CN115323045A - Gene sequencing reagent and gene sequencing method - Google Patents

Abstract

The invention relates to the technical field of gene sequencing, in particular to a gene sequencing reagent and a gene sequencing method. The method comprises: adding compound 1-4 and a polymerase into a system simultaneously to carry out a nucleotide polymerization reaction to obtain an intermediate state complex; Wash away the unreacted compound 1-4 to maintain the intermediate complex state, detect and record the fluorescent label of each incorporated nucleotide derivative, and determine the base at the corresponding position on the DNA template; compound 5-8 and polymerase Add the treated reaction system to carry out nucleotide polymerization reaction; add cleavage solution to the reaction system to wash off the cleavable protective group in compound 5-8, remove the solution phase and rinse with buffer; wash off the reaction The replaced compounds 1-4; the bases finally incorporated into the newly synthesized chain of the present invention are natural bases, no scar remains, and the flexibility of the newly synthesized chain is not affected, and the binding efficiency of the polymerase to the continuous extension of the chain is beneficial to Reduce the chance of base complementation mismatch and improve sequencing read length and data quality.

Description

Gene sequencing reagent and gene sequencing method

technical field

The invention relates to the technical field of gene sequencing, in particular to a gene sequencing reagent and a gene sequencing method.

Background technique

Gene sequencing is an important driving force for medical and biological discoveries. With the rapid development of gene sequencing technology, high-throughput gene sequencing technology has penetrated into various fields of life sciences. High-throughput gene sequencing adopts clonal amplification and sequencing while synthesizing (SBS) sequencing chemistry technology can achieve fast and accurate sequencing. After nearly a decade of rapid development, it has penetrated into various fields of life sciences, not only strongly promoting the development of basic research, but also occupying an important position in the clinical application stage. important role. In the current most popular high-throughput gene sequencing platform, in order to determine the base type, four reversible termination nucleotides (Reversible Termination Nucleotide, NRT) labeled with fluorescent dyes are added to the reaction system. Usually, each modified NRT The fluorescent dye label is connected to the base through a cleavable chain, and a cleavable protective group is added at the 3'-OH end. Different NRTs will emit unique fluorescent signals, which can be used to determine the sequence of DNA sequences. At present, after the fluorescent dye label modified on NRT is connected to the base through the cleavable chain, most of the chemical structures of the connecting chain will remain on the newly synthesized nucleotide chain during the elution process. These residual chemical bonds will It affects the flexibility of the newly synthesized chain, the binding efficiency of the polymerase to the continuous extension of the chain, and the probability of base complementary mismatches, thereby affecting the quality of sequencing.

Contents of the invention

In view of the deficiencies of the above-mentioned prior art, the present invention aims to provide a gene sequencing reagent and a gene sequencing method. The bases that are finally incorporated into the newly synthesized chain are natural bases, and there is no "scar" residue, which will not affect the DNA of the newly synthesized chain. Flexibility, the binding efficiency of the polymerase to the continuous extension of the chain, is conducive to reducing the probability of base complementary mismatches and improving the sequencing read length and data quality.

In order to solve the above problems, the present invention adopts the following technical solutions:

In one aspect, the present invention provides a gene sequencing method, comprising:

S1, prepare compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7 and compound 8 respectively;

S2, linking the nucleic acid template to be sequenced to the test carrier, and forming a cluster of nucleic acid molecules to be tested by amplification;

S3, adding compound 1, compound 2, compound 3, compound 4 and polymerase into the S2 system at the same time to carry out nucleotide polymerization reaction to obtain an intermediate state complex;

S4, wash away the unreacted compound 1, compound 2, compound 3, and compound 4 to maintain the intermediate complex state of S3, and detect and record the fluorescent label of each incorporated nucleotide derivative, and determine the corresponding position on the DNA template the base;

S5, adding compound 5, compound 6, compound 7, compound 8 and polymerase into the reaction system after S4 treatment to carry out nucleotide polymerization reaction;

S6, adding a cleavage solution to the system after the reaction in S5 to wash away the cleavable protective groups in compound 5, compound 6, compound 7, and compound 8, remove the solution phase and rinse with buffer;

S7, wash away compound 1, compound 2, compound 3, compound 4 replaced after the reaction in S6;

S8, repeat steps S3 to S7 one or more times;

The compound 1, compound 2, compound 3 and compound 4 are 2', 3'-dideoxytriphosphate nucleotide derivatives with the structure of formula (I);

The compound 5, compound 6, compound 7 and compound 8 are 2'-deoxynucleotide derivatives, having the structure of formula (II);

The structures of the formula (I) and the formula (II) are as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive active group, or do not exist;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

R ₅ are each independently a protecting group capable of performing an orthogonal cleavage reaction;

L ₁ is one of a linking group or a cleavable linking group.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ are each independently a fluorophore that can emit different fluorescent signals;

_R3 is H or absent;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently a fluorescent group or do not exist; wherein, in compound 1, R ₂ is a fluorescent group, and R ₃ does not exist; in compound 2, R ₂ is a different fluorescent group, which can emit the same The fluorescent signal of the R ₂ fluorophore is different, and R3 does not exist; R ₂ in compound 3 is a fluorophore that is the same as the R ₂ fluorophore in compound 1, or can emit the same fluorescence as the R ₂ fluorophore in compound 1 Signal fluorophore, R ₃ in compound 3 is the same fluorophore as R ₂ in compound 2, or a fluorophore that can emit the same fluorescent signal as the R ₂ fluorophore in compound 2; in compound 4 R2 and _R3 are H or absent _;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

Further, said S4 includes:

S4a, wash away unreacted compound 1, compound 2, compound 3, compound 4;

S4b, adding a fluorophore-labeled active group that can quickly combine with the R ₃ reactive active group in compound 3, thereby introducing a second fluorophore into compound 3, the fluorophore labeled on the active group The same as R2 in compound ₂ , or a fluorescent group that can emit the same fluorescent signal as the R2 fluorescent group in compound ₂ ;

S4c, add scanning buffer, excite the light source to detect and record the fluorescent signal, and then wash away the scanning buffer;

The structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive group, or do not exist; wherein, in compound 1, R ₂ is a fluorescent group, and R ₃ does not exist; in compound 2, R ₂ is Different fluorescent groups can emit different fluorescent signals from the R ₂ fluorescent group in compound 1, and R ₃ does not exist; the R ₂ fluorescent group in compound 3 is the same as the R ₂ fluorescent group in compound 1, or can emit A fluorophore with the same fluorescent signal as the R ₂ fluorophore in compound 1; R ₃ in compound 3 is a reactive active group that can quickly combine with the active group labeled with the fluorophore, and the fluorescent label on the active group The group is the same as R2 in compound ₂ , or a fluorescent group that can emit the same fluorescent signal as the R2 fluorescent group in compound _{2 ;} R2 and _R3 in compound 4 are H or do not exist;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or is absent.

