WO2023060858A1

WO2023060858A1 - Monochromatic fluorescence mrt gene sequencing reagent and method based on environment-sensitive dye

Info

Publication number: WO2023060858A1
Application number: PCT/CN2022/086205
Authority: WO
Inventors: 陈鑫; 卓少春; 冯越; 周蓉; 伍建
Original assignee: 深圳铭毅智造科技有限公司
Priority date: 2021-10-15
Filing date: 2022-04-12
Publication date: 2023-04-20
Also published as: CN114250283A; CN114250283B

Abstract

Provided are a monochromatic fluorescence MRT gene sequencing reagent and method based on an environment-sensitive dye. The reagent comprises polymerase and four different labeled nucleotide analogues. Hydroxyl groups at 3' positions of nucleotides of the four nucleotide analogues are all modified by protecting groups or cleavable linking groups, the protecting groups or the cleavable linking groups can be cleaved and separated to expose 3'-OH again, and the cleavable linking groups are linked with environment high-sensitivity fluorescent groups or environment high-sensitivity fluorescent group markers by means of covalent bonds or non-covalent bonds. The four nucleotide analogues are nucleotide derivatives that can or cannot emit fluorescence. Moreover, further disclosed is a method for performing gene sequencing by using the reagent.

Description

Single-color fluorescent MRT gene sequencing reagents and methods based on environmentally sensitive dyes

technical field

The invention relates to the field of gene sequencing, in particular to an environment-sensitive dye-based monochromatic fluorescent MRT gene sequencing reagent and method.

Background technique

Sequencing technology is used to determine the primary structure of biological macromolecules such as nucleic acids, proteins, polysaccharides, etc. It is a basic tool for life science research and the main means of obtaining biological information data, especially DNA sequencing technology, which is to obtain the base sequence of the target DNA fragment Obtaining the sequence of the target DNA fragment is the basis for further molecular biology research and genetic modification, enabling researchers to study and better understand health and disease, and is an essential technology for the personalized precision medicine paradigm. According to different principles, the development of sequencing technology can be roughly divided into four historical stages: first-generation sequencing, second-generation sequencing, third-generation sequencing, and fourth-generation sequencing. Since the four-generation sequencing technology has its own advantages and disadvantages, and its application fields are also different, the first-generation sequencing technology has not been eliminated. The current sequencing market is a situation where four-generation sequencing technologies coexist. Among them, the most widely used and fastest-growing is second-generation sequencing, also known as high-throughput sequencing. Different from traditional first-generation sequencing, it has the characteristics of fast speed, high throughput, and low cost. Next-generation sequencing technology uses the sequencing chemistry of clone amplification and sequencing by synthesis (SBS) to achieve rapid and accurate sequencing. This process recognizes deoxynucleoside triphosphate (dNTPs) derivatives modified with recognition signal groups while incorporating them into nucleic acid strands. Usually, the base of each modified dNTP has a fluorescent label connected by a cleavable chain, and a cleavable protective group (such as azide, vinyl, disulfide bond group, etc., US20010972364 ; US20130079232A1; US20160040225A1; Ju.J.et al.PNAS, 2006,103,19635-19640), this base derivative is also a fluorescently labeled reversible terminator nucleotide (NRT), and different NRTs will emit unique A fluorescent signal that can be used to determine the order of a DNA sequence. Studies have shown (Chen.F.et al.GPB,2013,34–40; Stupi.B.P.et al.Angew Chem Int Ed Engl,2012,51,1724-1727; Ondrus.M.et al.NAR.2020, 11982–11993) An important reason for affecting the read length in the second-generation SBS sequencing method is that the reversible terminator nucleotide analogues developed so far cannot bind the linker between the fluorophore and the base after cutting the fluorophore. If the group is completely removed, there will be a small molecular group of about a certain length remaining at the base (such as the propargylamino part modified by ethylene glycol), and with each round of SBS, it will continue to form on the newly formed DNA double strand Accumulated, to a certain extent, these residues may be enough to interfere with the stability and secondary structure of the DNA double helix structure, thereby affecting the recognition of DNA polymerase and terminating the chain growth reaction, limiting the length of the sequencing read. For this reason, some researchers have designed a single-modified reversible terminator (MRT), that is, the reversible blocking protection group and the fluorescent group are simultaneously modified on the 3'-OH group. This modification can play a dual role, both as a fluorescent The reporter of the signal acts as a reversible terminator. This modification method can theoretically successfully avoid the residues left on the base during NRT sequencing, thereby reducing the impact on DNA double-strand stability and polymerase recognition, so as to improve Sequencing read length. The main challenge of this approach is that, in the complex state formed by the polymerase with the complementary nucleotide and the DNA template, the 3'-OH position of the nucleotide is very close to the amino acid residue at the active site of the DNA polymerase. Therefore, it is difficult for DNA polymerase to accept 3'-0 macromolecular dye-modified nucleotides as substrates.

Contents of the invention

Aiming at the existing deficiencies, the present invention provides a single-color fluorescent MRT gene sequencing reagent and method based on environmentally sensitive dyes.

