotide mixture 1 and nucleotide mixture 2 prepared in a 3:2 ratio. The sequencing was performed using the MGISEQ-2000RS sequencing platform according to... Figure 2 The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0205] Experimental Group 3: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 were removed. The reagent in well #1 was replaced with synthetic reagent 3-2 from the test group; the reagent in well #2 was replaced with synthetic reagent 4 from the test group. Polymerization was performed in two cycles. The first cycle used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio. The second cycle used a dideoxynucleotide mixture. Sequencing was performed using the MGISEQ-2000RS platform according to… Figure 3 The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0206] Experimental Group 4: Using the MGISEQ-2000RS high-throughput sequencing kit, remove reagents from wells #1, #2, and #17. Replace reagent #1 with synthetic reagent 3-2 from the test group; replace reagent #2 with synthetic reagent 2 from the test group; and replace reagent #17 with synthetic reagent 4 from the test group. Sequencing is then performed using the MGISEQ-2000RS sequencing platform according to... Figure 4 The experimental procedure for SE100 sequencing was performed, with three rounds of polymerization per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio; the second round used nucleotide mixture 2; and the third round used dideoxynucleotide mixture. In short, the MGISEQ-2000RS high-throughput sequencing kit was followed according to the instructions: elution with elution reagents to free nucleotides, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0207] Experimental Group 5: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 were removed. Well #1 was replaced with the synthetic reagent 3-2 from the test group; well #2 was replaced with the synthetic reagent 5-1 from the test group. Polymerization was performed in two cycles. In the first cycle, the polymerized nucleotides were a mixture of nucleotide mixture 1 and nucleotide mixture 2 prepared at a 3:2 ratio. In the second cycle, the polymerized nucleotides were a mixture of nucleotide mixture 2 and dideoxynucleotide mixture prepared at a 1:100 ratio. The sequencing was performed using the MGISEQ-2000RS sequencing platform according to… Figure 5The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0208] Experimental Group Six: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 were removed. Well #1 was replaced with synthetic reagent 3-2 from the test group; well #2 was replaced with synthetic reagent 5-2 from the test group. Polymerization was performed in two cycles. In the first cycle, the polymerized nucleotides were a mixture of nucleotide mixture 1 and nucleotide mixture 2 prepared at a 3:2 ratio. In the second cycle, the polymerized nucleotides were a mixture of nucleotide mixture 2 and dideoxynucleotide mixture prepared at a 100:1 ratio. The sequencing was performed using the MGISEQ-2000RS sequencing platform according to… Figure 5 The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0209] Experimental Group 7: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 were removed. The reagent in well #1 was replaced with synthetic reagent 3-2 from the test group; the reagent in well #2 was replaced with synthetic reagent 5-3 from the test group. Polymerization was performed in two cycles. In the first cycle, the polymerized nucleotides were a mixture of nucleotide mixture 1 and nucleotide mixture 2 prepared at a 3:2 ratio. In the second cycle, the polymerized nucleotides were a mixture of nucleotide mixture 2 and dideoxynucleotide mixture prepared at a 50:1 ratio. The sequencing was performed using the MGISEQ-2000RS sequencing platform according to… Figure 5 The experimental procedure for SE100 sequencing was performed following the instructions of the MGISEQ-2000RS high-throughput sequencing kit: elution of free nucleotides with elution reagents, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0210] Experimental Group 8: Using the MGISEQ-2000RS high-throughput sequencing kit, remove reagents from wells #1, #2, and #17. Replace reagent #1 with synthetic reagent 3-1 from the test group; replace reagent #2 with synthetic reagent 2 from the test group; and replace reagent #17 with synthetic reagent 4 from the test group. Sequencing is then performed using the MGISEQ-2000RS sequencing platform according to... Figure 6 The experimental procedure for SE100 sequencing was performed, with three rounds of polymerization per cycle. The first round used a 1:1 mixture of nucleotide mixture 1 and nucleotide mixture 2; the second round used nucleotide mixture 2; and the third round used dideoxynucleotide mixture 2. In short, the MGISEQ-2000RS high-throughput sequencing kit was followed according to the instructions: elution with elution reagents to free nucleotides, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0211] Experimental Group Nine: Using the MGISEQ-2000RS high-throughput sequencing kit, remove reagents from wells #1, #2, and #17. Replace reagent #1 with synthetic reagent 3-3 from the test group; replace reagent #2 with synthetic reagent 2 from the test group; and replace reagent #17 with synthetic reagent 4 from the test group. Sequencing is then performed using the MGISEQ-2000RS platform according to... Figure 6 The experimental procedure for SE100 sequencing was performed, with three rounds of polymerization per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 prepared at a 1:10 ratio; the second round used nucleotide mixture 2; and the third round used dideoxynucleotide mixture. In short, the MGISEQ-2000RS high-throughput sequencing kit was followed according to the instructions: elution with elution reagents to free nucleotides, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0212] Experimental Group 10: Using the MGISEQ-2000RS high-throughput sequencing kit, remove reagents from wells #1, #2, and #17. Replace reagent #1 with synthetic reagents 3-4 from the test group; replace reagent #2 with synthetic reagent 2 from the test group; and replace reagent #17 with synthetic reagent 4 from the test group. Sequencing is then performed using the MGISEQ-2000RS platform according to... Figure 6The experimental procedure for SE100 sequencing was performed, with three rounds of polymerization per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 20:1 ratio; the second round used nucleotide mixture 2; and the third round used dideoxynucleotide mixture. In short, the MGISEQ-2000RS high-throughput sequencing kit was followed according to the instructions: elution with elution reagents to free nucleotides, signal acquisition with imaging reagents, removal of protecting groups with excision reagents, and washing with elution reagents. The decrease in Q30 for each cycle and the sequencing error rate curve for each cycle were then analyzed to evaluate sequencing quality.