Further, said S4 includes:

S4a, wash away the unreacted compound 1, compound 2, compound 3, and compound 4, and then add a scanning buffer to adjust the reaction environment so that compound 1 and compound 2 can be excited to emit fluorescence, detect and record the fluorescence signal;

S4b, washing away the scanning buffer, adding the active group of the fluorescent group labeled by compound 1, introducing the fluorescent group into compound 3 so that compound 3 can emit a fluorescent signal, and at the same time adjust the reaction environment to quench the fluorescent group on compound 2 cluster fluorescence;

S4c, add scanning buffer, excite the light source to detect and record the fluorescent signal, and then wash away the scanning buffer;

The structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ are each independently one of a fluorescent group and a reactive active group, or do not exist; wherein, in compound 1, R ₂ is a fluorescent group; in compound 2, R ₂ is an environmental high-sensitivity fluorescent dye group, the Under certain conditions, the fluorophore can emit the same fluorescent signal as the fluorophore of compound 1, and can quickly respond to the reaction system environment change to cause fluorescence quenching; in compound 3, R ₂ is a reactive active group; in compound 4 R2 is H or absent _;

_R3 is H or absent;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

In another aspect, the present invention provides a gene sequencing reagent, including polymerase, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7 and compound 8;

The compound 1, compound 2, compound 3, and compound 4 are 2', 3'-dideoxytriphosphate nucleotide derivatives, which have the structure of formula (I);

The compound 5, compound 6, compound 7, and compound 8 are 2'-deoxynucleotide derivatives with a structure of formula (II);

The structures of the formula (I) and the formula (II) are as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive active group, or do not exist;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

R ₅ are each independently a protecting group capable of performing an orthogonal cleavage reaction;

L1 is one of a linking group or a cleavable linking group.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ are each independently a fluorophore that can emit different fluorescent signals;

_R3 is H or absent;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently a fluorescent group or do not exist; wherein, in compound 1, R ₂ is a fluorescent group, and R ₃ does not exist; in compound 2, R ₂ is a different fluorescent group, which can emit the same The fluorescent signal of the R ₂ fluorophore is different, and R3 does not exist; R ₂ in compound 3 is a fluorophore that is the same as the R ₂ fluorophore in compound 1, or can emit the same fluorescence as the R ₂ fluorophore in compound 1 Signal fluorophore, R ₃ in compound 3 is the same fluorophore as R ₂ in compound 2, or a fluorophore that can emit the same fluorescent signal as the R ₂ fluorophore in compound 2; in compound 4 R2 and _R3 are H or absent _;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive group, or do not exist; wherein, in compound 1, R ₂ is a fluorescent group, and R ₃ does not exist; in compound 2, R ₂ is Different fluorescent groups can emit different fluorescent signals from the R ₂ fluorescent group in compound 1, and R ₃ does not exist; the R ₂ fluorescent group in compound 3 is the same as the R ₂ fluorescent group in compound 1, or can emit A fluorophore with the same fluorescent signal as the R ₂ fluorophore in compound 1; R ₃ in compound 3 is a reactive active group that can quickly combine with the active group labeled with the fluorophore, and the fluorescent label on the active group The group is the same as R2 in compound ₂ , or a fluorescent group that can emit the same fluorescent signal as the R2 fluorescent group in compound _{2 ;} R2 and _R3 in compound 4 are H or do not exist;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

Further, the structure of the formula (I) is as follows:

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ are each independently one of a fluorescent group and a reactive active group, or do not exist; wherein, in compound 1, R ₂ is a fluorescent group; in compound 2, R ₂ is an environmental high-sensitivity fluorescent dye group, the Under certain conditions, the fluorophore can emit the same fluorescent signal as the fluorophore of compound 1, and can quickly respond to the reaction system environment change to cause fluorescence quenching; in compound 3, R ₂ is a reactive active group; in compound 4 R2 is H or absent _;

_R3 is H or absent;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

Each L ₁ is independently a linking group or absent.

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

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

Further, the cleavable linking group is a group with any of the following structures:

Wherein, R1', R2' are each independently one of halogen, -H, and C1-C5 aliphatic chain.

The beneficial effect of the present invention is that: a gene sequencing reagent and a gene sequencing method provided by the present invention include 2', 3'-dideoxytriphosphate nucleotide derivatives, the first feature of which is the 5'-end, the first , the O between the two phosphate groups is replaced by the _CH2 group, and its second feature is that the base of the nucleotide polyphosphate derivative is marked with a fluorescent group or a reactive active group. This type of nucleoside In the process of SBS sequencing, acid polyphosphate derivatives can be recognized by DNA polymerase and incorporated into the DNA chain template to be tested. Due to the presence of _CH2 groups, phosphodiester bonds cannot be formed normally, and the DNA chain elongation is terminated. , and the deletion of the 2'-OH on the 2',3'-dideoxytriphosphate nucleotide also ensures that only a single nucleotide derivative is incorporated at a time; at this time, the nucleotide polyphosphate derivative and the polymerase and The nucleic acid template strand forms an intermediate complex, and the nucleotide derivatives incorporated at the 3' end of the nucleic acid chain are identified by detecting the fluorescent label of each incorporated nucleotide derivative and taking pictures and storing the image, according to This determines the base at the corresponding position on the DNA template; after taking pictures, add a 3'-protecting group-2'-deoxytriphosphate nucleotide that can normally form a phosphodiester bond to competitively replace the above 2', 3 '-Dideoxytriphosphate nucleotide derivatives, to complete this round of SBS chain growth, the 3'-protecting group ensures that only one base is increased each time, and after each round of SBS sequencing, the 3'-protecting group-2 The protective group of '-deoxy-triphosphate nucleotides can be cleaved to generate natural 3'-OH-2'-deoxy-triphosphate nucleotides, so as not to affect the next round of SBS reaction, and finally incorporated into the base of the newly synthesized chain The base is a natural base, and there is no "scar" residue, which is conducive to improving the sequencing read length and data quality. In addition, in addition to providing four-color fluorescent and two-color fluorescent SBS sequencing reagents and methods, the sequencing method of the present invention also provides single-color fluorescent reagents and sequencing methods. The sequencer only needs to be equipped with one excitation light source and one camera, thereby reducing the manufacturing cost of the sequencer. The cost and volume reduce the start-up threshold of hospitals, which is conducive to the spread of sequencers to wider hospitals and research institutions in third- and fourth-tier cities, and is suitable for localized testing in hospitals at all levels.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments.