The technical solution adopted by the present invention to solve its technical problems is: a single-color fluorescent MRT gene sequencing reagent and method based on environmentally sensitive dyes, including polymerase, four different labeled nucleotide analogs, four kinds of The hydroxyl group at the 3' position of the nucleotide analogs is modified by a protecting group or a cleavable linking group, and the protecting group or a cleavable linking group can be cleaved off to expose the 3'-OH again out, the cleavable linking group is linked with the environment-highly sensitive fluorescent group or the environment-highly sensitive fluorescent group label through a covalent bond or a non-covalent bond; the four kinds of nucleotide analogs are respectively compound 1 and compound 2 , compound 3 and compound 4, wherein,

Compound 1 is a nucleotide derivative that cannot emit a fluorescent signal or does not contain a fluorescent group;

Compound 2 is a nucleotide derivative carrying a fluorescent group capable of emitting a fluorescent signal;

Compound 3 is a nucleotide derivative carrying an environmentally sensitive fluorescent group. Under one condition, it cannot emit a fluorescent signal or can emit a fluorescent signal and the fluorescent signal emitted is different from that of Compound 2. In another A fluorescent signal can be emitted under the conditions and the fluorescent signal emitted is the same as that emitted by compound 2, and the structure of the environmentally sensitive fluorescent group is different from that of the fluorescent group in compound 2;

Compound 4 is a nucleotide derivative that cannot emit fluorescent signals but carries a reactive group that can undergo a linking reaction, and the reactive group can emit a The same fluorescent signal as compound 1; or compound 4 is a nucleotide derivative that can emit the same fluorescent signal as compound 2, but carries a fluorophore that is different from compound 2. The cleavable chain fluorescence The group can be cleaved and removed under certain conditions, which has no effect on compound 2.

Preferably, said compound 1, compound 2, compound 3, and compound 4 are compounds each independently having a structure of formula (I) or formula (II) or formula (III),

Among them, "----" is a non-covalent bond;

B represents different bases or analogs, and said bases or analogs refer to adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) and analog;

Each R1 is independently selected from hydrogen, a monophosphate group, a diphosphate group, a triphosphate group, a tetraphosphate group, or more phosphate groups;

R2 are each independently a protective group capable of carrying out an orthogonal cleavage reaction;

R3 are each independently selected from halogen, -H, -OH, N3 or C2-C10 flexible chain;

L is a cleavable linking group or is absent;

R4 are each independently an environmentally sensitive fluorophore that can emit a fluorescent signal, a fluorophore that can emit the same fluorescent signal, and a reactive group that can perform a linking reaction.

R5 are each independently a reactive group labeled with an environmentally sensitive fluorophore capable of emitting a fluorescent signal or a reactive group labeled with a fluorophore capable of emitting the same fluorescent signal.

Preferably, the base or analog is a compound of any of the following structures,

Preferably, the protecting group is a 3'-OH modification group that can be effectively recognized by the polymerase, can be incorporated into the growing DNA chain, and can be cut off after each round of SBS sequencing; The cleavage linking group refers to the small molecular group between the linking 3'-OH and the highly sensitive fluorescent group in the environment, the fluorescent group that can emit the same fluorescent signal, or the reactive group that can carry out the connection reaction; The fluorophore with the same fluorescent signal is a non-environmental high-sensitivity fluorescent dye that can emit the same or close emission wavelength as the selected environmental high-sensitivity fluorescent dye under the same excitation light source wavelength, and is detected and judged as the same fluorescent signal.

Preferably, the protecting group is a group with any of the following structures:

The cleavable linking group is a group of any of the following structures:

Disulfide chain group

cyanoethyl chain group

Azide chain group

Dialkyl ketal chain group

Allyl chain group

1-(4,4-Dimethyl-2,6-dioxocyclohex-1-yl)ethyl chain group

Nitrobenzyl chain group

Azo chain group

Wherein R _1' , R _2' are each independently selected from halogen, -H, C1-C5 aliphatic chain;

The fluorescent group that can emit the same fluorescent signal is from any one or more of the following fluorescent dyes: AF488, AF532, AF633, AF680, AF660, AF700, AF647, AF 594, AF 555, AF568, CY3, CY5, CY5 .5, CY7, CY7.5, ROX, R6G, ATTO 495, ATTO532, ATTO700, ATTO680, ATTO655, ATTO647N, ATTO594, ATTO Rho101, ATTO 590, ATTO Thio12, FAM, VIC, TET, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red635, iFluor 488, iFluor 514, iFluor 532, iFluor 546, iFluor 555, iFluor 568, iFluor 590, iFluor610, iFluor 633, 4iFluor7, iFluor 70, 06 iFluor710, Quasar705, Quasar670.

Preferably, the environmentally sensitive fluorescent group is selected from environmentally sensitive fluorescent dyes, and the environmentally sensitive fluorescent dyes are pH-sensitive fluorescent dyes.

Preferably, the pH-sensitive fluorescent dye is a compound comprising any of the following structural formulas,

Wherein: X is each independently selected from halogen, -H, -OH, N3, NH2, S, Si or C2-C10 flexible chain;

R6 are each independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol chain amino, polyethylene glycol maleimide, polyethylene glycol Mercapto, C1-C10 saturated alkyl, saturated alkylalkynyl, C1-C10 saturated alkyl chain carboxyl, C1-C10 alkyl chain azido, C1-C10C1-C10 alkyl chain amino, C1-C10 alkyl Chain mercapto, C1-C10 alkyl chain sulfonic acid group, C1-C10 alkyl chain maleimide;

R7 are each independently selected from hydrogen, C1-C6 saturated alkyl;

Ring A and ring B are each independently selected from heterocyclic groups having formulas (VI), (VII), (VIII), (IX),

Wherein; Y is each independently selected from hydrogen, oxygen, carbon, nitrogen, sulfur;

R8, R9 and R12 are each independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol chain amino, polyethylene glycol maleimide, Polyethylene glycol mercapto, C1-C10 saturated alkyl, saturated alkyl alkynyl, C1-C10 saturated alkyl chain carboxyl, C1-C10 alkyl chain azido, C1-C10C1-C10 alkyl chain amino, C1 -C10 alkyl chain mercapto group, C1-C10 alkyl chain sulfonic acid group, C1-C10 alkyl chain maleimide;

R10 and R11 are each independently selected from hydrogen, carboxyl, phosphoric acid and sulfonic acid.

