Methods for analyzing the sequence of target polynucleotides

Abstract

The present invention relates to a method for analyzing the sequence of a target polynucleotide, which achieves complete polymerization by polymerizing a nucleotide mixture and a polymerase multiple times, thereby meeting the requirements of sequencing and improving read length and accuracy.Furthermore, the present invention relates to a reagent test kit, which is used for analyzing or sequencing polynucleotides.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the sequence of a target polynucleotide, and by polymerizing a nucleotide mixture multiple times according to the need for sequencing, efficient and sufficient polymerization is achieved. Furthermore, the present invention relates to a kit that can be used for the analysis or sequencing of polynucleotides.

Background Art

[0002] High-throughput sequencing, in which sequencing by synthesis (SBS) is the commercial mainstream, mainly uses DNA polymerase, nucleotides with reversible blocking and fluorescent labels to identify DNA sequences. Hundreds of thousands to millions of DNA molecules can be sequenced in parallel at one time, and it has the advantages of high throughput, high-speed detection, flexibility and versatility, and low cost.

[0003] Currently, the specific process of sequencing by synthesis includes generating a large number of DNA templates by adopting amplification or rolling circle replication, then immobilizing specific sequencing primers, and simultaneously adding DNA polymerase and fluorescently labeled nucleotides to the reaction system, or simultaneously adding a mixture of DNA polymerase, fluorescently labeled nucleotides and non-fluorescently modified nucleotides to the reaction system. Since the 3'-OH of these dNTPs is protected, only one dNTP can be added at a time. Each time a dNTP is added, the DNA strand replication reaction stops, then the fluorescent signal is excited and collected, and then a chemical reagent is added to extinguish the fluorescent signal, the 3'-OH protecting group of the dNTP is removed, and the next round of sequencing reaction can be carried out.

[0004] However, this sequencing technology requires particularly high synthesis efficiency. If the synthesis of multiple copies is incomplete, the signal becomes confused, affecting sequencing accuracy and sequencing read length. This is also one of the major technical problems currently facing second-generation sequencers. While there is a demand for shorter sequencing times and higher throughput, there are limits to the reaction efficiency of polymerases, and in particular, there are limits to the polymerization ability of nucleotides with fluorescent groups, and these limitations affect the quality of sequencing due to polymerization efficiency. Prior art has described improving polymerization efficiency by co-polymerizing a mixed solution of fluorescently labeled and unfluorescently labeled nucleotides in the reaction system, but its effect is still limited. When these polymerization efficiencies cannot keep up with the sequencing rate, incomplete polymerization occurs in multiple copies, leading to signal confusion during sequencing and limiting sequencing read length and accuracy.

[0005] Therefore, there is a need to provide a method for improving the read length and accuracy of sequencing in polynucleotide sequencing. [Brief explanation of the drawing]

[0006] [Figure 1] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental group 1. [Figure 2] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental group 2. [Figure 3] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental group 3. [Figure 4] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental group 4. [Figure 5] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental groups 5, 6, and 7. [Figure 6] This is a flowchart showing the synthesis and sequencing of E. coli DNA in experimental groups 8, 9, and 10. [Figure 7] This shows the Q30(%) ratio for each cycle of the four experiments in experimental groups 1-4. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30(%) ratio for each cycle. [Figure 8] This graph shows the sequencing error rate (%) for each cycle of the four experiments in experimental groups 1-4. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle. [Figure 9] This graph shows the Q30 (%) ratio for each cycle in five experiments: experimental groups 2, 3, 5, 6, and 7. Experimental group 2 served as the control. In the other experimental groups, two rounds of polymerization were performed in each reaction cycle. The nucleotides polymerized in the first round were a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, while the nucleotides polymerized in the second round were a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in different ratios. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) ratio for each cycle. [Figure 10] This graph shows the sequencing error rate (%) for each cycle of five experiments in experimental groups 2, 3, 5, 6, and 7. Experimental group 2 served as the control group. In the other experimental groups, two rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, while the nucleotide polymerized in the second round was a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in different ratios. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle. [Figure 11]The results for the Q30 (%) ratio in each cycle of five experiments, experimental groups 2, 4, 8, 9, and 10, are shown. Experimental group 2 served as the control. In all other experimental groups, three rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared from nucleotide mixed solution 1 and nucleotide mixed solution 2 of different concentrations, the nucleotide polymerized in the second round was nucleotide mixed solution 2, and the nucleotide polymerized in the third round was a dideoxynucleotide mixed solution. In these, the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) ratio for each cycle. [Figure 12] The results of the sequencing error rate (%) in each cycle of five experiments in experimental groups 2, 4, 8, 9, and 10 are shown. Experimental group 2 served as the control group. In all other experimental groups, polymerization was carried out in three rounds in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared from nucleotide mixed solution 1 and nucleotide mixed solution 2 of different concentrations, the nucleotide polymerized in the second round was nucleotide mixed solution 2, and the nucleotide polymerized in the third round was a dideoxynucleotide mixed solution. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle. [Modes for carrying out the invention]

[0007] By increasing the number of polymerization reactions and adjusting the nucleotide components involved in the polymerization reaction, the polymerization rate can be adjusted to meet sequencing needs, improving the lead length and quality of the sequencing, thus completing the present invention.