It should be noted that these examples are only used to illustrate the present invention, rather than to limit the present invention, and the simple improvement of the method under the premise of the present invention all belongs to the protection scope of the present invention.

A gene sequencing reagent, including polymerase, compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8;

The compound 1, compound 2, compound 3, and compound 4 are 2', 3'-dideoxytriphosphate nucleotide derivatives with the structure of formula (I), and the compound 5, compound 6, compound 7, and compound 8 It is a 2'-deoxynucleotide derivative with a structure of formula (II):

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive group, or not present; the fluorescent group includes an environmentally sensitive fluorescent group that can emit the same fluorescent signal, and can emit the same or different Fluorophores for fluorescent signals;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

R ₅ are each independently a protecting group capable of performing an orthogonal cleavage reaction;

L ₁ is one of a linking group or a cleavable linking group.

In the embodiments of 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 acid polymerase, such as DNA (Deoxyribonucleic Acid, Desoxyribonucleic Acid) polymerase, RNA (Ribonucleic Acid, Ribonucleic Acid) polymerase, and reverse transcriptase.

Polymerases can use RNA or single-stranded DNA as templates to synthesize new DNA strands. According to actual needs, an appropriate polymerase can be selected for nucleotide polymerization, or a mixture of multiple polymerases can be selected for use.

In the embodiment of the present invention, DNA polymerase is used for nucleotide polymerization (hereinafter referred to as polymerase M).

Each compound in the present invention is the derivative of nucleotide A, (T/U), C and G respectively, and nucleotide refers to nucleoside-5'-polyphosphoric acid compound or its structural analogue, has complementary base pairing Ability, which can be incorporated by nucleic acid polymerases to extend growing nucleic acid strands, nucleotides can be modified at one or more base, sugar or phosphate groups, nucleotides can be labeled with fluorescent dyes or reactive active group.

The protective 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 cleavable linking group is Refers to any small molecular group between the fluorescent group linked to the 5' phosphate or base, the environmentally sensitive fluorescent group, the fluorescent group that can emit the same fluorescent signal, and the reactive active group that can carry out the connection reaction. The fluorophore that can emit the same fluorescent signal refers to the non-environmental high-sensitivity fluorescence that can emit fluorescence at 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. dye. Protecting groups can be deprotected in response to cutting agents including but not limited to Na2S2O4), THP), TEC, DTT, weak acids, Pd(0) or light irradiation (such as ultraviolet irradiation), and protecting groups include but are not limited to the following Several:

In the embodiment of the present invention, the nucleotide derivative is modified with a cleavable chain group, and the cleavable chain group refers to a cleavable chain group that responds to external stimuli (for example, enzymes, nucleophilic/alkaline reagents, reducing agents, light irradiation, electrophilic/ Acidic reagents, organometallic and metallic reagents or oxidizing agents) are orthogonally cleavable (eg, specifically cleavable) chain groups. In the orthogonal cleavage reaction, the cleavage agent used includes but not limited to Na2S2O4), THP), TEC, DTT, weak acid, Pd(0) or light irradiation (such as ultraviolet irradiation), etc., and the cleavable linking group has A group with any of the following structures:

Wherein, R _1' and R _2' are each independently one of halogen, -H, and C ₁ -C ₅ aliphatic chain.

The reactive active group (Active Group) is a conjugated reactive group capable of performing a specific orthogonal connection 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.

The fluorescent group comes 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, ATTO590, ATTO Thio12, FAM, VIC, TET, JOE, HEX, CAL Fluor Orange 560, TAMRA, CAL Fluor Red610、TEXAS RED、CAL Fluor Red635、iFluor 488、iFluor 514、iFluor 532、iFluor 546、iFluor 555、iFluor 568、iFluor 590、iFluor610、iFluor 633、iFluor 647、iFluor 680、iFluor700、iFluor710、Quasar705、Quasar670。

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.

A gene sequencing method, comprising the steps of:

S1, respectively prepare the aforementioned compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, the compound 1, compound 2, compound 3, compound 4 are 2', 3'-bis Deoxyribonucleotide triphosphate derivatives have a structure of formula (I), and the compound 5, compound 6, compound 7, and compound 8 are 2'-deoxynucleotide derivatives with a structure of formula (II):

Wherein, R is _a different base or analogue, and the base or analogue is one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ are each independently one of a fluorescent group and a reactive group, or not present; the fluorescent group includes an environmentally sensitive fluorescent group that can emit the same fluorescent signal, and can emit the same or different Fluorophores for fluorescent signals;

R ₄ are each independently one of a monophosphate group and a polyphosphate group;

R ₅ are each independently a protecting group capable of performing an orthogonal cleavage reaction;

L is _one of a linking group or a cleavable linking group;

S2, linking the nucleic acid template to be sequenced to the test carrier, and forming a cluster of nucleic acid molecules to be tested by amplification;

S3, adding compound 1, compound 2, compound 3, compound 4 and polymerase into the S2 system at the same time to carry out nucleotide polymerization reaction to obtain an intermediate state complex;

S4, wash away unreacted compound 1, compound 2, compound 3, and compound 4, maintain the state of the intermediate complex in S3, and detect and record the fluorescent label of each incorporated nucleotide derivative to determine the corresponding the base of the position;

S5, adding compound 5, compound 6, compound 7, compound 8 and polymerase to the reaction body after S4 treatment to perform nucleotide polymerization reaction;

S6, adding a cleavage solution to the system after the reaction in S5 to wash away the cleavable protective groups in compound 5, compound 6, compound 7, and compound 8, remove the solution phase and rinse with buffer;

S7, wash away compound 1, compound 2, compound 3, compound 4 replaced after the reaction in S6;

S8, repeat steps S3 to S7 one or more times.

According to different sequencing requirements, different methods can be used to achieve it. Each method is different in individual steps. Their differences are:

The first is a sequencing method with four-color fluorescent dyes:

In step S1, compound 1, compound 2, compound 3, and compound 4 are prepared respectively, so that compound 1, compound 2, compound 3, and compound 4 are respectively labeled with fluorescent groups with different excitation and emission wavelengths, and compounds 1-4 are independently Possess formula (I) structure:

At this time, R ₂ is each independently a fluorescent group that can emit different fluorescent signals; R ₃ is; L ₁ is H or does not exist.