Preferably, the reactive group can be recognized by a polymerase, and can be incorporated into a growing DNA chain to perform an orthogonal connection reaction with a corresponding complementary group through a covalent bond or a non-covalent bond. A mosaic structure that can be cleaved from a group that regenerates 3'-OH after each round of SBS sequencing; the reactive fluorophore is a fluorophore that carries an environmentally sensitive fluorophore or can emit the same fluorescent signal group, and the reactive group can be produced by covalent or non-covalent bond to carry out orthogonal linkage reaction to stabilize the group of splicing structure.

Preferably, the reactive group is any one of the following structures,

Amino

Cycloalkynyl

Azide group

Maleimide

Biotin

Phenylboronic acid

digoxin; phenol

Cycloalkenyl

The complementary group is any one of the following structures:

Tetrafluorophenyl ester

Cycloalkynyl

Streptavidin, 4-phenyluronazole

Azide group

Salicyl isohydroxylic acid group, digoxin antibody, sulfhydryl group

Tetrazine

A monochrome fluorescent MRT gene sequencing method based on environmentally sensitive dyes, characterized in that: using the sequencing reagent as described in any one of the preceding, its testing steps are as follows,

S1, preparing compound 1, compound 2, compound 3 and compound 4 respectively;

S2, connecting the nucleic acid template strand to be sequenced to the chip or microspheres to form a nucleic acid double-stranded molecular cluster reaction system;

S3, compound 1, compound 2, compound 3, compound 4 and polymerase are simultaneously added to the nucleic acid double-stranded molecular cluster reaction system to carry out nucleotide polymerization reaction, so that any one of them is incorporated into the 3' end of the growing nucleic acid chain and cease to grow;

S4, wash away unreacted compound 1, compound 2, compound 3, compound 4, then add buffer and adjust the pH value, detect the fluorescent label of each incorporated nucleotide derivative and take pictures and store images to identify the Nucleotide derivatives incorporated at the 3' end of a nucleic acid strand; wherein:

4a, if compound 2 and compound 3 can emit fluorescent signals, then remove the solution phase in the reaction system of the previous step, keep the duplex connected to the support, and detect the duplex or the growing Whether the nucleic acid chain emits the fluorescent signal; and then it is processed, which has no effect on compound 1 and compound 2, but can make compound 3 no longer emit fluorescent signal or the fluorescent signal emitted is different from compound 2, and at the same time make compound 4 occur The ligation reaction emits the same fluorescent signal as compound 2, and finally adjusts the pH value, and detects again whether the duplex or the growing nucleic acid chain emits the fluorescent signal;

4b, if compound 2 and compound 4 can emit fluorescent signals, then remove the solution phase in the reaction system of the previous step, retain the duplex attached to the support, and detect the duplex or the growing nucleic acid Whether the chain emits the fluorescent signal; and then it is processed, which has no effect on compound 1 and compound 2, but can make the fluorophore of compound 4 break the chain and remove the fluorophore, and at the same time make compound 3 emit the same the same fluorescent signal, and detect whether the duplex or the growing nucleic acid chain emits the fluorescent signal again;

S5, fluorescent signal image processing and analysis, to judge the nucleotide derivatives incorporated into the nucleic acid chain, the judgment principle is as follows:

5a, if in step S3, compound 1 is incorporated into the 3' end of the growing nucleic acid chain, then, since compound 4 itself does not carry a fluorescent group or other reactive groups, it is not subject to the treatment described in step S4 Therefore, no fluorescent signal will be detected in step S4;

5b, if in step S3, compound 2 is incorporated into the 3' end of the growing nucleic acid chain, then, since compound 2 carries a fluorescent group and is not affected by the treatments described in steps 4a and 4b in step S4, Fluorescent signals will be detected in step S4;

5c, if in step S3, compound 3 is incorporated into the 3' end of the growing nucleic acid chain, since compound 3 itself carries an environmentally sensitive fluorescent group, a fluorescent signal can be detected in step S4 in 4a, after processing and adjustment, no Send out a fluorescent signal again, or send out a fluorescent signal that is different from compound 2, and no fluorescent signal can be detected in step S4 under a given filter; in step S4, no fluorescent signal can be detected in 4b, and after processing and adjustment, it can send out the same compound 2 the same fluorescence, the fluorescence signal can be detected in step S4;

5d, if in step S3, compound 4 is incorporated into the 3' end of the growing nucleic acid chain, if compound 4 itself does not carry a fluorescent group, but carries a reactive group, no fluorescent signal can be detected in step S4 , add the corresponding complementary group carrying the same fluorescent group as compound 2 while processing and adjusting, after treatment, it can emit the same fluorescence as compound 2, and a fluorescent signal can be detected in step S4; if compound 4 itself carries the same fluorescent group as compound 2 Compound 2 has the same fluorescent group with different linking chain groups, and the fluorescent signal can be detected in step S4. A specific cleavage reagent is added while the treatment is adjusted. After the treatment, the linking group is broken and the fluorescent group is removed to cause fluorescence. The signal is lost, and no fluorescent signal can be detected in step S4.