[0213] Result: as follows Figure 7 - Figure 12 As shown, where Figure 7 The results show the Q30 (%) percentage for each cycle in the four experimental groups (groups 1-4). The horizontal axis represents the sequencing cycle number, and the vertical axis represents the Q30 (%) percentage for each cycle.

[0214] Figure 8 The results show the sequencing error rate (%) percentage for each cycle in the four experimental groups (groups 1-4). The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) percentage for each cycle.

[0215] Figure 9 The results of five experimental groups (groups 2, 3, 5 to 7) are shown, with group 2 as the control. The other experiments involved two rounds of polymerization per cycle. The first round of polymerization used a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio. The second round used mixtures of nucleotide mixture 2 and dideoxynucleotide mixture in different ratios. The horizontal axis represents the sequencing cycle number, and the vertical axis represents the Q30 (%) percentage for each cycle.

[0216] Figure 10 The results of five experimental groups (groups 2, 3, 5 to 7) are shown, with group 2 as the control. The other experiments involved two rounds of polymerization per cycle. The nucleotides polymerized in the first round were a mixture of nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio. The second round was a mixture of nucleotide mixture 2 and dideoxynucleotide mixture in different ratios. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the percentage of sequencing error rate (%) in each cycle.

[0217] Figure 11The results of five experimental groups, namely experimental groups 2, 4, 8 to 10, are shown. Experiment 2 serves as the control. The other experiments involved three rounds of polymerization per cycle. The first round used a mixture of nucleotide mixture 1 and nucleotide mixture 2 with different concentrations. The second round used nucleotide mixture 2. The third round used dideoxynucleotide mixture. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) percentage of each cycle.

[0218] Figure 12 The results of five experimental groups, namely experimental groups 2, 4, 8 to 10, are shown. Experiment 2 serves as the control. The other experiments involved three rounds of polymerization per cycle. The first round used a mixture of nucleotides with different concentrations of nucleotide mixture 1 and nucleotide mixture 2. The second round used nucleotide mixture 2. The third round used dideoxynucleotide mixture. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the percentage of sequencing error rate (%) for each cycle.

[0219] A higher percentage of Q30 (%) in each cycle indicates better quality, and a smaller decrease in Q30 percentage also indicates better quality. A lower sequencing error rate (%) in each cycle indicates better sequencing quality.

[0220] The results show that, in summary Figure 7 and Figure 8 The results showed that experimental group four had the best sequencing quality and error rate. Experimental group three was the next best, and both results were significantly better than experimental groups two and one. This confirms that the polymerization method proposed in this application is superior to existing polymerization methods. Figure 9 and Figure 10 Experimental groups 3, 5 to 7 were significantly better than experimental group 2 (control), with experimental group 6 showing the best results (its Q30 decreased the least). This confirms that the two-round polymerization method of this application is superior to the single-round polymerization method.

[0221] comprehensive Figure 11 Figure 12 and 12 Experimental groups four, eight, nine, and ten were all better than experimental group two (the control group), with experimental group four being the best. This confirms that the three-round polymerization method of this application is superior to the single-round polymerization method.

