HK40063186A

HK40063186A - Method for sequencing polynucleotides on basis of optical signal dynamics of luminescent label and secondary luminescent signal

Info

Publication number: HK40063186A
Application number: HK62022052519.5A
Authority: HK
Inventors: 陈曦; 廖莎; 章文蔚; 陈奥; 赵杰
Original assignee: 青岛华大智造科技有限责任公司
Filing date: 2019-08-20
Publication date: 2022-06-17

Description

Method for sequencing polynucleotide based on luminescent marker optical signal dynamics and secondary luminescent signal

Technical Field

The present invention relates to the field of nucleic acid sequencing. In particular, the present invention provides a method for sequencing polynucleotides based on the kinetics of luminescent label light signals and secondary luminescent signals, wherein the sequential incorporation of different nucleotides is resolved by using different luminescent forms and luminescent timings, thereby achieving the determination of polynucleotide sequence.

Background

The DNA sequencing technology includes a first generation DNA sequencing technology represented by Sanger (Sanger) sequencing method and a second generation DNA sequencing technology represented by Illumina Hiseq2500, Roche 454, ABI Solid, BGISEQ-500 and the like. In 1977, sanger invented dideoxy end-termination sequencing, which was representative of the first generation of sequencing technology. In 2001, human genome sketches were completed by means of the first generation sequencing technology. The Sanger sequencing method has the characteristics of simple experimental operation, intuitive and accurate result, short experimental period and the like, and is widely applied to the fields of clinical gene mutation detection, genotyping and the like with high requirements on timeliness of detection results. However, sanger sequencing has the disadvantages of low throughput and high cost, which limits its application in large-scale gene sequencing. In order to overcome the defects of the Sanger sequencing method, the second generation sequencing technology is developed. Compared with the first generation DNA sequencing technology, the second generation DNA sequencing technology has the characteristics of large sequencing flux, low cost, high automation degree and single molecule sequencing. Taking the sequencing technology of Hiseq2500V2 as an example, one experimental flow can generate data of 10-200G bases, the average sequencing cost of each base is less than 1/1000 of the sequencing cost of Sanger sequencing method, and the obtained sequencing result can be directly processed and analyzed by a computer. Therefore, second generation DNA sequencing technologies are well suited for large scale sequencing.

Over the past 10 years, the second generation gene sequencing technology has gradually grown from the emerging technology to the mainstream sequencing means, and gradually becomes an important detection tool in the clinical field, and plays an increasingly important role in the fields of infectious disease defense and control, genetic disease diagnosis, noninvasive prenatal screening and the like. In order to further expand the sequencing market, the sequencing instrument is civilized, and the development of a low-cost miniaturized sequencing instrument gradually becomes a development trend in the sequencing field. As a classic means of the second-generation sequencing technology, three sequencing methods based on four channels, two channels and a single channel are thousands of years, but compared with the three methods, single-channel sequencing has gradually become a development trend in the sequencing field due to the advantages of less material consumption, lower cost, easier realization of miniaturization and portability of instruments and the like. The current products based on single color channel on the market mainly include the sequencer of ion torrent series, 454 sequencer and Iseq100 newly introduced by illumina corporation.

The monochromatic signal sequencing is a method which utilizes chemiluminescent markers on bases to emit the same signals under specific conditions as required to identify four base types so as to achieve the purpose of sequencing. The method has the characteristics of simple signal identification, rapid biochemical reaction, high sequencing flux and the like, and is a mainstream means for second-generation sequencing at present. The technology is utilized by a small rapid sequencer based on monochromatic fluorescence imaging of Illumina. However, due to the inherent deficiencies of various DNA enrichment approaches, the sensitive response of the signal response to environmental and instrumental conditions, the uniformity of the luminescent marker signal intensity in both the time dimension and the spatial dimension of the test procedure is lacking. The monochromatic signal sequencing method is to determine four corresponding bases by using 1/0, 1/1, 0/1 and 0/0 four luminescence modes through twice detection and control of luminescence conditions. The detection mode does not distinguish the frequency of the optical signal, and only distinguishes the signal intensity and the signal generation time. The method has high dependence on signal intensity, so that the sequencing result is dispersed, and the accuracy of base identification is reduced.

Therefore, there is still a need in the art for a monochromatic signal sequencing method with greater base resolution accuracy.

Summary of The Invention

In order to solve the above technical problems, the inventors of the present application developed a new sequencing method that uses different luminescence forms and luminescence timings to resolve four bases, a, (T/U), C, and G. The sequencing method is based on a chemiluminescence reaction kinetic curve, a chemiluminescence marker marked on a specific base is inactivated by a selective chemical bond breaking method, and secondary luminescence judgment is carried out by utilizing the luminescence capacity of the rest of the luminescence markers, so that the base is identified. As only two characteristics of Flash and Glow are required to be selected for the chemiluminescence kinetic curve, confusion caused by unobvious kinetic curve characteristics can be greatly reduced. Meanwhile, for the second luminescence, only the signal intensity is required to exceed the background value, and the dynamic curve characteristics are not required, so that the influence of side reactions on signal identification in the reaction process can be reduced, and the base resolution precision is improved.

In one aspect, the invention provides a method of sequencing a nucleic acid molecule comprising monitoring the sequential incorporation of nucleotides complementary to the nucleic acid molecule, wherein the nucleotides are each attached to a chemiluminescent label that elicits a different luminescent kinetics, wherein each incorporated nucleotide is identified by detecting the luminescent kinetics of a chemiluminescent reaction in which the chemiluminescent label is involved and subsequently removing a portion of the chemiluminescent label.

In a specific embodiment, the ribose or deoxyribose moiety of each of the nucleotides comprises a protecting group attached through a 2' or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of each nucleotide so as to expose a 3' -OH group.

In a specific embodiment, a portion of the chemiluminescent label is removed alone and another portion of the chemiluminescent label and the protecting group are removed under the same conditions.

In a specific embodiment, the nucleotide is selected from nucleotide A, G, C and T or U.

In particular embodiments, detection of the luminescent kinetics of a chemiluminescent reaction in which a chemiluminescent label is involved comprises contacting the chemiluminescent label with a suitable substrate to trigger the chemiluminescent reaction, and detecting the luminescent kinetics of the light emitted thereby.

In a specific embodiment, the chemiluminescent markers are selected from the group consisting of biochemical luminescent markers that elicit different luminescent kinetics and any combination thereof.

In a specific embodiment, the chemiluminescent label is selected from the group consisting of luciferases that elicit different luminescence kinetics and any combination thereof.

In a specific embodiment, the chemiluminescent label is a combination of two luciferases that elicit different luminescence kinetics.

In particular embodiments, the lighting types include flash type (flash) and glow type (glow).

In one aspect, the invention provides a method of sequencing a nucleic acid molecule, comprising:

(1) providing four nucleotides, wherein a first nucleotide is labeled with a first chemiluminescent label via a second linking group, a second nucleotide is labeled with a first chemiluminescent label via a first linking group, a third nucleotide is labeled with a second chemiluminescent label via a third linking group, and a fourth nucleotide is not labeled with a chemiluminescent label;

(2) incorporating one of four nucleotides onto a complementary strand of the nucleic acid molecule;

(3) detecting a chemiluminescent label of the nucleotide of (2);

(4) adding a cleavage reagent to cleave the second linking group;

(5) detecting chemiluminescent labels of the cleaved nucleotides to determine the type of nucleotide incorporated;

(6) removing the remaining chemiluminescent label; and

(7) optionally, repeating steps (2) - (5) or (2) - (6) one or more times to determine the sequence of the nucleic acid molecule.

In a specific embodiment, detecting the chemiluminescent label of the nucleotides of (2) and (5) comprises contacting the chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the kinetics of the luminescence emitted thereby.

In a specific embodiment, the ribose or deoxyribose moiety of each of the nucleotides comprises a protecting group attached through a 2' or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of the nucleotide so as to expose the 3' -OH group.

In particular embodiments, the first linking group and the third linking group can be the same or different, and the second linking group and the third linking group are different.

In various aspects, the attachment between the nucleotide and the chemiluminescent label comprises an attachment mediated by affinity interaction.

In particular embodiments, affinity interactions include antigen-antibody interactions and biotin-avidin (e.g., streptavidin) interactions.

In particular embodiments, the chemiluminescent label is attached to the nucleotide by an affinity interaction between members involved in the affinity interaction by attaching the chemiluminescent label to one of the members and the nucleotide to the other member involved in the affinity interaction.

In a specific embodiment, the member attached to the nucleotide is biotin and the member attached to the chemiluminescent label is avidin (e.g., streptavidin).

In a specific embodiment, the member attached to the nucleotide is digoxigenin and the member attached to the chemiluminescent label is an anti-digoxigenin antibody.

In a specific embodiment, the member attached to the nucleotide is biotin and the member attached to the chemiluminescent label is avidin (e.g., streptavidin), wherein digoxin is affinity bound to avidin by an anti-digoxin antibody attached to biotin.

In a specific embodiment, the first nucleotide is attached to the first luciferase via a second linking group (linker2), the second nucleotide is attached to the first luciferase via a first linking group (linker1), the third nucleotide is attached to the second luciferase via a third linking group (linker1), and the fourth nucleotide is not attached to any luciferase.

Wherein the first linking group and the third linking group may be the same or different and the second linking group and the third linking group are different.

