Methods and kits for determining nucleotide sequences by elongation sequencing reactions

Abstract

This application relates to a method and kit for determining nucleotide sequences via extension sequencing reactions. The method includes: A) providing a single-stranded nucleic acid template disposed on the surface of a solid support; B) determining the type of nucleotide at a detection site in the single-stranded nucleic acid template in each cycle of the extension sequencing reaction, including: i) incorporating nucleotides carrying modifying groups into the complementary strand of the single-stranded nucleic acid template, each nucleotide being labeled with a different signal marker; ii) detecting first signal kinetic information of a detectable signal generated by the reaction of the signal marker with a signal activator; iii) changing the reaction conditions of the signal marker with the signal activator; iv) detecting second signal kinetic information of a detectable signal generated by the reaction of the signal marker with the signal activator; v) determining the type of the incorporated nucleotide based on the first and second signal kinetic information, and / or determining the type of nucleotide at the detection site in the single-stranded nucleic acid template. The aforementioned method can rapidly distinguish bases through a single sequencing cycle, reducing sequencing steps, shortening sequencing time, and improving sequencing efficiency.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more specifically, to methods and kits for determining nucleotide sequences via extension sequencing reactions. Background Technology

[0002] Monochrome signal sequencing is a method that uses signal markers on bases to emit the same signal on demand under specific conditions to identify the four base classes and achieve sequencing. Due to its simple signal recognition, rapid biochemical reaction, and high sequencing throughput, it has become the mainstream method for next-generation sequencing. Illumina's small, rapid sequencer based on monochrome fluorescence imaging utilizes this technology. However, due to the inherent limitations of various DNA enrichment methods and the sensitivity of signal response to environmental and instrument conditions, the uniformity of signal intensity of signal markers in both the temporal and spatial dimensions during the testing process is somewhat lacking.

[0003] Monochromatic signal sequencing controls the luminescence conditions and identifies four corresponding bases through two detections. Specifically, after introducing a first-type luminescence activator, labeled base types I and III emit light signals, while base types II and IV do not emit light. Then, after washing or quenching, a second-type luminescence activator is introduced. At this point, labeled base types II and III emit light signals, while base types I and IV do not emit light. The four corresponding bases are identified using four luminescence modes: 1 / 0, 1 / 1, 0 / 1, and 0 / 0. Figure 1a Because of the intermediate cleaning step between the two detections, the sequencing logic cannot significantly compress the sequencing time, limiting the improvement of sequencing efficiency. Furthermore, the two independent detections correspond to different biochemical systems, which increases the complexity of the entire sequencing system, indicating the complexity of mass production and increasing hidden costs in terms of manpower and operations.

[0004] Therefore, existing sequencing methods still need improvement. Summary of the Invention

[0005] This application aims to address at least one of the existing problems. To this end, this application proposes a method based on the difference in signal dynamics information associated with nucleotides labeled by signal markers in extended sequencing reactions, enabling rapid and accurate identification of nucleotides in a single detection in single-channel sequencing.

[0006] Specifically, this application provides the following technical solution.

[0007] In a first aspect, this application proposes a method for determining nucleotide sequences through an extension sequencing reaction. According to embodiments of this application, the aforementioned method includes: A) providing a single-stranded nucleic acid template disposed on the surface of a solid support; B) determining the type of nucleotide at a detection location in the single-stranded nucleic acid template in each cycle of the extension sequencing reaction, including: i) incorporating nucleotides carrying modifying groups into the complementary strand of the single-stranded nucleic acid template, each nucleotide being labeled with a different signal marker; ii) detecting first signal kinetic information of a detectable signal generated by the reaction of the signal marker with a signal activator; iii) changing the reaction conditions of the signal marker and the signal activator; iv) detecting second signal kinetic information of a detectable signal generated by the reaction of the signal marker with the signal activator; v) determining the type of the incorporated nucleotide based on the first and second signal kinetic information, and / or determining the type of nucleotide at the detection location in the single-stranded nucleic acid template.

[0008] According to the embodiments of this application, the aforementioned method can quickly distinguish bases through a single sequencing cycle detection, reducing sequencing steps, shortening sequencing time, and improving sequencing efficiency.

[0009] It should be noted that the term "single sequencing cycle detection" includes: in one sequencing cycle, different signal markers (such as type I signal markers and type II signal markers) are simultaneously introduced into the single-strand extension reaction system; in the signal detection stage, the corresponding signal exciters of different signal markers are simultaneously introduced into the single-strand extension reaction system, and by changing the reaction conditions of different signal markers and their corresponding signal exciters, signal kinetic information with the same or different characteristics is obtained, thereby realizing the identification of nucleotide types.

[0010] According to an embodiment of this application, the solid support is a semiconductor chip.

[0011] It should be noted that the term "signal kinetic information" refers to the signal characteristics generated during the interaction between signal markers and signal elicitors in the extension sequencing reaction. These characteristics include signal intensity, the timing of emission peaks (i.e., the chronological order of signal peak appearance), and the intensity of the signal peaks. Changes in these signal kinetic information can reflect the characteristics of different nucleotide types during the sequencing process. By detecting these changes, the types of incorporated nucleotides can be accurately distinguished and identified, thereby inferring the nucleotide sequence of the single-stranded nucleic acid template.

[0012] According to embodiments of this application, the aforementioned signal kinetic information can be used to determine the type of incorporated nucleotide. Specifically, each incorporated nucleotide interacts with a signal activator through its specific signal marker, generating characteristic signal kinetic information. By detecting this signal kinetic information, the type of incorporated nucleotide can be identified. Subsequently, based on the base complementary pairing principle between the incorporated nucleotide and the template nucleotide, the specific type of the template nucleotide is inferred.

[0013] According to other embodiments of this application, the aforementioned signal dynamics information can also be used to directly determine the nucleotide type at the detection position in the single-stranded nucleic acid template. Specifically, after the template nucleotide binds to the incorporated nucleotide labeled with a signal marker, the signal marker generates a signal through a specific interaction with a signal activator. Since the type of the signal marker is related to the specific binding mode of the template nucleotide, the generated signal type can be converted into a signal type reflecting the template nucleotide, thereby directly obtaining the sequence information of the template nucleotide.

[0014] According to embodiments of this application, the aforementioned method for determining nucleic acid sequences further includes: C) removing the modifying groups carried by the nucleotides to allow the complementary strand to continue extending. Furthermore, to prevent signal markers from interfering with subsequent sequencing, step C) further includes removing the signal markers that label the aforementioned nucleotides. Steps B)-C) only determine one type of nucleotide; by repeating steps B)-C) until the complementary strand is fully extended, the aforementioned single-stranded nucleic acid template sequence is determined.

[0015] According to embodiments of this application, the aforementioned nucleotides are selected from dATP, dCTP, dGTP, dTTP, or dUTP.

[0016] According to embodiments of this application, the aforementioned modifying group is selected from reversible blocking modifying groups.

[0017] As will be understood by those skilled in the art, nucleotides themselves do not possess optical properties (e.g., fluorescence properties), and the aforementioned luminescence kinetic information is obtained by detecting signal markers on labeled nucleotides.