Claims

1. A method for analyzing the sequence of a target polynucleotide, comprising: (a) Provide target polynucleotides, (b) Under conditions allowing hybridization or annealing, the target polynucleotide is contacted with a primer to form a partial double strand comprising the target polynucleotide and a primer used as the growth chain. (c) Under conditions allowing the polymerase to perform nucleotide polymerization, the partially duplexed polymerase is contacted with a mixture of polymerase and a first nucleotide to extend the growth chain, wherein... The first nucleotide mixture contains at least one nucleotide labeled with a marker; Each nucleotide in the first nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can reversibly block the elongation of the nucleic acid chain. (d) Under conditions that allow the polymerase to perform nucleotide polymerization, the product of the previous step is contacted with the polymerase and a mixture of the second nucleotides to extend the growth chain, wherein the second nucleotide mixture comprises: at least one (1) unlabeled nucleotide, or (2) irreversible blocking nucleotide, or (3) a combination of the unlabeled nucleotide and the irreversible blocking nucleotide. Each unlabeled nucleotide in the second nucleotide mixture contains a protective group in its ribose or deoxyribose moiety that can reversibly block the elongation of the nucleic acid chain; (e) If at least one nucleotide in the second nucleotide mixture contains an irreversible blocking nucleotide, this step is not performed; If the second nucleotide mixture does not contain an irreversible blocking nucleotide, then, under conditions that allow the polymerase to perform nucleotide polymerization, the product of the previous step is contacted with the polymerase and the third nucleotide mixture to extend the growth chain; the third nucleotide mixture contains at least one irreversible blocking nucleotide. (f) Detect the presence of the marker in the product of the previous step. (g) Remove protecting groups and markers contained in the product of the previous step. (h) Repeat steps (c)-(g) once or more. Thus, the sequence information of the target polynucleotide is obtained.

2. The method of claim 1, wherein, In step (c), the extension is a target polynucleotide extension.

3. The method of claim 1, wherein, The protecting group is a protecting group attached by a 2' or 3' oxygen atom.

4. The method of claim 1, wherein, The first nucleotide mixture also contains at least one unlabeled nucleotide.

5. The method of claim 1, wherein, The first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide; or The first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, a fourth nucleotide labeled with a fourth label, an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

6. The method of claim 5, wherein, The method has one or more features selected from the following: (1) The ratio of the first nucleotide labeled with the first marker to the unlabeled first nucleotide is 20:1 to 1:10; (2) The ratio of the second nucleotide labeled with the second marker to the unlabeled second nucleotide is 20:1 to 1:10; (3) The ratio of the third nucleotide labeled with the third marker to the unlabeled third nucleotide is 20:1 to 1:10; (4) The ratio of the third nucleotide co-labeled by the first and second markers to the unlabeled third nucleotide is 20:1 to 1:10; (5) The ratio of the fourth nucleotide labeled with the fourth marker to the unlabeled fourth nucleotide is 20:1 to 1:

10.

7. The method of claim 1, wherein, The second nucleotide mixture contains at least one unlabeled nucleotide.

8. The method of claim 1, wherein, In step (d), the second nucleotide mixture comprises: (1) Unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, and unlabeled fourth nucleotide; or, (2) The first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide; or, (3) It includes an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, and a first irreversible blocking nucleotide, a second irreversible blocking nucleotide, a third irreversible blocking nucleotide, and a fourth irreversible blocking nucleotide.

9. The method of claim 8, wherein, The method has one or more features selected from the following: (1) The ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1 to 1:100; (2) The ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1 to 1:100; (3) The ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1 to 1:100; (4) The ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1 to 1:

100.

10. The method of claim 5, wherein, The first marker, the second marker, the third marker, and the fourth marker are each independently identical or different.

11. The method of claim 10, wherein the first marker, the second marker, the third marker, and the fourth marker are fluorescent markers.

12. The method of claim 10, wherein the first marker, the second marker, the third marker, and the fourth marker are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, or Texas red.

13. The method of claim 1, wherein the target polynucleotide comprises DNA, RNA, or any combination thereof.

14. The method of claim 1, wherein the target polynucleotide is obtained from a sample derived from eukaryotes, prokaryotes, viruses, or any combination thereof.