The beneficial effect of the present invention is that: the present invention utilizes an environmental high-sensitivity fluorescent dye, or simultaneously uses a second kind of fluorescent dye that can emit the same fluorescent signal under the same excitation condition to distinguish 4 kinds of dNTPs, and the fluorescent labels of these dNTPs are not Like the traditional NRT tag on the base, but on the 3'-OH by covalent bond or non-covalent bond, these tagged dNTPs can be recognized by polymerase and integrated into the DNA double strand to complete SBS sequencing, And after each round of SBS sequencing, the connected fluorescent group chain can be cleaved to regenerate 3'-OH, so as not to affect the next round of SBS reaction. Substrates, T9 series modified polymerases obtained by targeted design, modification and high-throughput screening, can effectively recognize and catalyze dATP, dTTP/dUTP, dCTP and dGTP undergoes polymerization reaction and SBS sequencing, which eliminates the problem of affecting the sequencing read length caused by the accumulation of residues on the base in the previous second-generation SBS sequencing method. In addition, the sequencing method of the present invention uses a monochromatic fluorescent reagent, and the sequencer only It needs to be equipped with an excitation light source and a camera, which greatly reduces the manufacturing cost and volume of the sequencer, is convenient for transportation and portability, and is conducive to the spread of the sequencer to a wider third- and fourth-tier urban hospitals and research institutions, and decentralized use.

Description of drawings

Fig. 1 is the schematic diagram of image A in the utility model embodiment 1;

Fig. 2 is the schematic diagram of image B in the utility model embodiment 1;

Fig. 3 is the schematic diagram of image C in the utility model embodiment 2;

Fig. 4 is a schematic diagram of image D in Embodiment 2 of the utility model;

Detailed ways

In order to more clearly illustrate the purpose, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the embodiments, and a clear and complete description will be made. Obviously, the described embodiments are part of the embodiments of the present invention. rather than all examples. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In the present invention, polymerase is used to carry out nucleotide polymerization reaction, "polymerase" refers to any naturally or non-naturally occurring enzyme or other catalyst capable of catalyzing polymerization, including various known natural and modified nucleic acids Polymerase, such as DNA polymerase (using MEB202T9 in the embodiment of the present invention), RNA polymerase, and reverse transcriptase, the polymerase can use RNA or single-stranded DNA as a template to synthesize a new DNA chain, which can be used according to actual needs , choose the appropriate polymerase to carry out the nucleotide polymerization reaction, and you can also choose a mixture of multiple polymerases to use.

In the method of the present invention, the four compounds used in step S3 are derivatives of nucleotides A, (T/U), C and G, respectively, and "nucleotide" refers to nucleoside-5'-poly Phosphate compounds or structural analogs thereof, having complementary base pairing capabilities, which can be incorporated by nucleic acid polymerases to extend growing nucleic acid strands, which can pair nucleotides at one or more base, sugar, or phosphate groups Modifications, nucleotides can carry fluorescent groups, or reactive groups.

In the present invention, the hydroxyl group (-OH) at the 3' position of these four nucleotide derivatives has a protective group or a linking chain with a protective group, including but not limited to the following, such as allyl

o-nitrobenzene

Azidomethyl

Disulfide

Methoxyalkyl, azoalkyl, disulfide chain groups

cyanoethyl chain group

Azide chain group

Dialkyl ketal chain group

Allyl chain group

1-(4,4-Dimethyl-2,6-dioxocyclohex-1-yl)ethyl chain group

Nitrobenzyl chain group

Azo chain group

In the present invention, the 3'-O terminal protecting groups or fluorescent groups or reactive groups of these four kinds of nucleotide derivatives are composed of single or multiple identical or different orthogonally cleavable chain groups group connection. The chain-cleavable group refers to an orthogonally cleavable group in response to external stimuli (e.g., enzymes, nucleophilic/alkaline reagents, reducing agents, light irradiation, electrophilic/acidic reagents, organometallic and metallic reagents, or oxidizing agents). (e.g., specifically cleavable) chain group, the 3'-O and the fluorescent group or the reactive group are separated into different fragments by cleavage, and after the cleavable chain group is orthogonally cleaved, the two The separated fragments (eg fluorescent dyes, bioreactive groups) do not react further and also do not form new orthogonal scissile chain groups. In orthogonal cutting, the cutting agents used include but not limited to Na2S2O4), THP), TEC, DTT, weak acid, Pd(0) or light irradiation (such as ultraviolet irradiation), etc.

In the present invention, the reactive group is a conjugate reactive group capable of performing a specific orthogonal linking reaction with a complementary group carrying a fluorescent group. The chemical reactions of the orthogonal linkage reaction include but are not limited to: Staudinger linkage reaction, copper ion-catalyzed cycloaddition reaction of azide and alkynyl, ring tension-driven azide and alkynyl cycloaddition Synthesis reaction, binding reaction between digoxin and digoxin antibody, Diels-Alder reaction, Suzuki cross-coupling reaction, disulfide bond formation reaction between sulfhydryl and sulfhydryl derivatives, sulfhydryl and maleimide The reaction of amines to form thioethers, the photocatalytic free radical addition reaction of sulfhydryl and alkenyl derivatives, the photocatalytic free radical addition reaction of sulfhydryl and alkynyl derivatives, the exchange reaction of sulfonyl fluoride, biotin and streptavidin The binding reaction between and the prime, the reaction between the amino group and the activated ester.