[0018] In order to obtain luminescence dynamics information associated with different nucleotides, according to embodiments of this application, the selected signal markers include: type I signal markers and type II signal markers.

[0019] According to embodiments of this application, the signal exciter corresponding to the aforementioned signal marker includes: a type I signal exciter and a type II signal exciter; wherein, the type I signal exciter interacts with the type I signal marker to generate a type I detectable signal, and the type II signal exciter interacts with the type II signal marker to generate a type II detectable signal.

[0020] According to a preferred embodiment of this application, the type II signaling evokor enhances or inhibits the interaction between the type I signaling evokor and the type I signaling marker. By causing the activity of the nucleotide-labeled signaling marker to be lost / enhanced or inhibited / enhanced over time, different nucleotides exhibit different luminescence kinetic curves, thereby enabling the differentiation of nucleotide types.

[0021] According to embodiments of this application, the single-chain extension reaction system further includes an inhibitor, which inhibits the interaction between the type I signaling agent and the type I signaling marker or the interaction between the type II signaling agent and the type II signaling marker. The purpose of adding the inhibitor is also to enable different nucleotides to have different signal dynamics information, thereby achieving nucleotide type differentiation.

[0022] According to embodiments of this application, the single-chain extension reaction system further includes an enhancer, which enhances the interaction between the type I signal elicitor and the type I signal marker, or the interaction between the type II signal elicitor and the type II signal marker. The purpose of adding the enhancer is also to enable different nucleotides to have different signal dynamics information, thereby achieving nucleotide type differentiation.

[0023] According to embodiments of this application, the inhibitor or enhancer is selected from type III signaling activators, wherein the type I and type II signaling markers in the single-chain extension reaction system do not have activity that interacts with the type III signaling activator (e.g., catalytic activity). This avoids interfering with the resolution of signal dynamics information.

[0024] According to embodiments of this application, the inhibitor or enhancer is selected from type III signaling activators, wherein the type I and type II signaling activators in the single-chain extension reaction system do not have activity interacting with the type III signaling activator. This avoids interfering with the resolution of signal dynamics information.

[0025] According to embodiments of this application, the aforementioned type I and type II signal markers are placed in the same buffer system; during the single-strand extension reaction, the type I and type II signal markers are simultaneously introduced into the reaction system. According to a preferred embodiment of this application, the type I and type II signal activators are introduced into the system simultaneously with the type I and type II signal markers. According to a preferred embodiment of this application, the type I and type II signal activators are introduced into the reaction system after the type I and type II signal markers are introduced. This allows for determination of the nucleotide type through a single sequencing cycle.

[0026] According to embodiments of this application, type I and type II signaling activators are placed in the same buffer system; during the single-chain extension reaction, type I and type II signaling activators are simultaneously introduced into the reaction system. According to embodiments of this application, type III signaling activators are simultaneously introduced into the reaction system along with type I and type II signaling activators. According to embodiments of this application, type III signaling activators are introduced into the reaction system after type I and type II signaling activators.

[0027] This application employs single-channel sequencing, meaning all signals are detected within the same sequencing channel. Signal differentiation does not rely on physically separate optical channels, but rather on the dynamic information of the signals acquired in a single channel (such as signal intensity changes and the timing of emission peaks). [Reference] Figure 1b During the signal acquisition phase, type I and type II signal markers are simultaneously introduced into the reaction system, along with their corresponding signal elicitors. During signal acquisition, a single signal channel captures the emissive signals from the time series, and the kinetic characteristics of these signals (including signal intensity and the timing of signal peaks) reflect the incorporation of different nucleotide types. This approach enables parallel detection of multiple signals within the same channel, accurately distinguishing nucleotide types, reducing sequencing steps, shortening sequencing time, and improving sequencing efficiency.

[0028] According to embodiments of this application, the signal is an optical signal. This application does not specifically limit the type of optical signal; those skilled in the art can choose according to experimental needs, such as fluorescence, phosphorescence, chemiluminescence, bioluminescence, electroluminescence, thermoluminescence, or radioluminescence.

[0029] According to a preferred embodiment of this application, the light signal is a chemiluminescent signal or a bioluminescent signal.

[0030] According to embodiments of this application, the signal marker is an optical signal marker or an electrical signal marker. According to a preferred embodiment of this application, the optical signal marker is selected from any one of chemical signal markers, biological signal markers, and fluorescent probe markers. This application does not specifically limit the type of signal marker. Those skilled in the art can choose according to experimental needs. For example, chemical signal markers may include acridinium ester, luminol or its derivatives; biological signal markers may include luciferase, insect-derived luciferases such as firefly luciferase (FLuc) and click beetle luciferase; bacterial-derived luciferases such as bacterial luciferase (Lux); marine-derived luciferases such as renal luciferase (RLuc), deep-sea shrimp luciferase (OLuc), Gaussia luciferase (GLuc), Aequorea victoria luciferase, and variants artificially mutagenized by commercial companies, such as Promega's Nanoluc (NLuc) and BGI's G2L; fluorescent probe markers may include fluorescent protein-coupled probes, etc.

[0031] According to an embodiment of this application, in the nucleotides, the first nucleotide is labeled by the type I signal marker; the second nucleotide is labeled by the type II signal marker; the third nucleotide is labeled by both the type I and type II signal markers; and the fourth nucleotide is not labeled by a signal marker.

[0032] It should be noted that the aforementioned simultaneous labeling includes: in the single-chain extension reaction system, a portion of the third nucleotide is labeled by the type I signal marker, and another portion of the third nucleotide is labeled by the type II signal marker; wherein, the third nucleotide labeled with the type I signal marker and the third nucleotide labeled with the type II signal marker coexist in the single-chain extension reaction system. The aforementioned simultaneous labeling also includes: in the single-chain extension reaction system, each nucleotide in the third nucleotide is simultaneously labeled by both the type I signal marker and the type II signal marker, that is, the third nucleotide can be simultaneously labeled by the signal markers.

[0033] According to embodiments of this application, the aforementioned labeling includes: labeling before or after the single-chain extension reaction. Specifically, three of the four nucleotides (ribonucleotides or deoxyribonucleotides) are pre-labeled with at least one of a type I signal marker and a type II signal marker, then the ligation product pairs complementaryly with the nucleic acid to be tested, and a signal activator is added for signal detection. The second method involves labeling after the single-base extension reaction, where the four nucleotides introduced first pair complementaryly with the nucleic acid to be tested, then a signal marker is added, and once the signal marker is attached to the nucleotide, a signal activator is added for signal detection.

[0034] According to embodiments of this application, the aforementioned markings include direct markings and indirect markings.

[0035] According to embodiments of this application, the aforementioned indirect labeling is achieved through affinity interactions. These affinity interactions include antigen-antibody interactions or antigen-binding fragment interactions, and biotin-avidin interactions. The aforementioned signal marker carries an antibody or antigen-binding fragment capable of recognizing nucleotides. Labeling of nucleotides carrying modifying groups is achieved through the recognition function of the nucleotide antibody or its antigen-binding fragment.

[0036] According to other embodiments of this application, the nucleotide carrying the modifying group is further modified with biotin or an antigen. The biotin or antigen is then labeled with a signal marker. In some specific examples of this application, the aforementioned nucleotide structure is as follows: Figure 5 As shown in AE.