15. The method according to any one of claims 5-9, wherein the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.

16. The method of claim 8, wherein the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.

17. The method of claim 1, wherein the irreversible blocking nucleotide is a dideoxynucleotide.

18. The method of claim 1, wherein the first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; and the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

19. A reagent kit comprising: (a) A first nucleotide mixture, wherein the first nucleotide mixture comprises at least one nucleotide labeled with a marker; Each nucleotide in the first nucleotide mixture contains a protective group in its ribose or deoxyribose portion that can reversibly block the elongation of the nucleic acid chain. (b) A second nucleotide mixture comprising: at least one (1) an unlabeled nucleotide, or (2) an irreversible blocking nucleotide, or (3) a combination of the unlabeled nucleotide and the irreversible blocking nucleotide; Each unlabeled nucleotide in the second nucleotide mixture contains a protective group in its ribose or deoxyribose moiety that can reversibly block the elongation of the nucleic acid chain; (c) If the second nucleotide mixture does not contain an irreversible blocking nucleotide, the kit contains a third nucleotide mixture containing at least one irreversible blocking nucleotide; If at least one nucleotide in the second nucleotide mixture contains an irreversible blocking nucleotide, then the kit does not contain a third nucleotide mixture.

20. The kit of claim 19, wherein the irreversible blocking nucleotide is a dideoxynucleotide.

21. The kit of claim 19, wherein, The first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label or an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide; or The first nucleotide mixture comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, a fourth nucleotide labeled with a fourth label, an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

22. The kit of claim 21, wherein, The kit has one or more of the following characteristics: (1) The ratio of the first nucleotide labeled with the first marker to the unlabeled first nucleotide is 20:1 to 1:10; (2) The ratio of the second nucleotide labeled with the second marker to the unlabeled second nucleotide is 20:1 to 1:10; (3) The ratio of the third nucleotide labeled with the third marker to the unlabeled third nucleotide is 20:1 to 1:10; (4) The ratio of the third nucleotide co-labeled by the first and second markers to the unlabeled third nucleotide is 20:1 to 1:10; (5) The ratio of the fourth nucleotide labeled with the fourth marker to the unlabeled fourth nucleotide is 20:1 to 1:

10.

23. The kit of claim 21, wherein, When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and a fourth nucleotide labeled with a fourth label; the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or When the first nucleotide mixture contains a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide labeled with a third label, and an unlabeled fourth nucleotide, or a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with the first and second labels, and an unlabeled fourth nucleotide, the second nucleotide mixture contains an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

24. The kit of claim 19, wherein: The second nucleotide mixture comprises: (1) Unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, and unlabeled fourth nucleotide; or, (2) The first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide; or, (3) Unlabeled first nucleotide, unlabeled second nucleotide, unlabeled third nucleotide, unlabeled fourth nucleotide, and first irreversible blocking nucleotide, second irreversible blocking nucleotide, third irreversible blocking nucleotide, and fourth irreversible blocking nucleotide.

25. The kit of claim 24, wherein, The kit has one or more of the following characteristics: (1) The ratio of the unlabeled first nucleotide to the first irreversible blocking nucleotide is 100:1 to 1:100; (2) The ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1 to 1:100; (3) The ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1 to 1:100; (4) The ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1 to 1:100; (5) The irreversible blocking nucleotide is a dideoxynucleotide.

26. The kit of claim 19, wherein, The kit also contains one or more of the following: nucleic acid polymerase, reagents for amplification, reagents for sequencing, or any combination thereof.

27. The kit of claim 26, wherein the reagents for amplification comprise one or more selected from: enzyme working buffer, water, ion-containing solutions, single-stranded DNA binding proteins, or any combination thereof.

28. The kit of claim 26, wherein the reagent for sequencing comprises one or more selected from: sequencing slides, reagents for removing protecting groups and markers from nucleotides, and reagents for detecting the luminescent signal of the markers.

29. The kit of claim 27, wherein the enzyme is a nucleic acid polymerase.

30. The kit of claim 27, wherein the kit is used for analyzing polynucleotides; or, the kit is used for sequencing polynucleotides.

31. The kit of claim 27, wherein the presence of the marker is detected by one or more luminescent signals.

32. The kit of claim 21, wherein the first marker, the second marker, the third marker, and the fourth marker are luminescent markers.

33. The kit of claim 32, wherein the first, second, third, and fourth markers are each independently selected from coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, or Texas red.

34. The kit according to any one of claims 21-25, wherein the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.

35. The kit of claim 25, wherein the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are A, T, C, and G, respectively.

Images