In the present invention, the environmental high-sensitivity fluorescent dye is a fluorescent dye that can quickly respond to changes in the environment, such as polarity, pH, voltage, light source and viscosity, etc. to change the color of light emitted, the wavelength or intensity of fluorescence emission. The chemical reactions of the orthogonal connection reaction include but are not limited to various known environmentally sensitive fluorescent dyes widely used in the fields of fluorescent probes, chemical sensors, microenvironmental change detection, biological imaging, molecular switches, and phase separation visualization. , the environment highly sensitive fluorescent dyes include but not limited to the following fluorescent dyes: Cy-7, Dylight 800, IRDye 800, Alexa Fluor 790, HiLyte Fluor 750, Ovster 800, Rhodamine isothiocyanate, Texas Red derivatives, Alexa Fluor 680, DyLight 680 , Cy5.5 NHS ester (～67O nm, Lumiprobe), Alexa Fluor 546, DyLight 549, Oregon Green 514, Carboxylic Acid, pHrodoTM Red, 6-Carboxynaphthofluorescein, 7-Hydroxycoumarin-3-carboxylic acid, SNARFR-5F, SNARFB- 4F, SNARFR-1, BCECF, CyPHER5E, HCyC-647, Square-650-pH, 6-Carboxy-4,5'-Dichloro-2',7'-Dimethoxyfluorescein.

In the present invention, the fluorescent groups and their detection methods are known, and can be selected according to actual needs. Appropriate excitation light conditions and optical filters can also be selected to make different compounds emit the same or substantially the same fluorescence signals.

In the method of the present invention, the single-color fluorescent sequencing is realized by using the fluorophore and the highly sensitive fluorophore in the environment. The fluorophore and the highly sensitive fluorophore are not marked on the bases like the traditional NRT, but by Covalently or non-covalently labeled on the 3'-OH of dNTPs, these labeled dNTPs can be recognized by a modified DNA polymerase (MEB202T9) and integrated into the DNA double strand to complete SBS sequencing, and each round of SBS sequencing The post-connected fluorophore chain can be cleaved to regenerate 3'-OH, which will not affect the next round of SBS reaction, and can eliminate the problem of affecting the sequencing read length caused by the accumulation of residues on the base in the previous second-generation SBS sequencing method. The environmentally sensitive fluorescent group is different in structure from the correspondingly selected non-environmentally sensitive fluorescent group, and given a system environment, the same fluorescent signal can be emitted or the fluorescent signal emitted under suitable excitation light conditions and optical filters is essentially The above is the same, given another system environment, the environmental high-sensitivity fluorescent group loses its fluorescence performance and cannot emit fluorescent signals, or the fluorescent signal emitted under the same excitation light conditions and optical filters is essentially the same as that of the selected non-environmental high-sensitivity fluorescent group. The fluorescent signals emitted by the groups are different but not detected. In such a system environment, the pH value of the system is adjusted according to the characteristics of the base derivatives in the gene to meet the corresponding requirements. Four kinds of bases can be distinguished by single-color fluorescent sequencing Based on this, the sequencer only needs to be equipped with an excitation light source and a camera, which greatly reduces the manufacturing cost and volume of the sequencer, is convenient for transportation and portability, and facilitates the spread of the sequencer to hospitals and research institutions in third- and fourth-tier cities. Decentralized use.

Example 1:

Preparation of compound 1, the preparation of dGTP derivatives (3-N3-dGTP), the compound was purified by semi-preparative HPLC to obtain a product with a purity greater than 97%, HRMS: 561.0004, and its synthetic route and compound structure are as follows:

Preparation of compound 2, the preparation of dTTP derivatives (3'-HCyC-647-LN-dTTP) modified by 3'OH-environmentally sensitive dyes, the compound was purified by semi-preparative HPLC to obtain a product with a purity greater than 97%, MALDI-TOF: 1209.10, its synthetic route and compound structure are as follows:

Phase 1:

Phase 2:

Preparation of compound 3, 3'OH-dye-modified dATP derivative (3'-Cy5-LN-dATP), the compound was purified by semi-preparative HPLC to obtain a product with a purity greater than 97%, MALDI-TOF: 1213.54, which was synthesized The routes and compound structures are as follows:

Preparation of compound 4, the preparation of dCTP derivatives (3'-Biotin-LN-dCTP) modified by 3'OH-reactive groups, the compound was purified by semi-preparative HPLC to obtain a product with a purity greater than 97%, HRMS: 776.0504, which The synthetic route and compound structure are as follows:

The sequencing method used involves the following steps:

a) immobilizing the DNA template to be tested on the chip, and constructing nucleic acid double-stranded molecular clusters through bridge amplification;

b) Four dNTPs (3'-N3-dGTP, 3'-LN3-HCyC647-dTTP, 3'-LN3-Cy5-dATP, 3'-LN3-Biotin-dCTP,) and polymerase MEB202T9 in Example 1 At the same time, add the reaction system to carry out the nucleotide polymerization reaction, so that one of them is incorporated into the 3' end of the growing nucleic acid chain and terminates and continues to grow;

c) Wash away unreacted dNTPs, add scanning buffer, adjust the pH value to 6.8, use a 640nm light source as the excitation wavelength to detect the fluorescent signal, take pictures, and store image A, as shown in Figure 1;

d) wash off the scanning buffer, add water-soluble Cy5-streptavidin, the treatment has no effect on G, A and T base derivatives, and Cy5-streptavidin can specifically interact with C The base derivative is bound to Biotin, so that the fluorescent group Cy5 is introduced into the A base derivative, so that it emits a fluorescent signal;

e) Adjust the pH value to 7.5. The weak alkaline environment has no effect on the G, C and A base derivatives, but it can quench the fluorescence of HCyC-647 on the T base derivatives, and no fluorescence signal can be detected under the 640nm light source ;

f) adding scanning buffer, using a 640nm light source as the excitation wavelength to detect fluorescent signals, taking pictures, and storing image B, as shown in Figure 2;

g) Wash off the scanning buffer, add cutting reagent THP to treat the chip, remove the azide group or azide group chain at the 3'-position of dNTPs to regenerate free 3'-OH, and remove the fluorescent group at the same time or reactive groups.

