CN114250283B - Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye - Google Patents
Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye Download PDFInfo
- Publication number
- CN114250283B CN114250283B CN202111203792.9A CN202111203792A CN114250283B CN 114250283 B CN114250283 B CN 114250283B CN 202111203792 A CN202111203792 A CN 202111203792A CN 114250283 B CN114250283 B CN 114250283B
- Authority
- CN
- China
- Prior art keywords
- compound
- group
- fluorescent
- chain
- fluorescent signal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to a monochromatic fluorescence MRT gene sequencing reagent and a method based on an environment-sensitive dye, and the reagent comprises polymerase and four different labeled nucleotide analogs, wherein hydroxyl groups at the 3 'positions of nucleotides of the four nucleotide analogs are modified by protecting groups or cleavable connecting groups, the protecting groups or the cleavable connecting groups can be cleaved and separated to expose 3' -OH again, and the cleavable connecting groups are linked with environment high-sensitivity fluorescent groups or environment high-sensitivity fluorescent group markers through covalent bonds or non-covalent bonds; four nucleotide analogs are nucleotide derivatives that may or may not fluoresce, and methods for gene sequencing using the reagents are also disclosed. The method can effectively identify and catalyze the polymerization reaction when the 3' -O part is modified by macromolecular groups and carry out SBS sequencing, and solves the problem that the sequencing read length is influenced due to the accumulation of residues on the bases in the conventional second-generation SBS sequencing method.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to a monochromatic fluorescent MRT gene sequencing reagent and method based on an environment-sensitive dye.
Background
The sequencing technology is used for determining the primary structure of biological macromolecules such as nucleic acid, protein, polysaccharide and the like, is a basic tool for life science research and a main means for acquiring biological information data, particularly is a DNA sequencing technology, namely a technology for acquiring the base sequence of a target DNA fragment, and the acquisition of the sequence of the target DNA fragment is the basis for further molecular biology research and gene modification, so that researchers can research and better understand health and diseases, and is a necessary technology for a personalized precise medical paradigm. According to different principles, the development of sequencing technology can be roughly divided into 4 historical stages of first generation sequencing, second generation sequencing, third generation sequencing and fourth generation sequencing. Because the four-generation sequencing technologies have the advantages and the disadvantages and the application fields are different, the first-generation sequencing technology is not eliminated, and the current sequencing market is the situation of coexistence of the four-generation sequencing technologies. The second generation sequencing, also called high-throughput sequencing, has the characteristics of wide application and the highest development speed, is different from the traditional first generation sequencing, and has the characteristics of high speed, high throughput and low cost. The second-generation sequencing technology adopts a sequencing chemical technology of clone amplification and sequencing while synthesis (SBS), and realizes rapid and accurate sequencing. This process allows deoxynucleoside triphosphate (dNTPs) derivatives modified with an identifying signal group to be recognized while incorporated into a nucleic acid strand. Typically, each modified dNTP is base-tagged with a cleavable-strand-linked fluorescent label and a cleavable protecting group at the 3' -OH terminus (e.g., azide, vinyl, disulfide linkage, etc., US20010972364; US20130079232A1; US20160040225A1; ju.J.et al.PNAS,2006,103, 19635-19640), and this base derivative, i.e., the fluorescent-tagged reversible terminator Nucleotide (NRT), different NRTs will emit unique fluorescent signals that can be used to determine the order of the DNA sequence. Studies have shown that one important reason for influencing the read length in the two-generation SBS sequencing method (chen.f. et al.gpb,2013,34-40, stupi.b.p.et al.angelw Chem Int Ed engl,2012,51,1724-1727, ondrus.m.et al.nar.2020, 11982-11993) is that the reversible terminator nucleotide analogs developed so far, after cleaving the carried fluorophore, cannot completely remove the linker between the fluorophore and the base, leaving small molecular groups (such as ethylene glycol-modified propargylamino moieties) of about a certain length at the base, and as SBS accumulates on the newly formed DNA duplex, to a certain extent, these residues may be sufficient to interfere with the stability and secondary structure of the DNA duplex structure, thereby influencing the recognition of the DNA polymerase to terminate the chain extension reaction, and limiting sequencing read length. Therefore, researchers design a single-Modified Reversible Terminator (MRT), namely, a reversible blocking protective group and a fluorescent group are simultaneously modified on a 3' -0H group, the modification can play a dual role, and is a reporter of a fluorescent signal and a reversible terminator, and the modification mode can successfully avoid residues left on a base in NRT sequencing theoretically, so that the influences on DNA double-strand stability, polymerase recognition and the like are reduced, and the reading length of sequencing is improved. The major challenge of this approach is that DNA polymerases have difficulty accepting 3'-0 macromolecular dye-modified nucleotides as substrates because the 3' -OH position of the nucleotide is very close to the amino acid residue at the active site of the DNA polymerase in the state of a complex formed by the polymerase with the complementary nucleotide and the DNA template.
Disclosure of Invention
Aiming at the existing defects, the invention provides a monochromatic fluorescence MRT gene sequencing reagent based on an environment-sensitive dye and a method thereof.
