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WO2022105640A1 - Procédé de séquençage d'acide nucléique - Google Patents

Procédé de séquençage d'acide nucléique Download PDF

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WO2022105640A1
WO2022105640A1 PCT/CN2021/129461 CN2021129461W WO2022105640A1 WO 2022105640 A1 WO2022105640 A1 WO 2022105640A1 CN 2021129461 W CN2021129461 W CN 2021129461W WO 2022105640 A1 WO2022105640 A1 WO 2022105640A1
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polymerase
nucleic acid
alkyl
dna
cysteines
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Chinese (zh)
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白净卫
杜娟娟
徐扬
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Tsinghua University
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Tsinghua University
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the invention belongs to the field of biotechnology, and relates to a method for determining a nucleic acid sequence.
  • DNA deoxyribonucleic acid is the genetic material that composes an organism. It is composed of A, T, C, and G in different sequences. These genetic materials composed of A, T, C, and G in different sequences contain rich genetic information. and the meaning of life. Nucleic acid sequences of different genetic materials can be obtained through nucleic acid sequencing technology to help people understand the differences between individual genetic materials, which is of great significance to life science research, disease diagnosis, and personalized medicine.
  • the first generation sequencing technology is represented by the dideoxy chain end termination method invented by Sanger. This method has disadvantages such as high cost, slow speed and low throughput, which cannot meet the needs of research and application.
  • the second-generation sequencing technology Illumina's Solexa sequencing technology has relatively low cost, so it is widely used, but this technology takes 6 days from the beginning of library construction to the completion of sequencing, which is time-consuming.
  • SMRT of Pacific Bioscience is the only commercially applied technology. SMRT technology realizes single-molecule sequencing and has the advantages of no amplification and long read length. Although this technology is compared with Solexa sequencing technology from library construction The time to complete sequencing was shorter, but it still took about two days to complete.
  • Patent US5302509 describes a method of sequencing a polynucleotide template comprising a multiple extension reaction using DNA polymerase or DNA ligase to successively incorporate labeled nucleotides or polynucleotides complementary to the template strand.
  • a new nucleotide base-paired to the template strand is constructed in a 5' to 3' orientation by successively incorporating single nucleotides complementary to the template strand chain.
  • the nucleoside triphosphate substrate used in the sequencing reaction is blocked to prevent overincorporation, and the nucleoside triphosphate substrate is differentially labeled to enable determination of the species of incorporated nucleotide that is added as a subsequent nucleotide.
  • Patent CN106244712A discloses a DNA sequencing method, including the following steps: (1) adding a tag sequence at the 3' end of the DNA to be tested to form the DNA to be tested containing the tag sequence; the nucleotide sequence of the tag sequence and the sequencing primer (2) mixing the DNA to be tested containing the tag sequence and the sequencing primer to form a product in the form of a double-stranded main body at the 5' end; (3) completing step (2) Then, the products were mixed with dATP, dCTP, dTTP and dGTP respectively to obtain four systems, and each system was added to a specific DNA polymerase-modified single-molecule device to read the electrical signal.
  • the synthase chain extension reaction (Sequencing by synthesis), the core of Illumina's sequencing technology, is a three-substrate reaction, mainly including template-primer hybrid, dNTP, and DNA synthase, and the reaction kinetics is a secondary reaction; the present invention
  • the provided method uses dNTP-DNA synthase and reversible ligation complexes as reaction substrates, reacts with template-primer hybrids, and the reaction kinetics are first-order reactions, so it is faster than Illumina's SBS technology.
  • Illumina's SBS fluorescent molecule is connected to the base of dNTP, and there is usually only one fluorescent molecule, so the optical signal intensity is limited; the method provided by the present invention connects fluorescent molecules on the synthase, which can provide more connection sites and connect More than one fluorescent group can therefore increase the optical signal intensity.
  • WO2007076057A2 provides compositions comprising polymerases having features of improved entry of nucleotide analogs into the active site region and coordination of nucleotide analogs in the active site region. Also provided are methods of making such polymerases and using such polymerases for sequencing and DNA replication and amplification, as well as kinetic models of polymerase activity and computer-implemented methods using such models.
  • TdT terminal deoxynucleotidyl transferase
  • the system has a repulsive effect on other TdT-dNTPs, severing the connection between TdT and dNTPs to release the primer and allow subsequent extension, thus reversibly terminating the extension of a dNTP at the 3' end of single-stranded DNA.
  • this method only uses the extension reaction of single-stranded nucleic acid, that is, DNA synthesis, and does not involve identifying the sequence of template DNA, nor does it involve connecting fluorescent molecules or other luminescent groups to nucleic acid synthase.
  • the present invention provides a method capable of rapidly determining a nucleic acid sequence.
  • a first aspect of the present invention provides a method for determining a nucleic acid sequence, the sequencing method comprising the following steps:
  • the polymerase is connected to the dNTP through a cleavable group (linker), and the polymerase can emit a light signal;
  • the attached polymerase prevents the reaction of another dNTP-polymerase with the 3' end of the newly formed primer, resulting in chain termination.
  • the nucleic acid to be detected in the present invention can be DNA or RNA, and preferably the nucleic acid sequence to be detected is DNA, such as genomic DNA, cDNA and DNA fragments.
  • the polymerase can be a currently known DNA polymerase and its variants, for example, the polymerase is selected from Bst DNA Pol and its variants, DNA Pol I and its variants, DNA Pol ⁇ and its variants. , T3 DNA Pol and its variants, T5 DNA Pol and its variants, L5 DNA Pol and its variants, DNA Pol II and its variants, DNA Pol B and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol III and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol ⁇ and its variants, DNA Pol One or more of IV and its variants, DNA Pol V and its variants, terminal transferase and its variants; wherein, the terminal transferase is terminal deoxynucleotidyl transfera
  • the polymerase is DNA Pol ⁇ and a variant thereof, and the sequence of the DNA Pol ⁇ is SEQ ID NO: 1.
  • the DNA Pol ⁇ variant is a mutation of the DNA Pol ⁇ at one or more (for example, 2, 3, 4 or 5) amino acid sequence positions, the mutation comprising a substitution, deletion and/or insertion, or At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 with DNA Pol ⁇ coding sequence % or more sequence identity, and the DNA Pol ⁇ variant has polymerase activity.
  • the mutation site of the DNA Pol ⁇ variant is in the region near the catalytic center of DNA synthesis, and is not the amino acid residue position necessary for the catalytic reaction, which can ensure that the catalytic activity is not affected, and that the dNTP is away from the catalytic center. More recently, it does not require a particularly long cleavable group to contact the catalytic center to participate in the reaction.
  • the variant of the DNA Pol ⁇ is to mutate any one, two or three cysteines at positions 145-355 of SEQ ID NO: 1 to non-cysteines, preferably, the SEQ ID NO: 1 mutates any one, two or three cysteines at positions 145-355 to serines, and mutates any one, two or three non-cysteines at positions 1-236 to cysteine.
  • the variant of DNA Pol ⁇ is to mutate any one, two or three cysteines at positions 155-294 of SEQ ID NO: 1 to serines, and mutate any of positions 20-154 into serines.
  • One, two or three non-cysteines are mutated to cysteines.
  • the variant of the DNA Pol ⁇ is to mutate the cysteine at the 178th position, the 239th position and/or the 267th position of SEQ ID NO: 1 to serine, and mutate the 28th position, the 28th position, the 267th position Any one, two or three non-cysteines at positions 33 and/or 149 are mutated to cysteine.
  • the polymerase is a variant of DNA Pol ⁇ , and the variant of DNA Pol ⁇ is selected from one or a combination of two or more of the following variants:
  • the amino acid sequence of the polymerase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NOs: 3-6 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the DNA Pol ⁇ variant has polymerase activity.
  • amino acid sequence of the polymerase is shown in any of SEQ ID NOs: 3-6.
  • the polymerase is Bst DNA Pol and a variant thereof, and the sequence of the Bst DNA Pol is SEQ ID NO: 2.
  • the Bst DNA Pol variant is a mutation of the Bst DNA Pol at one or more (for example, 2, 3, 4 or 5) amino acid sequence positions, the mutation comprising a substitution, deletion and/or insertion , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the Bst DNA Pol coding sequence %, more than 99.5% sequence identity, and the Bst DNA Pol variant has polymerase activity.
  • the mutation site of the Bst DNA Pol variant is in the region near the catalytic center of DNA synthesis, and is not the amino acid residue position necessary for the catalytic reaction, which can ensure that the catalytic activity is not affected, and that the dNTP is separated from the catalytic reaction.
  • the center is relatively close, and it does not require a particularly long cleavable group to contact the catalytic center to participate in the reaction.
  • the variant of the Bst DNA Pol is to mutate any one, two or three cysteines at positions 85-100 or 540-570 of SEQ ID NO: 2 to non-cysteines,
  • any one, two or three cysteines at positions 85-100 or 540-570 of SEQ ID NO: 2 are mutated to serines, and any one of positions 250-280 or 320-380 is mutated , two or three non-cysteines are mutated to cysteines.
  • the variant of the Bst DNA Pol is to mutate any one, two or three cysteines at positions 90-95 or 549-555 of SEQ ID NO: 2 to serine, and mutate the 260- Any one, two or three non-cysteines at positions 270 or 330-370 were mutated to cysteine.
  • the variant of the Bst DNA Pol is to mutate the cysteine at the 93rd position and/or the 550th position of SEQ ID NO:2 to serine, and the 264th position, the 334th position and/ Or any one, two or three non-cysteines at position 364 are mutated to cysteine.
  • the polymerase is a variant of Bst DNA Pol, and the variant of Bst DNA Pol is selected from one or a combination of two or more of the following variants:
  • the amino acid sequence of the polymerase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NOs: 7-10 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the DNA Pol ⁇ variant has polymerase activity.
  • amino acid sequence of the polymerase is shown in any of SEQ ID NOs: 7-10.
  • the dNTP linked to the polymerase by the cleavable group is selected from any one of dATP or modified dATP, dGTP or modified dGTP, dCTP or modified dCTP, dTTP or modified dTTP, dUTP or modified dUTP .
  • the cleavable group is used to link polymerase and modified dNTP, or link polymerase and dNTP.
  • the cleavable group contains a cleavable structure X, and the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • the cleavable group has the structure of L1-L2-X-L3-L4, wherein L1 is an end group bound to dNTP or modified dNTP, L2 and L3 do not exist or are non-cleavable linking groups, L4 is the terminal group that binds to the polymerase, and X is the cleavable group.
  • the modified dNTP is a base-modified dNTP
  • the base-modified dNTP is a base-modified dNTP containing an N atom. More preferably, the base-modified dNTP is an amino-containing base.
  • Modified dNTPs are those whose bases are Modified dNTP
  • the base-modified dNTPs of the present invention can be either 3'-modified dNTPs or 3'-unmodified dNTPs, and the 3'-modified dNTPs can block the extension of nucleic acid chains.
  • the base-modified dNTP is selected from:
  • the linking site is selected from the following formula A, formula B, formula C or formula D.
  • the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • said L1 or L4 is independently selected from maleimide group, carboxyl group, mercapto group, azido group, alkynyl group, cyclooctynyl group and its derivatives, tetrazinyl group, dithiopyridyl group, vinyl group , alkenyl sulfone group, succinimidyl group, aldehyde group, hydrazide group, amineoxy group and ⁇ -halogenated carbonyl group.
  • said L2 or L3 is independently selected from -O(CH 2 CH 2 O) i -, -(CH 2 ) i -, -(CH 2 ) i NH(CH 2 ) i -, -(CH 2 ) i COO(CH 2 ) i -, -(CH 2 ) i CONH(CH 2 ) i -, -(CH 2 ) i O(CH 2 ) i -, -(CH 2 ) i CO(CH 2 ) i -or
  • the i is selected from an integer of 0-10.
  • the cleavable group is selected from:
  • the cleavable group linking the polymerase to the dNTP can be cleaved under the action of a method commonly used in the prior art, for example, the group is cleaved after being irradiated by ultraviolet light or by the action of a chemical substance, thereby separating the polymerase from the dNTP. .
  • the polymerase of the present invention can be a luminescent polymerase, for example, the polymerase is mutated into a luminescent polymerase, or a traditional polymerase is fused with a luminescent protein to form a fusion protein to become a luminescent polymerase.
  • the polymerase of the present invention can also be a modified polymerase containing one or more fluorescent markers, phosphorescent markers or chemiluminescent markers, the modification does not affect the activity of the polymerase, the fluorescent markers, Phosphorescent labels or chemiluminescent labels emit a light signal upon exposure to light.
  • the fluorescent markers include fluorescein, Cy2, Cy3, Cy5, Cy7, Alexa Fluor series dyes, fluorescein isothiocyanate, 5-hexachlorofluorescein phosphoramidate, 6-carboxy-2',4,7 ,7'-Tetrachlorofluorescein succinimide ester, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide ester, Texas Red , rhodamine 110, fluorescein maleimide dye, fluoroborodipyrrole, xanthene, carbocyanine, 1,1'-bis(octadecyl)-3,3,3',3'-tetra Methyl indocarbocyanine perchlorate, 3,3'-bis(octadecyl)-oxacarbocyanine perchlorate, pyrene, phthalocyanine, 6-carboxyrhodamine 6G
  • Said phosphorescent label includes transition metal iridium (Ir) or ruthenium (Ru) aza-heteroaryl complexes, preferably as shown in the following formula:
  • the polymerase provided in the step (1) connected to dNTP through a cleavable group is to provide four types of polymerases connected with different dNTPs, and the four types of polymerases connected with different dNTPs
  • the enzymes are linked with different fluorescent, phosphorescent or chemiluminescent labels.
  • the nucleic acid to be detected is immobilized on the support.
  • the supports can be obtained commercially, such as supports prepared from glass, ceramic, silica and silicon materials, and supports having a gold surface can also be used.
  • the support typically comprises a flat surface (plane), or at least a structure in which the polynucleic acids to be linked are substantially in the same plane.
  • the solid support may be non-planar, such as microbeads. Any suitable size can be used.
  • the support may be on the order of 1 to 10 cm in each direction.
  • the amplification reaction can be carried out substantially as described in WO98/44151. Briefly, after primer attachment, the solid support is brought into contact with the template to be amplified under conditions that allow hybridization of the template and the immobilized primer.
  • the template is typically added to the free solution under suitable hybridization conditions, as will be apparent to those skilled in the art. Typical hybridization conditions are eg 40°C, 5xSSC after an initial denaturation step.
  • Solid phase amplification can then be performed, the first step of which is a primer extension step in which nucleotides are added to the 3' end of the immobilized primer hybridized to the template to generate a fully extended complementary strand.
  • the complementary strand contains at its 3' end a sequence capable of binding to the second primer molecule immobilized on the solid support. Further rounds of amplification result in the formation of clusters or clusters of template molecules bound to the solid support.
  • the step (2) further includes the step of adding primers to the nucleic acid to be detected.
  • the polymerase is excited by light to emit a light signal.
  • the light signal emitted on the polymerase is detected by a detection system commonly used in the prior art.
  • the polymerase can be detected by CCD or other suitable detection methods.
  • the light signal emitted on it can be used to measure the fluorescent signal. Either by adding enzymatic chemiluminescence, direct chemiluminescence substrates and or catalytic reagents, or by applying voltage, electrocatalytic chemiluminescence is performed, and the luminescent signal is detected.
  • the nucleic acid sequencing method of the present invention further comprises step (4) of cleaving between the polymerase and the dNTP.
  • the cleavage between the polymerase and the dNTP is to break the cleavable group connecting the polymerase and the dNTP, and the cleavage of the cleavable group is performed by a method commonly used in the prior art. This is accomplished, for example, by cleavage of the group linking the polymerase and the dNTP by UV irradiation or by chemical substances, such as the action of reducing agents, pH changes.
  • the ultraviolet rays can be ultraviolet rays of 365nm-410nm;
  • the reducing agent can be DTT, TCEP, THPP, and the reagents for changing the solution include organic acids, inorganic acids, organic bases, inorganic alkali.
  • the step (4) of the present invention also includes removing the polymerase with an eluent.
  • the nucleic acid sequencing method provided by the present invention further comprises step (5), that is, repeating steps (1)-(4).
  • the nucleic acid sequencing method provided by the present invention further includes nucleic acid sample pretreatment, and the nucleic acid sample pretreatment includes nucleic acid sample library construction and amplification.
  • the nucleic acid sample library construction includes adding adapters at both ends of the nucleic acid to obtain the nucleic acid to be tested.
  • the nucleic acid sample library construction includes adding a linker at both ends of the nucleic acid after fragmenting the nucleic acid sample to obtain the nucleic acid to be tested.
  • Linkers are typically short oligonucleotides that can be synthesized by conventional methods. Adapters can be attached to the 5' and 3' ends of target nucleic acid fragments in a variety of ways. Preferably, two different linker sequences are attached to the nucleic acid to be amplified, such that one linker is attached to one end of the nucleic acid molecule and the other linker molecule is attached to the other end of the nucleic acid molecule.
  • Adapters contain sequences that allow amplification of nucleic acid using amplification primer molecules immobilized on a solid support.
  • one single strand of the template construct must contain a sequence complementary to the sequence in the forward amplification primer (so that the forward primer molecule can bind and initiate complementary strand synthesis) and a reverse The sequence corresponding to the sequence in the amplification primer molecule (so that the reverse primer molecule can bind to the complementary strand).
  • the sequences in the linker that allow hybridization to the primer molecule are typically about 20-30 nucleotides in length, although the invention is not limited to sequences of this length.
  • sequences in the amplification primers and the corresponding sequences in the adapters are generally not critical to the present invention, so long as the primer molecules can interact with the amplification sequences to direct bridge amplification.
  • the principles of primer design are generally familiar to those skilled in the art.
  • the amplification is the amplification of the nucleic acid to be detected by performing bridge PCR or rolling circle amplification technology with primers immobilized on the carrier.
  • a second aspect of the present invention provides a polymerase, the polymerase is a variant of DNA Pol ⁇ , and the variant of DNA Pol ⁇ is a combination of any one or two of positions 145-355 of SEQ ID NO: 1 Or three cysteines are mutated to non-cysteines, preferably, any one, two or three cysteines at positions 145-355 of SEQ ID NO: 1 are mutated to serine, and the first Any one, two or three non-cysteines at positions 1-236 were mutated to cysteine.
  • the variant of DNA Pol ⁇ is to mutate any one, two or three cysteines at positions 155-294 of SEQ ID NO: 1 to serines, and mutate any one of positions 20-154 of SEQ ID NO: 1 , two or three non-cysteines are mutated to cysteines. More preferably, the variant of the DNA Pol ⁇ is to mutate the cysteine at the 178th position, the 239th position and/or the 267th position of SEQ ID NO: 1 to serine, and mutate the 28th position, the 28th position, the 267th position Any one, two or three non-cysteines at positions 33 and/or 149 are mutated to cysteine.
  • the polymerase is a variant of DNA Pol ⁇ , and the variant of DNA Pol ⁇ is selected from one or a combination of two or more of the following variants:
  • the amino acid sequence of the polymerase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NOs: 3-6 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the DNA Pol ⁇ variant has polymerase activity.
  • amino acid sequence of the polymerase is shown in any of SEQ ID NOs: 3-6.
  • the third aspect of the present invention provides a polymerase, the polymerase is Bst DNA Pol and a variant thereof, and the sequence of the Bst DNA Pol is SEQ ID NO: 2.
  • the Bst DNA Pol variant is a mutation of the Bst DNA Pol at one or more (for example, 2, 3, 4 or 5) amino acid sequence positions, the mutation comprising a substitution, deletion and/or insertion , or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the Bst DNA Pol coding sequence %, more than 99.5% sequence identity, and the Bst DNA Pol variant has polymerase activity.
  • the mutation site of the Bst DNA Pol variant is in the region near the catalytic center of DNA synthesis, and is not the amino acid residue position necessary for the catalytic reaction, which can ensure that the catalytic activity is not affected, and that the dNTP is separated from the catalytic reaction.
  • the center is relatively close, and it does not require a particularly long cleavable group to contact the catalytic center to participate in the reaction.
  • the variant of the Bst DNA Pol is to mutate any one, two or three cysteines at positions 85-100 or 540-570 of SEQ ID NO: 2 to non-cysteines,
  • any one, two or three cysteines at positions 85-100 or 540-570 of SEQ ID NO: 2 are mutated to serines, and any one of positions 250-280 or 320-380 is mutated , two or three non-cysteines are mutated to cysteines.
  • the variant of the Bst DNA Pol is to mutate any one, two or three cysteines at positions 90-95 or 549-555 of SEQ ID NO: 2 to serine, and mutate the 260- Any one, two or three non-cysteines at positions 270 or 330-370 were mutated to cysteine. More preferably, the variant of the Bst DNA Pol is to mutate the cysteine at the 93rd position and/or the 550th position of SEQ ID NO: 2 to serine, and mutate the 264th position, the 334th position and the /or any one, two or three non-cysteines at position 364 are mutated to cysteine.
  • the polymerase is a variant of Bst DNA Pol, and the variant of Bst DNA Pol is selected from one or a combination of two or more of the following variants:
  • the amino acid sequence of the polymerase has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% with SEQ ID NOs: 7-10 %, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the DNA Pol ⁇ variant has polymerase activity.
  • amino acid sequence of the polymerase is shown in any of SEQ ID NOs: 7-10.
  • the fourth aspect of the present invention also provides a polymerase complex prepared by linking the polymerase and dNTP through a cleavable group.
  • the cleavable group is used to connect the polymerase and the modified dNTP, or, the polymerase and the dNTP.
  • the cleavable group contains a cleavable structure X, and the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • the cleavable group has a structure of L1-L2-X-L3-L4, wherein L1 is an end group bound to dNTP or modified dNTP, and L2 and L3 do not exist or are non-cleavable linking groups. , L4 is the end group bound to the polymerase, and X is the cleavable group.
  • the modified dNTP is a base-modified dNTP
  • the base-modified dNTP is a base-modified dNTP containing an N atom. More preferably, the base-modified dNTP is an amino-containing base.
  • Modified dNTPs are those whose bases are Modified dNTP
  • the base-modified dNTPs of the present invention can be either 3'-modified dNTPs or 3'-unmodified dNTPs, and the 3'-modified dNTPs can block the extension of nucleic acid chains.
  • the base-modified dNTP is selected from:
  • the linking site is selected from the following formula A, formula B, formula C or formula D.
  • the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • said L1 or L4 is independently selected from maleimide group, carboxyl group, mercapto group, azido group, alkynyl group, cyclooctynyl group and its derivatives, tetrazinyl group, dithiopyridyl group, vinyl group , alkenyl sulfone group, succinimidyl group, aldehyde group, hydrazide group, amineoxy group and ⁇ -halogenated carbonyl group.
  • said L2 or L3 is independently selected from -O(CH 2 CH 2 O) i -, -(CH 2 ) i -, -(CH 2 ) i NH(CH 2 ) i -, -(CH 2 ) i COO(CH 2 ) i -, -(CH 2 ) i CONH(CH 2 ) i -, -(CH 2 ) i O(CH 2 ) i -, -(CH 2 ) i CO(CH 2 ) i -or
  • the i is selected from an integer of 0-10.
  • the cleavable group is selected from:
  • the cleavable group linking the polymerase and the dNTP can be cleaved under the action of methods commonly used in the prior art, for example, the group is cleaved after being irradiated by ultraviolet light or by the action of a chemical substance, so that the polymerase is separated from the dNTP. .
  • the polymerase complexes of the present invention may also include fluorescent labels, phosphorescent labels or chemiluminescent labels.
  • the fluorescent labels include fluorescein, Cy2, Cy3, Cy5, Cy7, Alexa Fluor series dyes, fluorescein isothiocyanate, 5-hexachlorofluorescein phosphoramidate, 6-carboxyl -2',4,7,7'-Tetrachlorofluorescein succinimide ester, 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide Ester, Texas Red, Rhodamine 110, Fluorescein Maleimide Dye, Fluoroborodipyrrole, Xanthene, Carbocyanine, 1,1'-Di(octadecyl)-3,3, 3',3'-Tetramethylindocarbocyanine perchlorate, 3,3'-bis(octadecyl)-oxacarbocyanine perchlorate, pyrene, phthalocyanine, 6-carboxy Rhodamine 6G, Fluor
  • Said phosphorescent label includes transition metal iridium (Ir) or ruthenium (Ru) aza-heteroaryl complexes, preferably as shown in the following formula:
  • a fifth aspect of the present invention provides a nucleic acid synthesis method, the nucleic acid synthesis method comprising:
  • the polymerase is connected to the dNTP through a cleavable group
  • the dNTP on the polymerase is complementary to the bases on the sequence of the nucleic acid to be tested and enzymatically catalyzed by the polymerase
  • the reaction was added to the 3' end of the primer.
  • the attached polymerase prevents the reaction of another dNTP-polymerase with the 3' end of the newly formed primer, resulting in chain termination.
  • the nucleic acid synthesis method further comprises step (3) cleavage between the polymerase and dNTP; and (4) repeating steps (1)-(3).
  • the cleavable group is used for linking polymerase and modified dNTP, or linking polymerase and dNTP.
  • the cleavable group contains a cleavable structure X, and the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • the cleavable group has the structure of L1-L2-X-L3-L4, wherein L1 is an end group bound to dNTP or modified dNTP, L2 and L3 do not exist or are non-cleavable linking groups, L4 is the terminal group that binds to the polymerase, and X is the cleavable group.
  • the modified dNTP is a base-modified dNTP
  • the base-modified dNTP is a base-modified dNTP containing an N atom. More preferably, the base-modified dNTP is an amino-containing base.
  • Modified dNTPs are those whose bases are Modified dNTP
  • the base-modified dNTPs of the present invention can be either 3'-modified dNTPs or 3'-unmodified dNTPs, and the 3'-modified dNTPs can block the extension of nucleic acid chains.
  • the base-modified dNTP is selected from:
  • the linking site is selected from the following formula A, formula B, formula C or formula D.
  • the X is selected from:
  • j is an integer from 1 to 3
  • R is selected from H, C 1-10 straight or branched chain alkyl, C 2-10 alkenyl , C 2-10 alkynyl , C 3-10 cycloalkyl, aryl, heterocyclic containing N, O or S Cycloaryl, heterocycloalkyl containing N, O or S, -N(C 0-10 alkyl)(C 0-10 alkyl), -CON(C 0-10 alkyl)(C 0-10 alkyl), -N(C 0-10 alkyl) CO(C 0-10 alkyl), COR 1 , CN, C 1 - 10 alkoxy, aryloxy, containing N, O or S heteroaryloxy group, the H in the above groups can be substituted by the following groups: halogen, -CN, -OCH 2 F, -OCHF 2 , -OCF 3 , -OH, C 1-10 straight or branched chain alkyl, -N (C 0-10 alkyl) (
  • R' is selected from N or O.
  • said L1 or L4 is independently selected from maleimide group, carboxyl group, mercapto group, azido group, alkynyl group, cyclooctynyl group and its derivatives, tetrazinyl group, dithiopyridyl group, vinyl group , alkenyl sulfone group, succinimidyl group, aldehyde group, hydrazide group, amineoxy group and ⁇ -halogenated carbonyl group.
  • said L2 or L3 is independently selected from -O(CH 2 CH 2 O) i -, -(CH 2 ) i -, -(CH 2 ) i NH(CH 2 ) i -, -(CH 2 ) i COO(CH 2 ) i -, -(CH 2 ) i CONH(CH 2 ) i -, -(CH 2 ) i O(CH 2 ) i -, -(CH 2 ) i CO(CH 2 ) i -or
  • the i is selected from an integer of 0-10.
  • the cleavable group is selected from:
  • the primers can be free or immobilized on a support.
  • the cleavage between the polymerase and dNTP in the step (3) is to break the cleavable group between the ligation polymerase and the dNTP, and the cleavage of the cleavable group between the ligation polymerase and the dNTP is This is achieved by methods commonly used in the prior art, for example, by UV irradiation or by the cleavage of the group linking the polymerase and the dNTP by chemical substances, such as the action of reducing agents, pH changes.
  • the polymerase used in the nucleic acid sequencing method provided by the present invention is connected to the dNTP through a cleavable group, and contains a fluorescent marker, a chemiluminescent marker or a phosphorescent marker, and the nucleic acid sequencing method implemented by the polymerase can realize the nucleic acid sequence. Quick determination.
  • Figure 1 Structural formula of linker connecting dNTP and polymerase, specifically PC Mal-NHS carbonate lipid;
  • Figure 2 Purification results of DNA polymerase Bst DNA Pol and its variants, in which lane M represents Marker; lane CF represents Bst DNA Pol Cys-Free mutation site is C93S/C550S, lane D264C represents mutation site is C93S /C550S/D264C Bst DNA Pol variant, lane R334C represents the Bst DNA Pol variant with the mutation site C93S/C550S/R334C, and lane L364C represents the Bst DNA Pol variant with the mutation site C93S/C550S/L364C;
  • Figure 3 DNA polymerase Bst DNA Pol polymerase activity detection prepared in Example 1, wherein, lane M represents Marker, lane PC represents positive control, lane NC represents negative control, and lane CF represents Bst DNA Pol Cys-Free mutation
  • the site is C93S/C550S
  • lane D264C represents the Bst DNA Pol variant with the mutation site C93S/C550S/D264C
  • lane R334C represents the Bst DNA Pol variant with the mutation site C93S/C550S/R334C
  • lane L364C represents the mutation site
  • the point is the Bst DNA Pol variant of C93S/C550S/L364C;
  • Figure 4A dATP base modification structural formula, specifically 7-deaza-Propargylamino-dATP, with a molecular weight of 543.26;
  • Figure 4B dCTP base modification structural formula, specifically Propargylamino-dCTP, with a molecular weight of 520.22;
  • Figure 4C dGTP base modification structural formula, specifically 7-deaza-Propargylamino-dGTP, with a molecular weight of 559.26;
  • Figure 4D The structural formula of dUTP base modification, specifically Propargylamino-dUTP, the molecular weight is 521.20;
  • Figure 5 The junction site of dNTP and linker
  • FIG. 6A Mass spectrometry detects the connection of DNA polymerase Bst DNA Pol and its variants to small molecules, specifically the connection mass spectrum of Bst DNA Pol Cys-Free and dUTP;
  • FIG. 6B Mass spectrometry detects the connection of DNA polymerase Bst DNA Pol and its variants to small molecules, specifically the mass spectrogram of the Bst DNA Pol variant whose mutation site is C93S/C550S/L364C;
  • FIG. 6C Mass spectrometry detects the connection of DNA polymerase Bst DNA Pol and its variants to small molecules, specifically the connection mass spectrum of Bst DNA Pol variants with C93S/C550S/L364C mutation sites and dUTP;
  • Figure 6D Mass spectrometry detects the connection of DNA polymerase Bst DNA Pol and its variants to small molecules, specifically the connection mass spectrum of Bst DNA Pol variants with C93S/C550S/R334C mutation sites and dUTP;
  • Figure 6E Mass spectrometry detects the connection of DNA polymerase Bst DNA Pol and its variants to small molecules, specifically the connection mass spectrum of Bst DNA Pol variants with C93S/C550S/D264C mutation sites and dUTP;
  • Figure 7 The reaction mode diagram of the sequencing process, specifically designing a template-primer complex, carrying out an extension reaction with dNTP-linker-polymerase, washing away the unbound dNTP-linker-polymerase, and performing fluorescence or luminescence detection; Catalytic cleavage of the linker frees the polymerase from the primer-template complex. Use buffer to wash away the polymerase that has been cut off by light, and repeat the above steps to add the next nucleotide;
  • Figure 8 Single nucleotide extension detection electropherogram, in which lane P is the primer alone; lane P+T is the primer-template complex; lane 3nt is the extension of the primer-template complex with a separate polymerase and dTTP Reaction to increase the primer length by 3 nucleotides; mark +365 lane as primer-template complex and dUTP-linker-polymerase for extension reaction, since the polymerase and dUTP are linked by the linker, the primer will not be detached after the extension is completed -Template complex, which prevents other dUTP-linker-polymerase from binding to the primer-template complex, and cannot carry out the subsequent extension reaction, so it can only extend by 1 nucleotide.
  • the primers are separated from the polymerase, so the bands in the lane are primers that extend only 1 nucleotide in length; the reaction conditions in the marker-365 lane are the same as those in the marker+365 lane, because 365nm light is not used for cleavage, the primers and the polymerase The enzyme is covalently bound, and the denaturing gel cannot be opened, so the molecular weight of the primer band is extremely high.
  • nucleic acid in the present invention refers to at least two nucleotides covalently linked together.
  • dNTP in the present invention refers to deoxy-ribonucleotide triphosphate or ribonucleoside triphosphate, and usually includes dATP, dGTP, dTTP, dCTP or dUTP and the like.
  • mutant refers to an enzyme or protein with the same function formed by changing one or more amino acid sites in the wild-type polymerase.
  • the "combination of groups" mentioned in the present invention refers to a new group formed by connecting one or more substituent groups in the form of covalent bonds.
  • Modification refers to the substitution of one or more groups in the molecular structure of a substance.
  • C 0-10 alkyl refers to H, therefore, C 0-10 alkyl includes H, C 1 alkyl, C 2 alkyl, C 3 alkyl, C 4 Alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl.
  • C 1-10 straight chain/branched chain alkyl described in the present invention includes methyl, ethyl, C 3 straight chain/branched chain alkyl, C 4 straight chain/branched chain alkyl, C 5 straight chain /branched alkyl, C6 linear/branched alkyl, C7 linear/branched alkyl, C8 linear/branched alkyl, C9 linear/branched alkyl, C10 linear /branched alkyl.
  • C 3-10 branched chain alkyl described in the present invention includes isopropyl, isobutyl, tert-butyl, and isoamyl.
  • C 3-10 cycloalkyl described in the present invention includes C 3 cycloalkyl, C 4 cycloalkyl, C 5 cycloalkyl, C 6 cycloalkyl, C 7 cycloalkyl, C 8 cycloalkane group, C 9 cycloalkyl, C 10 cycloalkyl.
  • halogen described in the present invention includes fluorine, chlorine, bromine and iodine.
  • heterocycloalkyl refers to a non-aromatic saturated mono- or polycyclic ring system containing 3-10 ring atoms, preferably 5-10 ring atoms, one or more of which is not carbon atoms, but for example nitrogen, oxygen or sulfur atoms.
  • Preferred heterocycloalkyl groups contain 5-6 ring atoms.
  • aza, oxa or thia before heterocycloalkyl means that there is at least one nitrogen, oxygen or sulfur atom respectively as a ring atom.
  • heterocyclic aromatic group in the present invention refers to an aromatic monocyclic or polycyclic ring system containing 5-14 ring atoms, preferably 5-10 ring atoms, wherein one or more ring atoms are not carbon atoms, and are, for example, nitrogen, oxygen or sulfur atoms.
  • Preferred heterocyclic aromatic groups contain 5-6 ring atoms.
  • heterocyclic aromatic groups include pyrazinyl, furyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, pyrrolyl, pyrazolyl , triazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, 2,3-diazanaphthyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl , indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidinyl, pyrrolopyridyl, imidazolyl Pyridyl, isoquinolinyl, 1,2,4-triazin
  • the expression construct pET15-Bst DNA Pol for the production of N-terminal 6His-tagged Bst DNA Pol and its variants and variants thereof was used.
  • N-terminal 6His-tagged Bst DNA Pol and its variants are expressed in the cytoplasm due to increased yield.
  • N-terminal 6His-tagged Bst DNA Pol and its variants were introduced into the expression strain of BL21 (DE3) for expression. Bacteria were disrupted and centrifuged using buffer A (50 mM Tris-HCl pH 8.0, 800 mM NaCl, 1 mM EDTA, 1 mM DTT). DNA and other impurity proteins in the bacterial supernatant were removed using PEI solution and saturated ammonium sulfate solution in sequence. The N-terminal 6His-tagged Bst DNA Pol and its variant proteins were purified using His-trap FF column and AKTA protein purification system (GE Healthcare).
  • the protein eluate was collected and purified using a Heparin HP column (GE Healthcare). The protein eluate was collected and used SDS-PAGE gel to detect the protein purity. The results are shown in Figure 2.
  • the purity of the purified DNA polymerase Bst DNA Pol and its variants is greater than 95%, and lane M is the protein Marker (Thermo Fisher), Lane CF is DNA polymerase Bst DNA Pol Cys-Free, lane D264C is DNA polymerase Bst DNA Pol Cys-Free D264C mutant, lane R334C is DNA polymerase Bst DNA Pol Cys-Free R334C mutant, lane L364C is DNA polymerase Enzyme Bst DNA Pol Cys-Free L364C mutant. Measure the concentration of purified DNA polymerase Bst DNA Pol and its variants, and store in aliquots and freeze at -80°C ultra-low temperature until the dNTP-linker-polymerase complex is prepared.
  • the purified DNA polymerase Bst DNA Pol and its variants need to be tested for its activity first, and can be linked with small molecules after confirming that it has polymerase activity.
  • the purified DNA polymerase Bst DNA Pol and its variants were mixed with annealed bound primer-template complex, 10 mM MgCl 2 , dNTP mix and buffer (50 mM NaCl, 10 mM Tris-HCl pH7.9, 1 mM DTT), and the reaction The volume of the system is 10uL. Incubate at 37°C for 30 min, and then use non-denaturing polyacrylamide gel electrophoresis for detection.
  • lane M is DNA Marker (Thermo Fisher)
  • lane PC is a positive control, using polymerase Klenow (NEB)
  • lane NC is a negative control
  • lane CF is DNA polymerase Bst DNA Pol Cys-Free
  • lane D264C is DNA polymerase Bst DNA Pol Cys-Free D264C mutant
  • lane R334C is DNA polymerase Bst DNA Pol Cys-Free R334C mutant
  • lane L364C is DNA polymerase Bst DNA Pol Cys-Free L364C mutant.
  • a higher-purity polymerase is obtained by the above method, and the obtained polymerase has a higher polymerase activity.
  • the above-mentioned ligation product small molecule was dissolved in DMSO, mixed with the aforementioned expressed and purified N-terminal 6His-labeled Bst DNA Pol and its variant polymerase protein at a small molecule molar ratio of 10:1, and incubated at room temperature for 2 hours in the dark. . Afterwards, Zeba Spin Desalting Column (Thermo) was used to remove excess small molecules in the solution. Among them, the junction site of dNTP and linker is shown in Figure 5.
  • Figures 6A-6E show that the dNTP is linked to the polymerase through the linker, and the polymerase linked with the dNTP modified by the single molecule base is obtained.
  • a DNA template design a random sequence of 60 bp in length.
  • the 20-22nd position from the 3' end of the template is three consecutive adenines (A), and at the 5' end of the template, add Biotin tag, design a primer sequence complementary to the 3' end of the template, the length is 19nt, the sequence is shown in Table 1.
  • the extension reaction was performed by incubating the pre-annealed primers and templates with dNTP-linker-polymerase at the optimum temperature for enzymatic activity, 60°C, for 5 minutes.
  • the reacted solution was placed on ice and irradiated with an LED lamp with a wavelength of 365nm-405nm for 15 minutes, to perform photocleavage of the linker connected to the polymerase and dNTP, so that the polymerase was separated from the primer-template complex.
  • the reaction system after irradiation was incubated with streptavidin-labeled magnetic beads for 5 minutes to allow the extended primer-template complexes to bind to the magnetic beads. Photocleaved polymerase and unbound dNTP-linker-polymerase were washed away using magnetic bead wash buffer (0.5 M NaCl, 20 mM Tris-HCl pH 7.5, 1 mM EDTA).
  • the primer-template complex is opened with 50 mM KOH solution, the primer is eluted, neutralized with an equal volume of 50 mM HCl solution, and then used 15% urea denatured PAGE gel for detection. Since only the 5' end of the primers has a fluorophore (FAM), the size of the primers on the urea-denatured PAGE gel can be detected by using the Typhoon fluorescent gel scanner of GE Company, and photographed for comparative analysis.
  • FAM fluorophore
  • Lane P is a separate primer
  • lane P+T is a primer-template complex
  • lane 3nt is an extension reaction of the primer-template complex with a separate polymerase Bst L364C and dTTP to increase the primer length by 3 nucleotides
  • mark +365 lane is the primer-template complex and the polymerase Bst L364C-dUTP for extension reaction.
  • the bands in the lanes are primers that only extend 1 nucleotide; the reaction conditions in the marker-365 lanes are the same as those in the marker+365 lanes. Since 365nm light is not used for cleavage, the primers are covalently bound to the polymerase, and the denatured gel cannot be used. open, so the molecular weight of the primer band is extremely high.

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Abstract

La présente invention concerne un procédé de séquençage d'acide nucléique, comprenant les étapes suivantes : mise à disposition d'une polymérase liée au dNTP au moyen d'un groupe clivable et capable d'émettre un signal optique ; mise en contact de la polymérase avec l'extrémité 3' d'une amorce d'un complexe matrice-acide nucléique-amorce à mesurer, appariement complémentaire du dNTP sur la polymérase avec un groupe de base sur une séquence d'acide nucléique à mesurer, et ajout de l'extrémité 3' de l'amorce dans une réaction enzymatique de polymérase ; et détection du signal optique émis par la polymérase. Par la suite, le dNTP et la polymérase sont brisés, et le nouveau complexe matrice-amorce après la sortie de la polymérase peut entrer dans le cycle de séquençage suivant.
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