h) washing away the cleavage buffer, and then repeating steps (c)-(h);

i) After the images obtained by taking pictures twice during each cycle test step, compare the fluorescent signals of the nucleic acid double-stranded molecular clusters at the same position, if there are fluorescent signals in the scanning image A and scanning image B (image Shown in the square boxes in A and B), then the incorporated nucleotide derivative is 3'-LN3-Cy5-dATPC, correspondingly, it can be determined that the base at the corresponding position on the DNA template is T; if in There is no fluorescent signal in scanning image A and scanning image B (shown by the diamond box in image A and B), then the incorporated nucleotide derivative is 3'-N3-dGTP, and correspondingly, the DNA template can be determined The base at the corresponding position above is C; if there is a signal in the scanning image A, but no fluorescent signal in the scanning image B (shown by the circle box in the images A and B), the incorporated nucleotide derivative is 3'-LN3-HCyC-647-dTTP, correspondingly, it can be determined that the base on the corresponding position on the DNA template is A; if there is no signal in scanning image A, but there is a fluorescent signal in scanning image B (image A and B), then the incorporated nucleotide derivative is 3'-LN3-Biotin-dCTP, and correspondingly, the base at the corresponding position on the DNA template can be determined as T. As shown in table 1, table 1: embodiment 1 detection result and corresponding base,

Example 2:

Phase 1:

Phase 2:

Preparation of compound 4, 3'OH-reactive group-modified dCTP derivative (3'-Cy5-SS-dCTP), the compound was purified by semi-preparative HPLC to obtain a product with a purity greater than 97%, MALDI-TOF: 1225.89 , its synthetic route and compound structure are as follows:

The sequencing method used involves the following steps:

b) Four kinds of dNTPs (3'-N3-dGTP, 3'-LN3-HCyC-647-dTTP, 3'-LN3-Cy5-dATP, 3'-SS-Cy5-dCTP,) in Example 1 were combined with The enzyme MEB202T9 is added to the reaction system at the same time to carry out the nucleotide polymerization reaction, so that one of them is incorporated into the 3' end of the growing nucleic acid chain and terminates to continue growing;

c) washing away unreacted dNTPs, adding scanning buffer, adjusting the pH value to 7.4, using a 640nm light source as the excitation wavelength to detect fluorescent signals, taking pictures, and storing image C, as shown in Figure 3;

d) wash off the scanning buffer, add an orthogonal cleavage reagent (low concentration TECP) to break the disulfide bond of 3'-SS-Cy5-dCTP and remove its fluorescent group, and the treatment is effective for 3'-N3-dGTP, 3' -LN3-HCyC-647-dTTP, 3'-LN3-Cy5-dATP has no effect (or has little effect, does not affect image recognition and signal differentiation).

e) Adjust the pH value to 6.8 at the same time. The weakly acidic environment has no effect on the G, C and A base derivatives, but it can make the pH-sensitive fluorescent group HCyC-647 on the T base emit the same or in the same filter as CY5. Substantially the same fluorescent signal can be detected under the light sheet;

f) adding scanning buffer, using a light source with an excitation wavelength of 640nm to detect fluorescent signals, taking pictures, and storing image D, as shown in Figure 4;

h) washing away the cleavage buffer, and then repeating steps (c)-(h);

i) After the images obtained by taking pictures twice during each cycle test step, compare the fluorescent signals of the nucleic acid double-stranded molecular clusters at the same position, if there are fluorescent signals in the scanning image A and scanning image B (image As shown in the square boxes in A and B), then the incorporated nucleotide derivative is 3'-LN3-Cy5-dATPC, correspondingly, it can be determined that the base at the corresponding position on the DNA template is T; if in There is no fluorescent signal in scanning image A and scanning image B (shown by the diamond box in image A and B), then the incorporated nucleotide derivative is 3'-N3-dGTP, and correspondingly, the DNA template can be determined The base at the corresponding position above is C; if there is a signal in the scanning image A, but no fluorescent signal in the scanning image B (shown by the circle box in the images A and B), the incorporated nucleotide derivative is 3'-SS-Cy5-dCTP, correspondingly, it can be determined that the base on the corresponding position on the DNA template is G; if there is no signal in scanning image A, but there is a fluorescent signal in scanning image B (images A and B shown in the middle triangle box), then the incorporated nucleotide derivative is 3'-LN3-HCyC-647-dTTP, and correspondingly, the base at the corresponding position on the DNA template can be determined as A. As shown in table 2, table 2: embodiment 2 detection result and corresponding base

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims

A kind of monochrome fluorescent MRT gene sequencing reagent based on environment-sensitive dye, it is characterized in that: comprise polymerase, four kinds of different labeled nucleotide analogs, the nucleotide 3 of four kinds of described nucleotide analogs The hydroxyl groups at the ' position are all modified by protecting groups or cleavable linking groups, and the protecting groups or cleavable linking groups can be cleaved off to expose the 3'-OH again, and the cleavable linking groups are A valence bond or a non-covalent bond is linked with an environmental high-sensitivity fluorescent group or an environmental high-sensitivity fluorescent group label; the four kinds of nucleotide analogs are respectively compound 1, compound 2, compound 3 and compound 4, wherein,

Compound 1 is a nucleotide derivative that cannot emit a fluorescent signal or does not contain a fluorescent group;

Compound 2 is a nucleotide derivative carrying a fluorescent group capable of emitting a fluorescent signal;

Compound 3 is a nucleotide derivative carrying an environmentally sensitive fluorescent group. Under one condition, it cannot emit a fluorescent signal or can emit a fluorescent signal and the fluorescent signal emitted is different from that of Compound 2. In another A fluorescent signal can be emitted under the conditions and the fluorescent signal emitted is the same as that emitted by compound 2, and the structure of the environmentally sensitive fluorescent group is different from that of the fluorescent group in compound 2;