The technical scheme adopted by the invention for solving the technical problem is as follows: a monochromatic fluorescence MRT gene sequencing reagent and a method based on an environment-sensitive dye comprise polymerase and four different labeled nucleotide analogs, wherein hydroxyl groups at the 3 'positions of nucleotides of the four nucleotide analogs are modified by protecting groups or cleavable connecting groups, the protecting groups or the cleavable connecting groups can be cleaved and separated to expose 3' -OH again, and the cleavable connecting groups are linked with an environment high-sensitivity fluorescent group or an environment high-sensitivity fluorescent group marker through covalent bonds or non-covalent bonds; the four nucleotide analogs are compound 1, compound 2, compound 3, and compound 4, respectively, wherein,
compound 1 is a nucleotide derivative that does not emit a fluorescent signal or has no fluorophore;
the compound 2 is a nucleotide derivative which can emit a fluorescent signal and carries a fluorescent group;
the compound 3 is a nucleotide derivative carrying an environment high-sensitivity fluorescent group, cannot emit a fluorescent signal or can emit a fluorescent signal under one condition, the emitted fluorescent signal is different from that emitted by the compound 2, the emitted fluorescent signal can emit a fluorescent signal under the other condition and is the same as that emitted by the compound 2, and the structure of the environment high-sensitivity fluorescent group is different from that of the fluorescent group in the compound 2;
the compound 4 is a nucleotide derivative which cannot emit a fluorescent signal and carries a reactive group capable of undergoing a connection reaction, and the reactive group and the reactive fluorescent group can emit the same fluorescent signal as the compound 1 after the connection reaction; or compound 4 is a nucleotide derivative capable of emitting the same fluorescent signal as compound 2 but carrying a different cleavable chain fluorophore than compound 2, which is cleavable under conditions which do not affect compound 2.
Preferably, the compound 1, the compound 2, the compound 3 and the compound 4 are compounds which have the structure of formula (I), formula (II) or formula (III),
wherein "- - - -" is a non-covalent bond;
b represents different bases or analogues, which refer to adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) and analogues thereof;
each R1 is independently selected from hydrogen, a monophosphate group, a diphosphate group, a triphosphate group, a tetraphosphate group, or more phosphate groups;
each R2 is independently a protecting group capable of undergoing an orthogonal cleavage reaction;
each R3 is independently selected from halogen, -H, -OH, N3 or a C2-C10 flexible chain;
l is a cleavable linking group or absent;
r4 is independently an environment-sensitive fluorescent group capable of emitting a fluorescent signal, a fluorescent group capable of emitting the same fluorescent signal and a reactive group capable of carrying out a connection reaction.
And R5 is a reactive group marked by an environment-sensitive fluorescent group capable of emitting a fluorescent signal or a reactive group marked by a fluorescent group capable of emitting the same fluorescent signal.
Preferably, the base or the analogue is a compound having any one of the following structures,
preferably, the protecting group is a 3' -OH modifying group that is efficiently recognized by a polymerase, incorporated into a growing DNA strand, and cleaved off after each round of SBS sequencing; the cleavable connecting group is a micromolecule group which is linked between 3' -OH and an environment high-sensitivity fluorescent group, a fluorescent group capable of emitting the same fluorescent signal or a reactive group capable of carrying out a connecting reaction; the fluorescent groups capable of emitting the same fluorescent signal can emit emission wavelengths which are consistent with or close to those of the selected environment high-sensitivity fluorescent dye under the same excitation light source wavelength, and are detected and judged to be non-environment high-sensitivity fluorescent dyes of the same fluorescent signal.
Preferably, the protecting group is a group having any one of the following structures:
the cleavable connecting group is a group having any one of the following structures:
disulfide group
Cyanoethyl chain radical
Azido chain groups
Dialkyl ketal chain groups
Allyl chain radical
1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-yl) ethyl chain radical
Nitrobenzyl chain radical
Azo chain group
Wherein R is 1’ ,R 2’ Each independently selected from halogen, -H, C1-C5 fatty chain;
the fluorescent groups capable of emitting the same fluorescent signal are derived 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, jeo, HEX, 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 Fluor 680, CAlFluor Fluor 670, QUiFluor 700, QUiFluor Fluor 670.
Preferably, the environment-sensitive fluorescent group is selected from environment-sensitive fluorescent dyes, and the environment-sensitive fluorescent dye is a pH value-sensitive fluorescent dye.
Preferably, the pH sensitive fluorescent dye is a compound comprising any one of the following structural formulas,
wherein: each X is independently selected from halogen, -H, -OH, N3, NH2, S, si or a C2-C10 flexible chain;
r6 is independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol amino, polyethylene glycol maleimide, polyethylene glycol sulfydryl, 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 sulfydryl, C1-C10 alkyl chain sulfonic group and C1-C10 alkyl chain maleimide;
r7 is independently selected from hydrogen, C1-C6 saturated alkyl;
ring A and ring B are each independently selected from heterocyclic groups having the formulae (VI), (VII), (VIII), (IX),
wherein; each Y is 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 alkylamino, polyethylene glycol maleimide, polyethylene glycol sulfhydryl, 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 sulfhydryl, C1-C10 alkyl chain sulfo, C1-C10 alkyl chain maleimide;
r10 and R11 are each independently selected from hydrogen, carboxyl, phosphate, sulfonate.
Preferably, the reactive group is a group that is recognized by a polymerase, incorporated into a growing DNA strand, and orthogonally linked to a corresponding complementary group by covalent or non-covalent bonds to stabilize the splice structure, and cleaved off to regenerate 3' -0H after each round of SBS sequencing; the reactive fluorescent group is a group which carries an environment high-sensitivity fluorescent group or a fluorescent group capable of emitting the same fluorescent signal and performs orthogonal connection reaction with the reactive group through covalent bond or non-covalent bond production to stabilize a splicing structure.