In one aspect, the present invention provides a method of sequencing a nucleic acid molecule, comprising the steps of:

(1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching a nucleic acid molecule to be sequenced to a support;

(2) adding a primer for initiating a nucleotide polymerization reaction, a polymerase for performing the nucleotide polymerization reaction, and four nucleotides, thereby forming a reaction system containing a solution phase and a solid phase; wherein, the four nucleotides are respectively derivatives of nucleotide A, (T/U), C and G, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first nucleotide is linked to a first molecular tag by a cleavable linker2,

the second nucleotide is linked to a first molecular tag by a cleavable linker1,

the third nucleotide is linked to a second molecular tag by a cleavable linker1,

the fourth nucleotide is not connected with a molecular marker;

(3) annealing a primer to a nucleic acid molecule to be sequenced, said primer acting as an initial growing nucleic acid strand, together with said nucleic acid molecule to be sequenced, forming a support-attached duplex;

(4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform the nucleotide polymerization reaction, thereby incorporating one of the four nucleotides into the 3' end of the growing nucleic acid strand;

(5) contacting the duplex of the previous step with two different luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively, and performing a binding reaction, and then subjecting the luciferases to a fluorescence reaction in the presence of a substrate to detect the emitted fluorescent signal;

(6) adding lysis solution to lyse the linker2, then enabling the luciferase to perform a fluorescence reaction again in the presence of a substrate, and detecting the emitted fluorescence signal;

(7) removing the molecular marker of each nucleotide;

(8) optionally repeating steps (3) - (7) to obtain sequence information of said nucleic acid molecule.

In a specific embodiment, the first molecular tag is biotin, linked to the first luciferase is streptavidin, and the attachment of the nucleotide to the first luciferase is via the affinity interaction of biotin and streptavidin.

In a specific embodiment, the second molecular tag is digoxin, linked to the second luciferase is an anti-digoxin antibody, and the attachment of the nucleotide to the second luciferase is achieved by affinity interaction of digoxin and the anti-digoxin antibody.

In an embodiment of the invention, the molecular marker is connected with nucleotide derivatives through different linkers, then luciferase capable of specifically binding the molecular marker is added, and the sequential incorporation of different nucleotides is distinguished by detecting fluorescent signals emitted by the luciferase before and after the linker cleavage, thereby realizing the determination of the polynucleotide sequence. In particular embodiments. In specific embodiments, the first nucleotide is attached to the first molecular tag by linker2, or by linker1-linker 2; the second nucleotide is linked to the first molecular label by linker 1; the third nucleotide is linked with a second molecular label through a linker 1; the fourth nucleotide is not linked to a molecular tag. Detecting a first luminescent signal by adding a luciferase and a corresponding substrate capable of specifically binding to the first and second molecular labels, respectively; and then the linker is fragmented according to needs, a secondary luminescence signal after the fragmentation is detected, and the base recognition can be carried out according to the luminescence conditions of the four nucleotides.

Thus, in an exemplary embodiment, the method of sequencing a nucleic acid molecule of the present invention comprises the steps of:

the first nucleotide is linked to a first molecular tag by a cleavable linker2,

the fourth nucleotide is not connected with a molecular marker;

(5) removing the solution phase of the reaction system of the previous step, retaining the duplexes attached to the support, and adding two different luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively, to perform a binding reaction;

(6) removing unbound luciferase with an elution buffer;

(7) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(8) removing the solution of the previous step;

(9) adding lysis solution to crack linker 2;

(10) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(11) optionally removing linker1 and 3' protecting groups from each nucleotide;

(12) optionally removing the solution of the previous step;

(13) optionally repeating steps (3) - (12) or (3) - (10) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

In a specific embodiment, the linkage of the two luciferases to the nucleotide can be performed separately, for example, a first luciferase labeled with a first molecule is added to perform a binding reaction with the nucleotide labeled with the first molecule, then the unbound first luciferase is removed with an elution buffer, a substrate of the first luciferase is added, and a fluorescent signal is detected; then adding a second luciferase labeled with a second molecule to perform a binding reaction with the nucleotide labeled with the second molecule, removing the unbound second luciferase with an elution buffer, adding a substrate of the second luciferase, and detecting a fluorescent signal. In particular, the present invention provides a method of sequencing a nucleic acid molecule comprising the steps of:

the first nucleotide is linked to a first molecular tag by a cleavable linker2,

the fourth nucleotide is not connected with a molecular marker;

(5) removing the solution phase of the reaction system of the previous step, retaining the duplexes attached to the support, and adding a first luciferase capable of specifically binding to the first molecular label to perform a binding reaction;

(6) removing unbound first luciferase with an elution buffer;

(7) adding a substrate of the first luciferase and detecting a curve of the change of the fluorescent signal along with time;

(8) removing the solution of the previous step;

(9) adding a second luciferase capable of specifically binding to the second molecular marker to perform a binding reaction;

(10) removing unbound second luciferase with an elution buffer;

(11) adding a substrate of a second luciferase and detecting a curve of the change of the fluorescence signal along with time;

(12) optionally removing the solution of the previous step;

(13) adding lysis solution to crack linker 2;

(14) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(15) optionally removing linker1 and 3' protecting groups from each nucleotide

(16) Optionally removing the solution of the previous step;

(17) optionally repeating steps (3) - (16) or (3) - (14) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

In another aspect, the invention also relates to a kit for sequencing a polynucleotide, comprising:

(a) four nucleotides which are derivatives of nucleotides A, (T/U), C and G, respectively, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first nucleotide is linked to a first molecular tag by a cleavable linker2,

the fourth nucleotide is not connected with a molecular marker;

(b) two luciferases capable of specifically binding to the first molecular marker and the second molecular marker respectively, the two luciferases may be different mutants of the same luciferase or different luciferases; and

(c) lysis solution for lysis of linker 2.

In certain preferred embodiments, the kits of the invention further comprise: reagents and/or devices for extracting nucleic acid molecules from a sample; reagents for pretreating nucleic acid molecules; a support for attaching nucleic acid molecules to be sequenced; reagents for attaching (e.g., covalently or non-covalently attaching) a nucleic acid molecule to be sequenced to a support; a primer for initiating a nucleotide polymerization reaction; a polymerase for performing a nucleotide polymerization reaction; one or more buffer solutions; one or more wash solutions; or any combination thereof.

Drawings

FIG. 1 shows a flow chart of a sequencing method of the invention.

FIG. 2 shows the sequencing results of an example of a sequencing method according to the invention, showing the sequencing signal curve for each sequencing cycle.

FIG. 3 shows the sequencing results of an example of a sequencing method according to the invention, showing the sequencing signal curve for each sequencing cycle.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, and other publications mentioned herein are incorporated by reference in their entirety. If a definition set forth herein conflicts or disagrees with a definition set forth in a patent, application, or other publication incorporated by reference, the definition set forth herein controls.

As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogs thereof. Polynucleotides may be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. The polynucleotide molecules may be derived from double stranded DNA (dsDNA) forms (e.g., genomic DNA, PCR and amplification products, etc.), or may be derived from single stranded forms of DNA (ssdna) or RNA and may be converted to dsDNA forms, and vice versa. The exact sequence of the polynucleotide molecule may be known or unknown. The following are illustrative examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer, or amplified copy of any of the foregoing.

The polynucleotide may comprise a nucleotide or nucleotide analog. Nucleotides generally contain a sugar (e.g., ribose or deoxyribose), a base, and at least one phosphate group. The nucleotide may be abasic (i.e., lacking bases). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, Adenosine Monophosphate (AMP), Adenosine Diphosphate (ADP), Adenosine Triphosphate (ATP), Thymidine Monophosphate (TMP), Thymidine Diphosphate (TDP), Thymidine Triphosphate (TTP), cytidine diphosphate (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 diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTTP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP TP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), Deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP). Nucleotide analogs comprising modified bases may also be used in the methods described herein. Exemplary modified bases that can be included in a polynucleotide, whether having a natural backbone or a similar structure, include, for example, inosine, xanthine (xathanine), hypoxanthine (hypoxathanine), 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-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azacytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-amino adenine or guanine, 8-thio adenine or guanine, 8-sulfanyl adenine or guanine, 8-hydroxy adenine or guanine, 5-halogen substituted uracil or cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine and the like. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, for example, nucleotide analogs such as adenosine 5' -phosphosulfate.

Generally, a nucleotide includes nucleotide A, C, G, T or U. The term "nucleotide a" as used herein refers to a nucleotide containing adenine (a) or a modification or analog thereof, e.g., ATP, dATP. "nucleotide G" refers to a nucleotide containing guanine (G) or a modification or analog thereof, e.g., GTP, dGTP. "nucleotide C" refers to a nucleotide containing cytosine (C) or a modification or analog thereof, e.g., CTP, dCTP. "nucleotide T" refers to a nucleotide containing thymine (T) or a modification or analog thereof, e.g., TTP, dTTP. "nucleotide U" refers to a nucleotide containing uracil (U) or a modification or analog thereof, e.g., UTP, dUTP.

Labelling of nucleotides

The present invention relates to labeling nucleotides with different labels by different linkers, thereby linking different luciferases to the nucleotides.

As used herein, the term "chemiluminescent label" refers to any compound that can be attached to a nucleotide that can trigger a chemiluminescent reaction by contact with a suitable substrate to produce a detectable light signal without the need for excitation light. In general, any component that participates in a chemiluminescent reaction may be used as a chemiluminescent label as described herein, and correspondingly, the other components that participate in the chemiluminescent reaction are referred to herein as substrates for the chemiluminescent label. Examples of suitable chemiluminescent labels commonly used include, but are not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, isoluminol, acridinium esters, sulfonamides, and the like. The substrate for the chemiluminescent label will depend on the particular chemiluminescent label used, for example the substrate for alkaline phosphatase may be AMPPD (adamantyl 1, 2-dioxan aromatic phosphate), the substrate for luciferase may be luciferin, the substrate for acridinium ester may be a mixture of sodium hydroxide and H2O2, and the like. A detailed description of Chemiluminescent markers and their corresponding substrates can be found, for example, in Larry J.Kricka, Chemimenscent and Bioluminescence technologies, CLIN.CHEM.37/9, 1472-.

In a preferred embodiment, the chemiluminescent label as used herein is a biochemical chemiluminescent label.

As used herein, the term "chemiluminescent label" refers to any compound that can be attached to a nucleotide that can trigger a bioluminescent reaction by contact with a suitable substrate to produce a detectable light signal without the need for excitation light. Bioluminescence is one type of chemiluminescence that is light produced by a chemical reaction that occurs in the body or in the secretions of certain types of organisms. Examples of the chemiluminescent label may include, for example, luciferase, aequorin, glucose dehydrogenase, glucose oxidase, and the like. The substrate for the chemiluminescent label will depend on the particular chemiluminescent label used, for example the substrate for luciferase may be luciferin and the substrate for aequorin may be calcium. A detailed description of the Chemiluminescent markers and their corresponding substrates can be found, for example, in Larry J.Kricka, Chemimencent and Bioluminescence Techniques, CLIN.CHEM.37/9, 1472-.

In a preferred embodiment, the biochemical luminescent marker as used herein is luciferase. In particular embodiments, the luciferase is selected from the group consisting of a daphnia magna (Gaussia) luciferase, a Renilla (Renilla) luciferase, a dinoflagellate luciferase, a firefly luciferase, a fungal luciferase, a bacterial luciferase, and a glowworm (varula) luciferase.

As used herein, "labeling a nucleotide with a chemiluminescent label" means attaching a chemiluminescent label to a nucleotide. Specific ways of attaching chemiluminescent labels to nucleotides are known to those skilled in the art, for example, see the relevant description in the following documents (all incorporated herein by reference): sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989), Chapter 10; U.S. Pat. nos. 4,581,333,5,283,174,5,547,842,5,656,207 and 5,658,737. In one embodiment, the chemiluminescent label may be attached directly to the nucleotide by a covalent bond. In another embodiment, the chemiluminescent label may be attached to the nucleotide by a linking group.

As used herein, the molecular marker used to label the nucleotides and the marker on the luciferase may be any pair of molecules capable of specifically binding to each other. Specific binding between the paired members effects linkage of the nucleotide to the luciferase. Exemplary pairing members include, but are not limited to: (a) haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, e.g. digoxin-digoxin antibody, N3G-N3G antibody, FITC-FITC antibody; (b) aptamers and proteins; (c) non-immunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-neutral antibiotic); (d) hormone-hormone binding proteins; (e) a receptor-receptor agonist or antagonist; (f) lectin-carbohydrate; (g) enzyme-enzyme cofactors; (h) an enzyme-enzyme inhibitor; and (i) a complementary pair of oligonucleotides or polynucleotides capable of forming a nucleic acid duplex.

In specific embodiments, the first molecular marker and the second molecular marker are small molecule markers selected from the group consisting of biotin, digoxigenin, N3G, or FITC. Two luciferases are capable of specifically binding to the first molecular marker and the second molecular marker, respectively. For example, in one embodiment, where the first molecular tag is biotin, the first luciferase may be a streptavidin-labeled luciferase; the second molecular marker is digoxigenin, and the second luciferase may be a different luciferase labelled with digoxigenin antibody to the first luciferase, or a different mutant of the same luciferase labelled with digoxigenin antibody to the first luciferase. The different luciferases can correspond to the same substrate or different substrates, as long as the luminescence form of the reaction between the two enzymes and the substrate is different; different mutants of the same luciferase may also correspond to the same substrate or different substrates, as long as the different mutants react with different luminescent forms of the substrate, preferably the same substrate is selected. Sources of such luciferases include, but are not limited to, firefly, gaussia, Renilla, Oplophorus, and the like. For example, the Streptavidin-labeled luciferase may be SA-Gluc from Advity corporation, Streptavidin-Gaussia priceps luciferase; or a nanoKAZ from Promega; or nanoLuc from nanolight corporation; and a corresponding Glow morphological mutant or Flash morphological mutant. The digoxin antibody-labeled luciferase may be digoxin antibody-Gluc or digoxin antibody-Nluc or digoxin antibody-nanoKAZ, as well as the corresponding Glow morphology mutant or Flash morphology mutant. Its corresponding substrate may be coelenterazine; or dehydroxylated coelenterazine; or fluoro-substituted coelenterazine; or a furan ring substituted coelenterazine; or other similar modified coelenterazine.

In an embodiment of the invention, the first molecular marker and the second molecular marker are linked to the nucleotide derivative by a cleavable linker. The linker ensures cleavage as required in the reaction. For example, in one particular embodiment, the first nucleotide is linked to a first molecular tag by a cleavable linker2, or the first nucleotide is linked to a first molecular tag by a cleavable linker1-linker 2; the second nucleotide is linked to a first molecular tag by a cleavable linker 1; the third nucleotide is linked to a second molecular tag by a cleavable linker 1; the fourth nucleotide is not linked to a molecular tag in a specific embodiment. Wherein said linker includes but is not limited to disulfide bond, azide, cis-aconitic anhydride (cis-aconitic anhydride) -containing linker, etc.

Sequencing of polynucleotides

Preferably, the nucleotides of the invention to which different luciferases are linked are suitable for sequencing by synthesis. Sequencing-by-synthesis as used herein is a variety of sequencing-by-synthesis methods well known in the art. Basically, sequencing by synthesis involves first hybridizing a nucleic acid molecule to be sequenced to a sequencing primer, and then polymerizing nucleotides linked to different luciferases as described herein at the 3' end of the sequencing primer in the presence of a polymerase using the nucleic acid molecule to be sequenced as a template. Following polymerization, nucleotides are identified by detecting the fluorescent signal emitted by the luciferase. After removing luciferase from the labeled nucleotides, the next cycle of polymerase sequencing is performed.

The method for determining the sequence of a target polynucleotide can be performed by: denaturing the target polynucleotide sequence, contacting the target polynucleotides with different nucleotides, respectively, to form complements of the target nucleotides, and detecting incorporation of the nucleotides. The method utilizes polymerization such that a polymerase extends the complementary strand by incorporating the correct nucleotide that is complementary to the target polynucleotide. The polymerization reaction also requires special primers to initiate polymerization.

For each round of reaction, the incorporation of the nucleotide is performed by a polymerase, and the incorporation event is then determined. Many different polymerases exist and the most suitable polymerase is readily determined by one of ordinary skill in the art. Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7DNA polymerase, Taq polymerase or vent polymerase. Polymerases engineered to have specific properties may also be used.

The sequencing method is preferably performed on target polynucleotides arrayed on a solid support. The plurality of target polynucleotides may be immobilized on the solid support by linker molecules, or may be attached to particles, such as microspheres, which may also be attached to a solid support material.

The polynucleotides may be attached to the solid support by a variety of methods, including the use of biotin-streptavidin interactions. Methods for immobilizing polynucleotides on a solid support are well known in the art and include lithographic techniques and spotting each polynucleotide at a specific location on the solid support. Suitable solid supports are well known in the art and include glass slides and beads, ceramic and silicon surfaces, and plastic materials. The support is generally planar, although microbeads (microspheres) may also be used, and the latter may also be attached to other solid supports by known methods. The microspheres may be of any suitable size and are typically 10-100 nm in diameter. In a preferred embodiment, the polynucleotide is directly attached to a flat surface, preferably to a flat glass surface. The linkage is preferably by means of a covalent bond. The array used is preferably a single molecule array comprising polynucleotides located in distinct optically distinguishable regions, for example as described in international application No. WO 00/06770.

The conditions necessary to carry out the polymerization are well known to those skilled in the art. In order to carry out the polymerase reaction, it is generally first necessary to anneal to the target polynucleotide a primer sequence which is recognized by the polymerase and which serves as a starting site for subsequent extension of the complementary strand. The primer sequence may be added as a separate component to the target polynucleotide. Alternatively, the primer and target polynucleotide may each be part of a single-stranded molecule, with the primer portion and the target portion forming an intramolecular duplex, i.e., hairpin loop structure. The structure may be immobilised to the solid support at any site on the molecule. Other conditions necessary to carry out the polymerase reaction are well known to those skilled in the art and include temperature, pH, buffer composition.

Subsequently, the labeled nucleotides of the invention are contacted with the target polynucleotide to enable polymerization. The nucleotides may be added sequentially, i.e., each type of nucleotide (A, C, G or T/U) is added separately, or simultaneously.

Allowing the polymerization step to proceed for a time sufficient to incorporate one nucleotide.

Unincorporated nucleotides are then removed, for example, by removing the solution phase of the reaction system from the previous step, leaving the duplexes attached to the support.

Two luciferases containing different luciferases, each capable of specifically binding to the molecular marker for labeling the nucleotide, may then be added to perform a binding reaction, thereby effecting linkage of the luciferase to the incorporated nucleotide. Then by adding a substrate for the corresponding luciferase and detecting the fluorescent signal. It is also possible to add two luciferases containing different mutants of the same luciferase to perform a binding reaction, the luciferases being respectively capable of specifically binding to molecular markers for labeling nucleotides, thereby achieving linkage of the luciferases to the incorporated nucleotides. Then by adding a substrate for the corresponding luciferase and detecting the fluorescent signal.

Then adding a lysis solution to lyse the linker2, so that the molecular marker connected with the nucleotide through the linker2 is cut off; and removing the cracking agent by using an elution buffer solution, adding a luciferase substrate again, and detecting a signal of secondary luminescence by using an enzyme-labeling instrument so as to judge the base sequence.

In one embodiment, the four deoxyribonucleotide analogs are labeled with different small molecule markers biotin (abbreviated as B) and digoxigenin (abbreviated as D), respectively, for example, nucleotide A is labeled with B by linker1-linker2, nucleotide C is labeled with B by linker1, nucleotide T is labeled with D by linker1, and nucleotide G is not labeled. The 3' end hydroxyl of the four deoxyribonucleotide analogues marked with different small molecules is blocked, so that only one deoxyribonucleotide is bound in each sequencing reaction. In the sequencing reaction process, the mixture of the four deoxyribonucleotide analogs and sequencing polymerase is firstly introduced, and under the action of the polymerase, one deoxyribonucleotide analog is doped into the 3' end of a growing nucleic acid chain according to the base complementary pairing principle. Unbound deoxyribonucleotide analogs can be removed by removing the solution phase of the reaction system from the previous step, leaving the duplexes attached to the support. Then, two luciferases containing different luciferases are added, the first luciferase is labeled with streptavidin and combined with the nucleotide A or the nucleotide C labeled with the small molecule B, and the second luciferase is labeled with digoxin antibody and combined with the nucleotide T labeled with the small molecule D. After removing unbound luciferase with the elution buffer, the substrate to which luciferase has been added detects the signal with a detector, from which a first fluorescent signal is derived. Then a lysis solution capable of selectively lysing the linker2 was added, the linker2 was lysed and the linker1 was not lysed. The luciferase substrate is added again and the signal is detected using a detector, from which a second fluorescent signal is derived. The base recognition can be carried out according to the reaction signals of two times.

In one embodiment, the attachment of two luciferases containing different luciferases or different mutants of the same luciferase to the deoxynucleotide analogue and the signal detection may be performed separately. First luciferase, labeled with streptavidin, is added to bind to nucleotide a or nucleotide C labeled with small molecule B. After removing the unbound first luciferase with the elution buffer, a substrate of the first luciferase is added, and the nucleotide to which the first luciferase is linked emits light, and a signal is detected with a detector. And removing the reaction solution, adding a second luciferase marked by a digoxin antibody, combining with the nucleotide T marked with the small molecule D, removing the unbound second luciferase by using an elution buffer, adding a substrate of the second luciferase, allowing the base connected with the second luciferase to emit light, and detecting a signal by using a detector, thereby obtaining a first fluorescence signal. Then a lysis solution capable of selectively lysing the linker2 was added, the linker2 was lysed and the linker1 was not lysed. Adding a substrate of the first luciferase again, enabling the nucleotide connected with the first luciferase to emit light, and detecting a signal by using a detector; adding a substrate of the second luciferase, allowing the nucleotide linked with the second luciferase to emit light, and detecting the signal by using a detector, thereby obtaining a second fluorescent signal. The base recognition can be carried out according to the reaction signals of two times.

In another embodiment, the four deoxyribonucleotide analogs are labeled with different small molecule markers biotin (abbreviated as B) and digoxigenin (abbreviated as D), respectively, for example, nucleotide A is labeled with B by linker1-linker2, nucleotide C is labeled with B by linker1, nucleotide T is labeled with D by linker1, and nucleotide G is not labeled. The 3' end hydroxyl of the four deoxyribonucleotide analogues marked with different small molecules is blocked, so that only one deoxyribonucleotide is bound in each sequencing reaction. In the sequencing reaction process, the mixture of the four deoxyribonucleotide analogs and sequencing polymerase is firstly introduced, and under the action of the polymerase, one deoxyribonucleotide analog is doped into the 3' end of a growing nucleic acid chain according to the base complementary pairing principle. Unbound deoxyribonucleotide analogs can be removed by removing the solution phase of the reaction system from the previous step, leaving the duplexes attached to the support. Then, two luciferases containing different mutants of the same luciferase are added, the first luciferase is labeled with streptavidin, which binds to nucleotide a or nucleotide C labeled with small molecule B, and the second luciferase is labeled with digoxin antibody, which binds to nucleotide T labeled with small molecule D. After removing unbound luciferase with the elution buffer, the substrate to which luciferase has been added detects the signal with a detector, from which a first fluorescent signal is derived. Then a lysis solution capable of selectively lysing the linker2 was added, the linker2 was lysed and the linker1 was not lysed. The luciferase substrate is added again and the signal is detected using a detector, from which a second fluorescent signal is derived. The base recognition can be carried out according to the two reaction signals.

Detection of fluorescent signals

Means for detecting fluorescent signals are well known in the art. This can be achieved, for example, by means of a device that detects the wavelength of the fluorescence. Such devices are well known in the art. For example, such a device may be a confocal scanning microscope that scans the surface of a solid support with laser light in order to image fluorophores directly bound to nucleic acid molecules being sequenced. In addition, each of the signals generated can be observed, for example, with a sensitive 2-D detector, such as a Charge Coupled Detector (CCD). Other techniques such as scanning near-field optical microscopy (SNOM) may also be used, for example.

Removal of labels

After detection, the label on the nucleotide can be removed using suitable conditions.

In a specific embodiment, the nucleotide analogs of the invention also have a 3' protecting group. In some embodiments of the invention, the protecting group and the label are typically two different groups on a 3' blocked labeled nucleotide, but in other embodiments the protecting group and the label may be the same group.

As used herein, the term "protecting group" means a group that prevents a polymerase (which incorporates a nucleotide containing the group onto a polynucleotide strand being synthesized) from continuing to catalyze the incorporation of another nucleotide after the nucleotide containing the group is incorporated onto the polynucleotide strand being synthesized. Such protecting groups are also referred to herein as 3' -OH protecting groups. Nucleotides comprising such protecting groups are also referred to herein as 3' blocked nucleotides. The protecting group may be any suitable group that can be added to a nucleotide, provided that the protecting group prevents additional nucleotide molecules from being added to the polynucleotide chain while being easily removed from the sugar portion of the nucleotide without destroying the polynucleotide chain. Furthermore, nucleotides modified with protecting groups need to be resistant to polymerases or other suitable enzymes for incorporating the modified nucleotides into a polynucleotide chain. Thus, the desired protecting groups exhibit long-term stability, can be incorporated efficiently by polymerases, prevent secondary or further incorporation of nucleotides, and can be removed under mild conditions, preferably aqueous conditions, without disrupting the polynucleotide structure.

The prior art has described a variety of protecting groups which meet the above description. For example, WO 91/06678 discloses that 3' -OH protecting groups include esters and ethers, -F, -NH2, -OCH3, -N3, -OPO3, -NHCOCH3,2 nitrophenylcarbonate, 2, 4-sulfenyl dinitro and tetrahydrofuran ethers. Metzker et al (Nucleic Acids Research,22(20):4259-4267,1994) disclose the synthesis and use of eight 3' -modified 2-deoxyribonucleoside 5' -triphosphates (3 ' -modified dNTPs). WO2002/029003 describes the use of allyl protecting groups to cap 3' -OH groups on growing strands of DNA in polymerase reactions. Preferably, various protecting groups reported in international application publications WO2014139596 and WO2004/018497 may be used, including those protecting groups exemplified in figure 1A of WO2014139596 for example and those 3' hydroxy protecting groups (i.e. protecting groups) defined in the claims, and those protecting groups exemplified in figures 3 and 4 of WO2004/018497 for example and those protecting groups defined in the claims. The above references are all incorporated herein by reference in their entirety.

The skilled person will understand how to attach suitable protecting groups to the ribose ring in order to block the interaction with the 3' -OH. The protecting group may be attached directly to the 3' position or may be attached to the 2' position (the protecting group being of sufficient size or charge to block the interaction at the 3' position). In addition, the protecting groups may be attached at the 3' and 2' positions and may be cleaved to expose the 3' -OH group.

After successful incorporation of the 3 'blocked nucleotide into a growing nucleic acid strand, the sequencing protocol requires removal of the protecting group to create an available 3' -OH site for continuous strand synthesis. Reagents that can remove protecting groups from modified nucleotides as used herein depend largely on the protecting group used. For example, removal of the ester protecting group from the 3' hydroxyl functionality is typically achieved by basic hydrolysis. The ease of removal of the protecting group varies widely; generally, the greater the electronegativity of the substituents on the carbonyl carbons, the greater the ease of removal. For example, a highly electronegative trifluoroacetate group is capable of rapid cleavage from the 3' hydroxyl group in methanol at pH7 (Cramer et al, 1963) and is therefore unstable during polymerization at this pH. The phenoxyacetate group is cleaved in less than 1 minute, but requires a significantly higher pH, for example with NH-/methanol (Reese and Steward, 1968). A wide variety of hydroxyl protecting groups can be selectively cleaved using chemical methods other than alkaline hydrolysis. 2, 4-dinitrophenylthio-groups can be cleaved rapidly by treatment with nucleophiles such as thiophenol and thiosulfates (Letsinger et al, 1964). Allyl ether is cleaved by treatment with hg (ii) in acetone/water (Gigg and Warren, 1968). Tetrahydrothiopyranyl ethers are removed under neutral conditions using Ag (I) or Hg (II) (Cohen and Steele, 1966; Cruse et al, 1978). Photochemical deblocking can be used with photochemically cleavable protecting groups. Several protecting groups are available for this process. The use of o-nitrobenzyl ether as a protecting group for the 2' -hydroxy functionality of ribonucleosides is known and proven (Ohtsuka et al, 1978); it was removed by irradiation at 260 nm. The alkyl o-nitrobenzyl carbonate protecting group is also removed by irradiation at pH7 (Cama and Christensen, 1978). Enzymatic cleavage blocking of the 3' -OH protecting group is also possible. It has been demonstrated that T4 polynucleotide kinase can convert the 3 '-phosphate terminus to the 3' -hydroxy terminus and can then be used as a primer for DNA polymerase I (Henner et al, 1983). This 3 '-phosphatase activity was used to remove the 3' protecting group of those dNTP analogs that contain a phosphate as a protecting group.

Other reagents that can remove a protecting group from a 3 'blocked nucleotide include, for example, phosphines (e.g., tris (hydroxymethyl) phosphine (THP)), which can, for example, remove an azide-containing 3' -OH protecting group from a nucleotide (see, for this application of phosphines, for example, the description in WO2014139596, the entire contents of which are incorporated herein by reference). Other reagents which can remove the protecting group from the 3' blocked nucleotide also include the corresponding reagents described, for example, in WO2004/018497, at page 114-116 for removing 3' -allyl, 3, 4-dimethoxybenzyloxymethyl or fluoromethoxymethyl as protecting groups for 3' -OH.

In an embodiment of the invention, the label of the nucleotide is preferably removed together with the protecting group after detection.

In certain embodiments, the label may be incorporated into a protecting group, thereby allowing the 3' blocked nucleotide to be removed along with the protecting group after it is incorporated into the nucleic acid strand.

In other embodiments, the label may be attached to the nucleotide separately from the protecting group using a linker. Such labels may be attached, for example, to the purine or pyrimidine base of the nucleotide. In certain embodiments, the linking group used is cleavable. The use of a cleavable linker ensures that the label can be removed after detection, which avoids any signal interference with any subsequently incorporated labeled nucleotides. In other embodiments, a non-cleavable linker may be used because subsequent nucleotide incorporation is not required after incorporation of the labeled nucleotide into the nucleic acid strand, and thus removal of the label from the nucleotide is not required.

In further embodiments, the label and/or linking group may be of a size or structure sufficient to function to block incorporation of other nucleotides onto the polynucleotide strand (that is, the label itself may serve as a protecting group). The blocking may be due to steric hindrance or may be due to a combination of size, charge and structure.

Cleavable linkers are well known in the art and conventional chemical methods can be employed to attach the linker to the nucleotide base and the tag. The linker may be attached at any position of the nucleotide base, provided that Watson-Crick base pairing is still possible. For purine bases, it is preferred if the linking group is attached through the 7 position of the purine or preferred deazapurine analogue, through an 8-modified purine, through an N-6 modified adenine or an N-2 modified guanine. In the case of pyrimidines, the linkage is preferably via positions 5 on cytosine, thymine and uracil and the N-4 position on cytidine.

The use of the term "cleavable linking group" does not imply that the entire linking group needs to be removed (e.g., from the nucleotide base). When the label is attached to the base, the nucleoside cleavage site may be located at a position on the linking group which ensures that a portion of the linking group remains attached to the nucleotide base after cleavage.

Suitable linkers include, but are not limited to, disulfide linkers, acid labile linkers (including dialkoxybenzyl linkers, Sieber linkers, indole linkers, t-butyl Sieber linkers), electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, linkers that cleave under reducing conditions, oxidizing conditions, safety-catch linkers, and linkers that cleave by an elimination mechanism. Suitable linking groups may be modified with standard chemical protecting groups, as disclosed in the following references: greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Guillier et al disclose other suitable cleavable linkers for solid phase synthesis (chem. Rev.100: 2092-2157, 2000).

The linking group can be cleaved by any suitable means, including contact with an acid, base, nucleophile, electrophile, radical, metal, reducing or oxidizing agent, light, temperature, enzyme, and the like, suitable cleavage modes for the various cleavable linking groups being described below by way of example. Typically, the cleavable linking group can be cleaved under the same conditions as the protecting group, such that only one treatment is required to remove the label and protecting group.

Electrophilically cleavable linking groups are typically cleaved by protons and include acid-sensitive cleavage. Suitable electrophilically cleavable linking groups include modified benzyl systems such as trityl, p-hydrocarbyloxybenzyl and p-hydrocarbyloxybenzylamide. Other suitable linking groups include t-butyloxycarbonyl (Boc) groups and acetal systems. For the preparation of suitable linker molecules, it is also contemplated to use thiophilic metals such as nickel, silver or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups. Nucleophilically cleavable linking groups include groups that are labile in water (i.e., capable of simple cleavage at basic pH), such as esters, and groups that are labile to non-aqueous nucleophiles. Fluoride ions can be used to cleave siloxane bonds in groups such as Triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS). Photolytic linking groups are widely used in sugar chemistry. Preferably, the light required to activate cleavage does not affect other components in the modified nucleotide. For example, if a fluorophore is used as the label, it is preferred that the fluorophore absorbs light of a different wavelength than the light required to cleave the linker molecule. Suitable linking groups include those based on O-nitrobenzyl compounds and nitroveratryl compounds. Linking groups based on benzoin chemistry may also be used (Lee et al, J.org.chem.64:3454-3460, 1999). Various linking groups are known which are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art. Oxidation-based methods are well known in the art. These methods include oxidation of the hydrocarbyloxybenzyl group and oxidation of the sulfur and selenium linkages. It is also within the scope of the present invention to use iodine solutions (aquous iododine) to cleave disulfides and other sulfur or selenium based linkers. Safe-handle linkers are those which cleave in two steps. In a preferred system, the first step is the generation of reactive nucleophilic centers, followed by a second step involving intramolecular cyclization, which results in cleavage. For example, the levulinate linkage may be treated with hydrazine or photochemical procedures to release the active amine which is then cyclized to cleave the ester elsewhere in the molecule (Burgess et al, J.org.chem.62:5165-5168, 1997). Elimination reactions can also be used to cleave the linking group. Base-catalyzed elimination of groups such as fluorenylmethyloxycarbonyl and cyanoethyl and palladium-catalyzed reductive elimination of allyl systems can be used.

In certain embodiments, the linking group may comprise a spacer unit. The length of the linker is not critical as long as the label is kept at a sufficient distance from the nucleotide so as not to interfere with the interaction between the nucleotide and the enzyme.

In certain embodiments, the linking group may consist of a functional group similar to the 3' -OH protecting group. This would allow removal of the label and protecting group with only a single treatment. Particularly preferred linking groups are azide-containing linking groups that are cleavable by a phosphine.

The reagents that can remove the label from the modified nucleotide as used herein will depend to a large extent on the label used. For example, in the case where the label incorporates a protecting group, the label is removed using the protecting group-removing reagent described above. Alternatively, where the label is attached to the base of the nucleotide via a cleavable linker, the label is removed using a reagent that cleaves the linker as described above. In a preferred embodiment, the same reagents are used to remove the label and protecting group from the modified nucleotide, for example where the linking group consists of a functional group similar to the 3' -OH protecting group.

Reagent kit

The invention also provides a kit for sequencing a polynucleotide, comprising:

the first nucleotide is linked to a first molecular tag by a cleavable linker2,

the fourth nucleotide is not connected with a molecular marker;

(b) two different luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively; and

(c) lysis solution for lysis of linker 2.

In a specific embodiment, the molecular marker for labeling four nucleotides and the marker for labeling two luciferases are as defined above.

In certain preferred embodiments, the kit of the invention comprises two different luciferases comprising different labels capable of specifically binding to the four nucleotides, respectively, and the two different luciferases may comprise different luciferases or comprise different mutants of the same luciferase.

In certain preferred embodiments, the kits of the invention further comprise reagents and/or means for extracting nucleic acid molecules from a sample. Methods for extracting nucleic acid molecules from a sample are well known in the art. Thus, various reagents and/or devices for extracting nucleic acid molecules, such as a reagent for disrupting cells, a reagent for precipitating DNA, a reagent for washing DNA, a reagent for solubilizing DNA, a reagent for precipitating RNA, a reagent for washing RNA, a reagent for solubilizing RNA, a reagent for removing protein, a reagent for removing DNA (e.g., when the nucleic acid molecule of interest is RNA), a reagent for removing RNA (e.g., when the nucleic acid molecule of interest is DNA), and any combination thereof, may be provided in the kit of the present invention, as desired.

In certain preferred embodiments, the kits of the invention further comprise reagents for pretreating the nucleic acid molecules. In the kit of the present invention, the reagent for pretreating nucleic acid molecules is not additionally limited and may be selected according to actual needs. The reagent for pretreating a nucleic acid molecule includes, for example, a reagent for fragmenting a nucleic acid molecule (e.g., dnase I), a reagent for filling the ends of a nucleic acid molecule (e.g., DNA polymerase such as T4DNA polymerase, Pfu DNA polymerase, Klenow DNA polymerase), a linker molecule, a tag molecule, a reagent for linking a linker molecule to a nucleic acid molecule of interest (e.g., ligase such as T4DNA ligase), a reagent for repairing nicking of nucleic acid (e.g., DNA polymerase that loses 3'-5' exonuclease activity but exhibits 5'-3' exonuclease activity), a reagent for amplifying a nucleic acid molecule (e.g., DNA polymerase, primers, dntps), a reagent for separating and purifying a nucleic acid molecule (e.g., a chromatography column), and any combination thereof.

In certain preferred embodiments, the kits of the invention further comprise a support for attaching nucleic acid molecules to be sequenced. The support may have any of the technical features described in detail above for the support, as well as any combination thereof.

For example, in the present invention, the support may be made of various suitable materials. Such materials include, for example: minerals, natural polymers, synthetic polymers, and any combination thereof. Specific examples include, but are not limited to: cellulose, cellulose derivatives (e.g., nitrocellulose), acrylic resins, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene crosslinked with divinylbenzene and the like (see, for example, Merrifield Biochemistry 1964,3,1385-^TM) Agarose gel (Sepharose)^TM) And other supports known to those skilled in the art.

In certain preferred embodiments, the support for attaching nucleic acid molecules to be sequenced can be a solid support comprising an inert substrate or matrix (e.g., a glass slide, a polymer bead, etc.) that has been functionalized, for example, by application of an intermediate material containing reactive groups that allow covalent attachment of biomolecules such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly polyacrylamide hydrogels described in WO 2005/065814 and US 2008/0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecule (e.g., polynucleotide) may be covalently attached directly to the intermediate material (e.g., hydrogel), while the intermediate material itself may be non-covalently attached to the substrate or matrix (e.g., glass substrate). In certain preferred embodiments, the support is a glass slide or silicon wafer having a surface modified with a layer of avidin, amino, acrylamide silane, or aldehyde based chemical groups.

In the present invention, the support or solid support is not limited in its size, shape and configuration. In some embodiments, the support or solid support is a planar structure, such as a slide, chip, microchip and/or array. The surface of such a support may be in the form of a planar layer. In some embodiments, the support or surface thereof is non-planar, such as an inner or outer surface of a tube or container. In some embodiments, the support or solid support comprises a microsphere or bead. In certain preferred embodiments, the support for attaching the nucleic acid molecules to be sequenced is an array of beads or wells.

In certain preferred embodiments, the kits of the invention further comprise reagents for attaching (e.g., covalently or non-covalently) the nucleic acid molecule to be sequenced to a support. Such agents include, for example, agents that activate or modify a nucleic acid molecule (e.g., at its 5' end), such as a phosphate, thiol, amine, carboxylic acid, or aldehyde; a reagent for activating or modifying the surface of the support, such as amino-alkoxysilane (e.g., aminopropyltrimethoxysilane, aminopropyltriethoxysilane, 4-aminobutyltriethoxysilane, etc.); crosslinkers, such as succinyl anhydride, phenyl diisothiocyanate (Guo et al, 1994), maleic anhydride (Yang et al, 1998), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC), N-hydroxysuccinimide ester of m-maleimidobenzoic acid (MBS), N-succinimidyl [ 4-iodoacetyl ] aminobenzoic acid (SIAB), 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid Succinimide (SMCC), N-gamma-maleimidobutyryloxy-succinimide ester (GMBS), 4- (p-maleimidophenyl) butyric acid Succinimide (SMPB); and any combination thereof.

In certain preferred embodiments, the kits of the invention further comprise primers for initiating a nucleotide polymerization reaction. In the present invention, the primer is not additionally limited as long as it can specifically anneal to a region of the target nucleic acid molecule. In some exemplary embodiments, the length of the primer may be 5-50bp, such as 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 bp. In some exemplary embodiments, the primer may comprise a naturally occurring or non-naturally occurring nucleotide. In some exemplary embodiments, the primer comprises or consists of a naturally occurring nucleotide. In some exemplary embodiments, the primer comprises a modified nucleotide, such as a Locked Nucleic Acid (LNA). In certain preferred embodiments, the primer comprises a universal primer sequence.

In certain preferred embodiments, the kits of the invention further comprise a polymerase for performing a nucleotide polymerization reaction. In the present invention, various suitable polymerases can be used. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (e.g., a DNA polymerase) using DNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new DNA strand (e.g., a reverse transcriptase) using RNA as a template. In some exemplary embodiments, the polymerase is capable of synthesizing a new RNA strand using DNA or RNA as a template (e.g., RNA polymerase). Thus, in certain preferred embodiments, the polymerase is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

In certain preferred embodiments, the kits of the invention further comprise one or more buffer solutions. Such buffers include, but are not limited to, buffer solutions for dnase I, buffer solutions for DNA polymerase, buffer solutions for ligase, buffer solutions for eluting nucleic acid molecules, buffer solutions for solubilizing nucleic acid molecules, buffer solutions for performing nucleotide polymerization reactions (e.g., PCR), and buffer solutions for performing ligation reactions. The kit of the present invention may comprise any one or more of the above-described buffer solutions.

In certain preferred embodiments, the kits of the invention further comprise one or more wash solutions. Examples of such wash solutions include, but are not limited to, phosphate buffer, citrate buffer, Tris-HCl buffer, acetate buffer, carbonate buffer, and the like. The kit of the invention may comprise any one or more of the above-described wash solutions.

Advantageous effects of the invention

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the method of the invention connects the molecular marker and the nucleotide derivative through a cleavable linker, inactivates the chemiluminescent marker marked on the specific base through a selective chemical bond breaking method on the basis of a chemiluminescent reaction kinetic curve, and carries out secondary luminescence judgment by utilizing the luminous capacity of the rest of the luminescent markers so as to identify the base. As only two characteristics of Flash and Glow are required to be selected for the chemiluminescence kinetic curve, confusion caused by unobvious kinetic curve characteristics can be greatly reduced. Meanwhile, for the second luminescence, only the signal intensity is required to exceed the background value, and the dynamic curve characteristics are not required, so that the influence of side reactions on signal identification in the reaction process can be reduced, and the base resolution precision is improved.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Examples

Example 1

1. Sequencing library construction

(1) The following DNA sequences were designed: GATATCTGCAGGCATAGAATGAATATTATTGAATCAATAATTAAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAACAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTATAATGGGCTGGATACATGGAATGATTATAGATATATTAAGGAATAATGTTAATTAATGCCTAAATTAATTAATCTAAGGGGGTTAATACTTCAGCCTGTGATATC，For convenience of library construction, oligo sequences (bold type) are added at two ends of the sequence, a linker sequence (shaded part) of BGISEQ-500 is inserted in the middle part, and the bold part in italics is the first 10bp base of the sequence to be detected. The above sequence was synthesized by kasei biotechnology, and the synthesized sequence was inserted into pUC57 vector for unlimited use of the sequence and transformed into e.

(2) Coli bacteria containing known libraries were cultured in appropriate amounts, and plasmids were extracted, and the following pair of primers were designed: GATATCTGCAGGCAT (primer 1), GATATCACAGGCTGA (primer 2), the known sequences were amplified as follows (Table I) and scheme (Table II), and the PCR products were purified using magnetic beads. Adding the purified PCR product into split oligo (ATGCCTGCAGATATCGATATCACAGGCTGA) to perform cyclization library construction according to BGISEQ-500SE50 cyclization library construction kit (Huada Zhi) and the process for standby;

watch I (enzyme from BGI)

Table two:

2. amplification of library sequences

A96-well plate coated with Streptavidin from Thermo fisher is purchased, 1uM of primer GCCATGTCGTTCTGTGAGCCAAGG 100ul modified by biotin at the 5' end is incubated in one well at normal temperature for 30min, reaction liquid is removed, 6ng of the library constructed in the above section 1 and 20ul of DNB preparation buffer I in BGISEQ-500 kit (Chiense), the primer is hybridized with the primer modified by biotin at 60 ℃ for 5min, 40ul of DNB polymerase I and 4ul of DNB polymerase II in BGISEQ-500 sequencing kit (Chiense) are added, reaction is carried out at 30 ℃ for 60min, the reaction is terminated by heating to 65 ℃, and the reaction liquid is carefully removed. Adding 100ul of 5uM sequencing primer GCTCACAGAACGACATGGCTACGATCCGACTT, hybridizing at normal temperature for 30min, and carefully removing the reaction solution;

3. sequencing

(1) Acme Bioscience outsourced synthesis of 4 dNTPs as shown below, wherein Linker1 is an azide-containing Linker group, and Linker2 is a disulfide-containing Linker group:

dATP-Linker1-Linker2-biotin

dCTP-Linker1-biotin

dTTP-Linker1-digoxin

dGTP

(2) preparation of reagents:

the following reagents required in the sequencing reaction were prepared

Polymerization reaction solution: 50mM Tris-HCl, 50mM NaCl, 10mM (NH)₄) ₂SO ₄0.02mg/ml polymerase BG9(BGI), 3mM MgSO₄1mM EDTA, 1uM each of the four dNTPs mentioned above

Polymerization buffer: 50mM Tris-HCl, 50mM NaCl, Twen200.05%;

elution buffer: 5XSSC, Twen200.05%;

self-luminous enzymatic reaction solution: TBST buffer, 0.5M NaCl, 2ug/ml SA-NanoKAZ-window (promega), 2ug/ml anti-digoxin-Gluc (8990) -flash;

substrate luminescent liquid: preparing 50mM Tris-HCl 0.5M NaCl buffer, mixing 50X Coelenterazine (nanolight) and 50X Coelenterazine-f (nanolight)1:1, and diluting to 2X;

lysis buffer: 10Mm DTT, 50Mm Tris-HCl, 50mM NaCl, pH adjusted to 9.0 with 10M NaOH;

excision buffer: 20mM THPP, 0.5M NaCl, 0.05% tween 20;

(3) the sequencing procedure is shown in FIG. 1.

a. Polymerization: adding 100ul polymerase reaction solution into the well of the library amplified in the step (1), raising the temperature of an enzyme-labeling instrument to 55 ℃, reacting for 3min to polymerize four dNTPs onto the amplified temperature control, carefully removing the reaction solution, adding 100ul elution reaction solution, gently blowing and beating for several times, and removing the elution reaction solution

b. Luminescent label binding: 100ul of the autoluminase reaction solution 1 was added and incubated at 35 ℃ for 30min to bind SA-NanoKAZ-glow to dATP-linker1-linker 2-biotin and dCTP-linker 1-biotin, and anti digoxin-Gluc (8990) -flash to dTTP-linker 1-digoxin. Removing the reaction solution, adding the eluent, slightly blowing and beating for several times, and removing the eluent;

c. detection of spontaneous light 1: setting parameters of an enzyme-labeling instrument, adding substrate luminescent liquid, and detecting a self-luminescent curve; and according to the signal curve graph, performing first signal reading and recording. 200ul of eluent was added and gently tapped several times to remove the eluent.

d. Breaking of chemical bonds: adding 200ul of bond breaking buffer solution for reaction for 3min, and removing the bond breaking buffer solution; 200ul of elution buffer was added and washed three times to remove the eluent.

e. Self-luminescence detection 2: setting parameters of an enzyme-labeling instrument, adding substrate luminescent liquid, detecting spontaneous intensity, reading and recording signals for the second time. Adding 200ul of eluent, gently blowing and beating for several times, and removing the eluent

f. Cutting; removing the self-luminous reaction solution, adding 200ul of elution buffer solution, gently blowing and beating for several times, removing the elution buffer solution, adding 100ul of excision reaction solution, reacting at 55 ℃ for 3min, and removing the excision reaction solution; adding 200ul of elution buffer solution for cleaning, and repeating the cleaning for three times;

g. repeating the steps a-f, and performing next cycle sequencing;

(4) sequencing results

The a.10bp sequencing signal curve is shown in FIG. 2:

b. and (3) analyzing a sequencing result:

comparing the signal change curves of all cycles, as shown in the following chart, it can be judged according to the form of signal drop of each cycle:

nucleotide a, cycle 2, cycle 5, cycle 8;

nucleotide T, cycle 1, cycle 4, cycle 7

Nucleotide C, cycle 3, cycle 6, cycle 9

Nucleotide G cycle 10

And the base sequence of the first 10bp of the library to be tested: TACTACTACG are matched.

Example 2

1. Sequencing library construction

(1) The following DNA sequences were designed: GATATCTGCAGGCATAGAATGAATATTATTGAATCAATAATTAATAATGGGCTGGATACATGGAATGATTATAGATATATTAAGGAATAATGTTAATTAATGCCTAAATTAATTAATCTAAGGGGGTTAATACTTCAGCCTGTGATATC, for the convenience of library construction, oligo sequences (bold font) are added at both ends of the sequence, and the adaptor sequence (shaded part) of BGISEQ-500 is inserted in the middle part, the bold part in italics is the first 10bp base of the sequence to be tested. The above sequence was synthesized by kasei biotechnology, and the synthesized sequence was inserted into pUC57 vector for unlimited use of the sequence and transformed into e.

watch I (enzyme from BGI)

Table two:

2. amplification of library sequences

(1) acme Bioscience corporation outsourced synthesis of 4 dNTPs as shown below, wherein Linker1 is an azide-containing Linker, and Linker2 is a cis-aconitic anhydride (cis-aconic anhydride) -containing Linker:

dATP-Linker1-Linker2-biotin

dCTP-Linker1-biotin

dTTP-Linker1-digoxin

dGTP

(2) preparation of reagents:

the following reagents required in the sequencing reaction were prepared

Polymerization buffer: 50mM Tris-HCl, 50mM NaCl, Twen200.05%;

elution buffer: 5XSSC, Twen200.05%;

self-luminous enzymatic reaction solution: TBST buffer, 0.5M NaCl, 2ug/ml SA-Gluc (M2) -glow (nanolight), 2ug/ml anti digoxin-Gluc (8990) -flash;

substrate luminescent liquid: preparing 50mM Tris-HCl 0.5M NaCl buffer, and dissolving 50X Coelenterazine (nanolight) in the buffer to 3X;

lysis buffer: PBS buffer pH 3.550 mM;

excision buffer: 20mM THPP, 0.5M NaCl, 0.05% tween 20;

(3) the sequencing procedure is shown in FIG. 1.

a. Polymerization: adding 100ul polymerase reaction solution into the well of the library amplified in the step (1), raising the temperature of an enzyme-labeling instrument to 60 ℃, reacting for 3min to polymerize four dNTPs onto the amplified temperature control, carefully removing the reaction solution, adding 100ul elution reaction solution, gently blowing and beating for several times, and removing the elution reaction solution

b. Luminescent label binding: 100ul of the autoluminase reaction solution 1 was added and incubated at 35 ℃ for 30min to bind SA-Gluc (M2) -glow to dATP-linker1-linker 2-biotin and dCTP-linker 1-biotin, and anti digoxin-Gluc (8990) -flash to dTTP-linker 1-digoxin. Removing the reaction solution, adding the eluent, slightly blowing and beating for several times, and removing the eluent;

d. Breaking of chemical bonds: adding 200ul of bond breaking buffer solution for reaction for 30min, and removing the bond breaking buffer solution; 200ul of elution buffer was added and washed three times to remove the eluent.

f. Cutting; removing the self-luminous reaction solution, adding 200ul of elution buffer solution, gently blowing and beating for several times, removing the elution buffer solution, adding 100ul of excision reaction solution, reacting at 60 ℃ for 3min, and removing the excision reaction solution; adding 200ul of elution buffer solution for cleaning, and repeating the cleaning for three times;

g. repeating the steps a-f, and performing next cycle sequencing;

(4) sequencing results

The a.10bp sequencing signal curve is shown in FIG. 3:

b. and (3) analyzing a sequencing result:

nucleotide a, cycle 2, cycle 5, cycle 8;

nucleotide T, cycle 1, cycle 4, cycle 7

Nucleotide C, cycle 3, cycle 6, cycle 9

Nucleotide G cycle 10

Claims

A method of sequencing a nucleic acid molecule comprising monitoring the sequential incorporation of nucleotides complementary to the nucleic acid molecule,

wherein the nucleotides are attached via different linking groups to chemiluminescent labels which elicit different luminescent kinetics or types,

wherein the incorporated nucleotide is identified by detecting the kinetics of luminescence or the type of luminescence of the chemiluminescent reaction in which the chemiluminescent label participates before and after cleavage of the linking group of the moiety and subsequent removal of the chemiluminescent label.
The method of claim 1, wherein the ribose or deoxyribose moiety of each of the nucleotides comprises a protecting group attached through a 2' or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of each nucleotide so as to expose a 3' -OH group,

for example, the chemiluminescent label and the protecting group are removed under the same conditions,

for example, the nucleotide is selected from nucleotide A, G, C and T or U.
The method of claim 1 or 2, for example, the detection of the luminokinetics of a chemiluminescent reaction in which the chemiluminescent label is involved comprises contacting the chemiluminescent label with a suitable substrate to trigger the chemiluminescent reaction, and detecting the luminokinetics of the light emitted thereby,

for example, the chemiluminescent label is selected from the group consisting of biochemical chemiluminescent labels eliciting different luminescent kinetics and any combination thereof,

for example, the chemiluminescent label is selected from the group consisting of luciferases eliciting different luminescence kinetics and any combination thereof,

for example, the chemiluminescent label is a combination of two luciferases that elicit different luminescence kinetics,

for example, detection of the luminescence kinetics of a chemiluminescent reaction in which the chemiluminescent label is involved comprises contacting the chemiluminescent label with a suitable substrate to trigger the chemiluminescent reaction, and detecting the type of luminescence emitted thereby,

for example, the chemiluminescent label is selected from the group consisting of biochemical chemiluminescent labels eliciting different types of luminescence, and any combination thereof,

for example, the chemiluminescent label is selected from the group consisting of luciferases that elicit different luminescence types and any combination thereof,

for example, the chemiluminescent label is a combination of two luciferases that elicit different luminescence types,

for example, the lighting types include a flash type and a glow type,

for example, in the nucleotide, a first nucleotide is attached to a first chemiluminescent label by a second linking group, a second nucleotide is attached to the first chemiluminescent label by the first linking group, a third nucleotide is attached to a second chemiluminescent label by a third linking group, and a fourth nucleotide is not attached to any chemiluminescent label,

for example, in the nucleotide, a first nucleotide is attached to the first luciferase through the second linking group, a second nucleotide is attached to the first luciferase through the first linking group, a third nucleotide is attached to the second luciferase through the third linking group, and a fourth nucleotide is not attached to any luciferase.

Wherein the first linking group and the third linking group may be the same or different and the second linking group is different from the third linking group.
The method of any one of claims 1-3, attaching a chemiluminescent label to the nucleotide by affinity interaction,

for example, the affinity interaction includes antigen-antibody interaction and biotin-avidin (such as streptavidin) interaction,

for example, by attaching a chemiluminescent label to one of the members involved in the affinity interaction and a nucleotide to the other member involved in the affinity interaction, thereby attaching the chemiluminescent label to the nucleotide through the affinity interaction between the members,

for example, the member attached to the nucleotide is biotin, the member attached to the chemiluminescent label is avidin (e.g., streptavidin),

for example, the member linked to the nucleotide is digoxigenin, the member linked to the chemiluminescent label is an anti-digoxigenin antibody,

for example, the member attached to the nucleotide is digoxigenin and the member attached to the chemiluminescent label is avidin (e.g., streptavidin), wherein digoxigenin and avidin are affinity bound by an anti-digoxigenin antibody attached to biotin.
A method of sequencing a nucleic acid molecule comprising (a) providing one or more nucleotides, wherein the nucleotides are attached to a chemiluminescent label by a linking group, wherein

The chemiluminescent label to which each type of nucleotide is attached exhibits a different kinetics of luminescence or type of luminescence from the chemiluminescent labels to which the other types of nucleotides are attached, as measured before and after cleavage of a portion of the linking group;

(b) incorporating a nucleotide on the complementary strand of the nucleic acid;

(c) detecting a chemiluminescent label of the nucleotide of (b);

(d) removing the nucleotide moiety linking group of (b);

(e) detecting the chemiluminescent label of the nucleotide of step (b) treated in step (d) to determine the type of nucleotide incorporated;

(f) optionally, removing (b) the chemiluminescent label of the nucleotide; and

(e) optionally repeating steps (b) - (f) or (b) - (e) one or more times to determine the sequence of the target single-stranded polynucleotide.
The method of claim 5, wherein the ribose or deoxyribose moiety of each of the nucleotides comprises a protecting group attached through a 2' or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of the nucleotide so as to expose a 3' -OH group,

for example, the chemiluminescent label and the protecting group are removed under the same conditions,

for example, the nucleotide is selected from nucleotide A, G, C and T or U.
The method of claim 5 or 6, e.g., said detecting a chemiluminescent label of the nucleotide of (b) comprises contacting said chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the kinetics of the luminescence of the light emitted thereby,

for example, the chemiluminescent label is selected from the group consisting of biochemical chemiluminescent labels eliciting different luminescent kinetics and any combination thereof,

for example, the chemiluminescent label is selected from the group consisting of luciferases eliciting different luminescence kinetics and any combination thereof,

for example, the chemiluminescent label is a combination of two luciferases that elicit different luminescence kinetics,

for example, said detecting the chemiluminescent label of the nucleotide of (b) comprises contacting said chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the type of luminescence emitted thereby,

for example, the chemiluminescent label is selected from the group consisting of biochemical chemiluminescent labels eliciting different types of luminescence, and any combination thereof,

for example, the chemiluminescent label is selected from the group consisting of luciferases that elicit different luminescence types and any combination thereof,

for example, the chemiluminescent label is a combination of two luciferases that elicit different luminescence types,

for example, the lighting types include a flash type and a glow type,

for example, in the nucleotide, a first nucleotide is attached to a first chemiluminescent label by a second linking group, a second nucleotide is attached to the first chemiluminescent label by the first linking group, a third nucleotide is attached to a second chemiluminescent label by a third linking group, and a fourth nucleotide is not attached to any chemiluminescent label,

for example, in the nucleotides, a first nucleotide is attached to a first luciferase through a second linking group, a second nucleotide is attached to the first luciferase through the first linking group, a third nucleotide is attached to the second luciferase through a third linking group, and a fourth nucleotide is not attached to any luciferase,

wherein the first linking group and the third linking group may be the same or different and the second linking group and the third linking group are different.
The method of any one of claims 5-7, attaching a chemiluminescent label to the nucleotide by affinity interaction,

for example, the affinity interaction includes antigen-antibody interaction and biotin-avidin (such as streptavidin) interaction,

for example, by attaching a chemiluminescent label to one of the members involved in the affinity interaction and a nucleotide to the other member involved in the affinity interaction, thereby attaching the chemiluminescent label to the nucleotide through the affinity interaction between the members,

for example, the member attached to the nucleotide is biotin, the member attached to the chemiluminescent label is avidin (e.g., streptavidin),

for example, the member linked to the nucleotide is digoxigenin, the member linked to the chemiluminescent label is an anti-digoxigenin antibody,

for example, the member attached to the nucleotide is digoxigenin and the member attached to the chemiluminescent label is avidin (e.g., streptavidin), wherein digoxigenin and avidin are affinity bound by an anti-digoxigenin antibody attached to biotin.
The method of any one of claims 5-8, wherein each nucleotide is contacted with the target single-stranded polynucleotide sequentially, unincorporated nucleotides are removed prior to addition of the next nucleotide, and wherein the detection and removal of the chemiluminescent label is performed after addition of each nucleotide or after addition of all four nucleotides.
The method of claim 9, wherein one, two, three, or all four nucleotides are simultaneously contacted with the target single-stranded polynucleotide and unincorporated nucleotides are removed prior to detection, wherein detection and removal of the chemiluminescent label is performed after addition of the one, two, three, or all four nucleotides.
A kit, comprising: (a) one or more nucleotides selected from nucleotides A, G, C and T or U, wherein the nucleotides are attached to the chemiluminescent label by different linking groups, wherein a first nucleotide is attached to the first chemiluminescent label by a second linking group, a second nucleotide is attached to the first chemiluminescent label by a first linking group, a third nucleotide is attached to the second chemiluminescent label by a third linking group, and a fourth nucleotide is not attached to any chemiluminescent label; and (b) packaging materials therefor.
The kit of claim 11, wherein the first linking group and the third linking group are the same or different and the second linking group and the third linking group are different.
The kit of claim 11 or 12, further comprising an enzyme and a buffer suitable for the enzyme to function.
The kit of any one of claims 11-13, further comprising a suitable substrate for reaction with the chemiluminescent label.
A method of sequencing a nucleic acid molecule, comprising the steps of:

(1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching a nucleic acid molecule to be sequenced to a support;

(2) adding a primer for initiating a nucleotide polymerization reaction, a polymerase for performing the nucleotide polymerization reaction, and four nucleotides, thereby forming a reaction system containing a solution phase and a solid phase; wherein, the four nucleotides are respectively derivatives of nucleotide A, (T/U), C and G, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first nucleotide is linked to a first molecular tag by a cleavable second linker,

the second nucleotide is linked to a first molecular tag by a cleavable first linker,

the third nucleotide is linked to a second molecular tag by a cleavable third linker,

the fourth nucleotide is not connected with a molecular marker;

(3) annealing a primer to a nucleic acid molecule to be sequenced, said primer acting as an initial growing nucleic acid strand, together with said nucleic acid molecule to be sequenced, forming a support-attached duplex;

(4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform the nucleotide polymerization reaction, thereby incorporating one of the four nucleotides into the 3' end of the growing nucleic acid strand;

(5) contacting the duplex of the previous step with two different luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively, and performing a binding reaction, and then subjecting the luciferases to a fluorescence reaction in the presence of a substrate to detect the emitted fluorescent signal;

(6) adding a lysis solution to lyse the second connecting group, then enabling the luciferase to perform a fluorescence reaction again in the presence of a substrate, and detecting the emitted fluorescence signal;

(7) removing the molecular marker of each nucleotide;

(8) optionally repeating steps (3) - (7) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

Wherein the first linking group and the third linking group may be the same or different, and the second linking group and the third linking group are different.
The method of claim 15, wherein the duplex is contacted with two different luciferases in a one-step reaction and a binding reaction is performed in step (5); or the duplex is contacted with two luciferases in sequence in the step (5) and binding reaction is carried out.
The method according to claim 15 or 16, comprising the steps of:

(1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching a nucleic acid molecule to be sequenced to a support;

(2) adding a primer for initiating a nucleotide polymerization reaction, a polymerase for performing the nucleotide polymerization reaction, and four nucleotides, thereby forming a reaction system containing a solution phase and a solid phase; wherein, the four nucleotides are respectively derivatives of nucleotide A, (T/U), C and G, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first nucleotide is linked to a first molecular tag by a cleavable second linker,

the second nucleotide is linked to a first molecular tag by a cleavable first linker,

the third nucleotide is linked to a second molecular tag by a cleavable third linker,

the fourth nucleotide is not connected with a molecular marker;

(3) annealing a primer to a nucleic acid molecule to be sequenced, said primer acting as an initial growing nucleic acid strand, together with said nucleic acid molecule to be sequenced, forming a support-attached duplex;

(4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform the nucleotide polymerization reaction, thereby incorporating one of the four nucleotides into the 3' end of the growing nucleic acid strand;

(5) removing the solution phase of the reaction system of the previous step, retaining the duplexes attached to the support, and adding two different luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively, to perform a binding reaction;

(6) removing unbound luciferase with an elution buffer;

(7) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(8) removing the solution of the previous step;

(9) adding a lysis solution to lyse the second linking group;

(10) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(11) optionally removing the first linking group, the third linking group and the 3' protecting group of each nucleotide;

(12) optionally removing the solution of the previous step;

(13) optionally repeating steps (3) - (12) or (3) - (10) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

Wherein the first linking group and the third linking group may be the same or different, and the second linking group and the third linking group are different.
The method according to claim 15 or 16, comprising the steps of:

(1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching a nucleic acid molecule to be sequenced to a support;

(2) adding a primer for initiating a nucleotide polymerization reaction, a polymerase for performing the nucleotide polymerization reaction, and four nucleotides, thereby forming a reaction system containing a solution phase and a solid phase; wherein, the four nucleotides are respectively derivatives of nucleotide A, (T/U), C and G, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first nucleotide is linked to a first molecular tag by a cleavable second linker,

the second nucleotide is linked to a first molecular tag by a cleavable first linker,

the third nucleotide is linked to a second molecular tag by a cleavable third linker,

the fourth nucleotide is not connected with a molecular marker;

(3) annealing a primer to a nucleic acid molecule to be sequenced, said primer acting as an initial growing nucleic acid strand, together with said nucleic acid molecule to be sequenced, forming a support-attached duplex;

(4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform the nucleotide polymerization reaction, thereby incorporating one of the four nucleotides into the 3' end of the growing nucleic acid strand;

(5) removing the solution phase of the reaction system of the previous step, retaining the duplexes attached to the support, and adding a first luciferase capable of specifically binding to the first molecular label to perform a binding reaction;

(6) removing unbound first luciferase with an elution buffer;

(7) adding a substrate of the first luciferase and detecting a curve of the change of the fluorescent signal along with time;

(8) removing the solution of the previous step;

(9) adding a second luciferase capable of specifically binding to the second molecular marker to perform a binding reaction;

(10) removing unbound second luciferase with an elution buffer;

(11) adding a substrate of a second luciferase and detecting a curve of the change of the fluorescence signal along with time;

(12) optionally removing the solution of the previous step;

(13) adding a lysis solution to lyse the second linking group;

(14) adding a substrate of luciferase, and detecting a curve of a fluorescent signal changing along with time;

(15) optionally removing the first linking group, the third linking group and the 3' protecting group of each nucleotide

(16) Optionally removing the solution of the previous step;

(17) optionally repeating steps (3) - (16) or (3) - (14) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

Wherein the first linking group and the third linking group may be the same or different, and the second linking group and the third linking group are different.
The method of claim 17 or 18 wherein the first and second luciferases comprise different luciferases.
The method of any one of claims 15-19, wherein the first molecular tag is biotin and the first luciferase is streptavidin-labeled; the second molecular marker is digoxin and the second luciferase is labeled with an anti-digoxin antibody.
The method of any one of claims 15-20, wherein the first and third linking groups are azide-containing linking groups and the second linking group is a disulfide-containing linking group.
The method of any one of claims 15-20, wherein the first and third linking groups are azide-containing linking groups and the second linking group is a cis-aconitic anhydride (cis-aconitic anhydride) -containing linking group.
A kit for sequencing a polynucleotide, comprising:

(a) four nucleotides which are derivatives of nucleotides A, (T/U), C and G, respectively, and have base complementary pairing ability; and, the hydroxyl group (-OH) at the 3' -position of the ribose or deoxyribose of the four nucleotides is protected by a protecting group; and the number of the first and second electrodes,

the first compound is linked to a first molecular tag by a cleavable second linker,

the second nucleotide is linked to a first molecular tag by a cleavable first linker,

the third nucleotide is linked to a second molecular tag by a cleavable third linker,

the fourth compound is not linked to a molecular tag;

(b) two luciferases capable of specifically binding to the first molecular marker and the second molecular marker, respectively, which may be the same or different; and

(c) lysis solution for lysis of linker 2.
The kit according to claim 23, wherein the two different luciferases comprise the same luciferase or different luciferases.
The kit of claim 23, wherein the first linking group and the third linking group are the same or different and the second linking group and the third linking group are different.
The kit of any one of claims 23-25, wherein the first molecular tag is biotin and the first luciferase is labeled with streptavidin; the second molecular marker is digoxin and the second luciferase is labeled with an anti-digoxin antibody.
The kit of any one of claims 23-25, wherein the first and third linking groups are azide-containing linking groups and the second linking group is a disulfide-containing linking group.
The kit of any one of claims 23-25, wherein the first linking group is an azide-containing linking group, and the second linking group and the third linking group are cis-aconitic anhydride (cis-aconitic anhydride) -containing linking groups.
The kit of any one of claims 23-28, further comprising: reagents and/or devices for extracting nucleic acid molecules from a sample; reagents for pretreating nucleic acid molecules; a support for attaching nucleic acid molecules to be sequenced; reagents for attaching (e.g., covalently or non-covalently attaching) a nucleic acid molecule to be sequenced to a support; a primer for initiating a nucleotide polymerization reaction; a polymerase for performing a nucleotide polymerization reaction; one or more buffer solutions; one or more wash solutions; or any combination thereof.