WO2020073274A1

WO2020073274A1 - Method for sequencing polynucleotide

Info

Publication number: WO2020073274A1
Application number: PCT/CN2018/109809
Authority: WO
Inventors: 廖莎; 陈奥; 章文蔚; 徐崇钧
Original assignee: 深圳华大智造极创科技有限公司
Priority date: 2018-10-11
Filing date: 2018-10-11
Publication date: 2020-04-16
Also published as: CN112384632A

Abstract

Provided is a method for determining the sequence of a target single-chain polynucleotide, which method comprises monitoring the successive incorporation of nucleotides complementary to the target single-chain polynucleotide, wherein the nucleotides are respectively attached to chemiluminescence markers which can trigger different luminous dynamics or luminous types, wherein each incorporated nucleotide can be identified by detecting the luminous dynamics or luminous type of chemiluminescence reactions involving the chemiluminescence markers and following the removal of the chemiluminescence markers.

Description

Method for sequencing polynucleotide

Field of invention

The invention relates to a method for sequencing polynucleotides by monitoring the sequential incorporation of nucleotides labeled with chemiluminescent markers, wherein the monitoring involves the detection of the chemiluminescent markers of each incorporated nucleotide The luminescence kinetics or luminescence type of a chemiluminescence reaction.

Background of the invention

In the past 10 years, the second-generation gene sequencing technology has gradually developed from emerging technologies to mainstream sequencing methods, and has gradually become an important detection tool in the clinical field. It is used in the prevention and control of infectious diseases, diagnosis of genetic diseases, and non-invasive prenatal screening. Cha and other fields are playing an increasingly important role. In order to further expand the sequencing market and make sequencers popular, the development of low-cost miniaturized sequencers has gradually become a development trend in the field of sequencing. As a classic method of second-generation sequencing technology, three sequencing methods based on four-channel, two-channel and single-channel have their advantages, but compared to the three, single-channel sequencing has fewer consumables, lower costs, and is easier to achieve miniaturization and portable instruments. Such advantages have gradually become a development trend in the field of sequencing. Products based on monochrome channels currently on the market mainly include ion torrent series sequencers, 454 sequencers and Iseq100 newly launched by Illumin.

In the current single-channel sequencing technology, ion torrent series instruments have limited the scope of their use due to the high sequencing error rate of the polymer structure. Similarly, Roche ’s 454 instruments also have insufficient sequencing accuracy and high sequencing. The cost has gradually withdrawn from the sequencing market. Illumia's Iseq100 is based on monochromatic fluorescence technology and semiconductor technology, which realizes a miniaturized sequencer and maintains high sequencing quality. However, because its optical signal is excited by the laser, the instrument needs to be equipped with an additional laser, thereby increasing the volume of the instrument. In addition, in order to avoid the background value generated by the excitation light, it is necessary to perform special processing on the semiconductor chip to filter out the background generated by the excitation light. This processing will cause a high cost and increase the sequencing cost.

Therefore, there is still a need in the art for improved sequencing technology, which has lower sequencing costs and maintains higher sequencing quality.

Summary of the invention

The invention relates to labeling nucleotides with chemiluminescent markers and sequencing polynucleotides by monitoring the sequential incorporation of such labeled nucleotides. More specifically, the present invention relates to monitoring the sequential incorporation of nucleotides by detecting the luminescence kinetics or luminescence type of the chemiluminescence reaction in which the chemiluminescence label of each incorporated nucleotide participates.

In one aspect, the invention relates to a method for determining the sequence of a target single-stranded polynucleotide, which includes monitoring the sequential incorporation of nucleotides complementary to the target single-stranded polynucleotide, wherein the nucleotides are each attached To chemiluminescent labels that trigger different luminescence kinetics, where each incorporated nucleotide is identified by detecting the luminescence kinetics of the chemiluminescent reaction in which the chemiluminescent label participates and then removing the chemiluminescent label.

In specific embodiments, the respective ribose or deoxyribose moiety of the nucleotide contains a protecting group attached via a 2 'or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of each nucleotide Group to expose the 3'-OH group.

In a specific embodiment, the chemiluminescent label and the protecting group are removed under the same conditions.

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

In a specific embodiment, the detection of the luminescence kinetics of the chemiluminescence reaction involving the chemiluminescence label includes contacting the chemiluminescence label with a suitable substrate to trigger the chemiluminescence reaction, and detecting the light emitted thereby Luminous dynamics.

In a specific embodiment, the chemiluminescent label is selected from biochemiluminescent labels that induce different luminescence kinetics and any combination thereof.

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

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

In one aspect, the invention relates to a method for determining the sequence of a target single-stranded polynucleotide, which includes monitoring the sequential incorporation of nucleotides complementary to the target single-stranded polynucleotide, wherein the nucleotides are each attached To initiate chemiluminescent labels of different luminescence types, where each incorporated nucleotide is identified by detecting the luminescence type of the chemiluminescent reaction in which the chemiluminescent label participates and then removing the chemiluminescent label.

In a specific embodiment, the respective ribose or deoxyribose moiety of the nucleotide contains a protecting group attached via a 2 'or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of each nucleotide Group to expose the 3'-OH group.

In a specific embodiment, the detection of the luminescence kinetics of the chemiluminescence reaction involving the chemiluminescence label includes contacting the chemiluminescence label with a suitable substrate to trigger the chemiluminescence reaction, and detecting the light emitted thereby Luminous type.

In a specific embodiment, the chemiluminescent label is selected from biochemiluminescent labels that induce different types of luminescence and any combination thereof.

In a specific embodiment, the chemiluminescent label is selected from luciferases that trigger different types of luminescence and any combination thereof.

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

In a specific embodiment, the light-emitting type includes a flash type, a glow type, and a mixed type.

In one aspect, the invention relates to a method for determining the sequence of a target single-stranded polynucleotide, which includes (a) providing one or more nucleotides, wherein the nucleotides are each attached to a different chemiluminescence Markers, where the chemiluminescent marker attached to each type of nucleotide exhibits different luminescence kinetics when detected than the chemiluminescent marker attached to other types of nucleotides; (b) Incorporation of nucleotides into the complementary strand of the target single-stranded polynucleotide; (c) Detection of the chemiluminescent label of the nucleotides of (b) to determine the type of nucleotides incorporated; (d) Remove the chemiluminescent label of the nucleotide of (b); and (e) optionally repeat steps (b)-(d) one or more times in order to determine the sequence of the target single-stranded polynucleotide.

In a specific embodiment, detecting the chemiluminescent label of the nucleotide of (b) includes contacting the chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the luminous power of the light emitted thereby learn.

In a specific embodiment, the respective ribose or deoxyribose moiety of the nucleotide contains a protecting group attached via a 2 'or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of the nucleotide, In order to expose the 3'-OH group.

In a specific embodiment, each nucleotide is sequentially contacted with the target single-stranded polynucleotide, unincorporated nucleotides are removed before the next nucleotide is added, and wherein the chemiluminescent label The detection and removal are performed after adding each nucleotide or after adding all four nucleotides.