Preferably, the reactable group is any one of the following structures,
amino group
Cycloalkynyl radical
Azide radical
Maleimide
Biotin
Phenylboronic acids
Digoxin; phenol as the starting material
Cycloalkenyl radicals
The complementary group is any one of the following structures:
tetrafluorophenyl ester
Cycloalkynyl radical
Streptavidin, 4-phenylurazole
Azide radical
Salicyl isohydroxy-fatty acid radical, digoxin antibody, sulfhydryl group
Tetrazinyl radical
A monochromatic fluorescence MRT gene sequencing method based on environment-sensitive dye is characterized in that the sequencing reagent is adopted, the testing steps are as follows,
s1, preparing a compound 1, a compound 2, a compound 3 and a compound 4 respectively;
s2, connecting a nucleic acid template chain to be sequenced to a chip or a microsphere to form a nucleic acid double-stranded molecular cluster reaction system;
s3, simultaneously adding the compound 1, the compound 2, the compound 3, the compound 4 and polymerase into a nucleic acid double-stranded molecular cluster reaction system for nucleotide polymerization reaction, so that any one of the compounds is incorporated into the 3' end of a growing nucleic acid chain and stops growing continuously;
s4, washing away unreacted compound 1, compound 2, compound 3 and compound 4, adding a buffer solution, adjusting the pH value, detecting the fluorescent label of each incorporated nucleotide derivative, photographing and storing an image to identify the nucleotide derivative incorporated at the 3' end of the nucleic acid chain; wherein:
4a, if the compounds 2 and 3 are capable of emitting a fluorescent signal, removing the solution phase from the reaction system of the previous step, leaving the duplex attached to the support, and detecting whether the duplex or the growing nucleic acid strand emits the fluorescent signal; then, performing treatment, wherein the treatment has no influence on the compound 1 and the compound 2, but enables the compound 3 to emit no fluorescence signal or emit a fluorescence signal different from that of the compound 2, simultaneously enabling the compound 4 to perform a ligation reaction to emit a fluorescence signal identical to that of the compound 2, finally adjusting the pH value, and detecting whether the duplex or the growing nucleic acid chain emits the fluorescence signal again;
4b, if the compounds 2 and 4 are capable of emitting a fluorescent signal, removing the solution phase from the reaction system of the previous step, leaving the duplex attached to the support, and detecting whether the duplex or the growing nucleic acid strand emits the fluorescent signal; then a treatment is performed which has no effect on compound 1, compound 2 but which is capable of cleaving the cleavable chain fluorophores of compound 4 to remove the fluorophores while simultaneously causing compound 3 to emit the same fluorescent signal as compound 2, and again detecting whether the duplex or the growing nucleic acid strand emits the fluorescent signal;
s5, carrying out image processing and analysis on the fluorescence signal, and judging the nucleotide derivative incorporated into the nucleic acid chain according to the following judgment principle:
5a, if in step S3 compound 1 is incorporated at the 3' end of the growing nucleic acid strand, then in step S4 no fluorescent signal will be detected, since compound 4 does not itself carry a fluorescent group or other reactive group, nor is it affected by the treatment described in step S4;
5b, if in step S3 Compound 2 is incorporated at the 3' end of the growing nucleic acid strand, then in step S4 a fluorescence signal will both be detected, since Compound 2 carries a fluorophore and is not affected by the processing described in steps S4 a and 4 b;
5c, if in step S3, compound 3 is incorporated into the 3' end of the growing nucleic acid strand, since compound 3 itself carries an environmentally sensitive fluorophore, 4a can detect a fluorescent signal in step S4, and after processing and adjustment, no fluorescent signal is emitted, or a fluorescent signal different from compound 2 is emitted, and no fluorescent signal is detected in step S4 given the filter; 4b can not detect a fluorescence signal in step S4, can emit the same fluorescence as the compound 2 after being processed and adjusted, and can detect a fluorescence signal in step S4;
5d, if in step S3 compound 4 is incorporated at the 3' end of the growing nucleic acid strand, if compound 4 itself does not carry a fluorophore but a reactive group, no fluorescence signal is detected in step S4, the corresponding complementary group carrying the same fluorophore as compound 2 is added while the treatment is adjusted, and after the treatment, the same fluorescence as compound 2 can be emitted, and a fluorescence signal can be detected in step S4; if the compound 4 itself carries a fluorophore with the same fluorophore linking group as the compound 2, a fluorescent signal can be detected in step S4, a specific cleavage reagent is added while the treatment is adjusted, and after the treatment, the linking group is cleaved and the fluorophore is removed, so that the fluorescent signal is lost, and the fluorescent signal cannot be detected in step S4.
The invention has the beneficial effects that: the invention utilizes an environment high-sensitivity fluorescent dye or simultaneously uses a second fluorescent dye which can emit the same fluorescent signal under the same excitation condition to distinguish 4 kinds of dNTPs, the fluorescent labels of the dNTPs are not marked on a base like the traditional NRT but marked on 3' -OH in a covalent bond or non-covalent bond mode, the marked dNTPs can be identified and integrated into a DNA double chain by polymerase to complete SBS sequencing, and a connecting fluorescent group chain can be cut to regenerate 3' -OH after each round of SBS sequencing, so that the next round of SBS reaction is not influenced, the polymerase can effectively identify and catalyze dATP, dTTP/dUTP, dC and dGTP modified by macromolecular fluorescence modification at 3' -0 position as substrates by using dCTP and dGTP as well as pertinently designed, modified and screened by high throughput, thereby eliminating the problem that the sequencing is influenced by sequencing reading due to residue accumulation on the base in the conventional second-generation SBS sequencing method, the invention only needs to be a monochromatic fluorescence camera and is convenient for manufacturing, thereby greatly reducing the cost of a fluorescent light source and a wide-emission camera.
Drawings
Fig. 1 is a schematic view of image a in embodiment 1 of the present invention;
fig. 2 is a schematic view of image B in embodiment 1 of the present invention;
fig. 3 is a schematic view of an image C in embodiment 2 of the present invention;
fig. 4 is a schematic diagram of an image D in embodiment 2 of the present invention;
Detailed Description
To more clearly illustrate the objects, technical solutions and advantages of the embodiments of the present invention, the present invention will be further described in conjunction with the embodiments, which will be clearly and completely described. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, are within the scope of the present invention.
In the present invention, the "polymerase" refers to any naturally or non-naturally occurring enzyme or other catalyst capable of catalyzing a polymerization reaction, including various known naturally or modified nucleic acid polymerases, such as DNA polymerase (MEB 202T9 is used in the present embodiment), RNA polymerase, and reverse transcriptase, which can synthesize a new DNA strand using RNA or single-stranded DNA as a template, and can select a suitable polymerase for a nucleotide polymerization reaction according to actual needs, or a mixture of multiple polymerases.