Compound 4 is a nucleotide derivative that cannot emit fluorescent signals but carries a reactive group that can undergo a linking reaction, and the reactive group can emit a The same fluorescent signal as compound 1; or compound 4 is a nucleotide derivative that can emit the same fluorescent signal as compound 2, but carries a fluorophore that is different from compound 2. The cleavable chain fluorescence The group can be cleaved and removed under certain conditions, which has no effect on compound 2.
The monochromatic fluorescent MRT gene sequencing reagent based on environment-sensitive dye according to claim 1, is characterized in that: described compound 1, compound 2, compound 3, compound 4 have formula (I) or formula (II) independently respectively Or the compound of formula (III) structure,

Among them, "----" is a non-covalent bond;

B represents different bases or analogs, and said bases or analogs refer to adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) and analog;

Each R1 is independently selected from hydrogen, a monophosphate group, a diphosphate group, a triphosphate group, a tetraphosphate group, or more phosphate groups;

R2 are each independently a protective group capable of carrying out an orthogonal cleavage reaction;

R3 are each independently selected from halogen, -H, -OH, N3 or C2-C10 flexible chain;

L is a cleavable linking group or is absent;

R4 are each independently an environmentally sensitive fluorophore that can emit a fluorescent signal, a fluorophore that can emit the same fluorescent signal, and a reactive group that can perform a linking reaction.

R5 are each independently a reactive group labeled with an environmentally sensitive fluorophore capable of emitting a fluorescent signal or a reactive group labeled with a fluorophore capable of emitting the same fluorescent signal.
The monochromatic fluorescent MRT gene sequencing reagent based on environment sensitive dye according to claim 2, is characterized in that: described base or analogue is the compound of following any one structure,
The monochromatic fluorescent MRT gene sequencing reagent based on the environment-sensitive dye according to claim 1, characterized in that: the protective group can be effectively recognized by polymerase, can be incorporated into the growing DNA chain, and is present in each The 3'-OH modification group that can be cleaved and released after a round of SBS sequencing; the cleavable linking group refers to a fluorophore that links the 3'-OH and an environmental high-sensitivity fluorophore, can emit the same fluorescent signal, or can perform The small molecular group between the reactive groups of the connection reaction; the fluorescent group that can emit the same fluorescent signal can emit the same or close emission wavelength as the selected environmental high-sensitivity fluorescent dye under the same excitation light source wavelength, It is detected and judged as a non-environmental high-sensitivity fluorescent dye with the same fluorescent signal.
The monochromatic fluorescent MRT gene sequencing reagent based on environment-sensitive dye according to claim 4, is characterized in that: described protecting group is the group with following any one structure:

The cleavable linking group is a group of any of the following structures:

Disulfide chain group
cyanoethyl chain group
Azide chain group
Dialkyl ketal chain group
Allyl chain group
1-(4,4-Dimethyl-2,6-dioxocyclohex-1-yl)ethyl chain group
Nitrobenzyl chain group
Azo chain group

Wherein R 1' , R 2' are each independently selected from halogen, -H, C1-C5 aliphatic chain;

The fluorescent group that can emit the same fluorescent signal is from any one or more of the following fluorescent dyes: AF488, AF532, AF633, AF680, AF660, AF700, AF647, AF 594, AF 555, AF568, CY3, CY5, CY5 .5, CY7, CY7.5, ROX, R6G, ATTO 495, ATTO532, ATTO700, ATTO680, ATTO655, ATTO647N, ATTO594, ATTO Rho101, ATTO 590, ATTO Thio12, FAM, VIC, TET, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red 610, TEXAS RED, CAL Fluor Red635, iFluor 488, iFluor 514, iFluor 532, iFluor 546, iFluor 555, iFluor 568, iFluor 590, iFluor610, iFluor 633, 4iFluor7, iFluor 70, 06 iFluor710, Quasar705, Quasar670.
The monochromatic fluorescent MRT gene sequencing reagent based on environmentally sensitive dyes according to claim 1, wherein: the environmentally sensitive fluorescent group is selected from environmentally sensitive fluorescent dyes, and the environmentally sensitive fluorescent dyes are pH sensitive fluorescent dyes.
The monochromatic fluorescent MRT gene sequencing reagent based on environment sensitive dye according to claim 6, is characterized in that: described pH value sensitive fluorescent dye is to comprise the compound of following any one structural formula,

Wherein: X is each independently selected from halogen, -H, -OH, N3, NH2, S, Si or C2-C10 flexible chain;

R6 are each independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol chain amino, polyethylene glycol maleimide, polyethylene glycol Mercapto, C1-C10 saturated alkyl, saturated alkylalkynyl, C1-C10 saturated alkyl chain carboxyl, C1-C10 alkyl chain azido, C1-C10C1-C10 alkyl chain amino, C1-C10 alkyl Chain mercapto, C1-C10 alkyl chain sulfonic acid group, C1-C10 alkyl chain maleimide;

R7 are each independently selected from hydrogen, C1-C6 saturated alkyl;

Ring A and ring B are each independently selected from heterocyclic groups having formulas (VI), (VII), (VIII), (IX),

Wherein; Y is each independently selected from hydrogen, oxygen, carbon, nitrogen, sulfur;

R8, R9 and R12 are each independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol chain amino, polyethylene glycol maleimide, Polyethylene glycol mercapto, C1-C10 saturated alkyl, saturated alkyl alkynyl, C1-C10 saturated alkyl chain carboxyl, C1-C10 alkyl chain azido, C1-C10C1-C10 alkyl chain amino, C1 -C10 alkyl chain mercapto group, C1-C10 alkyl chain sulfonic acid group, C1-C10 alkyl chain maleimide;