In a specific embodiment, one, two, three, or all four nucleotides are contacted with the target single-stranded polynucleotide at the same time, and unincorporated nucleotides are removed before detection, where all The detection and removal of the chemiluminescent marker is performed after adding the one, two, three, or all four nucleotides.

In one aspect, the invention relates to a method for determining the sequence of a target single-stranded polynucleotide, which includes (a) providing one or more nucleotides, wherein the nucleotides are each attached to a different chemiluminescence Markers, where the chemiluminescent marker attached to each type of nucleotide exhibits a different type of luminescence from the chemiluminescent marker attached to other types of nucleotides when detected; (b) a nuclear Incorporation of nucleotides onto the complementary strand of the target single-stranded polynucleotide; (c) detection of the chemiluminescent label of the nucleotide of (b) to determine the type of nucleotide incorporated; (d) removal (b) the chemiluminescent label of the nucleotide; and (e) optionally repeating steps (b)-(d) one or more times in order to determine the sequence of the target single-stranded polynucleotide.

In a specific embodiment, detecting the chemiluminescent label of the nucleotide of (b) includes contacting the chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the type of light emitted thereby .

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

In various aspects, attachment between nucleotides and chemiluminescent labels includes attachment mediated by affinity interactions.

In specific embodiments, affinity interactions include antigen-antibody interactions and biotin-avidin (eg, streptavidin) interactions.

In specific embodiments, by linking the chemiluminescent label to one of the members participating in the affinity interaction, and linking the nucleotide to the other member participating in the affinity interaction, thereby passing the affinity between the members And interactions attach chemiluminescent labels to nucleotides.

In a specific embodiment, the member linked to the nucleotide is biotin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin).

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

In a specific embodiment, the member linked to the nucleotide is digoxin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin), wherein digoxin and avidin are linked to The biotin-linked anti-digoxin antibody binds affinity.

In a specific embodiment, of the nucleotides, the first nucleotide is attached to the first chemiluminescent label, the second nucleotide is attached to the second chemiluminescent label, and the third The nucleotide is attached to both the first chemiluminescent label and the second chemiluminescent label, and the fourth nucleotide is not attached to any chemiluminescent label.

In a specific embodiment, of the nucleotides, the first nucleotide is attached to the first luciferase, the second nucleotide is attached to the second luciferase, and the third nucleoside The acid is attached to both the first luciferase and the second luciferase, and the fourth nucleotide is not attached to any luciferase.

In other aspects, the invention also relates to a kit comprising: (a) one or more nucleotides selected from nucleotides A, G, C and T or U, wherein the nucleotides are each attached To different chemiluminescent labels, where the chemiluminescent labels attached to each type of nucleotide exhibit different luminescence kinetics and chemiluminescent labels attached to other types of nucleotides when detected / Or luminous type; and (b) their packaging materials.

In a specific embodiment, the kit also contains an enzyme and a buffer suitable for the enzyme to function.

In a specific embodiment, the kit also contains a suitable substrate that reacts with the chemiluminescent label.

BRIEF DESCRIPTION

Figure 1 shows examples of different luminescence kinetics.

Figure 2 shows a flow chart for sequencing polynucleotides according to one embodiment of the invention.

Figure 3 shows a signal curve for sequencing a polynucleotide according to an embodiment of the present invention.

Figure 4 shows a comparison of signal curves for sequencing polynucleotides according to one embodiment of the invention.

Detailed description of the invention

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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 the definitions set forth herein conflict or are inconsistent with the definitions described in the patents, applications, and other publications incorporated herein by reference, the definitions set forth herein shall control.

As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or the like. The polynucleotide may be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. The polynucleotide molecule may be derived from double-stranded DNA (dsDNA) form (eg, genomic DNA, PCR, and amplification products, etc.), or may be derived from single-stranded form DNA (ssDNA) or RNA and it may be converted to dsDNA form, And vice versa. The exact sequence of the polynucleotide molecule may be known or unknown. The following are illustrative examples of polynucleotides: genes or gene fragments (eg, probes, primers, EST or SAGE tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transport 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 of any of the above sequences Probes, primers or amplified copies.

Polynucleotides can include nucleotides or nucleotide analogs. Nucleotides usually contain sugars (such as ribose or deoxyribose), bases and at least one phosphate group. Nucleotides may be abasic (ie, lack bases). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleosides Acids and their mixtures. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thoracic acid Glycosine triphosphate (TTP), cytidine acid (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 (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCDP), deoxycytidine triphosphate (dCDP) dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine Glycoside triphosphate (dUTP). Nucleotide analogs containing modified bases can also be used in the methods described herein. Whether it has a natural backbone or a similar structure, exemplary modified bases that can be included in polynucleotides include, for example, inosine, xathanine, hypoxathanine, isocytosine, isobird Purine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2 -Propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propyne Cytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine Purine or guanine, 8-thioadenine or guanine, 8-sulfanyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine Purine, 7-methyladenine, 8-azaguanine, 8-azaguanine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine Wait. As is known in the art, certain nucleotide analogs cannot be introduced into polynucleotides, for example, nucleotide analogs, such as adenosine 5'-phosphorosulfuric acid.

As used herein, the term "nucleotide A" refers to a nucleotide containing adenine (A) or a modification or analogue thereof, such as ATP, dATP. "Nucleotide G" refers to a nucleotide containing guanine (G) or a modification or analogue thereof, such as GTP, dGTP. "Nucleotide C" refers to a nucleotide containing cytosine (C) or a modification or analogue thereof, such as CTP, dCTP. "Nucleotide T" refers to a nucleotide containing thymine (T) or a modification or analogue thereof, such as TTP, dTTP. "Nucleotide U" refers to a nucleotide containing uracil (U) or a modification or analogue thereof, such as UTP, dUTP.

Nucleotide labeling

The invention relates to the labeling of nucleotides with chemiluminescent markers, so that different nucleotides can be distinguished.

As used herein, the term "chemiluminescent label" refers to any compound that can be attached to a nucleotide, which can be triggered by contact with a suitable substrate to trigger a chemiluminescent reaction, thereby generating without the need for excitation light Detectable optical signal. Generally speaking, any component that participates in a chemiluminescent reaction can be used as a chemiluminescent marker as described herein, and correspondingly, other components that participate in a chemiluminescent reaction are referred to herein as the chemiluminescent marker Substrate. Examples of commonly used suitable chemiluminescent labels include, but are not limited to, peroxidase, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminal, luminol, Isoluminol, acridinium ester, sulfonamide, etc. The substrate of the chemiluminescent label will depend on the specific chemiluminescent label used, for example, the substrate of alkaline phosphatase may be AMPPD (adamantyl 1,2-dioxan aromatic phosphate), luciferase The substrate may be fluorescein, and the acridinium ester substrate may be a mixture of sodium hydroxide and H ₂ O _{2 and} the like. For a detailed description of chemiluminescent labels and their corresponding substrates, see, for example, Larry J. Kricka, Chemiluminescent and Bioluminescent Techniques, CLIN. CHEM. 37/9, 1472-1481 (1991) and edited by Tsuji, A. et al. (2005 ) Bioluminescence and chemiluminescence: Progress and perspectives. World Scientific: [sl] .ISBN 978-981-256-118-3.596pp ..