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, "nucleotide" means a nucleoside-5' -polyphosphate compound or a structural analog thereof, having a base complementary pairing ability, which can be incorporated by a nucleic acid polymerase to extend a growing nucleic acid strand, a nucleotide can be modified on one or more of a base, a sugar or a phosphate group, and a nucleotide can carry a fluorescent group, or a reactive group.
In the present invention, the hydroxyl group (-0H) at the 3' position of these four nucleotide derivatives has a protecting group or a linking chain having a protecting group, including but not limited to, those such as allyl group
O-nitrobenzene
Azidomethyl
Dithio radical
Methoxyalkyl, azoalkyl, dithio chain group
Cyanoethyl chain radical
Azido chain groups
Dialkyl ketal chain groups
Allyl chain radical
1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-yl) ethyl chain radical
Nitrobenzyl chain radical
Azo chain group
In the present invention, the 3' -O terminal protecting groups or fluorescent groups or reactive groups of these four nucleotide derivatives are linked by a single or a plurality of the same or different orthogonally cleavable chain groups. The cleavable chain group refers to a chain group that is orthogonally cleavable (e.g., specifically cleavable) in response to an external stimulus (e.g., an enzyme, a nucleophilic/basic reagent, a reducing agent, light irradiation, an electrophilic/acidic reagent, an organometallic, and a metallic reagent, or an oxidizing agent), 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 separated fragments (e.g., a fluorescent dye, a bio-reactive group) do not further react and do not form a new orthogonal cleavable chain group. In orthogonal cleavage, the cleavage agent used includes, but is not limited to, na2S2O 4), THP), TEC, DTT, weak acid, pd (0), or light irradiation (e.g., ultraviolet irradiation), and the like.
In the present invention, the reactive group is a conjugate reactive group capable of specific orthogonal linkage reaction with a complementary group carrying a fluorescent group. The chemical reactions of the orthogonal ligation reaction include, but are not limited to, a Staudinger ligation reaction, a cycloaddition reaction of azide and alkynyl catalyzed by copper ions, a cycloaddition reaction of azide and alkynyl driven by ring tension, a binding reaction between digoxin and digoxin antibody, a Diels-Alder reaction, a Suzuki cross-coupling reaction, a disulfide bond formation reaction of sulfhydryl and sulfhydryl derivatives, a reaction of sulfhydryl and maleimide to form thioether, a photocatalytic free radical addition reaction of sulfhydryl and alkene derivatives, a photocatalytic free radical addition reaction of sulfhydryl and alkynyl derivatives, a sulfonyl fluoride exchange reaction, a binding reaction between biotin and streptavidin, and a reaction between amino and activated ester.
In the present invention, the environment-sensitive fluorescent dye is a fluorescent dye capable of rapidly changing a light emission color, a fluorescence emission wavelength or intensity in response to a change in environment, such as polarity, pH, voltage, light source, viscosity, and the like. The chemical reactions of the orthogonal ligation reaction include, but are not limited to, various known environmentally sensitive fluorescent dyes widely used in the fields of fluorescent probes, chemical sensors, detection of micro-environmental changes, biological imaging, molecular switching, and phase separation visualization, including 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-Carboxyphoflrescein, 7-Hydroxymarin-3-Carboxylic Acid, SNARFR-5F, SNARFB-4F, SNARFR-1, BCECF, cyER 5E, HCyC-647, squale-650-pH, 6-carboloy-4, 5' -2-fluoro ',7' -2' -fluoro '.
In the present invention, the fluorescent group and the detection method thereof are known and can be selected according to actual needs. The excitation light conditions and optical filters may also be selected to provide the same or substantially the same fluorescent signal from different compounds.
In the method, the fluorescent group and the environment high-sensitivity fluorescent group are matched for use to realize monochromatic fluorescent sequencing, the fluorescent group and the environment high-sensitivity fluorescent group are not marked on a base like the traditional NRT, but are marked on dNTPs 3'-OH in a covalent bond or non-covalent bond mode, the marked dNTPs can be identified by modified DNA polymerase (MEB 202T 9) and integrated into a DNA double chain to complete SBS sequencing, and the connecting fluorescent group chain can be cut to regenerate 3' -OH after each round of SBS sequencing, so that the problem that sequencing reading length is influenced due to residue accumulation on the base in the conventional second-generation SBS sequencing method can be solved. The environment high-sensitivity fluorescent group has a structure different from that of a correspondingly selected non-environment high-sensitivity fluorescent group, the environment high-sensitivity fluorescent group can emit the same fluorescent signal under a given system environment or can emit the same fluorescent signal under a proper excitation light condition and an optical filter, the environment high-sensitivity fluorescent group loses the fluorescent property and cannot emit the fluorescent signal or the same excitation light condition and the fluorescent signal under the optical filter are different from the fluorescent signal emitted by the selected non-environment high-sensitivity fluorescent group and cannot be detected under the given system environment, the system environment adjusts the pH value of the system according to the characteristics of the base derivative in the gene so as to meet the corresponding requirements, 4 bases can be distinguished through monochromatic fluorescent sequencing, and the sequencer only needs to be provided with an excitation light source and a camera, so that the manufacturing cost and the volume of the sequencer are greatly reduced, the sequencer is convenient to transport and carry, and is favorable for the sequencer to diffuse to three-city hospitals and research institutions with wider range and is used in a decentralized mode.