In step S2, the nucleic acid template to be sequenced is connected to the test carrier, and the nucleic acid molecule cluster to be tested is formed by amplification, such as linking the sequenced nucleic acid template to a chip or a microsphere to form a nucleic acid molecule cluster to be tested;

In step S3, each compound and polymerase M are added to the S2 reaction system at the same time to carry out the nucleotide polymerization reaction. The polymerase M can recognize the corresponding compound and incorporate any of them into the 3' end of the growing nucleic acid chain. Due to the first, The O between the two phosphate groups is replaced by the CH2 group, the phosphodiester bond cannot be formed normally, the chain extension is terminated, and the structure of the 2',3'-dideoxytriphosphate nucleotide also ensures that only Incorporate a single nucleotide derivative; at this time, any compound in Compound 1, Compound 2, Compound 3, and Compound 4 forms an intermediate complex with the polymerase M and the nucleic acid template strand;

In step S4, unreacted compound 1, compound 2, compound 3, and compound 4 are washed away, and the state of the intermediate complex in step S3 is maintained; the fluorescent label of each incorporated nucleotide derivative is detected and the image is taken and stored for identification The nucleotide derivatives incorporated at the 3' end of the nucleic acid chain, due to the different fluorescent groups labeled by the compounds 1-4, the detected fluorescent signals are also different, which can be used to determine the base at the corresponding position on the DNA template. base;

In step S5, compound 5, compound 6, compound 7, and compound 8 can replace compound 1, compound 2, compound 3, and compound 4 in the intermediate state complex of step S3 and form a phosphodiester bond to achieve the purpose of chain extension. Due to the presence of R5 cleavable protective group in compound _5-8 , only a single base is incorporated into the DNA chain for each chain elongation;

In step S6, cleavage buffer is added to remove the cleavable protective group R2 in compound 5-8, the solution phase is removed and rinsed with buffer, and the replaced compound 1, compound 2, compound 3, and compound 4 are washed away, Obtain natural nucleotides without scars, which is conducive to the incorporation of the next base.

The second method is a sequencing method with two-color fluorescent dyes:

In step S1, compound 1, compound 2, compound 3, and compound 4 are prepared respectively; compounds 1-4 each independently have the structure of formula (I):

At this time, in compound ₁ , R2 is dye A, and _R3 does not exist; in compound ₂ , R2 is dye B, and _R3 does not exist; in compound ₃ , R2 is dye A, and _R3 is dye B; in compound ₄ , R2 and R ₃ do not exist; dye A and dye B have different structures and can emit different fluorescent signals; L ₁ is each independently a linking group or does not exist;

In step S2, the nucleic acid template to be sequenced is connected to the test carrier, and the nucleic acid molecule cluster to be tested is formed by amplification, such as fixing the accounting template on the chip, and constructing the nucleic acid molecule cluster to be tested by bridge amplification;

In step S3, the polymerase M can recognize the corresponding compound and incorporate any of them into the 3' end of the growing nucleic acid chain. Due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally. Chain elongation is terminated, and the structure of 2',3'-dideoxytriphosphate nucleotides also ensures that only a single nucleotide derivative is incorporated at a time; at this time, any one of compounds 1-3 and polymerase M and the nucleic acid template strand form an intermediate complex;

In step S4, the fluorescent label of each incorporated nucleotide derivative is detected and the image is taken and stored to identify the incorporated nucleotide derivative at the 3' end of the nucleic acid chain. At this time, compound 1 is on the filter Fluorescent signal can be detected in channel 1, but no fluorescent signal (or signal is very weak) can be detected in filter channel 2; no fluorescent signal (or signal is very weak) can be detected in filter channel 1 of compound 2, and in A fluorescent signal can be detected in filter channel 2, and a fluorescent signal can be detected in both filter channel 1 and filter channel 2 of compound 3; No fluorescent signal can be detected in filter channel 2, based on which the category of the incorporated nucleotide derivative and the base at the corresponding position on the DNA template can be judged.

The rest of the steps are the same as the first method.

The third method is also a sequencing method for two-color fluorescent dyes:

In step S1, compound 1, compound 2, compound 3, and compound 4 are prepared respectively; compounds 1-4 each independently have the structure of formula (I):

Now R in compound ₁ is dye A, _R does not exist; R in compound ₂ is dye B, _R does not exist; R in compound ₃ is dye A, _R is reactive group (Active Group , such as biotin biotin, biotin can quickly combine with fluorescently labeled avidin or streptavidin); R ₂ and R ₃ do not exist in compound 4; dye A and dye B have different structures and can emit different Fluorescent signal; each L ₁ is independently a linker or absent.

In step S2, the nucleic acid template to be sequenced is connected to the test carrier, and the nucleic acid molecule cluster to be tested is formed by amplification, such as fixing the accounting template on the chip, and constructing the nucleic acid molecule cluster to be tested by bridge amplification;

In step S3, the polymerase M can recognize the corresponding compound and incorporate any of them into the 3' end of the growing nucleic acid chain. Due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally. Chain elongation is terminated, and the structure of 2',3'-dideoxytriphosphate nucleotides also ensures that only a single nucleotide derivative is incorporated at a time; at this time, any one of compounds 1-3 and polymerase M and the nucleic acid template strand form an intermediate complex;

In step S4, it is also necessary to add an active substance carrying dye B that can specifically bind to the active group, such as a water-soluble dye B-streptavidin, introduce dye B into compound 3, and wash away excess substances after the binding reaction; detection Fluorescent labeling of each incorporated nucleotide derivative and photographing and storing the image to identify the incorporated nucleotide derivative at the 3′ end of the nucleic acid chain, at this time, compound 1 can be Fluorescent signal is detected, but no fluorescent signal (or very weak signal) is detected in filter channel 2; no fluorescent signal (or very weak signal) is detected in filter channel 1 for compound 2, and no fluorescent signal (or very weak signal) is detected in filter channel 1. Fluorescent signal can be detected in 2, and fluorescent signal can be detected in both filter channel 1 and filter channel 2 of compound 3; since compound 4 has no fluorescent group label, it can be detected in filter channel 1 and filter channel No fluorescent signal can be detected in 2, which can be used to determine the type of nucleotide derivative incorporated and the base at the corresponding position on the DNA template.