[0037] For example, the nucleotide carrying the modifying group is modified with biotin, and the signal marker is modified with avidin. Based on the interaction between biotin and avidin, the signal marker marks the nucleotide carrying the modifying group.

[0038] For example, a nucleotide carrying a modifying group is modified with an antigen, and a signal marker is modified with an antibody or its antigen-binding fragment. Based on the specific recognition function of the antigen and the antibody or its antigen-binding fragment, the signal marker marks the nucleotide carrying the modifying group.

[0039] According to embodiments of this application, the method of nucleotide labeling using the aforementioned antigen-antibody or antigen-binding fragment interaction and biotin-avidin interaction includes: the aforementioned type I signal marker is linked to avidin, at least a portion of the aforementioned first nucleotide and the aforementioned third nucleotide are linked to biotin, the aforementioned type II signal marker is linked to an anti-digoxigenin antibody or its antigen-binding fragment, and at least a portion of the aforementioned second nucleotide and the aforementioned third nucleotide are linked to digoxigenin; wherein, the aforementioned avidin has the activity of binding the aforementioned biotin, and the aforementioned anti-digoxigenin antibody or its antigen-binding fragment has the activity of binding the aforementioned digoxigenin.

[0040] For example, the first nucleotide is selected from dATP, the second nucleotide from dTTP, the third nucleotide from dCTP, and the fourth nucleotide from dGTP. dATP is linked to biotin, and the type I signal marker is linked to avidin, with the final ligation product having the general structural formula: dATP-biotin-avidin-type I signal marker; dTTP is linked to digoxigenin, and the type II signal marker is linked to digoxigenin antibody, with the final ligation product having the general structural formula: dTTP-dgoxigenin-dgoxigenin antibody-type II signal marker; the final ligation product of dCTP has the general structural formulas: dCTP-biotin-avidin-type I signal marker and dCTP-dgoxigenin-dgoxigenin antibody-type II signal marker; dGTP is not linked to any signal marker.

[0041] Those skilled in the art will understand that the selection of the aforementioned signal markers and nucleotide correspondences is arbitrary, and the above is only one example. The other markers are based on the same principle and will not be described in detail here.

[0042] According to other embodiments of this application, the aforementioned indirect labeling is achieved through nucleotide-nucleotide antibody or antigen-binding fragment interaction. In some embodiments, the aforementioned first nucleotide antibody or antigen-binding fragment and third nucleotide antibody or antigen-binding fragment are labeled with type I signal markers, and the second nucleotide antibody or antigen-binding fragment and third nucleotide antibody or antigen-binding fragment are labeled with type II signal markers; wherein the aforementioned nucleotide antibody or antigen-binding fragment has nucleotide recognition activity. According to embodiments of this application, the nucleotide-nucleotide antibody or antigen-binding fragment is a reversibly terminating nucleotide-antibody or antigen-binding fragment targeting a reversibly terminating nucleotide, such as a reversibly terminating guanine nucleotide-antibody or antigen-binding fragment targeting a reversibly terminating guanine nucleotide, a reversibly terminating adenine nucleotide-antibody or antigen-binding fragment targeting a reversibly terminating adenine nucleotide, a reversibly terminating thymine nucleotide-antibody or antigen-binding fragment targeting a reversibly terminating thymine nucleotide, or a reversibly terminating cytosine nucleotide-antibody or antigen-binding fragment targeting a reversibly terminating guanine cytosine nucleotide.

[0043] For example, the first nucleotide is selected from dATP, the second nucleotide is selected from dTTP, the third nucleotide is selected from dCTP, and the fourth nucleotide is selected from dGTP. The general structural formula of the final dATP ligation product is: dATP-anti-dATP antibody or its antigen-binding fragment-type I signal marker; the general structural formula of the final dTTP ligation product is: dTTP-anti-dTTP antibody or its antigen-binding fragment-type II signal marker; the general structural formula of the final dCTP ligation product is: dCTP-anti-dCTP antibody or its antigen-binding fragment-type I signal marker and dCTP-anti-dCTP antibody or its antigen-binding fragment-type II signal marker; dGTP is not ligated with a signal marker.

[0044] Those skilled in the art will understand that the selection of the aforementioned signal markers and nucleotide correspondences is arbitrary, and the above is only one example. The other markers are based on the same principle and will not be described in detail here.

[0045] According to embodiments of this application, the aforementioned single-base extension reaction is carried out in an amplification system, which further includes amplification primers and DNA polymerase. Those skilled in the art will understand that the aforementioned amplification system also includes some common sequencing reaction reagents, such as additives and buffer systems.

[0046] According to embodiments of this application, different signal markers interact with signal exciters under different suitable conditions to generate detectable signals. According to a preferred embodiment of this application, the aforementioned suitable conditions include at least one of a suitable temperature, a suitable pH, and a suitable ionic strength. According to a preferred embodiment of this application, the aforementioned suitable conditions are selected from a suitable temperature. By adjusting the suitable conditions for the reaction of different signal markers with signal exciters, distinguishable luminescence kinetic curves are obtained, thereby achieving the identification of nucleotides.

[0047] According to an embodiment of this application, the suitable temperature for the interaction between the type I signal exciter and the type I signal marker to generate a type I detectable signal is a first temperature, and the suitable temperature for the interaction between the type II signal exciter and the type II signal marker to generate a type II detectable signal is a second temperature.

[0048] According to an embodiment of this application, the aforementioned first temperature and the aforementioned second temperature are different.

[0049] According to embodiments of this application, the aforementioned first temperature is selected from 20°C to 30°C, and optionally from 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In a preferred embodiment of this application, the aforementioned first temperature is selected from 25°C.

[0050] According to embodiments of this application, the aforementioned second temperature is selected from 35°C to 45°C, and optionally from 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. In a preferred example of this application, the aforementioned second temperature is selected from 40°C.

[0051] According to an embodiment of this application, during the duration of a detectable signal, the first signal dynamics information (including signal strength, average signal strength, timing of emission peaks (i.e., the time order in which signal peaks appear) and the intensity of signal peaks) are detected in a first time period, and the second signal dynamics information (including signal strength, average signal strength, timing of emission peaks (i.e., the time order in which signal peaks appear) and the intensity of signal peaks, etc.) are detected in a second time period, wherein the end time of the first time period is not later than the start time of the second time period.

[0052] According to embodiments of this application, signal (e.g., fluorescence signal) detection is performed by simultaneously capturing images while a signal marker (e.g., luciferase) reacts with a signal elicitor (e.g., coelentin, fluorocoelentin). A signal is captured (e.g., one photograph is taken) every 0.5-9 seconds (optionally 0.5s, 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, or 9s), for a total of 2-80 consecutive captures (e.g., 2-80 photographs). In a preferred embodiment of this application, during fluorescence signal acquisition, the interval between each fluorescence signal acquisition (e.g., each photograph taken) is 0.5-3 seconds, for a total of 6-8 acquisitions (e.g., 6-8 consecutive photographs). In another preferred embodiment of this application, during fluorescence signal acquisition, the interval between each fluorescence signal acquisition (e.g., each photograph taken) is 0.5-1 seconds, for a total of 6-8 acquisitions (e.g., 6-8 consecutive photographs).