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

As used herein, the term "biochemiluminescent label" refers to any compound that can be attached to a nucleotide, which can be triggered by contact with a suitable substrate to trigger a bioluminescent reaction, without the need for excitation light Generate a detectable light signal. Bioluminescence is a type of chemiluminescence, which is light generated by chemical reactions that occur in the body or in the secretions of certain types of organisms. Examples of biochemiluminescent labels may include, for example, luciferase, aequorin, glucose dehydrogenase, glucose oxidase, and the like. The substrate of the biochemiluminescent label will depend on the specific biochemiluminescent label used, for example, the substrate of luciferase may be luciferin, and the substrate of aequorin may be calcium ion. For a detailed description of biochemiluminescent markers and their corresponding substrates, see, for example, Larry J. Kricka, Chemiluminescent and Bioluminescent Technologies, CLIN. CHEM. 37/9, 1472-1481 (1991) and Tsuji, A. et al. 2005) Bioluminescence and chemiluminescence: Progress and perspectives. World Scientific: [sl]. ISBN 978-981-256-118-3.596pp ..

In a preferred embodiment, the biochemiluminescent label as used herein is luciferase. In a specific embodiment, the luciferase is selected from the group consisting of Gaussia luciferase, Renilla luciferase, Waistworm luciferase, firefly luciferase, fungal luciferase, bacterial fluorescence Luciferase and vargula luciferase.

As used herein, "labeling nucleotides with chemiluminescent labels" means attaching chemiluminescent labels to nucleotides. Specific methods for attaching chemiluminescent labels to nucleotides are known to those skilled in the art, for example, reference may be made to the relevant descriptions in the following documents (both incorporated herein by reference): Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; US Patent Nos. 4,581,333, 5,283,174, 5,547,842, 5,656,207 and 5,658,737. In one embodiment, the chemiluminescent label can be directly attached to the nucleotide through a covalent bond. In another embodiment, the chemiluminescent label can be attached to the nucleotide via a linking group.

In a preferred embodiment, the chemiluminescent label is attached to the nucleotide through affinity interaction. As is well known to those skilled in the art, “affinity interaction” generally refers to the specific interaction between biological molecules (eg, protein molecules, such as enzymes, antibodies) and substances that they specifically recognize. Affinity interactions may include, for example, antigen-antibody interactions and biotin-avidin (e.g. streptavidin) interactions. An example of an antigen-antibody interaction may be, for example, digoxin-anti-digoxin antibody interaction.

In embodiments that utilize affinity interactions, the chemiluminescent label can be attached to one of the members involved in the affinity interaction, and the nucleotides can be attached to other members involved in the affinity interaction, thereby passing the member The affinity interaction between them attaches the chemiluminescent label to the nucleotide.

In an exemplary embodiment that utilizes digoxin-anti-digoxin antibody interaction, digoxin can be linked to a nucleotide and a chemiluminescent label can be linked to an anti-digoxin antibody to pass digoxin -Anti-digoxin antibody interaction attaches the chemiluminescent label to the nucleotide.

In an exemplary embodiment that utilizes biotin-avidin (eg, streptavidin) interaction, biotin can be linked to a nucleotide, and a chemiluminescent label can be linked to avidin (eg, streptavidin) ), Thereby attaching the chemiluminescent label to the nucleotide through biotin-avidin (eg, streptavidin) interaction. In another embodiment, the present invention also relates to multiple labeling of nucleotides, ie more than one chemiluminescent label is attached to the same nucleotide. In a preferred embodiment, more than one chemiluminescent label is attached to the same nucleotide through different affinity interactions. This can be done, for example, by simultaneously connecting members participating in different affinity interactions to nucleotides, and connecting different chemiluminescent labels to be attached to the nucleotides to other members participating in the different affinity interactions, Thus, attachment of more than one chemiluminescent label to the nucleotide is achieved through the different affinity interactions. In a preferred embodiment, two different chemiluminescent labels are attached to the same nucleotide through different affinity interactions.

In an exemplary embodiment where two different chemiluminescent labels are attached to nucleotides, the nucleotides can be simultaneously linked to one of the members participating in the first affinity interaction and participating in the second affinity interaction One of the members, and connect the first chemiluminescent label to other members participating in the first affinity interaction, and the second chemiluminescent label to other members participating in the first affinity interaction, thereby passing The first and second affinity interactions attach the first and second chemiluminescent labels to nucleotides.

In one embodiment, the first affinity interaction is a digoxin-anti-digoxin antibody interaction, and the second affinity interaction is biotin-avidin (eg, streptavidin) interaction. In a specific embodiment, the nucleotides can be linked to digoxin and biotin simultaneously, and the first chemiluminescent label can be linked to an anti-digoxin antibody, and the second chemiluminescent label can be linked to avidin (E.g. streptavidin), whereby the first chemiluminescent label and the first chemiluminescent label and the first digoxin-anti-digoxin antibody interaction and biotin-avidin (e.g. streptavidin) interaction The two chemiluminescent markers are attached to nucleotides.

In another specific embodiment, the nucleotides can be linked to digoxin and biotin simultaneously, and the first chemiluminescent label can be linked to an anti-digoxin antibody, and the second chemiluminescent label can be linked to the parent Heparin (e.g. streptavidin), whereby the first chemiluminescent label is interacted with digoxin-anti-digoxin antibody and biotin-avidin (e.g. streptavidin) interaction And a second chemiluminescent label attached to the nucleotide.

In addition, it is also possible to attach a chemiluminescent label to a nucleotide by combining different affinity interactions. In an exemplary embodiment, it is possible, for example, to link a nucleotide to one of the members involved in the first affinity interaction and one of the members involved in the second affinity interaction to participate in the first affinity interaction Other members of, and attaching the chemiluminescent label to other members participating in the second affinity interaction, thereby attaching the chemiluminescent label to the nucleoside through the first affinity interaction and the second affinity interaction acid.

In a specific embodiment, for example, nucleotides can be linked to digoxin, biotin can be linked to anti-digoxin antibodies, and chemiluminescent labels can be linked to avidin (eg, streptavidin), Thus, the chemiluminescent label is attached to the nucleotide through the digoxin-anti-digoxin antibody interaction and the biotin-avidin (eg, streptavidin) interaction.

As used herein, a "link" between a chemiluminescent label or nucleotide and a member involved in an affinity interaction may be any suitable form of link known in the art. Such connection may include, for example, direct connection or indirect connection, such as connection via a linker, and may also include, for example, non-covalent connection (such as connection mediated by hydrogen bonding, affinity interaction, etc.) and covalent connection, and may also For example, a recombinantly expressed fusion protein is formed to achieve such a connection.

Luminescence Kinetics of Chemiluminescence Reaction

The present invention relates to the detection of nucleotides labeled with chemiluminescent labels by detecting the luminescence kinetics of chemiluminescent reactions in which the chemiluminescent labels participate.

As used herein, "luminescence kinetics of a chemiluminescence reaction" refers to a characteristic spectrum of the intensity change of light emitted by a chemiluminescence reaction with time. This can be characterized, for example, by plotting the intensity of light emitted by the chemiluminescence reaction with time.

In the prior art, different chemiluminescent labels are usually detected and distinguished by detecting the light emitted by the chemiluminescent reaction at a specific wavelength. However, the present inventors have discovered that the luminescence kinetics by detecting chemiluminescence reactions can also be used to detect and distinguish different chemiluminescent labels. Such different chemiluminescent labels each elicit different luminescence kinetics. As used herein, "initiating" different luminescence kinetics means that the light emitted by the chemiluminescence reaction in which the chemiluminescent label participates has different luminescence kinetics.

Such a detection method of the present invention is advantageous because it is possible to distinguish chemiluminescent labels that have similar emission wavelengths but have different emission kinetics.

Therefore, in a specific embodiment, the present invention relates to a method for identifying nucleotides, which includes labeling nucleotides with chemiluminescent labels that induce different luminescence kinetics, and detecting chemiluminescent reactions in which the chemiluminescent labels participate Luminescence dynamics. The invention also relates to nucleotides labeled with chemiluminescent labels that induce different luminescence kinetics.

Therefore, in a specific embodiment, the first nucleotide can be labeled with a first chemiluminescent label that triggers a first luminescence kinetics, and the second core can be labeled with a second chemiluminescent label that triggers a second luminescence kinetics Glycosides, and the third nucleotide labeled with the third chemiluminescent label that triggers the third luminescence kinetics, and the fourth nucleotide are not labeled at all, so that the respective luminescent power of the chemiluminescent label can be detected by To identify the four nucleotides.

In another embodiment, the invention also relates to multiple labeling of nucleotides with a combination of chemiluminescent labels that induce different luminescence kinetics. For example, chemiluminescent labels that trigger different luminescence kinetics can be attached to the same nucleotide through different affinity interactions as described above. Such a technical solution is advantageous because the combination of chemiluminescent markers that initiate different luminescence kinetics will initiate luminescence kinetics that are different from the luminescence kinetics initiated by each chemiluminescent marker in the combination, thereby reducing costs .

In a specific embodiment, the invention relates to the dual labeling of nucleotides with a combination of two chemiluminescent labels that induce different luminescence kinetics. In a specific embodiment, the first nucleotide may be labeled with a first chemiluminescent label that triggers a first luminescence kinetics, and the second nucleotide may be labeled with a second chemiluminescent label that triggers a second luminescence kinetics, And double labeling the third nucleotide with the first chemiluminescent label and the second chemiluminescent label, and the fourth nucleotide without any labeling, so that the respective luminescence power of the chemiluminescent label can be detected To identify the four nucleotides, wherein the double labeling of the first chemiluminescent label and the second chemiluminescent label can trigger a different luminescence kinetics than the first luminescence kinetics and the second luminescence kinetics learn.

In a preferred embodiment, the nucleotide is selected from nucleotides A, G, C and T / U.

Chemiluminescence reaction type

In a further embodiment, the invention relates to the detection of nucleotides labeled by the chemiluminescent marker by detecting the type of luminescence of the chemiluminescent reaction in which the chemiluminescent marker participates.

As used herein, the "luminescence type of a chemiluminescent reaction" is divided according to the duration of light emitted by the chemiluminescent reaction, which generally includes a flash type and a glow type. The flash type emits light within a few seconds, as in the acridinium ester system. The glow time of the glow type is several minutes to several tens of minutes, such as horseradish peroxidase-luminol system, alkaline phosphatase-AMPPD system, xanthine oxidase-luminol system, etc. In the present invention, the light emission type between the flash type and the glow type is also called a hybrid type. Mixed-type luminescence is usually produced by mixing flash-type luminescence and glow-type luminescence together. For example, when the flash-initiated chemiluminescent marker and the glow-initiated chemiluminescent marker are mixed together and contact with their substrate and emit light at the same time, a mixed light-emitting column type between the flash type and the glow type will be generated . Typical examples of the emission characteristic spectrum of the flash type, the glow type, and the mixed type are shown in FIG. 1.

It is advantageous to detect chemiluminescent labels by detecting the luminescence type of the chemiluminescence reaction, because it is possible to distinguish chemiluminescent labels that have similar luminescence kinetics but significantly different luminescence types. Such detection is also particularly suitable for detecting multiple labels of chemiluminescent markers, because the combination of chemiluminescent markers with different luminescence types can result in a significantly different luminescence type from the individual chemiluminescent markers.

Therefore, in a specific embodiment, the present invention relates to a method of identifying nucleotides, which includes labeling nucleotides with chemiluminescent markers that trigger different luminescence types, and detecting Luminous type. The invention also relates to nucleotides labeled with chemiluminescent markers that trigger different types of luminescence.

In a specific embodiment, the first nucleotide can be labeled with a first chemiluminescent label that triggers a first luminescence type, the second nucleotide can be labeled with a second chemiluminescent label that triggers a second luminescence type, and The first chemiluminescent label and the second chemiluminescent label doubly label the third nucleotide and the fourth nucleotide without any labeling, so that they can be identified by detecting the respective luminescence types of the chemiluminescent label The four nucleotides, wherein the double labeling of the first chemiluminescent label and the second chemiluminescent label can initiate a different type of light emission than the first and second types of light emission.

In a specific embodiment, the first light-emitting type is a flash type and the second light-emitting type is a glow type. In a specific embodiment, the type of luminescence induced by the double labeling of the first chemiluminescent marker and the second chemiluminescent marker is between the first luminescent type (eg flash type) and the second luminescent type (eg glow Light type), which is referred to herein as a hybrid type.

Sequencing of polynucleotides

The nucleotides labeled with chemiluminescent labels that trigger different luminescence kinetics and the nucleotides labeled with chemiluminescent labels that trigger different luminescence types of the present invention can be used in various nucleic acid sequencing methods. Preferably, the nucleotides labeled with chemiluminescent labels that induce different luminescence kinetics and the nucleotides labeled with chemiluminescent labels that induce different luminescence types of the present invention are suitable for sequencing by synthesis. Synthetic sequencing as used herein is a variety of synthetic sequencing methods well known in the art. Basically, sequencing by synthesis involves first hybridizing the nucleic acid molecule to be sequenced with the sequencing primer, and then polymerizing the labeled nucleic acid molecule at the 3 'end of the sequencing primer as described herein in the presence of a polymerase as a template Nucleotides. After polymerization, the labeled nucleotide is identified by detecting the label. After removing the label from the labeled nucleotide (ie, the chemiluminescent label as described herein), the next polymerization sequencing cycle begins.

In addition, the nucleic acid sequencing method can also use the nucleotides described herein to perform the method disclosed in US Patent No. 5302509.

The method for determining the sequence of the target polynucleotide may be performed by denaturing the target polynucleotide sequence and contacting the target single-stranded polynucleotide with different nucleotides respectively to form a complement of the target nucleotide, And the incorporation of the nucleotide is detected. The method utilizes polymerization so that the polymerase extends the complementary strand by incorporating the correct nucleotide complementary to the target. The polymerization reaction also requires special primers to initiate polymerization.

For each round of reaction, the incorporation of the labeled nucleotide is performed by a polymerase, and the incorporation event is subsequently determined. There are many different polymerases, and it is easy for those of ordinary skill in the art to determine the most suitable polymerase. Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase or vent polymerase. Polymerases engineered to have specific properties can also be used.

The sequencing method is preferably performed on target polynucleotides arranged on a solid support. Multiple target polynucleotides can be immobilized on the solid support through a linker molecule, or can be attached to particles such as microspheres, which can also be attached to the solid support material.

The polynucleotide can be attached to the solid support by a variety of methods, including the use of biotin-streptavidin interactions. Methods for immobilizing polynucleotides on solid supports are well known in the art, and include lithography techniques and spotting each polynucleotide on a specific position 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 flat, although microbeads (microspheres) can also be used, and the latter can also be attached to other solid supports by known methods. The microspheres can have any suitable size, and their diameter is usually 10-100 nm. In a preferred embodiment, the polynucleotide is attached directly on a plane, preferably on a flat glass surface. The connection is preferably made in the form of a covalent bond. The array used is preferably a single molecule array, which includes polynucleotides located in unique optically distinguishable regions, for example as described in International Application No. WO00 / 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 usually necessary to first anneal the primer sequence to the target polynucleotide, the primer sequence is recognized by the polymerase and serves as the starting site for the subsequent extension of the complementary strand The role. The primer sequence may be added as an independent component with respect to the target polynucleotide. In addition, the primer and the target polynucleotide may be part of a single-stranded molecule, and the primer part and the target part form an intramolecular duplex, that is, a hairpin loop structure. The structure can be fixed on the solid support at any position of the molecule. Other conditions necessary to perform the polymerase reaction are well known to those skilled in the art, and these conditions include temperature, pH, and buffer composition.

Subsequently, the labeled nucleotide of the present invention is brought into contact with the target polynucleotide to enable polymerization. The nucleotides can be added sequentially, that is, each type of nucleotide (A, C, G, or T / U) is added separately, or simultaneously.

The polymerization step is allowed to proceed for a time sufficient to incorporate one nucleotide.

The unincorporated nucleotides are then removed, for example, by performing a washing step on the array, and then detection of the incorporation label can be performed.

The detection can be carried out by conventional methods, for example, the synthetic nucleic acid can be brought into contact with the corresponding substrate according to the specific chemiluminescent label carried by the nucleotide, and the luminescence kinetics or luminescence type of the light emitted thereby can be detected.

After detection, the label can be removed with suitable conditions.

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

In a specific embodiment, the labeled nucleotides of the present invention also have a 3 'protecting group. In some embodiments of the invention, the protecting group and the chemiluminescent label are generally two different groups on the 3'-blocked labeled nucleotide, but in other embodiments, the protecting group It may be the same group as the chemiluminescent label.

As used herein, the term "protecting group" means a group that prevents a polymerase (which incorporates a nucleotide containing the group into the polynucleotide chain being synthesized) from containing the group After the nucleotide of the group is incorporated into the polynucleotide chain being synthesized, it continues to catalyze the incorporation of another nucleotide. Such protecting groups are also referred to herein as 3'-OH protecting groups. Nucleotides containing 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 the nucleotide, as long as the protecting group can prevent additional nucleotide molecules from being added to the polynucleotide chain while not destroying the polynucleotide chain Is easily removed from the sugar portion of the nucleotide. In addition, the nucleotide modified by the protecting group needs to be resistant to polymerase or other suitable enzymes for incorporating the modified nucleotide into the polynucleotide chain. Therefore, the ideal protecting group exhibits long-term stability, can be efficiently incorporated by polymerase, prevents secondary incorporation or further incorporation of nucleotides, and can be used under mild conditions that do not disrupt the structure of polynucleotides It is preferably removed under aqueous conditions.

The prior art has described a variety of protecting groups consistent with the above description. For example, WO 91/06678 discloses that 3'-OH protecting groups include esters and ethers, -F, -NH ₂ , -OCH ₃ , -N ₃ , -OPO ₃ , -NHCOCH ₃ , 2 nitrobenzene carbonate, 2 , 4-sulfenyl dinitro and tetrahydrofuran ether. Metzker et al. (Nucleic Acids Research, 22 (20): 4259-4267, 1994) disclose the synthesis of eight 3'-modified 2-deoxyribonucleoside 5'-triphosphates (3'-modified dNTPs) And application. WO2002 / 029003 describes the use of allyl protecting groups to cap 3'-OH groups on the DNA growth chain in polymerase reactions. Preferably, various protecting groups reported in international application publications WO2014139596 and WO2004 / 018497 can be used, including, for example, those exemplified in FIG. 1A of WO2014139596 and those 3 ′ hydroxyl protecting groups defined in the claims (i.e. Protective groups), and those such as those exemplified in Figures 3 and 4 of WO2004 / 018497 and those defined in the claims. The above references are incorporated by reference in their entirety.

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

After successfully incorporating 3'-blocked nucleotides into the nucleic acid strand, the sequencing protocol needs to remove the protecting group to produce a usable 3'-OH site for continuous strand synthesis. As used herein, an agent that can remove a protecting group from a modified nucleotide depends largely on the protecting group used. For example, removal of the ester protecting group from the 3 'hydroxyl functional group is usually achieved by alkaline hydrolysis. The ease of removing protective groups varies greatly; in general, the greater the electronegativity of the substituent on the carbonyl carbon, the greater the ease of removal. For example, a highly electronegative trifluoroacetic acid group can be rapidly cleaved from 3 'hydroxyl groups in methanol at pH 7 (Cramer et al., 1963), so it is 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 variety of hydroxyl protecting groups can be selectively cleaved using chemical methods other than alkaline hydrolysis. 2,4-Dinitrophenylthio groups can be rapidly cleaved by treatment with nucleophiles such as thiophenol and thiosulfate (Letsinger et al., 1964). Allyl ether is cleaved by treatment with Hg (II) in acetone / water (Gigg and Warren, 1968). Tetrahydrothiopyranyl ethers were 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. There are several protecting groups available for this method. The use of o-nitrobenzyl ether as the protective group for the 2'-hydroxy functionality of the ribonucleoside is known and proven (Ohtsuka et al., 1978); it is removed by irradiation at 260 nm. The alkyl ortho-nitrobenzyl carbonate protecting group was also removed by irradiation at pH 7 (Cama and Christensen, 1978). Enzymatic cleavage of the 3'-OH protecting group is also possible. It has been shown that T4 polynucleotide kinase can convert the 3'-phosphate end to the 3'-hydroxyl end and can then be used as a primer for DNA polymerase I (Henner et al., 1983). This 3'-phosphatase activity is used to remove the 3 'protecting groups of those dNTP analogs that contain phosphates as protecting groups.