The rest of the steps are the same as the first method.

The fourth method is the single-color fluorescent sequencing method:

In step S1, compound 1, compound 2, compound 3, and compound 4 are prepared respectively; compounds 1-4 each independently have the structure of formula (I):

At this time, in compound 1, R ₂ is dye A, and R ₃ does not exist; in compound 2, R ₂ is dye B, and R ₃ does not exist; in compound 3, R ₂ is a reactive active group (Active Group, such as biotin biotin, Biotin can quickly combine with fluorescently labeled avidin or streptavidin), R ₃ does not exist; neither R ₂ nor R ₃ exists in compound 4; dye A and dye B have different structures, and dye B is environmentally sensitive The fluorophore, which can emit the same fluorescent signal as the dye A, can also quickly respond to the weak adjustment of the environment, and the phenomenon of fluorescence quenching occurs; L ₁ is independently a linking group or does not exist.

In step S2, the nucleic acid template to be sequenced is connected to the test carrier, and the nucleic acid molecular cluster to be tested is formed by amplification, such as fixing the accounting template on the chip, and constructing the first nucleic acid double-stranded molecular cluster through bridge amplification;

Step S3 is to add compound 1, compound 2, compound 3, compound 4 and polymerase into the S2 system at the same time for nucleotide recognition polymerization reaction, polymerase M can recognize the corresponding compounds and incorporate any of them into the growing At the 3' end of the nucleic acid chain, due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally, and the chain extension terminates, and the 2', 3'-dideoxytriphosphate nucleotide The structure also ensures that only a single nucleotide derivative is incorporated at a time; at this time, any one of the compounds 1-3 forms an intermediate complex with the polymerase M and the nucleic acid template strand;

Step S4 includes the following steps: S4a, wash away unreacted compound 1, compound 2, compound 3, and compound 4, and then add scanning buffer to adjust the reaction environment so that the dye B marked on compound 2 can be excited, and the dye B emits the same The same fluorescent signal as dye A, detect the fluorescent signal and record the fluorescent signal by taking pictures and storing the image; S4b, wash away the scanning buffer, add the active group labeled with dye A, which can react with the reactive group on compound 3 The active group undergoes a specific binding reaction, thereby introducing the fluorescent group A into compound 3 to make it emit a fluorescent signal; at the same time, the reaction environment is adjusted to quench the fluorescence of the fluorescent group on compound 2, which has no effect on compound 1 and compound 4. effect, but it can quench the fluorescence of the environmentally sensitive fluorophore on compound 2, and the fluorescence signal cannot be detected; S4c, add scanning buffer, excite the light source to detect the fluorescence signal and record the fluorescence signal by taking pictures and storing the image, and then wash away scan buffer;

Compare the results of the two photographs before and after to judge the category of nucleotide derivatives incorporated. At this time, the fluorescent signal can be detected in the two photographs of compound 1; the fluorescent signal can be detected in the first photograph of compound 2, No fluorescent signal (or very weak signal) could be detected in the second photoshoot; no fluorescent signal (or very weak signal) could be detected in compound 3 in the first photoshoot, but a fluorescent signal could be detected in the second photoshoot; Since compound 4 is not labeled with a fluorophore, no fluorescent signal can be detected in the two photographs before and after; this can be used to determine the type of nucleotide derivative incorporated and the base at the corresponding position on the DNA template.

The rest of the steps are the same as the first method.

Example 1

Compound 1, Compound 2, Compound 3, and Compound 4 have the structure of formula I-a:

Compound 5, Compound 6, Compound 7, and Compound 8 have the structure of formula IIa:

Base represents different bases, B in this embodiment are adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U)

Dye represents fluorescent dyes with different excitation and emission wavelengths. In this embodiment, Dye is AF532, Cy5, AF568, IF700,

Block is a cleavable protective group, in this embodiment Block is N3 or SS _- Et group

The synthetic route of compound 1, compound 2, compound 3, compound 4 with formula Ia is as follows:

The synthetic route of compound 5, compound 6, compound 7, compound 8 with formula IIa is as follows:

In its preferred embodiment the specific structures of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8:

The gene sequencing method is as follows:

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

b) Add compound 1, compound 2, compound 3, compound 4 and polymerase M to the reaction system at the same time to carry out nucleotide recognition polymerization reaction, polymerase M can recognize the corresponding compound and incorporate any of them into the growing nucleic acid At the 3' end of the chain, due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally, and the chain extension terminates. Moreover, this structure of 2',3'-dideoxytriphosphate nucleotide also ensures that only a single nucleotide derivative is incorporated at a time; Chains form an intermediate state complex;

c) wash away the unreacted compound 1, compound 2, compound 3, and compound 4, and maintain the state of the intermediate complex in step b; detect the fluorescent label of each incorporated nucleotide derivative and take pictures and store images to identify all Nucleotide derivatives incorporated into the 3' end of the nucleic acid chain; since compound compound 1, compound 2, compound 3, and compound 4 are marked with AF532, Cy5, AF568, and IF700 respectively, the detected fluorescent signals are completely different, which can be The nucleotide derivatives incorporated accordingly and the bases at the corresponding positions on the DNA template;

d) compound 5, compound 6, compound 7, compound 8 and polymerase are added to the above nucleic acid double-stranded molecular cluster reaction system to carry out nucleotide polymerization reaction. At this time, compound 5, compound 6, compound 7 and compound 8 can Compound 1, Compound 2, Compound 3, and Compound 4 in the intermediate state complex of step b replace and form a phosphodiester bond to achieve the purpose of chain extension. Due to the existence of R2 cleavable protective groups in Compound 5-8, each Secondary strand elongation incorporates only a single base into the DNA strand.

e) Add cleavage buffer to remove the 3'-terminal protective group in compound 5-8, remove the solution phase and wash it with buffer, and wash away the replaced compound 1, compound 2, compound 3, and compound 4 at the same time , which facilitates the incorporation of the next base.

f) Repeat steps a-e one or more times.

Example 2

Compound 1 and compound 2 have the structure of formula (I-a), compound 3 has the structure of formula (I-b); compound 4 has the structure of formula (I-c); compound 5, compound 6, compound 7 and compound 8 have the structure of formula (IIa), For example, compound 1 and compound 2 have the structure of formula Ia-1:

Compound 3 has the structure of formula I-b:

Compound 4 has the structure of formula I-c:

Compound 5, Compound 6, Compound 7, and Compound 8 have the structure of formula IIa:

Base represents different bases, and B in this embodiment is adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U).