[0053] In some preferred embodiments of this application, the aforementioned signal (e.g., fluorescence signal) data is detected by continuous acquisition (e.g., exposure).

[0054] According to embodiments of this application, the first time period and the second time period are each independently selected from 1s to 10s, optionally being 1s, 2s, 3s, 4s, 5s, 6s, 7s, 8s, 9s, or 10s. In some preferred embodiments of this application, the first time period and the second time period are each independently selected from 8s.

[0055] According to embodiments of this application, during the duration of the detectable signal, the detection of the first signal dynamics information in a first time period is performed at a third temperature, and the detection of the second signal dynamics information in a second time period is performed at a fourth temperature. According to a preferred embodiment of this application, the third temperature is a suitable temperature at which the type I signal marker reacts with the type I signal exciter to generate a detectable signal. According to a preferred embodiment of this application, the fourth temperature is a suitable temperature at which the type II signal marker reacts with the type II signal exciter to generate a detectable signal.

[0056] According to embodiments of this application, the nucleotide type is determined based on the kinetic information of a first signal in the first time period and a second signal in the second time period during which a detectable signal is generated by the reaction of a signal marker with a signal activator. According to a preferred embodiment of this application, determining the nucleotide type based on the first signal intensity in the first time period and the second signal intensity in the second time period during which a detectable signal is generated by the reaction of a signal marker with a signal activator includes:

[0057] A. The first signal intensity being higher than the second signal intensity is an indication of the nucleotide labeling type I signal marker; according to a preferred embodiment of this application, the first signal intensity being higher than the second signal intensity, and the first signal intensity being greater than the detected noise signal intensity, and the second signal intensity being greater than or equal to the detected noise signal intensity, is an indication of the nucleotide labeling type I signal marker;

[0058] B. The first signal intensity is lower than the second signal intensity, which is an indication of the nucleotide labeling type II signal marker; according to a preferred embodiment of this application, the first signal intensity is lower than the second signal intensity, and both the first signal intensity and the second signal intensity are greater than the detection noise signal intensity, which is an indication of the nucleotide labeling type II signal marker;

[0059] C. The first signal intensity and the second signal intensity are substantially the same, and are indicators of the nucleotide labeling type II signal marker and type I signal marker; According to a preferred embodiment of this application, the first signal intensity and the second signal intensity are substantially the same, and both the first signal intensity and the second signal intensity are greater than the detection noise signal intensity, and are indicators of the nucleotide labeling type II signal marker and type I signal marker;

[0060] D. The fact that the first signal intensity and the second signal intensity are less than or equal to the detected noise signal intensity indicates that the nucleotide is not labeled with a signal marker.

[0061] According to an embodiment of this application, the aforementioned signal is an optical signal, and the dynamic curve of the aforementioned optical signal A is as follows: Figure 2-A is shown; the dynamic curve of the optical signal in B mentioned above is as follows: Figure 2 -B is shown; the dynamic curve of the aforementioned optical signal C is as follows: Figure 2 -C is shown; the aforementioned dynamic curve of the optical signal of D is as follows Figure 2 -D is shown.

[0062] According to other embodiments of this application, the detection of the first signal dynamics information in a first time period is performed at a fourth temperature, and the detection of the second signal dynamics information in a second time period is performed at a third temperature. According to a preferred embodiment of this application, the fourth temperature is a suitable temperature at which the type II signal marker catalyzes the emission of the type II signal exciter. According to a preferred embodiment of this application, the third temperature is a suitable temperature at which the type I signal marker catalyzes the reaction of the type I signal exciter.

[0063] According to embodiments of this application, the nucleotide type is determined based on the kinetic information of a first signal in the first time period and a second signal in the second time period during which a detectable signal is generated by the reaction of a signal marker with a signal activator. According to a preferred embodiment of this application, determining the nucleotide type based on the first signal intensity in the first time period and the second signal intensity in the second time period during which a detectable signal is generated by the reaction of a signal marker with a signal activator includes:

[0064] E. The first signal intensity is higher than the second signal intensity, which is an indication of the nucleotide labeling type II signal marker; according to a preferred embodiment of this application, the first signal intensity is higher than the second signal intensity, and both the first signal intensity and the second signal intensity are greater than the detected noise signal intensity, which is an indication of the nucleotide labeling type II signal marker;

[0065] F. The first signal intensity and the second signal intensity are substantially the same, and are indicators of the nucleotide labeling type II signal marker and type I signal marker; According to a preferred embodiment of this application, the first signal intensity and the second signal intensity are substantially the same, and both the first signal intensity and the second signal intensity are greater than the detected noise signal intensity, and are indicators of the nucleotide labeling type II signal marker and type I signal marker;

[0066] G. The first signal intensity being lower than the second signal intensity is an indication of the nucleotide labeling type I signal marker; according to a preferred embodiment of this application, the first signal intensity being lower than the second signal intensity, and the first signal intensity being greater than or equal to the detected noise signal intensity, and the second signal intensity being greater than the detected noise signal intensity, is an indication of the nucleotide labeling type I signal marker;

[0067] H. The fact that the first signal intensity and the second signal intensity are less than or equal to the detected noise signal intensity is an indication that the nucleotide is not labeled with a signal marker.

[0068] According to an embodiment of this application, the aforementioned signal is an optical signal, and the dynamic curve of the aforementioned optical signal E is as follows: Figure 3 -A is shown; the aforementioned dynamic curve of the optical signal of F is as follows: Figure 3 -B is shown; the aforementioned dynamic curve of the optical signal of G is as follows: Figure 3 -C is shown; the aforementioned dynamic curve of the optical signal of H is as follows: Figure 3 -D is shown.

[0069] According to an embodiment of this application, X signal data are collected in a first time period, and Y signal data are collected in a second time period. The maximum value of the X signal data is the first signal strength, and the maximum value of the Y signal data is the second signal strength.

[0070] According to an embodiment of this application, X signal data are collected in a first time period, and Y signal data are collected in a second time period. The average value of the X signal data is the first signal strength, and the average value of the Y signal data is the second signal strength.

[0071] Those skilled in the art will understand that, once the type of nucleotide is known, the type of nucleotide at a predetermined position on the nucleic acid sequence to be tested can be determined based on complementary base pairing.

[0072] Those skilled in the art will understand that, based on the pairing pattern between nucleotides and single-stranded nucleic acid templates, the signal dynamics information of the incorporated nucleotides can also be directly mapped to the signal dynamics information of the nucleotide to be tested, thereby directly determining the specific type of the nucleotide to be tested that pairs with the nucleotide.

[0073] It should be noted that the aforementioned signal in the first time period and the signal in the second time period are basically the same means that their signal fluctuations are within a predetermined range. This predetermined range is determined by the fact that the signal deviation between the two time periods does not exceed ±5%.