Example 1:
preparation of the Compound 1, a derivative of dGTP (3-N3-dGTP) which is purified by semi-preparative HPLC to give a product with a purity of greater than 97%, HRMS:561.0004, the synthetic route and compound structure of which is shown below:
preparation of Compound 2,3 'OH-Environment sensitive dye modified dTTP derivative (3' -HCyC-647-LN-dTTP) which was purified by semi-preparative HPLC to give a product with a purity of greater than 97%, MALDI-TOF:1209.10, the synthetic route and compound structure of which are shown below:
stage 1: flow
Stage 2: mixing
Preparation of Compound 3,3 'OH-dye modified dATP derivative (3' -Cy 5-LN-dATP) which was purified by semi-preparative HPLC to give a product with a purity of greater than 97%, MALDI-TOF:1213.54, the synthetic route and the structure of the compound are shown below:
preparation of Compound 4,3 'preparation of an OH-reactive group-modified dCTP derivative (3' -Biotin-LN-dCTP) which was purified by semi-preparative HPLC to give a product with a purity of more than 97%, HRMS:776.0504, the synthetic route and compound structure of which are shown below:
the sequencing method used involves the following steps
a) Fixing a DNA template to be detected on a chip, and constructing a nucleic acid double-stranded molecular cluster through bridge amplification;
b) The four dNTPs (3 ' -N3-dGTP, 3' -LN3-HCyC647-dTTP, 3' -LN3-Cy5-dATP, 3' -LN 3-Biotin-dCTP) in example 1 and polymerase MEB202T9 are added into the reaction system at the same time to carry out nucleotide polymerization reaction, one of them is incorporated into the 3' end of the growing nucleic acid chain and stops growing;
c) Washing away unreacted dNTPs, adding a scanning buffer solution, adjusting the pH value to 6.8, detecting a fluorescence signal by taking a light source with 640nm as an excitation wavelength, taking a picture, and storing an image A as shown in figure 1;
d) Washing off the scanning buffer solution, adding water-soluble Cy 5-streptavidin, wherein the treatment has no influence on G, A and T base derivatives, and the treatment and the Cy 5-streptavidin can be specifically combined with Biotin on the C base derivative, so that a fluorescent group Cy5 is introduced into the A base derivative to enable the A base derivative to emit a fluorescent signal;
e) The pH value is adjusted to be 7.5, the weak alkaline environment has no influence on G, C and A base derivatives, but HCyC-647 fluorescence on the T base derivatives can be quenched, and no fluorescence signal can be detected under a 640nm light source;
f) Adding a scanning buffer solution, detecting a fluorescent signal by taking a 640nm light source as an excitation wavelength, photographing, and storing an image B as shown in figure 2;
g) And washing away the scanning buffer solution, adding a cleavage reagent THP to process the chip, removing the azide group or the azide group chain at the 3 '-position of the dNTPs to regenerate free 3' -OH, and removing the fluorescent group or the reactive group.
h) Washing off the cleavage buffer, and then repeating steps (c) - (h);
i) After images obtained by photographing twice in the process of each cycle test step, comparing fluorescence signals of the nucleic acid double-stranded molecular cluster at the same position, if the fluorescence signals are in both the scanned image A and the scanned image B (shown by square frames in the images A and B), the incorporated nucleotide derivative is 3' -LN3-Cy5-dATPC, and correspondingly, the base at the corresponding position on the DNA template can be determined to be T; if there is no fluorescence signal in both scan A and scan B (diamonds in both A and B), then the nucleotide derivative incorporated is 3' -N3-dGTP and, correspondingly, the base at the corresponding position on the DNA template can be determined to be C; if there is a signal in Scan image A and no fluorescence signal in Scan image B (indicated by the circled boxes in images A and B), 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 to be A; if there is no signal in scan A and a fluorescent signal in scan B (indicated by the triangular boxes in images A and B), the incorporated nucleotide derivative is 3' -LN3-Biotin-dCTP, and accordingly, the base at the corresponding position on the DNA template can be determined to be T. As shown in Table 1, the results of detection and the corresponding bases in example 1,
example 2:
preparation of the Compound 1, a derivative of dGTP (3-N3-dGTP) which is purified by semi-preparative HPLC to give a product with a purity of greater than 97%, HRMS:561.0004, the synthetic route and compound structure of which is shown below:
preparation of Compound 2,3 'OH-Environment sensitive dye modified dTTP derivative (3' -HCyC-647-LN-dTTP) which was purified by semi-preparative HPLC to give a product with a purity of greater than 97%, MALDI-TOF:1209.10, the synthetic route and compound structure of which are shown below:
stage 1: mixing
Stage 2: mixing
Preparation of Compound 3,3 'OH-dye modified dATP derivative (3' -Cy 5-LN-dATP) which was purified by semi-preparative HPLC to give a product with a purity of greater than 97%, MALDI-TOF:1213.54, the synthetic route and the structure of the compound are shown below:
preparation of Compound 4,3 'OH-reactive group-modified dCTP derivative (3' -Cy 5-SS-dCTP), which was purified by semi-preparative HPLC to give a product with a purity of more than 97%, MALDI-TOF:1225.89, the synthetic route and the structure of the compound are shown as follows:
the sequencing method used involves the following steps
a) Fixing a DNA template to be detected on a chip, and constructing a nucleic acid double-stranded molecular cluster through bridge amplification;
b) The four dNTPs (3 ' -N3-dGTP, 3' -LN3-HCyC-647-dTTP, 3' -LN3-Cy5-dATP, 3' -SS-Cy 5-dCTP) in example 1 and polymerase MEB202T9 were simultaneously added to the reaction system to carry out nucleotide polymerization reaction, one of them was incorporated into the 3' end of the growing nucleic acid chain and the growth was terminated;
c) Washing away unreacted dNTPs, adding a scanning buffer solution, adjusting the pH value to 7.4, detecting a fluorescence signal by taking a 640nm light source as an excitation wavelength, photographing, and storing an image C, wherein the image C is shown in figure 3;
d) The scanning buffer was washed away and the 3'-SS-Cy5-dCTP disulfide bond cleaved to remove its fluorophore by addition of an orthogonal cleavage reagent (low concentration of TECP) which had no effect (or little effect, no effect on image recognition and signal discrimination) on 3' -N3-dGTP, 3'-LN3-HCyC-647-dTTP, 3' -LN3-Cy 5-dATP.