Dye A and Dye B are each independently a fluorescent group capable of emitting different fluorescent signals.

Block is a cleavable protective group, and in this embodiment Block is N3 or SS _- Et group.

Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, and Compound 8 have the same synthetic routes as in Example 1.

The synthetic route of compound 3 having formula I-c is shown below:

In its preferred embodiment, the specific structures of compound 1, compound 2, compound 3 and compound 4 are as follows:

The method for using it to carry out gene sequencing is as follows:

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

b) Add compound 1, compound 2, compound 3, compound 4 and polymerase M to the reaction system at the same time to carry out nucleotide recognition polymerization reaction, polymerase M can recognize the corresponding compound and incorporate any of them into the growing nucleic acid At the 3' end of the chain, due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally, and the chain extension terminates. Moreover, this structure of 2',3'-dideoxytriphosphate nucleotide also ensures that only a single nucleotide derivative is incorporated at a time; Chains form an intermediate state complex;

c) wash away unreacted compound 1, compound 2, compound 3, compound 4, and maintain the state of the intermediate complex in step b; detect the fluorescent label of each incorporated nucleotide derivative and take pictures and store images to identify all Nucleotide derivatives incorporated into the 3′ end of the nucleic acid chain. At this time, the dye Cy5 labeled with compound 1 can detect a fluorescent signal in the filter channel 1, and no fluorescence can be detected in the filter channel 2. Signal (or the signal is very weak); the dye AF532 labeled with compound 2 cannot detect the fluorescent signal (or the signal is very weak) in the filter channel 1, and the fluorescent signal can be detected in the filter channel 2, and the compound 3 is simultaneously labeled There are Cy5 and AF532, so the fluorescent signal can be detected in both filter channel 1 and filter channel 2; since compound 4 is not labeled with a fluorophore, it cannot be detected in both filter channel 1 and filter channel 2 A fluorescent signal is detected; based on this, the incorporated nucleotide derivative and the base at the corresponding position on the DNA template;

d) compound 5, compound 6, compound 7, compound 8 and polymerase are added to the above nucleic acid double-stranded molecular cluster reaction system to carry out nucleotide polymerization reaction. At this time, compound 5, compound 6, compound 7 and compound 8 can Compound 1, Compound 2, Compound 3, and Compound 4 in the intermediate state complex of the S3 step replace and form a phosphodiester bond to achieve the purpose of chain extension. Due to the existence of R2 cleavable protective groups in Compound 5-8, each Secondary strand elongation incorporates only a single base into the DNA strand.

e) Add cleavage buffer to remove the cleavable protective group R2 in compound 5-8, remove the solution phase, rinse with buffer, and wash away the replaced compound 1, compound 2, compound 3, and compound 4 , to obtain natural nucleotides without scars, which is conducive to the incorporation of the next base.

f) Repeat steps a-e one or more times.

Example 3

Compound 1 and compound 2 have the structure of formula (I-a), compound 3 has the structure of formula (I-d); compound 4 has the structure of formula (I-c); compound 5, compound 6, compound 7 and compound 8 have the structure of formula (IIa), For example, compound 1 and compound 2 have the structure of formula Ia-1:

Compound 3 has the structure of formula I-d:

Compound 4 has the structure of formula I-c:

Compound 5, Compound 6, Compound 7, and Compound 8 have the structure of formula IIa:

Base represents different bases, and B in this embodiment is adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U).

Dye A and Dye B are independently fluorescent groups capable of emitting different fluorescent signals, and AG (ActiveGroup) is a reactive group capable of linking reactions.

Block is a cleavable protective group, and in this embodiment Block is N3 or SS _- Et group.

Compound 1, Compound 2, Compound 4, Compound 5, Compound 6, Compound 7, and Compound 8 have the same synthetic routes as in Example 1.

The synthetic route of compound 3 having formula 1-d is shown below:

In its preferred embodiment, the specific structures of compound 1, compound 2, compound 3 and compound 4 are as follows:

The method for using it to carry out gene sequencing is as follows:

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

b) Add compound 1, compound 2, compound 3, compound 4 and polymerase M to the reaction system at the same time to carry out nucleotide recognition polymerization reaction, polymerase M can recognize the corresponding compound and incorporate any of them into the growing nucleic acid At the 3' end of the chain, due to the presence of the β-CH2 group at the 5'-OH end, the phosphodiester bond cannot be formed normally, and the chain extension terminates. Moreover, this structure of 2',3'-dideoxytriphosphate nucleotide also ensures that only a single nucleotide derivative is incorporated at a time; Chains form an intermediate state complex;

c) Add water-soluble AF532-streptavidin, which specifically binds to the biotin group on compound 3, thereby introducing the AF322 fluorescent group into compound 3, and washing away unreacted compound 1, compound 2, and compound 3 , compound 4, and AF532-streptavidin, maintain the intermediate state complex state of step b; detect the fluorescent label of each incorporated nucleotide derivative and take pictures and store images to identify the 3' of the nucleic acid chain At this time, the dye Cy5 labeled with compound 1 can detect a fluorescent signal in the filter channel 1, but no fluorescent signal (or a very weak signal) can be detected in the filter channel 2 ); the dye AF532 labeled with compound 2 could not detect the fluorescent signal (or the signal was very weak) in the filter channel 1, and the fluorescent signal could be detected in the filter channel 2, and the compound 3 was labeled with Cy5 and AF532 at the same time, so Fluorescent signals can be detected in both filter channel 1 and filter channel 2; since compound 4 has no fluorophore label, no fluorescent signal can be detected in filter channel 1 and filter channel 2; according to The incorporated nucleotide derivatives and the bases at the corresponding positions on the DNA template;

d) compound 5, compound 6, compound 7, compound 8 and polymerase are added to the above nucleic acid double-stranded molecular cluster reaction system to carry out nucleotide polymerization reaction. At this time, compound 5, compound 6, compound 7 and compound 8 can Compound 1, Compound 2, Compound 3, and Compound 4 in the intermediate state complex of the S3 step replace and form a phosphodiester bond to achieve the purpose of chain extension. Due to the existence of R2 cleavable protective groups in Compound 5-8, each Secondary strand elongation incorporates only a single base into the DNA strand.

e) Add cleavage buffer to remove the cleavable protective group R2 in compound 5-8, remove the solution phase, rinse with buffer, and wash away the replaced compound 1, compound 2, compound 3, and compound 4 , to obtain natural nucleotides without scars, which is conducive to the incorporation of the next base.

a) Repeat steps a-e one or more times.