[0008] Therefore, in the first embodiment, the present application relates to a method for analyzing the sequence of a target polynucleotide, (a) the step of providing a target polynucleotide; (b) The steps of contacting the target polynucleotide with a primer under conditions that allow hybridization or annealing, thereby forming a partial double helix comprising the target polynucleotide and the primer to be used as a growth chain; (c) A step of contacting a partial double helix with a polymerase and a first nucleotide mixture under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that a growth chain is elongated, wherein the first nucleotide mixture comprises at least one nucleotide labeled with a label; Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide; for example, 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; The first nucleotide mixture includes, in each nucleotide, a protecting group (e.g., a protecting group bonded via a 2'- or 3'-oxygen atom group) on its ribose or deoxyribose moiety that can reversibly block nucleic acid chain elongation; (d) A step of contacting the product of the previous step with a polymerase and a second nucleotide mixture under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that a growth chain is elongated, wherein the second nucleotide mixture comprises at least one (e.g., 1, 2, 3, or 4) of (1) unlabeled nucleotides, or (2) irreversibly blocking nucleotides, or (3) a combination of unlabeled nucleotides and irreversibly blocking nucleotides; In certain embodiments, the second nucleotide mixture comprises at least one unlabeled nucleotide; for example, 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; Each unlabeled nucleotide in the second nucleotide mixture contains a protecting group (e.g., a protecting group bonded via a 2'- or 3'-oxygen atom) on its ribose or deoxyribose moiety that can reversibly block nucleotide chain elongation; (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, the step is to contact the product of the previous step with a polymerase and a third nucleotide mixture under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that a growth chain is elongated; the third nucleotide mixture contains at least one irreversible blocking nucleotide; (f) a step of detecting the presence of a label in the product of the previous step; (g) a step of removing protecting groups and labels contained in the product of the previous step; (h) Optionally, a step in which steps (c) through (g) are repeated one or more times; Includes, This provides a method for obtaining sequence information of a target polynucleotide.

[0009] Methods for sequencing target polynucleotides can be performed by denaturing the target polynucleotide sequence, contacting the target polynucleotide with different nucleotides to form complements of the target polynucleotide, and detecting the incorporation of the nucleotides. This method utilizes polymerization, in which polymerase elongates the growth chain by incorporating the correct nucleotide complementary to the target.

[0010] In each round of polymerization, nucleotide incorporation is carried out by polymerase. Many different polymerases exist, and determining the most suitable polymerase is readily apparent to those skilled 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 possess specific properties can also be used.

[0011] The conditions under which polymerization occurs are well known to those skilled in the art. To carry out the polymerase reaction, the primer is typically first annealed to the target polynucleotide, where it is recognized by the polymerase and subsequently serves as the starting site for the elongation of the growth chain. The primer may also be added as a separate component to the target polynucleotide. Other conditions necessary for carrying out the polymerase reaction are well known to those skilled in the art, and these conditions include temperature, pH, and buffer composition.

[0012] In certain embodiments, the first nucleotide mixture of the present invention is brought into contact with a primer and polymerase to enable the first round of polymerization. The nucleotides can be added sequentially (i.e., each type of nucleotide (A, C, G, or T / U) separately) or simultaneously. Depending on the sequencing rate required, the second nucleotide mixture of the present invention is brought into contact with the polymerase again to perform the second round of polymerization. The nucleotides can be added sequentially (i.e., each type of nucleotide (A, C, G, or T / U) separately) or simultaneously. Finally, depending on whether the second nucleotide mixture contains dideoxynucleotides or not, a third round of polymerization is either not performed or is performed. In certain embodiments, the second nucleotide mixture does not contain dideoxynucleotides, and the third nucleotide mixture of the present invention is brought into contact with the polymerase to enable the third round of polymerization. The nucleotides can be added sequentially (i.e., each type of nucleotide (A, C, G, or T / U) separately) or simultaneously.

[0013] In certain embodiments, nucleotides that are not incorporated are removed. For example, nucleotides that are not incorporated are removed by performing a washing step.

[0014] In certain embodiments, the label is detected. Detection can be performed by conventional methods, and means for detecting fluorescent labels or signals are well known in the art. For example, this can be achieved by an apparatus that detects the wavelength of fluorescence. Such apparatuses are well known in the art. For example, such an apparatus may be a confocal scanning microscope that scans the surface of a solid support with a laser to image the fluorophore directly attached to the nucleic acid molecule being sequenced. Further, each signal generated can be observed with a sensitive two-dimensional detector such as, for example, a charge-coupled detector (CCD). For example, other techniques such as scanning near-field optical microscopy (SNOM) can also be used.

[0015] In certain embodiments, after detection, the label can be removed using appropriate conditions.

[0016] The use of the labeled nucleotides of the present invention is not limited to DNA sequencing technology. The nucleotides of the present invention can also be used to perform other research forms including polynucleotide synthesis, DNA hybridization analysis, and single nucleotide polymorphism studies. Any technique involving the interaction between nucleotides and enzymes can utilize the molecules of the present invention. For example, the molecules can be used as substrates for reverse transcriptase or terminal transferase.

[0017] In certain embodiments, in step (c), the extension is an extension using the target polynucleotide as a template. In certain embodiments, the extension is an extension of one nucleotide.

[0018] In certain 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with a first label and a second label, and an unlabeled fourth nucleotide. Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide.

[0019] In certain 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, 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with a first label and a second label, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0020] In certain embodiments, the ratio of the first nucleotide labeled with the first label to the first nucleotide that is 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 the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 3:2.

[0021] In certain embodiments, the ratio of the second nucleotide labeled with the second label 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 certain embodiments, the ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 3:2.

[0022] In certain embodiments, the ratio of the third nucleotide labeled with the third label 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 certain embodiments, the ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 3:2.

[0023] In certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an 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 certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an unlabeled third nucleotide is 3:2.

[0024] In certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label 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 certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 3:2.

[0025] In certain embodiments, in step (d), the second nucleotide mixture is (1) an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an 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) an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, and the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide; Includes.

[0026] In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 100:1.

[0027] In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 100:1.

[0028] In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 100:1.

[0029] In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 100:1.

[0030] In certain embodiments, the first, second, third, and fourth signs are each independently identical or different.

[0031] In certain embodiments, the first, second, third, and fourth signs are different.

[0032] In certain embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).

[0033] In certain embodiments, the first, second, third, and fourth labels are each independently selected from the group consisting of coumarin, AlexaFluor, Bodipy, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas Red, and their derivatives.

[0034] In certain embodiments, the target polynucleotide includes or is DNA, RNA, or any combination thereof. In certain embodiments, the extension product of the nucleic acid molecule is DNA.

[0035] In certain embodiments, the target polynucleotide is obtained from a sample derived from a eukaryote (e.g., animal, plant, fungus), a prokaryote (e.g., bacteria, actinomycetes), a virus, a phage, or any combination thereof.