e) Simultaneously adjusting the pH value to 6.8, wherein the weak acidic environment has no influence on G, C and A base derivatives, but can enable a pH sensitive fluorescent group HCyC-647 on a T base to emit a signal fluorescent signal which is the same as that of CY5 or can be detected under the same filter;
f) Adding scanning buffer solution, detecting fluorescent signal with light source with excitation wavelength of 640nm, photographing, and storing image D as shown in FIG. 4;
g) And washing away the scanning buffer solution, adding a cleavage reagent THP to process the chip, removing the azide group or the azide group chain at the 3 '-position of the dNTPs to regenerate free 3' -OH, and removing the fluorescent group or the reactive group.
h) Washing off the cleavage buffer, and then repeating steps (c) - (h);
i) After images obtained by photographing twice in the process of each cycle test step, comparing fluorescence signals of the nucleic acid double-stranded molecular cluster at the same position, if the fluorescence signals are in both the scanned image A and the scanned image B (shown by square frames in the images A and B), the incorporated nucleotide derivative is 3' -LN3-Cy5-dATPC, and correspondingly, the base at the corresponding position on the DNA template can be determined to be T; if there is no fluorescence signal in both scan A and scan B (diamonds in both A and B), then the nucleotide derivative incorporated is 3' -N3-dGTP and, correspondingly, the base at the corresponding position on the DNA template can be determined to be C; if there is a signal in scan A and no fluorescent signal in scan B (indicated by the circled boxes in images A and B), then the incorporated nucleotide derivative is 3' -SS-Cy5-dCTP and, correspondingly, the base at the corresponding position on the DNA template can be determined to be G; if there is no signal in Scan image A and there is a fluorescent signal in Scan image B (indicated by the triangular boxes in images A and B), the incorporated nucleotide derivative is 3' -LN3-HCyC-647-dTTP, and accordingly, the base at the corresponding position on the DNA template can be determined to be A. As shown in Table 2, table 2 shows the results of detection and the corresponding bases in example 2
It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.
Claims (3)
1. The monochromatic fluorescence MRT gene sequencing reagent based on the environment-sensitive dye is characterized by comprising polymerase and four different labeled nucleotide analogs, wherein hydroxyl groups at the 3 'positions of nucleotides of the four nucleotide analogs are modified by protecting groups or cleavable connecting groups, the protecting groups or the cleavable connecting groups can be cleaved and separated to expose 3' -OH again, and the cleavable connecting groups are linked with environment-sensitive fluorescent groups or environment-sensitive fluorescent group markers through covalent bonds or non-covalent bonds; the four nucleotide analogs are compound 1, compound 2, compound 3, and compound 4, respectively, wherein,
compound 1 is a nucleotide derivative that emits no fluorescent signal or carries no fluorescent group;
the compound 2 is a nucleotide derivative which can emit a fluorescent signal and carries a fluorescent group;
the compound 3 is a nucleotide derivative carrying an environment high-sensitivity fluorescent group, cannot emit a fluorescent signal or can emit a fluorescent signal under one condition, the emitted fluorescent signal is different from that emitted by the compound 2, the emitted fluorescent signal can emit a fluorescent signal under the other condition and is the same as that emitted by the compound 2, and the structure of the environment high-sensitivity fluorescent group is different from that of the fluorescent group in the compound 2;
the compound 4 is a nucleotide derivative which cannot emit a fluorescent signal and carries a reactive group capable of undergoing a connection reaction, and the reactive group and the reactive fluorescent group can emit the same fluorescent signal as the compound 1 after the connection reaction; or the compound 4 is a nucleotide derivative which can emit the same fluorescence signal as the compound 2 and carries a breakable chain fluorescent group different from the compound 2, wherein the breakable chain fluorescent group can be broken and removed under a certain condition, and the condition has no influence on the compound 2;
the compound 1, the compound 2, the compound 3 and the compound 4 are compounds which have the structures of a formula (I), a formula (II) or a formula (III) independently,
wherein "- - - -" is a non-covalent bond;
b represents different bases or analogues, which refer to adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) and analogues thereof;
each R1 is independently selected from hydrogen, a monophosphate group, a diphosphate group, a triphosphate group, a tetraphosphate group, or more phosphate groups;
each R2 is independently a protecting group capable of undergoing an orthogonal cleavage reaction;
each R3 is independently selected from halogen, -H, -OH, N3 or a C2-C10 flexible chain;
l is a cleavable linking group or absent;
r4 is respectively and independently an environment-sensitive fluorescent group capable of emitting a fluorescent signal, a fluorescent group capable of emitting the same fluorescent signal and a reactive group capable of carrying out a connection reaction;
each R5 is independently an environment-sensitive fluorescent group-labeled reactable group capable of emitting a fluorescent signal or a fluorescent group-labeled reactable group capable of emitting the same fluorescent signal;
the protecting group is a 3' -OH modifying group which can be effectively recognized by polymerase, can be incorporated into a growing DNA chain and can be cut and detached after each round of SBS sequencing; the cleavable connecting group is a micromolecule group which is linked between 3' -OH and an environment high-sensitivity fluorescent group, a fluorescent group capable of emitting the same fluorescent signal or a reactive group capable of carrying out a connecting reaction; the fluorescent group capable of emitting the same fluorescent signal is a non-environment high-sensitivity fluorescent dye which can emit an emission wavelength consistent with or close to that of the selected environment high-sensitivity fluorescent dye under the same excitation light source wavelength and is detected and judged to be the same fluorescent signal, and the protecting group is a group with any one of the following structures:
