WO2022105640A1

WO2022105640A1 - Nucleic acid sequencing method

Info

Publication number: WO2022105640A1
Application number: PCT/CN2021/129461
Authority: WO
Inventors: 白净卫; 杜娟娟; 徐扬
Original assignee: 清华大学
Priority date: 2020-11-17
Filing date: 2021-11-09
Publication date: 2022-05-27
Also published as: CN112322715B; CN112322715A

Abstract

The present invention provides a nucleic acid sequencing method, comprising: providing a polymerase linked to dNTP by means of a cleavable group and capable of emitting an optical signal; making the polymerase in contact with 3' end of a primer of a nucleic acid template-primer complex to be measured, complementarily pairing dNTP on the polymerase with a base group on a nucleic acid sequence to be measured, and adding the 3' end of the primer into a polymerase enzymatic reaction; and detecting the optical signal emitted by the polymerase. Subsequently, dNTP and the polymerase are broken, and the new template-primer complex after the leaving of the polymerase may enter the next sequencing cycle.

Description

A nucleic acid sequencing method

technical field

The invention belongs to the field of biotechnology, and relates to a method for determining a nucleic acid sequence.

Background technique

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. In 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. Among the third-generation sequencing technologies, 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. In this "sequencing by synthesis" reaction, 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. Experiments show that DNA sequencing can be performed by using the method provided by the invention, which has important application value. 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. In addition, 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.

Sebastian Palluk et al. (Nature Biotechnology, 2018(36): 645-650) disclosed a method for synthesizing an oligonucleotide using terminal deoxynucleotidyl transferase (TdT) for nuclear Synthesis of Glycosides. The principle of synthesis is that TdT molecule can form a ligation complex with a single deoxyribonucleoside triphosphate (dNTP) molecule. After the primer is combined with the dNTP that forms the enzymatic ligation system, the 3' end of the primer is covalently combined with TdT to form a new complex. 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. However, 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.

Fei Chen et al. (Genomics Proteomics Bioinformatics, 2013(11): 34-40) introduced the classification of reversible terminators and the development history and working mechanism of the application of reversible terminators in gene sequencing technology.

In view of the deficiencies of the prior art, the present invention provides a method capable of rapidly determining a nucleic acid sequence.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a method for determining a nucleic acid sequence, the sequencing method comprising the following steps:

(1) providing a polymerase, the polymerase is connected to the dNTP through a cleavable group (linker), and the polymerase can emit a light signal;

(2) contacting the polymerase with the 3' end of the primer of the nucleic acid template-primer complex to be tested, the dNTPs on the polymerase are complementary to the bases on the sequence of the nucleic acid to be tested, and enzymatically react with the polymerase Add primer 3' end;

(3) Detecting the light signal emitted by the polymerase.

Here, after the enzymatic reaction forms an extension of one base, 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 Polγ 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 Polα and its variants, DNA Pol△ and its variants, DNA Polε and its variants, DNA Pol III and its variants, DNA Polβ and its variants, DNA Polσ and its variants, DNA Polλ and its variants, DNA Polμ 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 transferase (TdT).

Preferably, the polymerase is DNA Polβ and a variant thereof, and the sequence of the DNA Polβ is SEQ ID NO: 1. The DNA Polβ variant is a mutation of the DNA Polβ 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 Polβ coding sequence % or more sequence identity, and the DNA Polβ variant has polymerase activity.

Preferably, the mutation site of the DNA Polβ 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.

More preferably, the variant of the DNA Polβ 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.

More preferably, the variant of DNA Polβ 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.

More preferably, the variant of the DNA Polβ 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.

Particularly preferably, the polymerase is a variant of DNA Polβ, and the variant of DNA Polβ is selected from one or a combination of two or more of the following variants:

(1) mutate the cysteine at position 178 of SEQ ID NO: 1 to serine, the cysteine at position 239 to serine, the cysteine at position 267 to serine, and the cysteine at position 28 to serine Paragine is mutated to cysteine;

(2) mutate the cysteine at position 178 of SEQ ID NO: 1 to serine, the cysteine at position 239 to serine, the cysteine at position 267 to serine, the isotope at position 33 to serine Leucine is mutated to cysteine;

(3) mutate the cysteine at position 178 of SEQ ID NO: 1 to serine, the cysteine at position 239 to serine, the cysteine at position 267 to serine, the sperm at position 149 amino acid to cysteine.