Example 4

Compound 1 and compound 2 have the structure of formula (I-a), compound 3 has the structure of formula (I-f); compound 4 has the structure of formula (I-c); compound 5, compound 6, compound 7 and compound 8 have the structure of formula (IIa), For example, compound 1 and compound 2 have the structure of formula Ia-1:

Compound 3 has the structure of formula I-d:

Compound 4 has the structure of formula I-c:

Compound 5, Compound 6, Compound 7, and Compound 8 have the structure of formula IIa:

Base represents different bases, and B in this embodiment is adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U).

Dye represents an environment-sensitive fluorescent dye or a fluorescent group that can emit the same fluorescent signal, which are HCyC-647 and Cy5 in this example;

AG (Active Group) is a reactive group capable of linking reaction.

Block is a cleavable protective group, and in this embodiment Block is N3 or SS _- Et group.

Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, and Compound 8 have the same or similar synthetic routes as in Example 3.

In its preferred technical scheme, one of the structures of compound 1, compound 2, compound 3 and compound 4 is:

The method for using it to carry out gene sequencing is as follows:

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

c) Compound 1, compound 2, compound 3, compound 4 and polymerase M are added to the reaction system at the same time to carry out nucleotide recognition polymerization reaction, polymerase M can recognize the corresponding compound and incorporate any of them into the growing nucleic acid chain Due to the existence of the β-CH2 group at the 5'-OH end, the 3' end of the phosphodiester bond cannot be formed normally, and the chain extension is terminated. Moreover, this structure of 2',3'-dideoxytriphosphate nucleotide also ensures that only a single nucleotide derivative is incorporated at a time; Chains form an intermediate state complex;

d) washing away unreacted dNTPs, adding scanning buffer, adjusting the pH value to 6.8, using a 640nm light source as the excitation wavelength to detect fluorescent signals, taking pictures, and storing image A;

e) wash off the scanning buffer, add water-soluble Cy5-streptavidin, the treatment has no effect on compound 1, compound 2 and compound 4, and Cy5-streptavidin can specifically bind to compound 3 Combined with Biotin, the fluorescent group Cy5 was introduced into compound 3 to make it emit a fluorescent signal;

f) Adjusting the pH value to 7.5, the weakly alkaline environment has no effect on compound 1, compound 3 and compound 4, but can quench the fluorescence of HCyC-647 on compound 2, and no fluorescence signal can be detected under a 640nm light source; g) adding Scan buffer, use 640nm light source as excitation wavelength to detect fluorescent signal, take pictures, and store image B; h) Wash away the scan buffer, add compound 5, compound 6, compound 7, compound 8 and polymerase to the above nucleic acid double-stranded molecules at the same time The cluster reaction system carries out the nucleotide polymerization reaction. At this time, compound 5, compound 6, compound 7, and compound 8 can replace compound 1, compound 2, compound 3, and compound 4 in the intermediate state complex of step b to form phosphoric acid The diester bond achieves the purpose of chain extension. Due to the presence of the 3'-OH cleavable protective group in compound 5-8, only a single base is incorporated into the DNA chain for each chain extension.

i) Add cleavage reagent THP to treat the chip, remove the protective group at the 3'-position of compound 5, compound 6, compound 7, and compound 8 to regenerate free 3'-OH, and wash away the replaced compound 1, Compound 2, compound 3 and compound 4 are beneficial to the incorporation of the next base.

j) repeat steps (b)-(h);

k) 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 both scanning image A and scanning image B, then The incorporated nucleotide derivative is compound 1 (A base derivative, Cy5 label), correspondingly, it can be determined that the base at the corresponding position on the DNA template is T; If there is no fluorescent signal in any of the DNA templates, the incorporated nucleotide derivative is compound 4 (G base derivative, no fluorescent label). Correspondingly, it can be determined that the base at the corresponding position on the DNA template is C; if If there is a signal in scanning image A, but there is no fluorescent signal in scanning image B, then the incorporated nucleotide derivative is compound 2 (T base, labeled with environmental sensitive dye HCyC647), and correspondingly, the DNA template can be determined The base at the corresponding position above is A; if there is no signal in the scanning image A, but there is a fluorescent signal in the scanning image B, then the incorporated nucleotide derivative is compound 3 (C base, Biotin label), Correspondingly, it can be determined that the base at the corresponding position on the DNA template is T. As shown in Table 1.

Table 1: Example 4 detection results and corresponding bases

Sequencing verification was performed using the reagents and methods described in Example 4. The sequencing sample was a Lambda DNA fragment, and the sequencing mode was 30 bases at a single end. Table 2 shows the base sequences of some fragments, and Table 3 shows the test results and analysis .

Table 2: Base sequences of partial Lambda DNA fragments

Table 3 Sequencing results and analysis

The sequencing results showed that the overall error rate was 0.2847%, and the analysis showed that the sequencing reagents and methods in this specific example can accurately sequence the sample DNA fragments.

Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those skilled in the art should understand that it can be described in the form Various changes may be made in matter and details thereof without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A method of gene sequencing comprising:

s1, preparing a compound 1, a compound 2, a compound 3, a compound 4, a compound 5, a compound 6, a compound 7 and a compound 8 respectively;

s2, linking a nucleic acid template to be sequenced to a test carrier, and forming a nucleic acid molecular cluster to be sequenced through amplification;

s3, simultaneously adding the compound 1, the compound 2, the compound 3, the compound 4 and polymerase into an S2 system for nucleotide polymerization reaction to obtain an intermediate complex;

s4, washing away unreacted compound 1, compound 2, compound 3 and compound 4 to maintain the intermediate state complex state of S3, detecting and recording the fluorescent label of each incorporated nucleotide derivative, and judging the base at the corresponding position on the DNA template;

s5, adding the compound 5, the compound 6, the compound 7, the compound 8 and polymerase into the reaction system treated by the S4 to perform nucleotide polymerization reaction;

s6, adding a cutting fluid into the system after the reaction of the S5 to wash away the cleavable protecting groups in the compound 5, the compound 6, the compound 7 and the compound 8, removing a solution phase and washing the solution phase clean by using a buffer solution;

s7, washing off the compound 1, the compound 2, the compound 3 and the compound 4 which are replaced after the reaction in the S6;

s8, repeating the steps S3 to S7 for one or more times;

the compound 1, the compound 2, the compound 3 and the compound 4 are 2',3' -dideoxy triphosphate nucleotide derivatives and have a structure shown in a formula (I);

the compound 5, the compound 6, the compound 7 and the compound 8 are 2' -deoxynucleotide derivatives and have the structure of a formula (II);

the structures of formula (I) and formula (II) are as follows:

wherein R is ₁ Is different bases or analogues, and the bases or analogues are one of adenine, guanine, cytosine, thymine and uracil or analogues;

R ₂ and R ₃ Each independently is one of a fluorophore and a reactable reactive group, or is absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

R ₅ each independently a protecting group capable of undergoing an orthogonal cleavage reaction;

L ₁ is one of a linker or a cleavable linker.

2. The method for gene sequencing of claim 1, wherein the structure of formula (I) is as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ each independently is a fluorophore capable of emitting different fluorescent signals;

R ₃ is H or absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

L ₁ each independently a linking group or absent.

3. The method for gene sequencing of claim 1, wherein the structure of formula (I) is as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ and R ₃ Each independently a fluorophore or is absent; wherein, in the compound 1, R ₂ Is a fluorescent group, R ₃ Is absent; in the compound 2R ₂ Is a different fluorophore capable of emitting a fluorescent light similar to that of R in Compound 1 ₂ Fluorescence signals of different fluorophores, R3 is not existed; r in Compound 3 ₂ Is a fluorescent group and R in compound 1 ₂ The fluorescent group is the same as or capable of emitting the same as R in the compound 1 ₂ Fluorophores with identical fluorescence signals, R in Compound 3 ₃ Then is with R in compound 2 ₂ The same fluorescent group, or a group capable of emitting a light identical to that of R in Compound 2 ₂ Fluorophores with the same fluorescent signals as the fluorophores; r in Compound 4 ₂ And R ₃ Is H or absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

L ₁ each independently is a linking group or is absent.

4. The method for gene sequencing of claim 1, wherein S4 comprises:

s4a, washing to remove unreacted compounds 1, 2, 3 and 4;

s4b, addition of R which is capable of reacting with compound 3 ₃ A fluorophore-labeled active group to which a reactive group rapidly binds, fromAnd a second fluorescent group is introduced into the compound 3, the fluorescent group marked on the active group and R in the compound 2 ₂ Is the same as or capable of emitting the same as R in the compound 2 ₂ Fluorophores with the same fluorescent signals as the fluorophores;

s4c, adding a scanning buffer solution, detecting and recording a fluorescent signal by an excitation light source, and then washing away the scanning buffer solution;

the structure of formula (I) is as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ and R ₃ Each independently is one of a fluorophore and a reactable reactive group, or is absent; wherein, in the compound 1, R ₂ Is a fluorophore group, R ₃ Is absent; in the compound 2R ₂ Is a different fluorophore capable of emitting a fluorescent light similar to that of R in Compound 1 ₂ Fluorescent signals of different fluorophores, R ₃ Is absent; r in Compound 3 ₂ Fluorophores and R in Compound 1 ₂ The fluorescent group is the same as or capable of emitting the same as R in the compound 1 ₂ Fluorophores with the same fluorescent signals as the fluorophores; r in Compound 3 ₃ Is a reactive group, can be rapidly combined with a fluorophore-labeled reactive group, and the fluorophore labeled on the reactive group is combined with R in the compound 2 ₂ Is the same as or capable of emitting the same as R in the compound 2 ₂ Fluorophores with the same fluorescent signals as the fluorophores; r in Compound 4 ₂ And R ₃ Is H or absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

L ₁ each independently is a linking group or is absent.

5. The method for gene sequencing of claim 1, wherein S4 comprises:

s4a, washing away unreacted compounds 1, 2, 3 and 4, adding a scanning buffer solution to adjust the reaction environment so that the compounds 1 and 2 can be excited to emit fluorescence, and detecting and recording fluorescence signals;

s4b, washing off the scanning buffer solution, adding an active group of the fluorescent group marked by the compound 1, introducing the fluorescent group into the compound 3 to enable the compound 3 to emit a fluorescent signal, and simultaneously adjusting the reaction environment to quench the fluorescence of the fluorescent group on the compound 2;

s4c, adding a scanning buffer solution, detecting and recording a fluorescent signal by an excitation light source, and washing off the scanning buffer solution;

the structure of formula (I) is as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ each independently is one of a fluorophore and a reactable reactive group, or is absent; wherein, in the compound 1, R ₂ Is a fluorophore group; in the compound 2R ₂ The fluorescent group can emit a fluorescent signal which is the same as that of the fluorescent group of the compound 1 under a specific condition and can quickly respond to the environmental change of a reaction system to generate a fluorescence quenching phenomenon; r in Compound 3 ₂ Is a reactive group; r in Compound 4 ₂ Is H or absent;

R ₃ is H or absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

L ₁ each independently a linking group or absent.

6. A gene sequencing reagent, which is characterized by comprising polymerase, a compound 1, a compound 2, a compound 3, a compound 4, a compound 5, a compound 6, a compound 7 and a compound 8;

the compound 1, the compound 2, the compound 3 and the compound 4 are 2',3' -dideoxy triphosphate nucleotide derivatives and have a structure shown in a formula (I);

the compound 5, the compound 6, the compound 7 and the compound 8 are 2' -deoxynucleotide derivatives and have the structure of a formula (II);

the structures of formula (I) and formula (II) are as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ and R ₃ Each independently is one of, or absent from, a fluorophore and a reactable reactive group;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

R ₅ each independently a protecting group capable of undergoing an orthogonal cleavage reaction;

l1 is one of a linking group or a cleavable linking group.

7. The gene sequencing reagent of claim 6, wherein the structure of the formula (I) is as follows:

wherein R is ₁ Is different bases or analogs, and the bases or analogs are one of adenine, guanine, cytosine, thymine and uracil or analogs;

R ₂ each independently is a fluorescent group capable of emitting different fluorescent signals;

R ₃ is H or absent;

R ₄ each independently is one of a monophosphate group and a polyphosphate group;

L ₁ each independently a linking group or absent.

8. The gene sequencing reagent of claim 6, wherein the base or the analogue is a compound having any one of the following structures:

9. the gene sequencing reagent of claim 6, wherein the protecting group is a group having any one of the following structures:

10. the gene sequencing reagent of claim 6, wherein the cleavable linking group is a group having any one of the following structures:

wherein, R1 'and R2' are respectively and independently one of halogen, -H and C1-C5 fatty chain.