the cleavable linking group is a group of any one of the following structures:
disulfide chain group
Cyanoethyl chain radical
Azido chain groups
Dialkyl ketal chain groups
Allyl chain radical
1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-yl) ethyl chain radical
Nitrobenzyl chain radical
Azo chain group
Wherein R is 1’ ,R 2’ Each independently selected from halogen, -H, C1-C5 fatty chain;
the fluorescent groups capable of emitting the same fluorescent signal are derived 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, jeo, HEX, 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 Fluor 680, CAlFluor Fluor 670, QUiFluor 700, QUiFluor Fluor 670;
the environment high-sensitivity fluorescent group is selected from environment high-sensitivity fluorescent dyes, the environment high-sensitivity fluorescent dyes are pH value sensitive fluorescent dyes, the pH value sensitive fluorescent dyes are compounds with any one of the following structural formulas,
wherein: each X is independently selected from halogen, -H, -OH, N3, NH2, S, si or a C2-C10 flexible chain;
r6 is independently selected from hydrogen, polyethylene glycol carboxyl, polyethylene glycol alkynyl, polyethylene glycol azide, polyethylene glycol amino, polyethylene glycol maleimide, polyethylene glycol sulfydryl, 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 sulfydryl, C1-C10 alkyl chain sulfonic group and C1-C10 alkyl chain maleimide;
r7 is independently selected from hydrogen, C1-C6 saturated alkyl;
ring A and ring B are each independently selected from heterocyclic groups having the formula (VI), (VII), (VIII), (IX),
wherein; each Y is 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 alkylamino, polyethylene glycol maleimide, polyethylene glycol sulfhydryl, 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 sulfhydryl, C1-C10 alkyl chain sulfo, C1-C10 alkyl chain maleimide;
r10 and R11 are each independently selected from hydrogen, carboxyl, phosphate, sulfonate;
the reactable group is a group which can be recognized by polymerase, can be incorporated into a growing DNA chain, can perform orthogonal connection reaction with a corresponding complementary group through covalent bonds or non-covalent bonds to stabilize a splicing structure, and can be cut off to regenerate 3' -0H after each round of SBS sequencing; the reactable fluorescent group is a group which carries an environment sensitive fluorescent group or can emit the same fluorescent signal and carries out orthogonal connection reaction with the reactable group through covalent bond or non-covalent bond production to stabilize a splicing structure, and the reactable group is any one of the following structures,
amino group
Cycloalkynyl radical
Azide radical
Maleimide
Biotin
Phenylboronic acids
Digoxin; phenol and its preparation
Cycloalkenyl radicals
The complementary group is any one of the following structures:
tetrafluorophenyl ester
Cycloalkynyl radical
Streptavidin, 4-phenylurazole
Azide radical
Salicyl isohydroxy fatty acid radical, digoxin antibody, sulfhydryl group
Tetrazinyl radical
。
2. The monochromatic fluorescent MRT gene sequencing reagent based on environmental sensitive dye according to claim 1, characterized in that the base or analog is a compound of any one of the following structures,
3. a method for sequencing a monochromatic fluorescent MRT gene based on an environment-sensitive dye, characterized in that a sequencing reagent according to any one of claims 1 to 2 is used, and the testing steps are as follows,
s1, preparing a compound 1, a compound 2, a compound 3 and a compound 4 respectively;
s2, connecting the nucleic acid template chain to be sequenced to a chip or a microsphere to form a nucleic acid double-stranded molecular cluster reaction system;
s3, simultaneously adding the compound 1, the compound 2, the compound 3, the compound 4 and polymerase into a nucleic acid double-stranded molecular cluster reaction system for nucleotide polymerization reaction, so that any one of the compounds is incorporated into the 3' end of a growing nucleic acid chain and stops growing continuously;
s4, washing away unreacted compound 1, compound 2, compound 3 and compound 4, adding a buffer solution, adjusting the pH value, detecting the fluorescent label of each incorporated nucleotide derivative, photographing and storing an image to identify the nucleotide derivative incorporated at the 3' end of the nucleic acid chain;
wherein: 4a, if compound 2 and compound 3 are capable of emitting a fluorescent signal, removing the solution phase from the reaction system of the previous step, leaving the duplex attached to the support, and detecting whether the duplex or the growing nucleic acid strand emits the fluorescent signal; then, performing treatment, wherein the treatment has no influence on the compound 1 and the compound 2, but enables the compound 3 to emit no fluorescence signal or emit a fluorescence signal different from that of the compound 2, simultaneously enabling the compound 4 to perform a ligation reaction to emit a fluorescence signal identical to that of the compound 2, finally adjusting the pH value, and detecting whether the duplex or the growing nucleic acid chain emits the fluorescence signal again;
4b, if compound 2 and compound 4 are capable of emitting a fluorescent signal, removing the solution phase from the reaction system of the previous step, leaving the duplex attached to the support, and detecting whether the duplex or the growing nucleic acid strand emits the fluorescent signal; then, performing treatment which has no influence on the compound 1 and the compound 2 but can break the cleavable-chain fluorescent group of the compound 4 to remove the fluorescent group, simultaneously enabling the compound 3 to emit the same fluorescent signal as the compound 2, and detecting whether the duplex or the growing nucleic acid chain emits the fluorescent signal again;
s5, carrying out image processing and analysis on the fluorescence signal, and judging the nucleotide derivative incorporated into the nucleic acid chain according to the following judgment principle:
5a, if in step S3 Compound 1 is incorporated at the 3' end of the growing nucleic acid strand, then since Compound 4 does not itself carry a fluorescent group or other reactive group nor is it affected by the treatment described in step S4, then in step S3