In a specific embodiment of the present invention, 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 Polβ variant has polymerase activity.

In another specific embodiment of the present invention, the amino acid sequence of the polymerase is shown in any of SEQ ID NOs: 3-6.

Preferably, 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.

Preferably, 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.

More preferably, 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, Preferably, 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.

Preferably, 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 the 264th position, the 334th position and/ Or any one, two or three non-cysteines at position 364 are mutated to cysteine.

Particularly preferably, 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:

(1) mutate the cysteine at position 93 of SEQ ID NO: 2 to serine, and mutate the cysteine at position 550 to serine;

(2) mutating the cysteine at position 93 of SEQ ID NO: 2 to serine, the cysteine at position 550 to serine, and the aspartic acid at position 264 to cysteine;

(3) mutating the cysteine at position 93 of SEQ ID NO: 2 to serine, the cysteine at position 550 to serine, and the arginine at position 334 to cysteine;

(4) The cysteine at position 93 of SEQ ID NO: 2 is mutated to serine, the cysteine at position 550 is mutated to serine, and the leucine at position 364 is mutated to cysteine.

In a specific embodiment of the present invention, 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 Polβ variant has polymerase activity.

In another specific embodiment of the present invention, the 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. Preferably, the cleavable group contains a cleavable structure X, and the X is selected from:

where 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) (C _0-10 alkyl), -OC _0-10 alkyl, C _3-10 cycloalkyl, -O heterocycloalkyl, -N heterocycloalkyl, -S hetero Cycloalkyl, -N heterocyclic aromatic group, -O heterocyclic aromatic group, -S heterocyclic aromatic group, R ₁ is selected from H, O _0-10 alkyl group, C ₁ - ₁₀ straight chain or branched chain alkyl group,

R' is selected from N or O.

More preferably, 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, and 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. Particularly preferably, the base-modified dNTPs are those whose bases are

Modified dNTP, the n is selected from an integer of 0-12, preferably, the n is selected from an integer of 1-6, for example, n is selected from 1, 2, 3, 4, 5 or 6, the said G is selected from -COOH, -SH, _-NH2 , _-N3 , -CH= _CH2 , -C≡CH.

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.

Preferably, the base-modified dNTP is that the 7th position of the purine base or the 3rd position of the pyrimidine base is modified by the following groups:

Preferably, the dNTP modified at the 3' position can be substituted by the following group at the H atom at the 3' position:

In a specific embodiment of the present invention, the base-modified dNTP is selected from:

Propargylamino-3'-azidomethyl-dCTP;

Propargyl-3'-azidomethyl-dCTP;

Propargylamino-3'-azidomethyl-dUTP;

Propargyl-3'-azidomethyl-dUTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP;

7-deaza-propargyl-3'-azidomethyl-dGTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dATP;

7-deaza-propargyl-3'-azidomethyl-dATP;

Propargylamino dCTP, propargyl-dCTP;

Propargylamino dUTP, propargyl-dUTP;

7-Deaza-7-Propargylamino dATP, 7-deaza-propargyl-dATP;

7-Deaza-7-Propargylamino dGTP, 7-deaza-propargyl-dGTP.

Preferably, the linking site is selected from the following formula A, formula B, formula C or formula D.

Preferably, the X is selected from:

where j is an integer from 1 to 3,

R' is selected from N or O.

Preferably, 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 α-halogenated carbonyl group.

Preferably, 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

One or a combination of two or more groups in , the i is selected from an integer of 0-10.

Preferably, 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 chemiluminescent label is a group currently known to emit light signal under the irradiation of visible light and/or ultraviolet light, for example, horseradish peroxidase, alkaline phosphatase, luciferase and its derivatives, acridine Pyridinyl esters, peroxyoxalates, lofenacine, lucigenin, luminol and its derivatives, metal ion complexes of catalytic chemiluminescent markers, electrocatalytic chemiluminescent markers, benzodifuran, methine base, triphenylmethane, azine, triphenazine, naphthalimide, pyrazole, naphthoquinone, anthraquinone, monoazo and bisazo and derivatives of the above groups, with absorption bands in the visible light region Derivatives of benzene, C=C, C=O, -N=N-, -NO ₂ , -C=S, etc. with ultraviolet absorption bands.

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, isothiocyanate acid fluorescein, 6-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine B, rhodamine 6G, 7-amino- 4-Methylcoumarin, Dihydrorhodamine 123, Tetramethylrhodamine-6-maleimide, Tetramethylrhodamine-5-maleimide, 5-indoleacetamidofluorescein , bis[NN-bis(carboxymethyl)aminomethyl]fluorescein tetrasodium salt, fluorescein-5-maleimide, sulforhodamine G, 7-hydroxy-4-methylcoumarin, 3-cyano-7-hydroxycoumarin, fluorescein disodium salt, tetramethylrhodamine-6-isothiocyanate, 6-carboxy-X-rhodamine succinimide ester, 5-carboxy-X -Rhodamine succinimide ester, 6-carboxy-X-rhodamine, 5-carboxytetramethylrhodamine succinimide ester, 6-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine, Energy transfer dyes and fluorescent proteins such as one or more of GFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein) and derivatives are produced.

Said phosphorescent label includes transition metal iridium (Ir) or ruthenium (Ru) aza-heteroaryl complexes, preferably as shown in the following formula:

Preferably, 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.

Preferably, in the step (2), the nucleic acid to be detected is immobilized on the support.

Those skilled in the art can understand that 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. Alternatively, the solid support may be non-planar, such as microbeads. Any suitable size can be used. For example, the support may be on the order of 1 to 10 cm in each direction.

If only the primer mixture is grafted to the surface of the solid support, and the template to be amplified is present in free solution, 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. Thus, 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.

Preferably, the step (2) further includes the step of adding primers to the nucleic acid to be detected.

In the step (3), the polymerase is excited by light to emit a light signal.

In the step (3), the light signal emitted on the polymerase is detected by a detection system commonly used in the prior art. For example, by using a specific wavelength laser or using other suitable light sources, the polymerase can be detected by CCD or other suitable detection methods. the light signal emitted on it. For example, the method described in patent PCT/US07/007991 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.

Preferably, the nucleic acid sequencing method of the present invention further comprises step (4) of cleaving between the polymerase and the dNTP.

In the step (4), 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.

In the embodiment of the present invention, 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).

Preferably, 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.

Preferably, 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. In order to function as a nucleic acid amplification template, 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.

The 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 Polβ, and the variant of DNA Polβ 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.

Preferably, the variant of DNA Polβ 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 Polβ 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 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.

More preferably, 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, Preferably, 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. Preferably, 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 fourth aspect of the present invention also provides a polymerase complex prepared by linking the polymerase and dNTP through a cleavable group. Wherein, the cleavable group is used to connect the polymerase and the modified dNTP, or, the polymerase and the dNTP. Preferably, the cleavable group contains a cleavable structure X, and the X is selected from:

where j is an integer from 1 to 3,

R' is selected from N or O.

More preferably, 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 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 dNTP modified at the 3' position can be substituted by the following group at the H atom at the 3' position:

Propargylamino-3'-azidomethyl-dCTP;

Propargyl-3'-azidomethyl-dCTP;

Propargylamino-3'-azidomethyl-dUTP;

Propargyl-3'-azidomethyl-dUTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP;

7-deaza-propargyl-3'-azidomethyl-dGTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dATP;

7-deaza-propargyl-3'-azidomethyl-dATP;

Propargylamino dCTP, propargyl-dCTP;

Propargylamino dUTP, propargyl-dUTP;

7-Deaza-7-Propargylamino dATP, 7-deaza-propargyl-dATP;

7-Deaza-7-Propargylamino dGTP, 7-deaza-propargyl-dGTP.

Preferably, the X is selected from:

where j is an integer from 1 to 3,

R' is selected from N or O.

Preferably, 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 chemiluminescent label is a group currently known to emit light signal under the irradiation of visible light and/or ultraviolet light, for example, horseradish peroxidase, alkaline phosphatase, luciferase and its derivatives, acridine Pyridine esters, peroxyoxalates, lofenacine, lucigenin, luminol and their derivatives, metal ion complexes of catalytic chemiluminescent markers, electrocatalytic chemiluminescent markers, benzodifuran, methine base, triphenylmethane, azine, triphenazine, naphthalimide, pyrazole, naphthoquinone, anthraquinone, monoazo and bisazo and derivatives of the above groups, with absorption bands in the visible light region Derivatives of benzene, C=C, C=O, -N=N-, -NO ₂ , -C=S, etc. with ultraviolet absorption bands.

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, Fluorescein isothiocyanate, 6-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine B, rhodamine Ming 6G, 7-amino-4-methylcoumarin, dihydrorhodamine 123, tetramethylrhodamine-6-maleimide, tetramethylrhodamine-5-maleimide, 5 -Indoleacetamidofluorescein, bis[NN-bis(carboxymethyl)aminomethyl]fluorescein tetrasodium salt, fluorescein-5-maleimide, sulforhodamine G, 7-hydroxy-4 - Methylcoumarin, 3-cyano-7-hydroxycoumarin, fluorescein disodium salt, tetramethylrhodamine-6-isothiocyanate, 6-carboxy-X-rhodamine succinimide Ester, 5-carboxy-X-rhodamine succinimide ester, 6-carboxy-X-rhodamine, 5-carboxytetramethylrhodamine succinimide ester, 6-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine Carboxytetramethylrhodamine, produces energy transfer dyes and fluorescent proteins, such as GFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein) and one of its derivatives one or more.

A fifth aspect of the present invention provides a nucleic acid synthesis method, the nucleic acid synthesis method comprising:

(1) providing a polymerase, the polymerase is connected to the dNTP through a cleavable group;

(2) contacting the polymerase with the 3' end of the primer of the template-primer complex of the nucleic acid to be tested, 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.

Preferably, the nucleic acid synthesis method further comprises step (3) cleavage between the polymerase and dNTP; and (4) repeating steps (1)-(3).

Wherein, the cleavable group is used for linking polymerase and modified dNTP, or linking polymerase and dNTP.

Preferably, the cleavable group contains a cleavable structure X, and the X is selected from:

where j is an integer from 1 to 3,

R' is selected from N or O.

Propargylamino-3'-azidomethyl-dCTP;

Propargyl-3'-azidomethyl-dCTP;

Propargylamino-3'-azidomethyl-dUTP;

Propargyl-3'-azidomethyl-dUTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP;

7-deaza-propargyl-3'-azidomethyl-dGTP;

7-Deaza-7-Propargylamino-3'-azidomethyl-dATP;

7-deaza-propargyl-3'-azidomethyl-dATP;

Propargylamino dCTP, propargyl-dCTP;

Propargylamino dUTP, propargyl-dUTP;

7-Deaza-7-Propargylamino dATP, 7-deaza-propargyl-dATP;

7-Deaza-7-Propargylamino dGTP, 7-deaza-propargyl-dGTP.

Preferably, the X is selected from:

where j is an integer from 1 to 3,

R' is selected from N or O.

Preferably, 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.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

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, and 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;

Figure 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;

Figure 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;

Figure 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. After the extension reaction, after 365nm light irradiation , 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.

Detailed ways

The term "nucleic acid" in the present invention refers to at least two nucleotides covalently linked together.

The "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.

The "mutant" or "variant" in the present invention 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" as used in the present invention refers to the substitution of one or more groups in the molecular structure of a substance.

The term C _0-10 alkyl described in the present invention, C ₀ 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.

The term 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.

The term C _3-10 branched chain alkyl described in the present invention includes isopropyl, isobutyl, tert-butyl, and isoamyl.

The term 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.

The term halogen described in the present invention includes fluorine, chlorine, bromine and iodine.

The term heterocycloalkyl as used herein 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. The prefix aza, oxa or thia before heterocycloalkyl means that there is at least one nitrogen, oxygen or sulfur atom respectively as a ring atom.

The term 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. Representative 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-triazinyl, benzothiazolyl and the like.

Example 1 Preparation of polymerase

1. Cloning and Bst DNA Pol expression strains

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. For N-terminal 6His-tagged Bst DNA Pol and its variants are expressed in the cytoplasm due to increased yield.

2. Protein expression and purification

Using a general chemical transformation method, the expression plasmids of 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.

3. Detection of polymerase activity

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. In comparison with the negative control, the molecular weight of the primer-template complex will increase, indicating that the polymerase has enzymatic activity to extend the primer to the template length. As shown in Figure 3, 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.

As shown in Figures 2-3, a higher-purity polymerase is obtained by the above method, and the obtained polymerase has a higher polymerase activity.

Example 2 Preparation of dNTP-linker-polymerase

1. dNTP and linker connection

The above-mentioned dNTP (as shown in Figure 4A-4D) and the linker (as shown in Figure 1) were mixed in a molar ratio of 1:2, incubated in the dark at room temperature for 2 hours, the ligation product was precipitated with ethyl acetate, and the supernatant was evaporated by centrifugation. Store the pellet in a -20°C refrigerator.

2. Preparation of Small Molecules Linked to Polymerase

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.

Using mass spectrometry, a part of the ligation product was taken for detection of ligation effect and efficiency. Figures 6A-6E. 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.

Example 3 Sequencing

According to the reaction pattern diagram (Fig. 7), design a DNA template with 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). In order to detect whether a nucleotide is extended on the primer after one round of reaction, 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.

Table 1 Primer and template sequences

The reaction results are shown in Figure 8. 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. Since the polymerase and dUTP are linked by the linker, it will not detach from the primer-template complex after the extension is completed, hindering the Other dUTP-linker-polymerases are bound to the primer-template complex and cannot carry out the subsequent extension reaction, so only one nucleotide can be added for extension. Therefore, 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.

The results showed that the dUTP-linked polymerase Bst L364C had high polymerase activity, and the polymerase Bst L364C that could not escape the primer-template complex effectively hindered the subsequent extension reaction, and the hindering group could be cleaved by 365nm light. Remove obstacles.

Claims

A nucleic acid sequencing method comprising the steps of:

(1) providing a polymerase, the polymerase is connected to the dNTP through a cleavable group, and the polymerase can emit a light signal;

(2) contacting the polymerase with the 3' end of the primer of the nucleic acid template-primer complex to be tested, the dNTPs on the polymerase are complementary to the bases on the sequence of the nucleic acid to be tested, and enzymatically react with the polymerase Add primer 3' end;

(3) Detecting the light signal emitted by the polymerase.
A nucleic acid sequencing method according to claim 1, wherein the polymerase is selected from the group consisting of Bst DNA Pol and its variants, DNA Pol I and its variants, DNA Polγ and its variants, T3 DNA Pol and Its variants, T5 DNA Pol and its variants, L5 DNA Pol and its variants, DNA Po1 II and its variants, DNA Pol B and its variants, DNA Polα and its variants, DNA Pol△ and its variants , DNA Polε and its variants, DNA Pol III and its variants, DNA Polβ and its variants, DNA Polσ and its variants, DNA Polλ and its variants, DNA Polμ and its variants, DNA Pol IV and its variants One or more of DNA Pol V and its variants, terminal transferase and its variants.
A nucleic acid sequencing method according to claim 2, wherein the polymerase is a DNA Polβ variant, and the DNA Polβ variant is a mutation of DNA Polβ at one or more amino acid sequence positions, and the DNA Polβ variant is The mutation comprises substitution, deletion and/or insertion, or the DNA Polβ variant has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the DNA Polβ variant has polymerase activity.
A nucleic acid sequencing method according to claim 3, wherein the variant of the DNA Polβ is any one, two or three cysteines from positions 145-355 of SEQ ID NO: 1 Mutations to non-cysteines and any one, two or three non-cysteines at positions 1-236 are mutated to cysteines.
A nucleic acid sequencing method according to claim 4, wherein the variant of the DNA Polβ is any one, two or three cysteines at positions 145-355 of SEQ ID NO: 1 Mutation to serine and any one, two or three non-cysteines at positions 20-154 were mutated to cysteine.
A nucleic acid sequencing method according to claim 5, wherein the variant of the DNA Polβ is the cysteine at the 178th position, the 239th position and/or the 267th position of SEQ ID NO: 1 Acids were mutated to serines and any one, two or three non-cysteines at positions 28, 33 and/or 149 were mutated to cysteines.
A nucleic acid sequencing method according to claim 2, wherein the polymerase is a Bst DNA Pol variant, and the Bst DNA Pol variant is a mutation of the Bst DNA Pol at one or more amino acid sequence positions , the mutation comprises a substitution, deletion and/or insertion, or the Bst DNA Pol variant and the Bst DNA Pol coding sequence have at least 70%, 75%, 80%, 85%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more sequence identity, and the Bst DNA Pol variant has polymerase activity.
A nucleic acid sequencing method according to claim 7, wherein 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 mutating any one, two or three non-cysteines at positions 264, 334 and/or 364 to cysteines.
A nucleic acid sequencing method according to claim 1, wherein the cleavable group contains a cleavable structure X, and the X is selected from:

where 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, hetero N, O or S containing 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) (C 0-10 alkyl), -OC 0-10 alkyl, C 3-10 cycloalkyl, -O heterocycloalkyl, -N heterocycloalkyl, -S hetero Cycloalkyl, -N heterocyclic aryl, -O heterocyclic aryl, -S heterocyclic aryl, R 1 is selected from H, O 0-10 alkyl, C 1-10 straight or branched chain alkyl,

R' is selected from N or O.
The nucleic acid sequencing method according to claim 9, wherein the cleavable group has a structure of L1-L2-X-L3-L4, wherein L1 is an end group bound to dNTP or modified dNTP , L2 and L3 are absent or non-cleavable linking groups, and L4 is the end group bound to the polymerase.
A nucleic acid sequencing method according to claim 10, wherein said L1 or L4 is independently selected from maleimide, carboxyl, sulfhydryl, azide, alkynyl, cyclooctynyl and Its derivatives, tetrazinyl, dithiopyridyl, vinyl, alkenyl, succinimidyl, aldehyde, hydrazide, amineoxy and α-halogenated carbonyl.
The nucleic acid sequencing method according to claim 10, wherein 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
One or a combination of two or more groups in , the i is selected from an integer of 0-10.
The nucleic acid sequencing method according to claim 1, wherein the polymerase is linked with one or more fluorescent labels, phosphorescent labels or chemiluminescent labels.
A nucleic acid sequencing method according to claim 13, wherein the chemiluminescent marker is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, luciferase and derivatives thereof, acridine esters, Peroxyoxalates, Lofenine, Lucigenin, Luminol and its derivatives, Metal ion complexes of catalytic chemiluminescent markers, Electrocatalytic chemiluminescent markers, benzodifuran, methine, triphenyl Methane, azine, triphenazine, naphthalimide, pyrazole, naphthoquinone, anthraquinone, monoazo and bisazo and derivatives of the above groups, derivatives of benzene with absorption bands in the visible region , C=C, C=O, -N=N-, -NO 2 , -C=S with ultraviolet absorption band; the fluorescent label is selected from fluorescein, Cy2, Cy3, Cy5, Cy7, Alexa Fluor Series Dyes, Fluorescein isothiocyanate, 5-Hexachlorofluorescein phosphoramidate, 6-carboxy-2',4,7,7'-tetrachlorofluorescein succinimidyl ester, 6-carboxy-4',5'-Dichloro-2',7'-dimethoxyfluorescein succinimidyl ester, Texas red, Rhodamine 110, Fluorescein maleimide dye, fluoroborodipyrrole, xanthium Tonne, carbocyanine, 1,1'-bis(octadecyl)-3,3,3',3'-tetramethylindocarbocyanine perchlorate, 3,3'-bis(decyl) Octaalkyl)-oxacarbocyanine perchlorate, pyrene, phthalocyanine, 6-carboxyrhodamine 6G, fluorescein isothiocyanate, 6-carboxyfluorescein succinimidyl ester, 5-carboxyfluorescein Succinimidyl ester, 5-carboxyfluorescein, 6-carboxyfluorescein, rhodamine B, rhodamine 6G, 7-amino-4-methylcoumarin, dihydrorhodamine 123, tetramethylrhodamine- 6-maleimide, tetramethylrhodamine-5-maleimide, 5-indoleacetamidofluorescein, bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein tetra sodium salt, fluorescein-5-maleimide, sulforhodamine G, 7-hydroxy-4-methylcoumarin, 3-cyano-7-hydroxycoumarin, fluorescein disodium salt, Tetramethylrhodamine-6-isothiocyanate, 6-carboxy-X-rhodamine succinimide ester, 5-carboxy-X-rhodamine succinimide ester, 6-carboxy-X-rhodamine, 5-Carboxytetramethylrhodamine succinimide ester, 6-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine, produce energy transfer dyes and fluorescent proteins such as GFP (green fluorescent protein), CFP ( One or more of cyan fluorescent protein), BFP (blue fluorescent protein), YFP (yellow fluorescent protein) and derivatives; the phosphorescent label includes nitrogen of transition metal iridium (Ir) or ruthenium (Ru) Heteroaryl complexes.
A nucleic acid sequencing method according to claim 1, wherein the step (1) provides that the polymerase connected to the dNTP through the cleavable group is to provide four polymerases connected with different dNTPs, and The four polymerases linked with different dNTPs are linked with different fluorescent labels, chemiluminescent labels or phosphorescent labels.
The nucleic acid sequencing method according to claim 1, wherein in the step (2), the nucleic acid to be detected is immobilized on a support.
The nucleic acid sequencing method according to claim 1, wherein the nucleic acid sequencing method further comprises step (4) of cleaving between the polymerase and the dNTP.
The nucleic acid sequencing method according to claim 17, wherein the nucleic acid sequencing method further comprises step (5): repeating steps (1)-(4).
The nucleic acid sequencing method according to claim 1, wherein the nucleic acid sequencing method further comprises nucleic acid sample pretreatment, and the nucleic acid sample pretreatment includes nucleic acid sample library construction and amplification.
A polymerase, characterized in that the polymerase mutates any one, two or three cysteines at positions 145 to 355 of SEQ ID NO: 1 to non-cysteines, and mutates the first cysteines to non-cysteines. Any one, two or three non-cysteines at positions 1-236 were mutated to cysteine.
A polymerase according to claim 20, wherein the polymerase mutates any one, two or three cysteines from positions 145 to 355 of SEQ ID NO: 1 to serine, And mutate any one, two or three non-cysteines at positions 20-154 to cysteine.
A polymerase according to claim 21, wherein the polymerase mutates the cysteine at position 178, 239 and/or 267 of SEQ ID NO: 1 to serine , and any one, two or three non-cysteines at positions 28, 33 and/or 149 are mutated to cysteine.
A polymerase, characterized in that the polymerase mutates any one, two or three cysteines at positions 85-100 or 540-570 of SEQ ID NO: 2 to non-cysteine acid and mutate any one, two or three non-cysteines at positions 250-280 or 320-380 to cysteines.
The polymerase according to claim 23, wherein the polymerase mutates the cysteine at the 93rd position and/or the 550th position of SEQ ID NO: 2 to serine, and mutates the 264th position , any one, two or three non-cysteines at positions 334 and/or 364 are mutated to cysteine.
A polymerase complex is characterized in that, the polymerase complex is prepared by connecting the polymerase and dNTP through a cleavable group.
A polymerase complex according to claim 25, wherein the cleavable group contains a cleavable structure X, and the X is selected from:

Wherein, 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, containing N, O or S Heterocyclic aryl, 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 hetero Aryloxy, 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) (C 0-10 alkyl), -OC 0-10 alkyl, C 3-10 cycloalkyl, -O heterocycloalkyl, -N heterocycloalkyl, - S heterocycloalkyl, -N heterocyclic aryl, -O heterocyclic aryl, -S heterocyclic aryl, R 1 is selected from H, O 0-10 alkyl, C 1-10 straight chain or branched alkane base,

R' is selected from N or O.
A method for synthesizing nucleic acid, wherein the method for synthesizing nucleic acid comprises:

(1) providing a polymerase, the polymerase is connected to the dNTP through a cleavable group;

(2) contacting the polymerase with the 3' end of the primer of the template-primer complex of the nucleic acid to be tested, 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 method for synthesizing nucleic acid according to claim 27, wherein the method for synthesizing nucleic acid further comprises step (3) cleaving between the polymerase and dNTP; and (4) repeating (1)-( 3) step.