[0036] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from the group consisting of A, T, C, G, and U.

[0037] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each different.

[0038] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively. In certain 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 the group consisting of A, T, C, G, and U.

[0039] In a particular embodiment, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are different.

[0040] In a particular embodiment, 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.

[0041] In certain embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0042] In a particular embodiment, the first nucleotide mixture includes 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 includes an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide.

[0043] In certain embodiments, if 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 if it comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with the first and second labels, and an unlabeled fourth nucleotide, then the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0044] In a second embodiment, this application is a kit, (a) A first nucleotide mixture comprising at least one nucleotide labeled with a label; Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide; for example, 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; The first nucleotide mixture comprises a nucleotide in which each nucleotide has a protecting group (e.g., a protecting group bonded via a 2'- or 3'-oxygen atom) on its ribose or deoxyribose moiety that can reversibly block nucleic acid chain elongation; (b) A second nucleotide mixture comprising at least one (e.g., 1, 2, 3, or 4) of (1) unlabeled nucleotides, or (2) irreversibly blocking nucleotides, or (3) a combination of unlabeled nucleotides and irreversibly blocking nucleotides; In certain embodiments, the second nucleotide mixture comprises at least one unlabeled nucleotide; for example, 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; The second nucleotide mixture comprises: each unlabeled nucleotide in the second nucleotide mixture having a protecting group (e.g., a protecting group bonded via a 2'- or 3'-oxygen atom) on its ribose or deoxyribose moiety that can reversibly block nucleic acid chain elongation; (c) If the second nucleotide mixture does not contain an irreversible blocking nucleotide, the kit shall contain a third nucleotide mixture containing at least one irreversible blocking nucleotide; If at least one nucleotide in the second nucleotide mixture contains an irreversibly blocking nucleotide, the kit provides a kit that does not contain the third nucleotide mixture.

[0045] In certain embodiments, the second nucleotide mixture comprises at least one unlabeled nucleotide; for example, 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.

[0046] In certain embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0047] In certain 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with a first label and a second label, and an unlabeled fourth nucleotide. Optionally, the first nucleotide mixture further comprises at least one unlabeled nucleotide.

[0048] In certain 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, 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with a first label and a second label, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0049] In certain embodiments, the ratio of the first nucleotide labeled with the first label to the first nucleotide that is 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 the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 3:2.

[0050] In certain embodiments, the ratio of the second nucleotide labeled with the second label 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 certain embodiments, the ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 3:2.

[0051] In certain embodiments, the ratio of the third nucleotide labeled with the third label 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 certain embodiments, the ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 3:2.

[0052] In certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an 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 certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of a third nucleotide co-labeled with the first and second labels to an unlabeled third nucleotide is 3:2.

[0053] In certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label 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 certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 20:1, 1:10, 1:1, or 3:2. In certain embodiments, the ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 3:2.

[0054] In a particular embodiment, the first nucleotide mixture includes 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 includes an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide.

[0055] In a particular embodiment, if 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide co-labeled with a first label and a second label, and an unlabeled fourth nucleotide, then the second nucleotide mixture comprises an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

[0056] In certain embodiments, the second nucleotide mixture is (1) an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an 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) an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, an unlabeled fourth nucleotide, and the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide; Includes.

[0057] In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled first nucleotide to first irreversible blocking nucleotide is 100:1.

[0058] In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled second nucleotide to second irreversible blocking nucleotide is 100:1.

[0059] In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled third nucleotide to third irreversible blocking nucleotide is 100:1.

[0060] In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 100:1 to 1:100, or only irreversible blocking nucleotide is present. In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 1:100, 50:1, or 100:1. In certain embodiments, the ratio of unlabeled fourth nucleotide to fourth irreversible blocking nucleotide is 100:1.

[0061] In certain embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0062] In a particular embodiment, the kit further comprises one or more selected from the group consisting of nucleic acid polymerase, a reagent for extension, a reagent for sequencing, or any combination thereof.

[0063] In certain embodiments, the reagents for extension include a primer complementary to all or part of the polynucleotide, a DNB preparation buffer, a working buffer for an enzyme (e.g., a nucleic acid polymerase), water, and ions (e.g., Mg 2+ It consists of one or more selected from the group comprising a solution containing ), a single-stranded DNA-binding protein, or any combination thereof.

[0064] In certain embodiments, the sequencing reagent includes one or more selected from the group consisting of a sequencing slide, a reagent for removing protecting groups and labels from nucleotides, and a reagent for detecting the luminescence signal of the label (e.g., a fluorescent mixed solution).

[0065] In certain embodiments, the nucleic acid polymerase is a DNA polymerase such as a heat-stable DNA polymerase. In certain embodiments, the heat-resistant DNA polymerase is found in *Thermus aquaticus* (Taq), *Thermus thermophiles* (Tth), *Thermus filiformis*, *Thermis flavus*, *Thermococcus literalis*, *Thermus antranildanii*, *Thermus caldophllus*, *Thermus chliarophilus*, *Thermus flavus*, *Thermus igniterrae*, *Thermus lacteus*, *Thermus oshimai*, and *Thermus ruba*. Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus thermophllus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus litoralis, Thermococcus barossi, Thermococcus gorgonarius, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Pyrococcus uoesei Pyrococcus horikoshii, Pyrococcus abyssiiIt is obtained from *Abyssi*, *Pyrodictium occultum*, *Aquifex pyrophilus*, and *Aquifex aeolieus*.

[0066] In certain embodiments, the kit is used for the analysis of polynucleotides.

[0067] In certain embodiments, the kit is used for sequencing polynucleotides.

[0068] In certain embodiments, the presence of a label is detected by a light emission signal.

[0069] In certain embodiments, the presence of a label is detected by one or more (e.g., two, three, or four) luminescence signals.

[0070] In certain embodiments, the first, second, third, and fourth signs are each independently identical or different.

[0071] In certain embodiments, the first, second, third, and fourth signs are different.

[0072] In certain embodiments, the first, second, third, and fourth markers are luminescent markers (e.g., fluorescent markers).

[0073] In certain embodiments, the first, second, third, and fourth labels are each independently selected from the group consisting of coumarin, Alexafluoro, Bodypea, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, EF700, Texas Red, and their derivatives.

[0074] In certain embodiments, the target polynucleotide includes or is DNA, RNA, or any combination thereof. In certain embodiments, the extension product of the nucleic acid molecule is DNA.

[0075] In certain embodiments, the target polynucleotide is obtained from a sample derived from a eukaryote (e.g., animal, plant, fungus), a prokaryote (e.g., bacteria, actinomycetes), a virus, a phage, or any combination thereof.

[0076] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from the group consisting of A, T, C, G, and U.

[0077] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each different.

[0078] In certain embodiments, the first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively. In certain 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 the group consisting of A, T, C, G, and U.

[0079] In a particular embodiment, the first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are different.

[0080] In a particular embodiment, 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.

[0081] In certain embodiments, the irreversible blocking nucleotide is a dideoxynucleotide.

[0082] [Definition of Terms] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. All patents, applications, and other publications described herein are incorporated in their entirety by reference. In the event of any conflict or inconsistency between any definition described herein and any definition in any patent, application, or other publication incorporated herein by reference, the definition described herein shall prevail.

[0083] In this specification, the term “polynucleotide” refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), etc. Polynucleotides may be single-stranded, double-stranded, or may contain both single-stranded and double-stranded sequences. Polynucleotide molecules may be derived from double-stranded DNA (dsDNA) form (e.g., genomic DNA, PCR and amplification products, etc.), or derived from single-stranded DNA (ssDNA) form or RNA and converted to dsDNA form, and vice versa. The exact sequence of a polynucleotide molecule may be known or unknown. Examples of polynucleotides include genes or gene fragments (e.g., probes, primers, EST or SAGE tags, etc.), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transporter RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, and nucleic acid probes, primers or amplified copies of any of the aforementioned sequences.

[0084] Polynucleotides may contain nucleotides or nucleotide analogs. Nucleotides typically contain a sugar (e.g., ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides may be non-basic (i.e., lacking a base). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified sugar-phosphate 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), deoxyadenosine monophosphate (dAMP), deoxyadenosine Examples include xyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (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.Examples of modified bases that may be included in polynucleotides, whether having a native backbone or a similar structure, include, for example, inosine, xasanine, hypoxasanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halogenated uracil, 15-halogenated cytosine, and 5-propynyluracil. Examples include 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine. As is known in the art, certain nucleotide analogs, such as adenosine 5'-phosphoryl sulfate, cannot be incorporated into polynucleotides.

[0085] 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 variants or analogs, such as ATP or dATP. “nucleotide G” refers to a nucleotide containing guanine (G) or its variants or analogs, such as GTP or dGTP. “nucleotide C” refers to a nucleotide containing cytosine (C) or its variants or analogs, such as CTP or dCTP. “nucleotide T” refers to a nucleotide containing thymine (T) or its variants or analogs, such as TTP or dTTP. “nucleotide U” refers to a nucleotide containing uracil (U) or its variants or analogs, such as UTP or dUTP.

[0086] As used herein, the term "dideoxynucleotide" refers to a nucleotide deoxygenated at the 2'- and 3'-carbons of ribose, and is also known as a 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 its analogues, such as ddATP. "Dideoxynucleotide G" refers to a dideoxynucleotide containing guanine (G) or its analogues, such as ddGTP. "Dideoxynucleotide C" refers to a dideoxynucleotide containing cytosine (C) or its analogues, such as ddCTP. "Dideoxynucleotide T" refers to a dideoxynucleotide containing thymine (T) or its analogues, such as ddTTP. "Dideoxynucleotide U" refers to a dideoxynucleotide containing uracil (U) or its analogues, such as ddUTP. As used herein, ddNTP refers to 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 these, in which dideoxyuridine triphosphate and dideoxythymidine triphosphate do not appear simultaneously.

[0087] In this specification, the term "label" refers to a group that can emit a light-emitting signal under specific conditions.

[0088] As used herein, the term “luminescent label” refers to any substance that, when excited at a suitable excitation wavelength, can fluoresce at a specific emission wavelength. Such luminescent labels may be chemiluminescent labels, selected from, for example, biochemical luminescent labels that induce different luminescence dynamics, and any combination thereof, for example, chemiluminescent labels may be selected from luciferases that induce different luminescence dynamics, or any combination thereof; such luminescent labels may be phosphors selected from, for example, coumarin, Alexafluoro, Bodypea, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, EF700, AF532, Texas Red, and their derivatives.

[0089] In this specification, the term “protecting group” refers to a group that, after a nucleotide containing the group has been incorporated into a polynucleotide chain during synthesis, prevents polymerase (which incorporates the nucleotide containing the group into the polynucleotide chain during synthesis) from catalyzing the subsequent incorporation of another nucleotide. 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'-blocked nucleotide. The protecting group may be any suitable group that can be attached to a nucleotide, provided that the protecting group prevents the addition of another nucleotide molecule to the polynucleotide chain and can be readily removed from the sugar portion 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 used to incorporate the modified nucleotide into the polynucleotide chain. Therefore, an ideal protecting group exhibits long-term stability, can be efficiently incorporated by polymerase, can prevent secondary or further incorporation of nucleotides, and can be removed under mild conditions, preferably aqueous conditions, without damaging the polynucleotide structure.

[0090] Prior art describes various protecting groups that fit the above description. For example, International Publication No. 91 / 06678 discloses esters and ethers, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrobenzene carbonate, 2,4-sulfenyldinitro, and tetrahydrofuran ether as 3'-OH protecting groups. Metzker et al. (Nucleic Acids Research, 22(20):4259-4267, 1994) disclose the synthesis and application of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs). International Publication No. 2002 / 029003 describes the use of allyl protecting groups to cap 3'-OH groups on growing DNA strands in polymerase reactions. Preferably, various protecting groups reported in International Publication Nos. 2014139596 and 2004 / 018497 can be used, for example, the exemplary protecting group in Figure 1A and the 3'-hydroxyl protecting group (i.e., protecting group) specified in the claims of International Publication No. 2014 / 139596, as well as the protecting groups exemplified in Figures 3 and 4 and the protecting groups specified in the claims of International Publication No. 2004 / 018497. The above documents are incorporated in their entirety by reference.

[0091] As used herein, the term “reversible blocking group” refers to a group that, when incorporated into a polynucleotide chain during synthesis, prevents polymerase from proceeding to the next polymerization round, thereby causing termination of the polymerization reaction. In this case, only one base is incorporated into the growing nucleic acid chain in each round of polymerization. In addition, this group can be removed, after which the growing nucleic acid chain can proceed to the next polymerization round, and a base is reintroduced. Examples of reversible blocking groups include those in which the H of the 3'-OH group is substituted with an ester, ether, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3, 2-nitrobenzene carbonate, 2,4-sulfenyldinitro and tetrahydrofuran ether -CH2-CH=CH2, SS group, or those in which the base is blocked by a large sterically hindered group and a fluorescent group, and linked to fluorescence via SS.

[0092] As used herein, the term "irreversible blocking nucleotide" refers to a nucleotide that, when incorporated into a polynucleotide chain during synthesis, prevents the subsequent incorporation of other nucleotides into the polynucleotide chain due to its blocking effect. This blocking effect is irreversible. Irreversible blocking nucleotides typically include dideoxynucleotides, or nucleotides in which the 3'-OH group is replaced with a methoxy group at the 3' end (3'-OMe, or 3'-OCH3), and nucleotides having a 3'-terminal azide (3'-N3) and a 3'-terminal ethoxy (3'-OEt, 3'-OCH2CH3).

[0093] [Beneficial technical effects of the present invention] In sequencing, the present invention achieves a more efficient polymerization reaction by increasing the number of polymerization reactions, thereby meeting the polymerization rate requirements and improving the lead length and quality of the sequencing. Furthermore, the present invention achieves a more efficient polymerization reaction by shortening the first round of polymerization and rapidly carrying out the second round or the second and third rounds of polymerization while adjusting the nucleotide components participating in polymerization (for example, polymerizing nucleotide components without fluorescent labeling).

[0094] The method of the present invention can not only improve the efficiency of the polymerization reaction, but also ensure that the polymerization reaction is sufficient to improve the lead length and quality of the sequencing. Furthermore, it can reduce signal interference caused by the removal of unclean fluorescence or the elution of such removed fluorescence, thereby improving removal efficiency and reducing the sequencing error rate. [Examples]

[0095] Next, the present invention will be described with reference to the following examples, which are intended to illustrate the present invention and not to limit it.

[0096] The molecular biology experimental methods used in this invention are, unless otherwise specified, basically those described in J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Experimental Guide to Molecular Biology, 3 rd See Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will understand that the examples are illustrative and not intended to limit the scope of the invention.

[0097] 1. Main equipment used in this embodiment: MGISEQ-2000RS sequencer, MGIDL-200H loader, MGISEQ-2000RS sequencing slide.

[0098] 2. The main reagents used in this example are shown in Table 1 below:

[0099] [Table 1]

[0100] Example 1 In this example, various reagents were prepared in advance. 1) Preparation of nucleotide mixture solution 1 As shown in Table 2 below, all nucleotides in nucleotide mixture solution 1 possessed both a fluorescent dye and a reversible blocking group. A, T, G, and C were adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively. The fluorescent dyes present in the nucleotides were dATP-Cy5, dGTP-Cy3, dTTP-ROX, and dCTP-EF700, all of which also possessed a reversible blocking group.

[0101] [Table 2]

[0102] Of these, dATP-1 refers to adenine nucleotide having a reversible blocking group modification and Cy5 fluorescence modification, dTTP-1 refers to thymine nucleotide having a reversible blocking group modification and ROX fluorescence modification, dGTP-1 refers to guanine nucleotide having a reversible blocking group modification and Cy3 fluorescence modification, and dCTP-1 refers to cytosine nucleotide having a reversible blocking group modification and EF700 fluorescence modification.

[0103] 2) Preparation of Nucleotide Mixture Solution 2 As shown in Table 3 below, all nucleotides in nucleotide mixture solution 2 had only reversible blocking groups, and A, T, G, and C were adenine nucleotide, thymine nucleotide, guanine nucleotide, and cytosine nucleotide, respectively.

[0104] [Table 3]

[0105] Of these, Cold dATP refers to adenine nucleotide modified only with reversible blocking groups, Cold dTTP refers to thymine nucleotide modified only with reversible blocking groups, Cold dGTP refers to guanine nucleotide modified only with reversible blocking groups, and Cold dCTP refers to cytosine nucleotide modified only with reversible blocking groups.

[0106] 3) Preparation of Nucleotide Mixture Solution 3 As shown in Table 4 below, all nucleotides in nucleotide mixture solution 3 are dideoxynucleotides, where ddATP refers to adenine triphosphate dideoxynucleotide, ddTTP refers to thymine triphosphate dideoxynucleotide, ddGTP refers to guanine triphosphate dideoxynucleotide, and ddCTP refers to cytosine triphosphate dideoxynucleotide.

[0107] [Table 4]

[0108] 4) Preparation of synthetic reagent 1 Synthetic reagent 1 was prepared by mixing according to Table 5 below for later use. The nucleotides in synthetic reagent 1 were nucleotides modified with both a fluorescent dye and a reversible blocking group.

[0109] [Table 5]

[0110] 5) Preparation of synthetic reagent 2 Synthetic reagent 2 was prepared by mixing according to Table 6 below for later use. The nucleotides in synthetic reagent 2 were nucleotides modified only with reversible blocking groups.

[0111] [Table 6]

[0112] 6) Preparation of synthetic reagents 3-1, 3-2, 3-3, and 3-4. These nucleotide mixture solutions were prepared by varying the concentration ratios of nucleotide mixture solution 1 and nucleotide mixture solution 2. The specific concentration ratios are shown in Tables 7 to 10.

[0113] Synthetic reagent 3-1 was prepared by mixing according to Table 7 below for later use.

[0114] [Table 7]

[0115] 7) Preparation of synthetic reagent 3-2 Synthetic reagent 3-2 was prepared by mixing according to Table 8 below for later use.

[0116] [Table 8]

[0117] 8) Preparation of synthetic reagent 3-3 Synthetic reagent 3-3 was prepared by mixing according to Table 9 below for later use.

[0118] [Table 9]

[0119] 9) Preparation of synthetic reagents 3-4 Synthetic reagents 3-4 were prepared by mixing according to Table 10 below for later use.

[0120] [Table 10]

[0121] 10) Preparation of synthetic reagent 4 Synthetic reagent 4 was prepared by mixing according to Table 11 below for later use.

[0122] [Table 11]

[0123] 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 in different concentration ratios. The specific concentration ratios are shown in Tables 12 to 14.

[0124] Synthetic reagent 5-1 was prepared by mixing according to Table 12 below for later use.

[0125] [Table 12]

[0126] 12) Preparation of synthetic reagent 5-2 Synthetic reagent 5-2 was prepared by mixing according to Table 13 below for later use.

[0127] [Table 13]

[0128] 13) Preparation of synthetic reagent 5-3 Synthetic reagent 5-3 was prepared by mixing according to Table 14 below for later use.

[0129] [Table 14]

[0130] Example 2 In all of the following experiments, single-stranded circular DNA from E. coli (i.e., MGI's standard library reagent V3.0) was used as a template, and DNA nanospheres were prepared using the MGISEQ-2000RS high-throughput sequencing kit (MGI) and then loaded onto a chip for subsequent sequencing.

[0131] 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 synthetic reagent 1 prepared above, i.e., the reaction solution of a nucleotide mixture containing both fluorescent and reversible blocking modifying groups. In each reaction cycle, only one round of polymerization was performed, and the polymerized nucleotides were a nucleotide mixture containing both fluorescent and reversible blocking modifying groups. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 1. That is, according to the instructions of the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and washing was performed with the elution reagent in sequence. Subsequently, the Q30 reduction rate and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0132] Experimental Group 2: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagent in well #1 of the kit was removed and replaced with the synthetic reagent 3-2 prepared above, i.e., a reaction solution prepared by mixing nucleotide mixture solution 1, which has both fluorescent and reversible blocking modifying groups, and nucleotide mixture solution 2, which has only reversible blocking modifying groups, in a 3:2 ratio. In each reaction cycle, only one round of polymerization was performed, and the polymerized nucleotides were used as a mixed solution prepared by mixing nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 2. That is, according to the instructions of the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and washing was performed with the elution reagent in sequence. Subsequently, the Q30 reduction rate and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0133] Experimental Group 3: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 of the kit were removed. The reagent in well #1 was replaced with synthesis reagent 3-2 from the sequencing group, and the reagent in well #2 was replaced with synthesis reagent 4 from the sequencing group. Two rounds of polymerization were performed in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, and the polymerized nucleotide in the second round was a mixed solution of dideoxynucleotides. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 3. That is, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and washing was performed with the elution reagent in sequence. Subsequently, the Q30 reduction rate and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0134] Experimental Group 4: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1, #2, and #17 of the kit were removed. The reagent in well #1 was replaced with sequencing group synthesis reagent 3-2, the reagent in well #2 with sequencing group synthesis reagent 2, and the reagent in well #17 with sequencing group synthesis reagent 4. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 4. Three rounds of polymerization were performed in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, the polymerized nucleotide in the second round was mixed solution 2, and the polymerized nucleotide in the third round was a dideoxynucleotide mixture solution. Specifically, following the instructions for the MGISEQ-2000RS high-throughput sequencing kit, we sequentially eluted free nucleotides with the elution reagent, collected signals with the imaging reagent, removed protecting groups with the excision reagent, and washed with the elution reagent. Subsequently, we calculated the Q30 reduction rate and the sequencing error rate curve for each cycle to evaluate the sequencing quality.

[0135] Experimental Group 5: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 of the kit were removed. The reagent in well #1 was replaced with synthesis reagent 3-2 from the sequencing group, and the reagent in well #2 was replaced with synthesis reagent 5-1 from the sequencing group. Two rounds of polymerization were performed in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, and the polymerized nucleotide in the second round was a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in a 1:100 ratio. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 5. Specifically, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and the mixture was washed with the elution reagent. Subsequently, the Q30 degradation rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0136] Experimental group 6: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 of the kit were removed. The reagent in well #1 was replaced with synthesis reagent 3-2 of the sequencing group, and the reagent in well #2 was replaced with synthesis reagent 5-2 of the sequencing group. Two rounds of polymerization were performed in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, and the polymerized nucleotide in the second round was a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in a 100:1 ratio. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 5. Specifically, following the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and the mixture was washed with the elution reagent. Subsequently, the Q30 degradation rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0137] Experimental Group 7: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1 and #2 of the kit were removed. The reagent in well #1 was replaced with synthesis reagent 3-2 from the sequencing group, and the reagent in well #2 was replaced with synthesis reagent 5-3 from the sequencing group. Two rounds of polymerization were performed in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, and the polymerized nucleotide in the second round was a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in a 50:1 ratio. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 5. Specifically, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting groups were removed with the excision reagent, and the mixture was washed with the elution reagent. Subsequently, the Q30 degradation rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0138] Experimental group 8: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1, #2, and #17 of the kit were removed, and the reagent in well #1 was replaced with sequencing group synthesis reagent 3-1, the reagent in well #2 with sequencing group synthesis reagent 2, and the reagent in well #17 with sequencing group synthesis reagent 4. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 6, with three rounds of polymerization in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by mixing nucleotide solution 1 and nucleotide solution 2 in a 1:1 ratio, the polymerized nucleotide in the second round was nucleotide solution 2, and the polymerized nucleotide in the third round was dideoxynucleotide mixed solution. In other words, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting group was removed with the excision reagent, and washing was performed with the elution reagent in sequence. Subsequently, the Q30 degradation rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0139] Experimental group 9: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1, #2, and #17 of the kit were removed. The reagent in well #1 was replaced with sequencing group synthesis reagent 3-3, the reagent in well #2 with sequencing group synthesis reagent 2, and the reagent in well #17 with sequencing group synthesis reagent 4. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 6, with three rounds of polymerization in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by mixing nucleotide solution 1 and nucleotide solution 2 in a 1:10 ratio, the polymerized nucleotide in the second round was nucleotide solution 2, and the polymerized nucleotide in the third round was dideoxynucleotide mixed solution. In other words, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting group was removed with the excision reagent, and the mixture was washed with the elution reagent. Subsequently, the Q30 reduction rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0140] Experimental group 10: Using the MGISEQ-2000RS high-throughput sequencing kit, the reagents in wells #1, #2, and #17 of the kit were removed. The reagent in well #1 was replaced with synthesis reagents 3-4 of the sequencing group, the reagent in well #2 with synthesis reagent 2 of the sequencing group, and the reagent in well #17 with synthesis reagent 4 of the sequencing group. SE100 sequencing was performed using the MGISEQ-2000RS sequencing platform according to the experimental process shown in Figure 6, with three rounds of polymerization in each reaction cycle. The polymerized nucleotide in the first round was a mixed solution prepared by mixing nucleotide solution 1 and nucleotide solution 2 in a 20:1 ratio, the polymerized nucleotide in the second round was nucleotide solution 2, and the polymerized nucleotide in the third round was dideoxynucleotide solution. In other words, according to the instructions for the MGISEQ-2000RS high-throughput sequencing kit, free nucleotides were eluted with the elution reagent, the signal was collected with the imaging reagent, the protecting group was removed with the excision reagent, and washing was performed with the elution reagent. Subsequently, the Q30 reduction rate for each cycle and the sequencing error rate curve for each cycle were calculated to evaluate the sequencing quality.

[0141] Results: As shown in Figures 7 to 12, Figure 7 shows the Q30(%) ratio for each cycle in the four experiments, i.e., experimental groups 1 to 4. In this figure, the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30(%) ratio in each cycle.

[0142] Figure 8 shows the sequencing error rate (%) for each cycle in the four experiments, i.e., experimental groups 1-4. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle.

[0143] Figure 9 shows the results for five experiments, namely experimental groups 2, 3, and 5-7. Experimental group 2 served as the control. In all other experiments, two rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared by combining nucleotide mixture solution 1 and nucleotide mixture solution 2 in a 3:2 ratio, while the nucleotide polymerized in the second round was a mixed solution prepared by combining nucleotide mixture solution 2 and dideoxynucleotide mixture solution in different proportions. In these experiments, the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) ratio for each cycle.

[0144] Figure 10 shows the results for five experiments, namely experimental groups 2, 3, and 5-7. Experimental group 2 served as the control. In all other experiments, two rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixture prepared by combining nucleotide mixture 1 and nucleotide mixture 2 in a 3:2 ratio, while the nucleotide polymerized in the second round was a mixture prepared by combining nucleotide mixture 2 and dideoxynucleotide mixture in different proportions. In these figures, the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle.

[0145] Figure 11 shows the results of five experiments, namely experimental groups 2, 4, and 8-10. Experimental group 2 served as the control. In all other experiments, three rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared by combining nucleotide mixture 1 and nucleotide mixture 2 at different concentration ratios, the nucleotide polymerized in the second round was nucleotide mixture 2, and the nucleotide polymerized in the third round was a dideoxynucleotide mixture solution. The horizontal axis represents the number of sequencing cycles, and the vertical axis represents the Q30 (%) ratio for each cycle.

[0146] Figure 12 shows the results of five experiments, namely experimental groups 2, 4, and 8-10. Experimental group 2 served as the control. In all other experiments, three rounds of polymerization were performed in each reaction cycle. The nucleotide polymerized in the first round was a mixed solution prepared by combining nucleotide mixture 1 and nucleotide mixture 2 at different concentration ratios. The nucleotide polymerized in the second round was nucleotide mixture 2. The nucleotide polymerized in the third round was a dideoxynucleotide mixture. In this figure, the horizontal axis represents the number of sequencing cycles, and the vertical axis represents the sequencing error rate (%) for each cycle.

[0147] A higher percentage of Q30 in each cycle indicates better quality, and a lower percentage decrease in Q30 also indicates better quality. A lower sequencing error rate in each cycle indicates higher sequencing quality.

[0148] From the results in Figures 7 and 8, experimental group 4 showed the best sequencing quality and error rate, followed by experimental group 3, and both results were significantly better than those of experimental groups 2 and 1. It was confirmed that the polymerization method of this application is superior to existing polymerization methods. From Figures 9 and 10, experimental groups 3, 5-7 were significantly better than experimental group 2 (control), and experimental group 6 showed the best results (smallest decrease in Q30). It was confirmed that the effect of the two-step polymerization method of this application is superior to the one-step polymerization method.

[0149] Based on Figures 11 and 12, experimental groups 4, 8, 9, and 10 all performed better than experimental group 2 (control group), with experimental group 4 being the best. This confirms that the effect of the three-step polymerization method of this application is superior to that of the single-step polymerization method.

Claims

1. A method for analyzing the sequence of a target polynucleotide, (a) the step of providing a target polynucleotide; (b) The step of contacting the target polynucleotide with a primer under conditions that enable hybridization or annealing, thereby forming a partial double helix comprising the target polynucleotide and the primer to be used as a growth chain; (c) A step of bringing the partial double helix into contact with a polymerase and a mixture of first nucleotides under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that the growth chain is extended, The first nucleotide mixture described above comprises at least one nucleotide labeled with a label, The first nucleotide mixture contains or does not contain at least one unlabeled nucleotide. Each nucleotide in the first nucleotide mixture comprises a protecting group in its ribose or deoxyribose portion that can reversibly block nucleic acid chain elongation; (d) A step of contacting the product of the previous step with a polymerase and a mixture of second nucleotides under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that the growth chain is extended, The second nucleotide mixture comprises at least one of (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 comprises a protecting group on its ribose or deoxyribose portion that can reversibly block nucleotide chain elongation; (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, the step is to contact the product of the previous step with a polymerase and a third nucleotide mixture under conditions that allow the polymerase to carry out a nucleotide polymerization reaction so that the growth chain is elongated, wherein the third nucleotide mixture contains at least one irreversible blocking nucleotide; (f) The step of detecting the presence of a label in the product of the previous step; (g) the step of removing the protecting groups and labels contained in the product of the previous step; (h) A step in which steps (c) through (g) are repeated once or more; Includes, A method for obtaining sequence information of the target polynucleotide.

2. The method according to claim 1, characterized by one or more of the following: (1) The first nucleotide mixture comprises 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; (2) The protecting group is bonded via a 2'- or 3'-oxygen atom group; (3) The second nucleotide mixture comprises at least one unlabeled nucleotide; (4) The second nucleotide mixture includes 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.

3. The method according to claim 1, characterized by one or more of the following: (1) In step (c), the extension is an extension using the target polynucleotide as a template; (2) In step (c), the elongation is an elongation of one nucleotide; (3) 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with the first label and the second label, and an unlabeled fourth nucleotide, and the first nucleotide mixture further comprises or does not comprise at least one unlabeled nucleotide; or (4) 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 comprises a first nucleotide labeled with a first label, a second nucleotide labeled with a second label, a third nucleotide colabeled with the first and second labels, an unlabeled fourth nucleotide, an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

4. The method according to claim 3, characterized by one or more of the following: (1) The ratio of the first nucleotide labeled with the first label to the first nucleotide that is not labeled is between 20:1 and 1:10; (2) The ratio of the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 10:1 to 1:10, 5:1 to 1:5, or 3:1 to 1:3; (3) The ratio of the first nucleotide labeled with the first label to the first nucleotide that is not labeled is 20:1, 1:10, 1:1, or 3:2; (4) The ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is between 20:1 and 1:10; (5) The ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 10:1 to 1:10, 5:1 to 1:5, or 3:1 to 1:3; (6) The ratio of the second nucleotide labeled with the second label to the unlabeled second nucleotide is 20:1, 1:10, 1:1, or 3:2; (7) The ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 20:1 to 1:10; (8) The ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 10:1 to 1:10, 5:1 to 1:5, or 3:1 to 1:3; (9) The ratio of the third nucleotide labeled with the third label to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2; (10) The ratio of the third nucleotide colabeled with the first label and the second label to the unlabeled third nucleotide is 20:1 to 1:10; (11) The ratio of the third nucleotide colabeled with the first label and the second label to the unlabeled third nucleotide is 10:1 to 1:10, 5:1 to 1:5, or 3:1 to 1:3; (12) The ratio of the third nucleotide co-labeled with the first label and the second label to the unlabeled third nucleotide is 20:1, 1:10, 1:1, or 3:2; (13) The ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 20:1 to 1:10; (14) The ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 10:1 to 1:10, 5:1 to 1:5, or 3:1 to 1:3; (15) The ratio of the fourth nucleotide labeled with the fourth label to the unlabeled fourth nucleotide is 20:1, 1:10, 1:1, or 3:

2.

5. In step (d), the second nucleotide mixture (1) an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an 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 The method according to claim 1, including the method described in claim 1.

6. The method according to claim 5, characterized by one or more of 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 first nucleotide to the first irreversible blocking nucleotide is 1:100, 50:1, or 100:1; (3) The ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 100:1 to 1:100; (4) The ratio of the unlabeled second nucleotide to the second irreversible blocking nucleotide is 1:100, 50:1, or 100:1; (5) The ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 100:1 to 1:100; (6) The ratio of the unlabeled third nucleotide to the third irreversible blocking nucleotide is 1:100, 50:1, or 100:1; (7) The ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 100:1 to 1:100; (8) The ratio of the unlabeled fourth nucleotide to the fourth irreversible blocking nucleotide is 1:100, 50:1, or 100:

1.

7. The method according to claim 3, characterized by one or more of the following: (1) The first sign, the second sign, the third sign, and the fourth sign are each independently identical or different; (2) The first sign, the second sign, the third sign, and the fourth sign are different; (3) The first sign, the second sign, the third sign and the fourth sign are luminescent signs; (4) The first label, the second label, the third label and the fourth label are each independently selected from the group consisting of coumarin, Alexafluoro, Bodypea, fluorescein, tetramethylrhodamine, phenoxazine, acridine, Cy5, Cy3, AF532, Texas Red and their derivatives; (5) The target polynucleotide comprises or is DNA, RNA, or any combination thereof; (6) The target polynucleotide is obtained from a sample derived from a eukaryote, prokaryote, virus, phage, or any combination thereof; (7) The first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are each independently selected from the group consisting of A, T, C, G, and U; (8) The first nucleotide, the second nucleotide, the third nucleotide and the fourth nucleotide are different; (9) The first nucleotide, the second nucleotide, the third nucleotide, and the fourth nucleotide are A, T, C, and G, respectively.

8. The method according to claim 5, characterized by one or more of the following: (1) 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 the group consisting of A, T, C, G, and U; (2) The first irreversible blocking nucleotide, the second irreversible blocking nucleotide, the third irreversible blocking nucleotide, and the fourth irreversible blocking nucleotide are different; (3) 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.

9. The method according to claim 7, characterized by one or more of the following: (1) The light-emitting marker is a fluorescent marker; (2) The eukaryote is an animal, plant, or fungus; (3) The prokaryote is a bacterium or actinomycete; (4) The irreversible blocking nucleotide is a dideoxynucleotide; (5) If the first nucleotide mixture includes a first nucleotide labeled with the first label, a second nucleotide labeled with the second label, a third nucleotide labeled with the third label, and a fourth nucleotide labeled with the fourth label, then the second nucleotide mixture includes an unlabeled first nucleotide, an unlabeled second nucleotide, an unlabeled third nucleotide, and an unlabeled fourth nucleotide; or If the first nucleotide mixture includes 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 if it includes 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, then the second nucleotide mixture includes an unlabeled first nucleotide, an unlabeled second nucleotide, and an unlabeled third nucleotide.

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