[0074] It should be noted that the aforementioned detection noise signal refers to interference signals caused by various factors, such as steric hindrance, non-specific adsorption of reagent components, signal contamination due to premature or delayed reactions of DNA nanospheres (DNB) during sequencing, etc. These factors result in some nucleotides not following the base pairing principle and falling into regions of non-sequencing target nucleotides; indiscriminate adsorption of enzyme proteins in the system onto various target nucleic acid (e.g., DNB) sites or DNBs; enzyme proteins falling into areas on the chip that should not have undergone chemical treatment to bind DNBs or enzyme proteins; or the four nucleotides currently being detected being affected by premature or delayed reactions. The detection of detection noise signals is usually due to noise in the sequencing system, impurities in reagents, non-specific reactions, or unavoidable incomplete sequencing reactions. It is understood that detection values ​​may vary between different platforms (e.g., microplate readers, sequencers).

[0075] According to embodiments of this application, the aforementioned third temperature and the aforementioned fourth temperature are different.

[0076] According to embodiments of this application, the aforementioned third temperature is selected from 20°C to 30°C, and optionally 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. According to a preferred embodiment of this application, the aforementioned third temperature is selected from 25°C. At this temperature, the signal intensity generated by the reaction of the type I signal marker with the type I signal exciter is higher.

[0077] According to embodiments of this application, the aforementioned fourth temperature is selected from 35°C to 45°C, and optionally from 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. In a preferred example of this application, the aforementioned fourth temperature is selected from 40°C. At this temperature, the signal intensity generated by the reaction of the type II signal marker catalyzing the type II signal exciter is higher.

[0078] According to embodiments of this application, the aforementioned type I signal marker and the type II signal marker are selected from luciferase.

[0079] According to embodiments of this application, the type I signaling agent and the type II signaling agent are each independently selected from coelentrin, coelentrin derivatives (e.g., fluorocoelentrin), or isomers thereof. According to a preferred embodiment of this application, the type I signaling agent is selected from coelentrin. According to a preferred embodiment of this application, the type II signaling agent is selected from fluorocoelentrin.

[0080] According to a preferred embodiment of this application, the suitable temperature for the interaction between GLuc and coelentrin to generate a detectable signal is 25°C. According to a preferred embodiment of this application, the suitable temperature for the interaction between NLuc and fluorocoelentrin to generate a detectable signal is 40°C. According to a preferred embodiment of this application, fluorocoelentrin has an inhibitory effect on the interaction between GLuc and coelentrin.

[0081] For example, the aforementioned type I and type II signal markers are selected from GLuc and NLuc, respectively; the first nucleotide is selected from dATP and labeled with GLuc; the second nucleotide is selected from dTTP and labeled with NLuc; the third nucleotide is selected from dCTP and simultaneously labeled with both GLuc and NLuc (here, "simultaneously labeled" means that some dCTP is labeled with GLuc and some dCTP is labeled with NLuc, such as half of dCTP being labeled with GLuc and half being labeled with NLuc); the fourth nucleotide is selected from dGTP and is not labeled with luciferase; the type I signal activator is selected from coelentrin; the type II signal activator is selected from fluorocoelentrin; the first temperature is 25°C, and the second temperature is 40°C. During sequencing, if a fluorescence signal is detected, the kinetic curve is as follows: Figure 2 As shown in A, the luciferase is confirmed to be GLuc, and it can be concluded that dATP binds to the nucleic acid sequence to be tested, and the corresponding position of the nucleic acid to be tested is thymine nucleotide; the kinetic curve of the detected fluorescence signal is shown in Figure A. Figure 2 As shown in B, this confirms that luciferase is an indicator of NLuc, thus indicating that dTTP binds to the nucleic acid sequence being tested, and the corresponding position of the nucleic acid is adenine nucleotide; the kinetic curve for detecting the fluorescence signal is shown in Figure B. Figure 2 As shown in C, this confirms that the luciferase is a mixture of NLuc and GLuc, indicating that dCTP binds to the nucleic acid sequence being tested, and the corresponding position of the nucleic acid is guanine nucleotide; the kinetic curve of the detected fluorescence signal is shown in Figure C. Figure 2 As shown in D, it is confirmed that the luciferase substrate did not produce a fluorescent signal, thus confirming that dGTP bound to the nucleic acid sequence to be tested is dGTP, and the corresponding position of the nucleic acid to be tested is cytosine nucleotide. The combined curve of the above fluorescence signal kinetic curves AD is shown in Figure 1. Figure 2 As shown in E.

[0082] For example, the aforementioned type I and type II signal markers are selected from GLuc and NLuc, respectively; the first nucleotide is selected from dATP and labeled with GLuc; the second nucleotide is selected from dTTP and labeled with NLuc; the third nucleotide is selected from dCTP and simultaneously labeled with both GLuc and NLuc (here, "simultaneously labeled" means that some dCTP is labeled with GLuc and some dCTP is labeled with NLuc, such as half of dCTP being labeled with GLuc and half being labeled with NLuc); the fourth nucleotide is selected from dGTP and is not labeled with luciferase; the type I signal activator is selected from coelentrin; the type II signal activator is selected from fluorocoelentrin; the first temperature is 40°C, and the second temperature is 25°C. During sequencing, if a fluorescence signal is detected, the kinetic curve is as follows: Figure 3 As shown in Figure A, the luciferase is confirmed to be Nluc, and thus the binding to the nucleic acid sequence to be tested is dTTP, with the corresponding position of the nucleic acid being adenine nucleotide; the kinetic curve of the detected fluorescence signal is shown in Figure A. Figure 3 As shown in B, this confirms that the luciferase is a mixture of NLuc and GLuc, indicating that dCTP binds to the nucleic acid sequence being tested, and the corresponding position of the nucleic acid is guanine nucleotide; the kinetic curve of the detected fluorescence signal is shown in Figure B. Figure 3 As shown in C, the luciferase is confirmed to be GLuc, and it can be concluded that dATP binds to the nucleic acid sequence to be tested, and the corresponding position of the nucleic acid to be tested is thymine nucleotide; the kinetic curve of the detected fluorescence signal is shown in Figure C. Figure 3 As shown in D, it is confirmed that the luciferase substrate did not produce a fluorescent signal, thus confirming that dGTP bound to the nucleic acid sequence to be tested is dGTP, and the corresponding position of the nucleic acid to be tested is cytosine nucleotide. The combined curve of the above fluorescence signal kinetic curves AD is shown in Figure 1. Figure 3 As shown in E.

[0083] According to embodiments of this application, the type I and type II signal markers are selected from two luciferases or variants thereof with the same luminescence type. According to embodiments of this application, the type I and type II signal markers are selected from two luciferases or variants thereof with different luminescence types.

[0084] Specifically, the type of emission is classified according to the duration of the light emitted in the emission reaction, and generally includes flash type and glow type. In some embodiments, the emission time of flash type is within a few seconds. In some embodiments, the emission time of glow type is tens of seconds, minutes, or even more than tens of minutes. Flash and glow are usually relative terms, with flash type emission time being shorter than glow type emission time.

[0085] According to embodiments of this application, both the type I and type II signal markers are selected from glow-type luciferases or their variants.

[0086] According to other embodiments of this application, both the type I and type II signal markers described above are selected from flash luciferase or variants thereof.

[0087] According to other embodiments of this application, the type I signal marker and the type II signal marker are selected from flash luciferase and glow-type luciferase, respectively. In some embodiments, the type I signal marker is selected from flash luciferase and the type II signal marker is selected from glow-type luciferase. In some embodiments, the type I signal marker is selected from glow-type luciferase and the type II signal marker is selected from flash luciferase.

[0088] It should be noted that the combination of luciferase and nucleotides described above is not limited and can be set according to experimental needs. Similarly, the method for identifying single-stranded nucleic acid templates should also be adjusted accordingly. The above examples are for illustrative purposes only.

[0089] Secondly, this application proposes a kit for implementing the method of determining nucleotide sequences by extension sequencing reaction in the first aspect.

[0090] According to embodiments of this application, a kit is proposed, comprising: a nucleotide carrying a modifying group, at least two signal markers, and at least two signal exciters, wherein the signal markers and signal exciters interact to generate a detectable signal, and during the duration of the detectable signal, the signal dynamics of the detectable signal generated by the signal markers and signal exciters varies under different reaction conditions. According to embodiments of this application, this kit enables convenient adjustment of the signal dynamics curve, reduces sequencing steps, shortens sequencing time, and improves sequencing efficiency.

[0091] According to embodiments of this application, the aforementioned nucleotides are selected from dATP, dCTP, dGTP, dTTP, or dUTP.

[0092] According to embodiments of this application, the aforementioned modifying group is selected from reversible blocking modifying groups.

[0093] According to an embodiment of this application, the kit further includes a solid support. According to an embodiment of this application, the solid support is a semiconductor chip.

[0094] According to embodiments of this application, the aforementioned signal markers include: Type I signal markers and Type II signal markers.

[0095] According to an embodiment of this application, in the nucleotides, the first nucleotide is labeled by the type I signal marker; the second nucleotide is labeled by the type II signal marker; the third nucleotide is labeled by both the type I and type II signal markers; and the fourth nucleotide is not labeled by a signal marker.

[0096] According to embodiments of this application, the aforementioned type I signal marker and type II signal marker are selected from luciferases. Exemplarily, the aforementioned type I signal marker and type II signal marker are selected from GLuc and NLuc, respectively.

[0097] According to an embodiment of this application, the signal exciter includes: a type I signal exciter and a type II signal exciter; wherein the type I signal exciter interacts with a type I signal marker to generate a type I detectable signal, and the type II signal exciter interacts with a type II signal marker to generate a type II detectable signal.

[0098] According to a preferred embodiment of this application, the type II signal exciter enhances or inhibits the interaction between the type I signal exciter and the type I signal marker. This enhancement or inhibition results in different luminescence kinetic profiles after the substrate is catalyzed.

[0099] According to embodiments of this application, the aforementioned type I signal activator and type II signal activator are independently selected from coelentrin, coelentrin derivatives, or isomers thereof. Exemplarily, the type I signal activator is selected from coelentrin; the type II signal activator is selected from fluorocoelentrin.

[0100] According to embodiments of this application, the aforementioned kit further comprises: an inhibitor, said inhibitor having an inhibitory effect on the interaction between the type I signaling activator and the type I signaling marker or the interaction between the type II signaling activator and the type II signaling marker. Through this inhibitory effect, the reaction between the signaling marker and the signaling activator produces different signal dynamics curves.

[0101] According to embodiments of this application, the aforementioned kit further includes: an enhancer, said enhancer having an enhancing effect on the interaction between the type I signal elicitor and the type I signal marker, or the interaction between the type II signal elicitor and the type II signal marker. Through this enhancing effect, the reaction between the signal marker and the signal elicitor produces different signal dynamics curves.

[0102] According to embodiments of this application, the inhibitor or enhancer is selected from type III signaling activators, wherein the type I and type II signaling markers in the single-chain extension reaction system do not have activity that interacts with the type III signaling activator (e.g., catalytic activity), in order to avoid interference with the signal.

[0103] According to embodiments of this application, the inhibitor or enhancer is selected from type III signaling activators, wherein the type I and type II signaling activators in the single-chain extension reaction system do not have activity that interacts with the type III signaling activator, in order to avoid interference with the signal.

[0104] According to embodiments of this application, the aforementioned type I and type II signal markers are placed in the same buffer system; during the single-strand extension reaction, the type I and type II signal markers are simultaneously introduced into the reaction system. According to a preferred embodiment of this application, the type I and type II signal activators are introduced into the system simultaneously with the type I and type II signal markers. According to a preferred embodiment of this application, the type I and type II signal activators are introduced into the reaction system after the type I and type II signal markers are introduced. This allows for determination of the nucleotide type through a single sequencing cycle.

[0105] According to embodiments of this application, type I and type II signaling activators are placed in the same buffer system; during the single-chain extension reaction, type I and type II signaling activators are simultaneously introduced into the reaction system. According to embodiments of this application, type III signaling activators are simultaneously introduced into the reaction system along with type I and type II signaling activators. According to embodiments of this application, type III signaling activators are introduced into the reaction system after type I and type II signaling activators.

[0106] According to embodiments of this application, the signal marker is an optical signal marker or an electrical signal marker. According to a preferred embodiment of this application, the optical signal marker is selected from any one of chemical signal markers, biological signal markers, and fluorescent probe markers.

[0107] According to embodiments of this application, the reaction conditions include at least one of temperature, pH, and ionic strength. According to a preferred embodiment of this application, the reaction conditions are selected from temperature. By adjusting the reaction conditions between the signal marker and the signal exciter, distinguishable luminescence kinetic curves are obtained, thereby achieving the identification of nucleotides.

[0108] According to a preferred embodiment of this application, at a third temperature, the type I signal exciter and the type I signal marker generate first signal kinetic information of a type I detectable signal, and the type II signal exciter and the type II signal marker generate first signal kinetic information of a type II detectable signal; at a fourth temperature, the type I signal exciter and the type I signal marker generate second signal kinetic information of a type I detectable signal, and the type II signal exciter and the type II signal marker generate second signal kinetic information of a type II detectable signal, wherein the changes in the first and second signal kinetic information of the type I detectable signal are different from the changes in the first and second signal kinetic information of the type II detectable signal.

[0109] According to embodiments of this application, the aforementioned third temperature and the aforementioned fourth temperature are different.

[0110] According to embodiments of this application, the aforementioned third temperature is selected from 20°C to 30°C, and optionally from 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. According to a preferred embodiment of this application, the aforementioned third temperature is selected from 25°C.

[0111] According to embodiments of this application, the aforementioned fourth temperature is selected from 35°C to 45°C, and optionally from 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. According to a preferred embodiment of this application, the aforementioned fourth temperature is selected from 40°C.

[0112] According to a preferred embodiment of this application, the reaction conditions are suitable conditions under which the signal marker interacts with the signal exciter to generate a detectable signal.

[0113] According to embodiments of this application, different signal markers interact with signal exciters under different suitable conditions to generate detectable signals. According to a preferred embodiment of this application, the aforementioned suitable conditions include at least one of a suitable temperature, a suitable pH, and a suitable ionic strength. According to a preferred embodiment of this application, the aforementioned suitable conditions are selected from a suitable temperature. By adjusting the suitable conditions for the reaction of different signal markers with signal exciters, distinguishable luminescence kinetic curves are obtained, thereby achieving the identification of nucleotides.

[0114] According to embodiments of this application, the suitable temperature for the interaction between the type I signal exciter and the type I signal marker to generate a type I detectable signal is a first temperature, and the suitable temperature for the interaction between the type II signal exciter and the type II signal marker to generate a type II detectable signal is a second temperature. According to a preferred embodiment of this application, the aforementioned first temperature and the aforementioned second temperature are different.

[0115] According to a preferred embodiment of this application, the third temperature is selected from the first temperature. According to a preferred embodiment of this application, the fourth temperature is selected from the second temperature.

[0116] According to embodiments of this application, the aforementioned first temperature is selected from 20°C to 30°C, and optionally from 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. According to a preferred embodiment of this application, the aforementioned first temperature is selected from 25°C.

[0117] According to embodiments of this application, the aforementioned second temperature is selected from 35°C to 45°C, and optionally from 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. According to a preferred embodiment of this application, the aforementioned second temperature is selected from 40°C.

[0118] It should be understood that, within the scope of this application, the above-described technical features of this application and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0119] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0120] Figure 1a This is a schematic diagram of the traditional monochromatic signal sequencing workflow acquisition and analysis mode.

[0121] Figure 1b This is a schematic diagram of the single-channel monochrome signal sequencing workflow acquisition and analysis mode provided in the embodiments of this application;

[0122] Figure 2 This is a schematic diagram of the photodynamic curve of the reaction between the signal marker and the signal exciter provided in the embodiments of this application, wherein the detection temperature in the first time period is 25°C and the detection temperature in the second time period is 40°C.

[0123] Figure 3 This is a schematic diagram showing the effect of the dilution of luciferase and substrate on the luciferase luminescence kinetics curve provided in the embodiments of this application. The detection temperature in the first time period is 40°C, and the detection temperature in the second time period is 25°C.

[0124] Figure 4 This is a schematic diagram of the clustering detection results of different sequencing cycle base signals provided in the embodiments of this application, wherein A is C001; B is C050; and C is C100.

[0125] Figure 5This is a schematic diagram of the structure of a nucleotide carrying a modifying group and modified with biotin or antigen, provided in an embodiment of this application. Detailed Implementation

[0126] Embodiments of the present invention are described in detail below, with examples of the foregoing embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0127] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0128] The term "solid support" refers to a solid material with stable physical properties and a modifiable chemical surface, used to immobilize biomolecules such as nucleic acids, proteins, and cells, providing a reproducible platform to support the interaction, signal transduction, or functional detection of specific biomolecules with reagents in biological experiments or reaction systems. In this application, the solid support is used to immobilize single-stranded nucleic acid templates, providing a stable physical platform for extended sequencing reactions. Its surface is functionalized to effectively bind nucleic acid molecules and support multiple rounds of reactions and signal detection. Common types of solid supports include glass or silicon wafers, plastic microplates, magnetic beads, gel matrices, and porous membranes.

[0129] The term "fluorescence" refers to the phenomenon where certain substances rapidly emit lower-energy photons after absorbing high-energy photons (such as ultraviolet or blue light). The term "phosphorescence" refers to the phenomenon where certain substances emit light over a relatively long period (from milliseconds to hours or even longer) after absorbing photons. This is because electrons pass through a triplet state when returning from the excited state to the ground state, resulting in a delayed emission. The term "chemiluminescence" refers to the phenomenon where reactants release energy and emit light as a form during a chemical reaction. The term "bioluminescence" refers to the cold light phenomenon produced by certain organisms through chemical reactions. This process is usually produced by the reaction of luciferin with oxygen catalyzed by luciferase. More specifically, the term "fluorescence" refers to the cold light phenomenon produced by certain organisms through chemical reactions, usually produced by the reaction of luciferin with oxygen catalyzed by luciferase, which releases energy and emits light as a form. The term "electroluminescence" refers to the phenomenon where certain materials emit light under the influence of an electric field. Electrons transition to an excited state under the influence of an electric field, and then release photons upon returning to the ground state. The term "thermoluminescence" refers to the phenomenon where certain materials release previously absorbed energy as light when heated. The term "radioluminescence" refers to the light emitted by certain materials under the influence of radioactive radiation (such as alpha, beta, and gamma rays).

[0130] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0131] Example 1: Base resolution and sequencing applications based on luminescence kinetics curves

[0132] 1. Experimental Samples and Reagents

[0133] Sequencing sample: E. coli DNA double-stranded PCR product;

[0134] Reagent kit: DNBSEQ One-Step DNB Preparation Reagent Kit V2.0 (OS-SB) (Manufacturer: MGI);

[0135] The DNBSEQ-E25RS high-throughput sequencing kit (FCL SE100) (manufacturer: MGI, catalog number: 940-000570-00) contains luciferase SA-Gluc, luciferase Anti-Dig-Nluc; dilution buffer 1: signal factor buffer; signal reagent 1 (containing fluorocoelenthalpy); and signal reagent 2 (containing natural coelenthalpy).

[0136] 2. Experimental apparatus

[0137] DNBSEQ-E5RS gene sequencer (manufacturer: MGI);

[0138] DNBSEQ-E5 sequencing slide (manufacturer: MGI);

[0139] PCR amplification instrument (manufacturer: Life Pro gene amplification instrument).

[0140] 3. Experimental Procedure

[0141] 1) Following the instructions of the DNBSEQ-E25RS sequencing kit, the E. coli DNA double-stranded PCR product was circularized and rolled around to prepare DNA nanospheres. The DNA rolling circle replication time was 40 min.

[0142] 2) Mix signal reagent 1 and signal reagent 2 in a 1:1 ratio.

[0143] 3) Reagent kit preparation: Manually fill the reagents required for the sequencing process. The reagent components include Hot reagents (dNTPs-biotin). 、 The kit contains dNTPs-digoxin and BG9 polymerase, Cold reagent (dNTPs and BG9 polymerase), sequencing primers, and the signal reagent from step 2). Luciferase SA-Gluc and Luciferase Anti-Dig-Nluc are diluted using dilution buffer 1, and the diluted solutions are then loaded into the corresponding positions in the kit.

[0144] 4) Sequencing logic modification: The sequencing script of DNBSEQ-E5RS was modified to change the substrate reaction temperature during the sequencing process. Temperature control was achieved by modifying the E5 temperature control parameters. After the signal reagent was introduced, the reaction was run according to the following logic: 40℃ for 8s; 25℃ for 8s, imaging exposure time was 1 frame per second, and 1 image was captured per second.

[0145] 5) Chip loading: Select one chip from the QC (quality control) qualified batch and open the transparent vacuum packaging bag to load the chip.

[0146] 6) Set up the experimental program, correctly place the DNBSEQ-E5RS sequencing kit into the sequencer and run the reagent slot one-click loading function.

[0147] 7) Use a pipette and wide-mouth pipette tip to add the DNB loading reaction system prepared in step 2) into the DNB well of the sequencing slide.

[0148] 8) Start sequencing

[0149] 4. Experimental Results and Analysis

[0150] Based on affinity and labeling, the acquired photodynamic curves show a one-to-one correspondence with the four types of bases. The ideal state of the photodynamic curves for the four types of bases is as follows: Figure 3 。 Feature point extraction and downstream bioinformatics analysis of the acquired signals yielded the following results: Figure 4 Clustering results are shown in Table 1, along with the SE100 sequencing metrics.

[0151] Table 1 Sequencing Indicators

[0152] CycleNumber (number of loops) 100 Total Reads (M) 1 MappedReads(M) (M) (Comparison Reads) 0.86 MappingRate(%) (Match Rate, %) 86.07 AvgErrorRate(%) (Average Error Rate, %) 2.39 AvgErrorRate! N(%) (Average error rate, %) after removing N bits. 2.39

[0153] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0154] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for determining nucleotide sequences by extension sequencing, characterized in that, include: A) Provide single-stranded nucleic acid templates that can be arranged on the surface of a solid support; B) In each cycle of the extension sequencing reaction, determine the nucleotide type at the detection site in the single-stranded nucleic acid template, including: i) Incorporating nucleotides carrying modifying groups into the complementary strand of the single-stranded nucleic acid template, wherein each nucleotide is labeled with a different signal marker; ii) First signal dynamics information of the detectable signal generated by the reaction of the signal marker and the signal exciter; iii) Change the reaction conditions between the signal marker and the signal exciter; iv) Detect the second signal dynamics information of the detectable signal generated by the reaction of the signal marker and the signal exciter; v) Based on the first signal dynamics information and the second signal dynamics information, determine the type of incorporated nucleotide, and / or determine the type of nucleotide at the detection position in the single-stranded nucleic acid template.

2. The method according to claim 1, characterized in that, Further includes: C) Remove the modifying groups carried by the nucleotides to allow the complementary strand to continue to extend; Optionally, the modifying group is selected from reversible blocking modifying groups; Optionally, repeat steps B)-C) until the complementary strand is fully extended to determine the single-stranded nucleic acid template sequence; Optionally, the nucleotide is selected from dATP, dCTP, dGTP, dTTP, or dUTP.

3. The method according to claim 1, characterized in that, The signal markers include: Type I signal markers and Type II signal markers; Optionally, the signal exciter includes: a type I signal exciter and a type II signal exciter; wherein the type I signal exciter interacts with a type I signal marker to generate a type I detectable signal, and the type II signal exciter interacts with a type II signal marker to generate a type II detectable signal; Preferably, the type II signaling exciter enhances or inhibits the interaction between the type I signaling exciter and the type I signaling marker; Optionally, the extended sequencing reaction system further includes: an inhibitor or an enhancer, wherein the inhibitor inhibits the interaction between the type I signal activator and the type I signal marker or the interaction between the type II signal activator and the type II signal marker, and the enhancer enhances the interaction between the type I signal activator and the type I signal marker or the interaction between the type II signal activator and the type II signal marker; Optionally, the type I signal marker and the type II signal marker are placed in the same buffer system; Preferably, the signal is an optical signal; preferably, the optical signal is a chemiluminescent signal or a bioluminescent signal.

4. The method according to claim 3, characterized in that, In the nucleotides, the first nucleotide is labeled with the type I signal marker; The second nucleotide is labeled with the type II signal marker; The third nucleotide is simultaneously labeled by both the type I signal marker and the type II signal marker; The fourth nucleotide is not tagged by a signal marker.

5. The method according to any one of claims 1-4, characterized in that, Different signal markers interact with signal exciters under different suitable conditions to generate detectable signals; Preferably, the suitable conditions include at least one of: suitable temperature, suitable pH, and suitable ionic strength; Preferably, the suitable conditions are selected from suitable temperatures; Preferably, the suitable temperature for the interaction between the type I signal exciter and the type I signal marker to generate a type I detectable signal is a first temperature, and the suitable temperature for the interaction between the type II signal exciter and the type II signal marker to generate a type II detectable signal is a second temperature. Preferably, the first temperature is selected from 20℃ to 30℃, and the second temperature is selected from 35℃ to 45℃.

6. The method according to any one of claims 1-4, characterized in that, The reaction conditions include at least one of temperature, pH, and ionic strength; Preferably, the reaction conditions are selected from temperature; Preferably, the temperature at which the first signal dynamics information is detected is a third temperature, and the temperature at which the second signal dynamics information is detected is a fourth temperature; Preferably, the third temperature is selected from 20℃ to 30℃, and the fourth temperature is selected from 35℃ to 45℃.

7. The method according to claim 3, characterized in that, The type I signal marker and the type II signal marker are selected from luciferase; Optionally, the type I signaling agent and the type II signaling agent are each independently selected from coelentrin, coelentrin derivatives or isomers thereof.

8. A reagent kit, characterized in that, include: The invention comprises a nucleotide carrying a modifying group, at least two signal markers, and at least two signal activators, wherein the signal markers and signal activators interact to generate a detectable signal, and the signal dynamics of the detectable signal generated by the signal markers and signal activators vary under different reaction conditions during the duration of the detectable signal. Optionally, the modifying group is selected from reversible blocking modifying groups; Optionally, the kit further includes a solid support.

9. The reagent kit according to claim 8, characterized in that, The signal markers include: Type I signal markers and Type II signal markers; Optionally, the type I signal marker and the type II signal marker are selected from luciferase; Optionally, the signal exciter includes: a type I signal exciter and a type II signal exciter; wherein the type I signal exciter interacts with a type I signal marker to generate a type I detectable signal, and the type II signal exciter interacts with a type II signal marker to generate a type II detectable signal; Preferably, the type II signaling exciter enhances or inhibits the interaction between the type I signaling exciter and the type I signaling marker; Optionally, the type I signal activator and the type II signal activator are each independently selected from coelentrin, coelentrin derivatives or isomers thereof; Optionally, the kit further comprises: an inhibitor or an enhancer, the inhibitor having an inhibitory effect on the interaction between the type I signaling activator and the type I signaling marker or the interaction between the type II signaling activator and the type II signaling marker, and the enhancer having an enhancing effect on the interaction between the type I signaling activator and the type I signaling marker or the interaction between the type II signaling activator and the type II signaling marker; Optionally, the type I signal marker and the type II signal marker are placed in the same buffer system; Preferably, the signal marker is an optical signal marker or an electrical signal marker. More preferably, the optical signal marker is selected from any one of chemical signal markers, biological signal markers, and fluorescent probe markers.

10. The reagent kit according to claim 9, characterized in that, Reaction conditions include at least one of the following: temperature, pH, and ionic strength; Preferably, the reaction conditions are selected from temperature; Preferably, at a third temperature, the type I signal exciter and the type I signal marker generate first signal kinetic information for a type I detectable signal, and the type II signal exciter and the type II signal marker generate first signal kinetic information for a type II detectable signal; at a fourth temperature, the type I signal exciter and the type I signal marker generate second signal kinetic information for a type I detectable signal, and the type II signal exciter and the type II signal marker generate second signal kinetic information for a type II detectable signal, wherein the changes in the first and second signal kinetic information of the type I detectable signal are different from the changes in the first and second signal kinetic information of the type II detectable signal. Preferably, the reaction conditions are selected from suitable conditions under which the signal marker interacts with the signal exciter to generate a detectable signal; Preferably, different signal markers interact with signal exciters under different suitable conditions to generate detectable signals.

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