Other reagents that can remove the protecting group from the 3'-blocked nucleotide include, for example, phosphines (such as tris (hydroxymethyl) phosphine (THP)), which can, for example, replace the azide-containing 3'-OH protecting group The mass is removed from the nucleotide (for this application of phosphines, see for example the description in WO2014139596, the entire contents of which are incorporated herein by reference). Other reagents that can remove the protecting group from the 3'-blocked nucleotides also include, for example, the removal of the 3'-OH as a 3'-OH protecting group as described on pages 114-116 of the specification of WO2004 / 018497 The corresponding reagents for allyl, 3,4-dimethoxybenzyloxymethyl or fluoromethoxymethyl.

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

In certain embodiments, the chemiluminescent label can be incorporated into a protecting group, allowing it to be removed together with the protecting group after incorporating the 3'-blocked nucleotide into the nucleic acid strand. For example, radioactive substances such as C ¹⁴ or P ³² can be incorporated into the protecting group. Alternatively, the protecting group may contain a group that does not fluoresce itself, but can react with other substances in fluorescence. For example, the protecting group may contain a metal binding ligand such as a carboxylic acid group, which may react with added rare earth metal ions such as europium or terbium ions to generate a fluorescent substance.

In other embodiments, the chemiluminescent label can be attached to the nucleotide separately from the protecting group using a linking group. Such a chemiluminescent label may, for example, be linked to a purine or pyrimidine base of a nucleotide. In certain embodiments, the linking group used is cleavable. The use of a cleavable linking group ensures that the label can be removed after detection, which avoids any signal interference with any labeled nucleotides that are subsequently incorporated. In other embodiments, a non-cleavable linking group may be used, because after the labeled nucleotide is incorporated into the nucleic acid strand, no subsequent nucleotide incorporation is required, so there is no need to label the nucleotide Remove.

In other embodiments, the chemiluminescent label and / or linking group may have a size or structure sufficient to block the incorporation of other nucleotides into the polynucleotide chain (that is, the label itself is available As a protecting group). The blocking may be due to steric hindrance, or it may be due to a combination of size, charge, and structure.

Cleavable linking groups are well known in the art, and conventional chemical methods can be used to link the linking group to the nucleotide base and the chemiluminescent label. The linking group can be connected at any position of the nucleotide base, provided that Watson-Crick base pairing can still be performed. For purine bases, if the linking group is through position 7 of the purine or the preferred deazapurine analogue, through 8-modified purine, through N-6 modified adenine or N-2 Modified guanine linkages will be preferred. For pyrimidines, the connection is preferably via the 5th position on cytosine, thymine and uracil and the N-4 position on cytidine.

The use of the term "cleavable linking group" does not mean that the entire linking group needs to be removed (eg, from the nucleotide base). When the chemiluminescent label is attached to the base, the nucleoside cleavage site can be located on the linking group, which can ensure that a part of the linking group remains connected to the nucleotide base after cleavage.

Suitable linking groups include but are not limited to disulfide linking groups, acid labile linking groups (including dialkoxybenzyl linking groups, Sieber linking groups, indole linking groups, tert-butyl Sieber Linking group), electrophilic cleavable linking group, nucleophilic cleavable linking group, photo-cleavable linking group, linking group cleaved under reducing and oxidizing conditions, safety-catch ) Linking groups, and linking groups that are cleaved by elimination mechanisms. Suitable linking groups can be modified with standard chemical protecting groups, as disclosed in the following documents: Greene & Wuts, Protective Groups, Organic Synthesis, John Wiley & Sons. Guillier et al. Disclose other suitable cleavable linking groups for solid-phase synthesis (Chem. Rev. 100: 2092-2157, 2000).

The linking group can be cleaved by any suitable method, including contact with acids, bases, nucleophiles, electrophiles, free radicals, metals, reducing or oxidizing reagents, light, temperature, enzymes, etc. Suitable cleavage of a cleavable linking group. Generally, the cleavable linking group can be cleaved under the same conditions as the protecting group, so that only one treatment is required to remove the chemiluminescent label and the protecting group.

Electrophilic cleavage linking groups are typically cleaved by protons and include acid-sensitive cleavage. Suitable electrophilic cleavage linking groups include modified benzyl systems such as trityl, p-hydrocarbyloxybenzyl ester and p-hydrocarbyloxybenzylamide. Other suitable linking groups include tert-butoxycarbonyl (Boc) groups and acetal systems. In order to prepare suitable linking molecules, it is also conceivable to use thiophilic metals such as nickel, silver or mercury in the cleavage of thioacetals or other sulfur-containing protecting groups. Nucleophilic cleavage linking groups include groups that are unstable in water (ie, can be easily cleaved at alkaline pH), such as esters, and groups that are unstable to non-aqueous nucleophiles. Fluoride ions can be used to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS). Photodegradable 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 a label, preferably, the fluorophore absorbs light of a different wavelength than the light required to cleave the linking molecule. Suitable linking groups include those based on O-nitrobenzyl compounds and nitroresveryl compounds. Linking groups based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64: 3454-3460, 1999). Various linking groups sensitive to reductive cleavage are known. 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 the oxidation of hydrocarbyloxybenzyl groups and the oxidation of sulfur and selenium linking groups. It is also within the scope of the invention to use an iodine solution to cleave disulfides and other sulfur or selenium-based linking groups. Safety-catchlinkers are those that are cleaved in two steps. In the preferred system, the first step is the generation of reactive nucleophilic centers, and the subsequent second step involves intramolecular cyclization, which results in cleavage. For example, the levulinate linkage can be treated with hydrazine or photochemical methods 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 fluorenylmethoxycarbonyl and cyanoethyl groups and palladium-catalyzed reduction elimination of allyl systems can be used.

In certain embodiments, the linking group may include spacer units. The length of the linking group is not important, as long as the chemiluminescent 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 be composed of functional groups similar to the 3'-OH protecting group. This allows the chemiluminescent label and protecting group to be removed in a single process. A particularly preferred linking group is an azide-containing linking group cleavable by phosphine.

The reagents that can remove chemiluminescent labels from modified nucleotides as used herein depend largely on the chemiluminescent labels used. For example, in the case where the chemiluminescent label incorporates a protective group, the chemiluminescent label is removed using the reagent for removing the protective group described above. Alternatively, when the chemiluminescent label is connected to the base of the nucleotide through a cleavable linking group, the reagent that cleaves the linking group as described above is used to remove the chemiluminescent label. In a preferred embodiment, the same reagents are used to remove the chemiluminescent label and the protecting group from the modified nucleotide, for example in the case where the linking group consists of functional groups similar to the 3'-OH protecting group under.

An exemplary embodiment of the present invention

In a specific embodiment, the present invention relates to a method for differentiating four nucleotides using different types of light emission (flash and glow) to achieve gene sequencing. This method does not require an additional excitation light source and can meet the development of a low-cost portable sequencer. In this method, the characteristics of two different luciferases and their substrates to produce different luminescence forms are used to distinguish different nucleotides according to different luminescence curves. The sources of luciferase include but are not limited to firefly, gaussia, Renilla and other organisms. The luciferase can be attached to the four deoxynucleotide derivatives in an affinity interaction manner.

In a more specific embodiment, the invention relates to a method for determining the sequence of a target single-stranded polynucleotide, which includes

(a) Four nucleotides are provided, wherein the first nucleotide is attached to the first luciferase, the second nucleotide is attached to the second luciferase, and the third nucleotide is attached to Both the first luciferase and the second luciferase, the fourth nucleotide is not attached to any luciferase, wherein the first luciferase and the second luciferase behave when reacting with their substrate Different luminescence kinetics or luminescence types; in a preferred embodiment, the substrates of the first luciferase and the second luciferase are the same, for example selected from coelenterazine;

(b) Incorporating one nucleotide into the complementary strand of the target single-stranded polynucleotide;

(c) Detect the chemiluminescent label of the nucleotide of (b) to determine the type of nucleotide incorporated;

(d) the chemiluminescent label of nucleotides of (b) is removed; and

(e) Optionally repeat steps (b)-(d) one or more times in order to determine the sequence of the target single-stranded polynucleotide.

Examples

1. Construction of sequencing library

(1) a DNA sequence designed as follows: GATATCTGCAGGCAT AGAATGAATATTATTGAATCAATAATTAAAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAACTAGTACGTCAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTTTACAACTACGATAATGGGCTGGATACATGGAATGATTATAGATATATTAAGGAATAATGTTAATTAATGCCTAAATTAATTAATCTAAGGGGGTTAATACT TCAGCCTGTGATATC, convenience for the construction of the library, the sequence at both ends was added the same oligonucleotide sequence as the two ends underlined font, and the linker sequences inserted in BGI-SEQ500 The intermediate portion Italic marked sequence. The above sequence was handed over to Kingsray Biotechnology Corporation for synthesis. For the unlimited use of the sequence, the synthesized sequence was inserted into the pUC57 vector and transformed into E. coli.

(2) Cultivate an appropriate amount of E. coli containing a known library, and extract the plasmid, design the following pair of primers: GATATCTGCAGGCAT, GATATCACAGGCTGA, and amplify the known sequence according to the following system (Table 1) and procedure (Table 2) The PCR products were purified using magnetic beads. The purified PCR product is added to split oligo (ATGCCTGCAGATATCGATATCACAGGCTGA) according to the BGIseq-500 SE50 cyclization library construction kit and process to carry out the cyclization library construction and use;

Table 1 (Enzymes are produced by BGI)

Table II:

2. Amplification of library sequences

Purchase a 96-well plate coated with streptavidin from Thermofisher, incubate 100ul of 1uM 5 'end biotin modified primer (RCA) GCCATGTCGTTCTGTGAGCCAAGG with one of the wells at room temperature for 30min, remove the reaction liquid, add 6ng of Example 1 Prepare the library and the DLB in the 20ul BGISEQ-500 kit to prepare buffer I. After primer hybridization with the above biotin-modified primers at 60 ° C for 5min, add 40ul of the DNB polymerase I and BGISEQ-500 sequencing kit. 4ul DNB polymerase II, react at 30 ° C for 60min, heat to 65 ° C to terminate the reaction, and carefully remove the reaction solution. Add 100ul of 5uM sequencing primer GCTCACAGAACGACATGGCTACGATCCGACTT, hybridize at room temperature for 30min, and carefully remove the reaction solution;

3. Sequencing

(1) Acme Bioscience outsourced the synthesis of 4 kinds of dNTPs as shown in the figure:

dATP-biotin

dCTP-Biotin-Digoxin

dGTP

dTTP-Digoxin

(2) Reagent preparation:

Prepare the following reagents required in the sequencing reaction

Polymerization reaction solution: 50 mM Tris-HCl, 50 mM NaCl, 10 mM (NH4) ₂ SO ₄ , 0.02 mg / ml polymerase BG9 (BGI), 3 mM MgSO ₄ , 1 mM EDTA, 1 uM each of the above four dNTPs;

Polymerization buffer: 50mM Tris-HCl, 50mM NaCl, Tween20 0.05%;

Elution buffer: 5XSSC, Tween20 0.05%;

Antibody reaction solution: TBST buffer, 1uM anti-digoxin-s-s-biotin (Abcam);

Antibody eluent: TBST buffer

Self-luminous enzyme reaction solution 1: TBST buffer, 2ug / ml SA-gluc (M2) -glow (avidity);

Self-luminous enzyme reaction solution 2: TBST buffer, 2ug / ml SA-gluc (8990) -flash (avidity)

Substrate luminescent solution: prepare 50mM Tris-HCl 0.5mM NaCl buffer and dilute 50X Coelenterazine (nonolight) to 1X;

Excision buffer: 20mM THPP, 0.5M NaCl, 0.05% tween20;

(3) Sequencing reaction:

The sequencing process is shown in Figure 2.

a. Polymerization: add 100ul of polymerase reaction solution to the wells of the library amplified in (1), raise the temperature of the microplate reader to 55 ℃, and react for 3min to polymerize the four dNTPs to the amplified temperature control After carefully removing the reaction solution, add 100ul of the elution reaction solution, gently pipetting a few times to remove the elution reaction solution

b. Self-luminescence enzyme 1 binding: add 100ul of self-luminescence enzyme reaction solution 1, incubate at 35 ° C for 30min to bind SA-gluc (M2) -glow to dNTP-biotin, remove the reaction solution, add washing Deliquoring, gently pipetting several times to remove eluent;

c. Self-luminescence enzyme 2 binding: add 100ul antibody reaction solution, incubate at 35 ℃ for 3min to bind anti-digoxin-ss-biotin to dNTP-digoxin, remove the reaction solution, add 200ul antibody to elute Solution, gently pipetting several times to remove the antibody eluent; adding 100ul of the self-luminescent enzyme 2 reaction solution and reacting at 35 ° C for 2min to make SA-gluc (8990) -flash bind with anti-digoxin-ss-biotin Go to dNTP-digoxin and remove the reaction solution; add 200ul of eluent and gently pipette several times to remove the eluent;

d. Self-luminescence detection: set the parameters of the microplate reader, add substrate luminescent liquid, and detect the self-luminescence curve; read the corresponding base according to the signal curve;

e. Excision; remove the self-luminous reaction solution, add 200ul of elution buffer, after gently pipetting several times, remove the elution buffer, add 100ul of excision reaction solution, 55 ℃, after 3min reaction, remove the excision reaction solution; add 200ul Wash the elution buffer and repeat the washing three times;

f. Repeat steps a-e for next cycle sequencing;

(4) Sequencing results

a. The 10bp sequencing signal curve is shown in Figure 3 and Figure 4:

b. Analysis of sequencing results:

Comparing the signal change curves of all cycles, as shown in the figure below, it can be judged according to the form of the signal drop of each cycle:

Nucleotide A: cycle 1, cycle 4, cycle 5, cycle 8;

Nucleotide T: cycle 2, cycle 7

Nucleotide C: Cycle 3, Cycle 6, Cycle 9

Nucleotide G: Cycle 10

[Corrected according to Rule 26 28.11.2018]
Match the first 10bp base sequence of the library to be tested: TACAACTACG.

Claims

A method for determining the sequence of a target single-stranded polynucleotide, which includes monitoring the sequential incorporation of nucleotides complementary to the target single-stranded polynucleotide,

Where the nucleotides are each attached to chemiluminescent labels that trigger different luminescence kinetics or types,

Each of the incorporated nucleotides is identified by detecting the luminescence kinetics or luminescence type of the chemiluminescence reaction in which the chemiluminescence marker participates and subsequently removing the chemiluminescence marker.
The method of claim 1, wherein the respective ribose or deoxyribose moiety of the nucleotide comprises a protecting group attached via a 2 'or 3' oxygen atom, wherein the nucleotide is modified or removed after incorporation of each nucleotide Protecting the group so that the 3'-OH group is exposed,

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

For example, the nucleotide is selected from nucleotides A, G, C and T or U.
The method of claim 1 or 2, for example, the detection of the luminescence kinetics of the chemiluminescence reaction involving the chemiluminescence label includes contacting the chemiluminescence label with a suitable substrate to trigger the chemiluminescence reaction, and detecting The luminous dynamics of this emitted light,

For example, the chemiluminescent label is selected from biochemiluminescent labels that induce different luminescence kinetics and any combination thereof,

For example, the chemiluminescent label is selected from luciferase that induces different luminescence kinetics and any combination thereof,

For example, the chemiluminescent label is a combination of two luciferases that trigger different luminescence kinetics,

For example, the detection of the luminescence kinetics of the chemiluminescence reaction involving the chemiluminescence label includes contacting the chemiluminescence label with a suitable substrate to trigger the chemiluminescence reaction, and detecting the luminescence type of the light emitted thereby,

For example, the chemiluminescent label is selected from biochemiluminescent labels that induce different types of luminescence and any combination thereof,

For example, the chemiluminescent label is selected from luciferase that triggers different types of luminescence and any combination thereof,

For example, the chemiluminescent label is a combination of two luciferases that trigger different types of luminescence,

For example, the light-emitting type includes a flash type, a glow type, and a mixed type,

For example, in the nucleotides, the first nucleotide is attached to the first chemiluminescent label, the second nucleotide is attached to the second chemiluminescent label, and the third nucleotide is attached To both the first chemiluminescent marker and the second chemiluminescent marker, the fourth nucleotide is not attached to any chemiluminescent marker,

For example, among the nucleotides, the first nucleotide is attached to the first luciferase, the second nucleotide is attached to the second luciferase, and the third nucleotide is attached to the first Both a luciferase and a second luciferase, the fourth nucleotide is not attached to any luciferase.
The method of any one of claims 1 to 3, the chemiluminescent label is attached to the nucleotide by affinity interaction,

For example, the affinity interaction includes antigen-antibody interaction and biotin-avidin (e.g. streptavidin) interaction,

For example, by attaching a chemiluminescent label to one of the members participating in the affinity interaction, and attaching the nucleotide to the other member participating in the affinity interaction, the chemistry will be changed The luminescent marker is attached to the nucleotide,

For example, the member linked to the nucleotide is biotin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin),

For example, the member linked to the nucleotide is digoxin, and the member linked to the chemiluminescent label is an anti-digoxin antibody,

For example, the member linked to the nucleotide is digoxin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin), in which digoxin and avidin are linked to the biotin by Digoxin antibody affinity binding.
A method for determining the sequence of a target single-stranded polynucleotide, which includes

(a) providing one or more nucleotides, wherein the nucleotides are each attached to different chemiluminescent labels, wherein the chemiluminescent labels attached to each type of nucleotide behave when detected Showing different luminescence kinetics or luminescence types from chemiluminescent labels attached to other types of nucleotides;

(b) Incorporating one nucleotide into the complementary strand of the target single-stranded polynucleotide;

(c) Detect the chemiluminescent label of the nucleotide of (b) to determine the type of nucleotide incorporated;

(d) the chemiluminescent label of nucleotides of (b) is removed; and

(e) Optionally repeat steps (b)-(d) one or more times in order 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 via a 2 'or 3' oxygen atom, wherein the protecting group is modified or removed after incorporation of the nucleotide Group to expose the 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 nucleotides A, G, C and T or U.
The method of claim 5 or 6, for example, the detection of the chemiluminescent label of the nucleotide of (b) comprises contacting the chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting Luminous dynamics of light,

For example, the chemiluminescent label is selected from biochemiluminescent labels that induce different luminescence kinetics and any combination thereof,

For example, the chemiluminescent label is selected from luciferase that induces different luminescence kinetics and any combination thereof,

For example, the chemiluminescent label is a combination of two luciferases that trigger different luminescence kinetics,

For example, the detection of the chemiluminescent label of the nucleotide of (b) includes contacting the chemiluminescent label with a suitable substrate to trigger a chemiluminescent reaction, and detecting the type of light emitted thereby,

For example, the chemiluminescent label is selected from biochemiluminescent labels that induce different types of luminescence and any combination thereof,

For example, the chemiluminescent label is selected from luciferase that triggers different types of luminescence and any combination thereof,

For example, the chemiluminescent label is a combination of two luciferases that trigger different types of luminescence,

For example, the light-emitting type includes a flash type, a glow type, and a mixed type,

For example, in the nucleotides, the first nucleotide is attached to the first chemiluminescent label, the second nucleotide is attached to the second chemiluminescent label, and the third nucleotide is attached To both the first chemiluminescent marker and the second chemiluminescent marker, the fourth nucleotide is not attached to any chemiluminescent marker,

For example, among the nucleotides, the first nucleotide is attached to the first luciferase, the second nucleotide is attached to the second luciferase, and the third nucleotide is attached to the first Both a luciferase and a second luciferase, the fourth nucleotide is not attached to any luciferase.
The method of any one of claims 5-7, the chemiluminescent label is attached to the nucleotide via an affinity interaction,

For example, the affinity interaction includes antigen-antibody interaction and biotin-avidin (e.g. streptavidin) interaction,

For example, by attaching a chemiluminescent label to one of the members participating in the affinity interaction, and attaching the nucleotide to the other member participating in the affinity interaction, the chemistry will be changed by the affinity interaction between the members The luminescent marker is attached to the nucleotide,

For example, the member linked to the nucleotide is biotin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin),

For example, the member linked to the nucleotide is digoxin, and the member linked to the chemiluminescent label is an anti-digoxin antibody,

For example, the member linked to the nucleotide is digoxin, and the member linked to the chemiluminescent label is avidin (eg, streptavidin), in which digoxin and avidin are linked to the biotin Digoxin antibody affinity binding.
The method of any one of claims 5-8, wherein each nucleotide is sequentially contacted with the target single-stranded polynucleotide, unincorporated nucleotides are removed before the next nucleotide is added, and wherein The detection and removal of the chemiluminescent marker is performed after adding each nucleotide or after adding 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 The detection and removal of the chemiluminescent marker is performed after adding 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 each attached to different chemiluminescent labels , Where the chemiluminescent label attached to each type of nucleotide exhibits a different luminescence kinetics or type of luminescence than the chemiluminescent label attached to other types of nucleotides when detected; and (b ) Their packaging materials.
The kit of claim 11, further comprising an enzyme and a buffer suitable for the enzyme to function.
The kit of claim 11 or 12, further comprising a suitable substrate that reacts with the chemiluminescent label.