R10 and R11 are each independently selected from hydrogen, carboxyl, phosphoric acid and sulfonic acid.
The monochromatic fluorescent MRT gene sequencing reagent based on the environmental sensitive dye according to claim 1, it is characterized in that: described reactive group can be recognized by polymerase, and can be incorporated in the DNA chain in growth and corresponding Corresponding complementary groups carry out orthogonal connection reaction through covalent bonds or non-covalent bonds to stabilize the splicing structure, and can be cut off after each round of SBS sequencing to regenerate the 3'-OH group; the reactive The reactive fluorophore is a fluorophore carrying an environmentally sensitive fluorophore or a fluorophore that can emit the same fluorescent signal, and is produced by covalent or non-covalent bonds with the reactive group to perform an orthogonal connection reaction to stabilize the splicing structure. group.
The monochromatic fluorescent MRT gene sequencing reagent based on environment sensitive dye according to claim 8, is characterized in that: described reactive group is any one in following structure,

Amino
Cycloalkynyl

Azide group
Maleimide
Biotin
Phenylboronic acid
digoxin; phenol
Cycloalkenyl

The complementary group is any one of the following structures:

Tetrafluorophenyl ester
Cycloalkynyl

Streptavidin, 4-phenyluronazole
Azide group
Salicyl isohydroxylic acid group, digoxin antibody, sulfhydryl group
Tetrazine
A monochrome fluorescent MRT gene sequencing method based on environmentally sensitive dyes, characterized in that: using the sequencing reagent as described in any one of claims 1 to 9, its testing steps are as follows,

S1, preparing compound 1, compound 2, compound 3 and compound 4 respectively;

S2, connecting the nucleic acid template strand to be sequenced to the chip or microspheres to form a nucleic acid double-stranded molecular cluster reaction system;

S3, compound 1, compound 2, compound 3, compound 4 and polymerase are simultaneously added to the nucleic acid double-stranded molecular cluster reaction system to carry out nucleotide polymerization reaction, so that any one of them is incorporated into the 3' end of the growing nucleic acid chain and cease to grow;

S4, wash away unreacted compound 1, compound 2, compound 3, compound 4, then add buffer and adjust the pH value, detect the fluorescent label of each incorporated nucleotide derivative and take pictures and store images to identify the Nucleotide derivatives incorporated at the 3' end of a nucleic acid strand;

Wherein: 4a, if compound 2 and compound 3 can emit fluorescent signals, then remove the solution phase in the reaction system of the previous step, retain the duplex connected to the support, and detect the duplex or the Whether the growing nucleic acid chain emits the fluorescent signal; then, it is processed, which has no effect on compound 1 and compound 2, but can make compound 3 no longer emit fluorescent signal or the fluorescent signal emitted is different from compound 2, and simultaneously make compound 4. A ligation reaction occurs to emit the same fluorescent signal as compound 2, and finally adjust the pH value, and detect again whether the duplex or the growing nucleic acid chain emits the fluorescent signal;

4b, if compound 2 and compound 4 can emit fluorescent signals, then remove the solution phase in the reaction system of the previous step, retain the duplex attached to the support, and detect the duplex or the growing nucleic acid Whether the chain emits the fluorescent signal; and then it is processed, which has no effect on compound 1 and compound 2, but can make the fluorophore of compound 4 break the chain and remove the fluorophore, and at the same time make compound 3 emit the same the same fluorescent signal, and detect whether the duplex or the growing nucleic acid chain emits the fluorescent signal again;

S5, fluorescent signal image processing and analysis, to judge the nucleotide derivatives incorporated into the nucleic acid chain, the judgment principle is as follows:

5a, if in step S3, compound 1 is incorporated into the 3' end of the growing nucleic acid chain, then, since compound 4 itself does not carry a fluorescent group or other reactive groups, it is not subject to the treatment described in step S4 Therefore, no fluorescent signal will be detected in step S4;

5b, if in step S3, compound 2 is incorporated into the 3' end of the growing nucleic acid chain, then, since compound 2 carries a fluorescent group and is not affected by the treatments described in steps 4a and 4b in step S4, Fluorescent signals will be detected in step S4;

5c, if in step S3, compound 3 is incorporated into the 3' end of the growing nucleic acid chain, since compound 3 itself carries an environmentally sensitive fluorescent group, a fluorescent signal can be detected in step S4 in 4a, after processing and adjustment, no Send out a fluorescent signal again, or send out a fluorescent signal that is different from compound 2, and no fluorescent signal can be detected in step S4 under a given filter; in step S4, no fluorescent signal can be detected in 4b, and after processing and adjustment, it can send out the same compound 2 the same fluorescence, a fluorescent signal can be detected in step S4; 5d, if in step S3, compound 4 is incorporated into the 3' end of the growing nucleic acid chain, if compound 4 itself does not carry a fluorescent group, but carries a reactive When the fluorescent group is used, no fluorescent signal can be detected in step S4, and the corresponding complementary group carrying the same fluorescent group as compound 2 is added during the treatment and adjustment. After treatment, it can emit the same fluorescence as compound 2. Step S4 can detect the fluorescent signal; if compound 4 itself carries the same fluorescent group as compound 2 and the linking chain group is different, the fluorescent signal can be detected in step S4, and a specific cleavage reagent is added while processing and adjusting, and after processing , the linking group is broken and the fluorescent group is removed, resulting in the loss of the fluorescent signal, and no fluorescent signal can be detected in step S4.