No fluorescent signal will be detected in S4;
5b, if in step S3 compound 2 is incorporated at the 3' end of the growing nucleic acid strand, then in step S4 a fluorescence signal will both be detected, since compound 2 carries a fluorophore and is not affected by the processing described in steps 4a and 4b in step S4;
5c, if in step S3, compound 3 is incorporated into the 3' end of the growing nucleic acid strand, since compound 3 itself carries an environmentally sensitive fluorophore, 4a can detect a fluorescent signal in step S4, and after processing and adjustment, no fluorescent signal is emitted, or a fluorescent signal different from compound 2 is emitted, and no fluorescent signal is detected in step S4 given the filter; 4b can not detect a fluorescence signal in step S4, can emit the same fluorescence as the compound 2 after being processed and adjusted, and can detect a fluorescence signal in step S4; 5d, if in step S3 compound 4 is incorporated at the 3' end of the growing nucleic acid strand, if compound 4 itself does not carry a fluorophore but a reactive group, no fluorescence signal is detected in step S4, the corresponding complementary group carrying the same fluorophore as compound 2 is added while the treatment is adjusted, and after the treatment, the same fluorescence as compound 2 is emitted, and a fluorescence signal is detected in step S4; if the compound 4 itself carries a fluorophore with the same fluorophore linking group as the compound 2, a fluorescent signal can be detected in step S4, a specific cleavage reagent is added while the treatment is adjusted, and after the treatment, the linking group is cleaved and the fluorophore is removed, so that the fluorescent signal is lost, and the fluorescent signal cannot be detected in step S4.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111203792.9A CN114250283B (en) | 2021-10-15 | 2021-10-15 | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye |
| PCT/CN2022/086205 WO2023060858A1 (en) | 2021-10-15 | 2022-04-12 | Monochromatic fluorescence mrt gene sequencing reagent and method based on environment-sensitive dye |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111203792.9A CN114250283B (en) | 2021-10-15 | 2021-10-15 | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114250283A CN114250283A (en) | 2022-03-29 |
| CN114250283B true CN114250283B (en) | 2022-10-04 |
Family
ID=80790394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111203792.9A Active CN114250283B (en) | 2021-10-15 | 2021-10-15 | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN114250283B (en) |
| WO (1) | WO2023060858A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114250283B (en) * | 2021-10-15 | 2022-10-04 | 深圳铭毅智造科技有限公司 | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye |
| CN114574571A (en) * | 2022-05-05 | 2022-06-03 | 深圳铭毅智造科技有限公司 | Nucleotide derivative and gene sequencing method |
| CN115232867B (en) * | 2022-07-21 | 2024-01-30 | 深圳赛陆医疗科技有限公司 | Rapid second-generation gene sequencing method |
| CN116854749B (en) * | 2023-09-01 | 2023-11-21 | 深圳赛陆医疗科技有限公司 | Method for synthesizing nucleotide intermediate |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6951947B2 (en) * | 2000-07-13 | 2005-10-04 | The Scripps Research Institute | Labeled peptides, proteins and antibodies and processes and intermediates useful for their preparation |
| RU2009121089A (en) * | 2006-12-05 | 2011-01-20 | Лазержен, Инкорпорэйтед (Сша) (Us) | PHOTOGRAPHABLE TAGGED NUCLEOTIDES AND NUCLEOSIDES AND TAGGED NUCLEOTIDES AND NUCLEOSIDES, AND WAYS OF THEIR USE IN DNA SEQUENCING |
| CN101575639B (en) * | 2009-06-19 | 2012-04-18 | 无锡艾吉因生物信息技术有限公司 | DNA sequencing method capable of verifying base information for second time |
| KR101107315B1 (en) * | 2009-09-29 | 2012-01-20 | 한국과학기술연구원 | A DNA sequencing method using novel nucleoside triphosphates with a fluorescent 3'-O-blocking group as reversible terminators |
| US8603741B2 (en) * | 2010-02-18 | 2013-12-10 | Pacific Biosciences Of California, Inc. | Single molecule sequencing with two distinct chemistry steps |
| JP5781589B2 (en) * | 2013-12-20 | 2015-09-24 | レーザーゲン インコーポレイテッド | Photocleavable labeled nucleotides and nucleosides and labeled nucleotides and nucleosides and their use in DNA sequencing |
| CN106281306B (en) * | 2015-06-13 | 2019-08-09 | 复旦大学 | The near-infrared fluorescent group of reversible instruction physiological phenomenon environment |
| US11384391B2 (en) * | 2016-04-04 | 2022-07-12 | The Trustees Of Columbia University In The City Of New York | Flourescene energy transfer-based single molecule/ensemble DNA sequencing by synthesis |
| CN112218640A (en) * | 2018-03-15 | 2021-01-12 | 哥伦比亚大学董事会 | Nucleotide analogs and their use in nucleic acid sequencing and analysis |
| CN112601825B (en) * | 2018-07-09 | 2023-11-28 | 青岛华大智造科技有限责任公司 | Nucleic acid sequencing method |
| CN110818757A (en) * | 2018-08-07 | 2020-02-21 | 深圳华大生命科学研究院 | Nucleotide analogs and method for screening DNA polymerase |
| CN114981283A (en) * | 2019-07-09 | 2022-08-30 | 纽约市哥伦比亚大学信托人 | Novel nucleotide analogs and methods of use |
| CN114250283B (en) * | 2021-10-15 | 2022-10-04 | 深圳铭毅智造科技有限公司 | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye |
-
2021
- 2021-10-15 CN CN202111203792.9A patent/CN114250283B/en active Active
-
2022
- 2022-04-12 WO PCT/CN2022/086205 patent/WO2023060858A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| WO2023060858A1 (en) | 2023-04-20 |
| CN114250283A (en) | 2022-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114250283B (en) | Monochromatic fluorescent MRT gene sequencing reagent and method based on environment-sensitive dye | |
| US11827932B2 (en) | Methods and compositions for nucleic acid sequencing | |
| US20080032307A1 (en) | Use of Single-Stranded Nucleic Acid Binding Proteins In Sequencing | |
| TWI622580B (en) | Polymethine compounds and their use as fluorescent labels | |
| US8993784B2 (en) | Polymethine compounds and their use as fluorescent labels | |
| US20050239085A1 (en) | Methods for nucleic acid sequence determination | |
| CN115323045A (en) | Gene sequencing reagent and gene sequencing method | |
| CN114250282B (en) | Gene sequencing reagent and method based on pH value sensitive dye | |
| US20240141428A1 (en) | Methods and compositions for nucleic acid sequencing | |
| US20230304086A1 (en) | Labeled avidin and methods for sequencing | |
| CN115803457A (en) | Methods, systems, and compositions for nucleic acid sequencing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |