WO2023185795A1 - 荧光检测试剂及其制备方法及应用 - Google Patents

荧光检测试剂及其制备方法及应用 Download PDF

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WO2023185795A1
WO2023185795A1 PCT/CN2023/084270 CN2023084270W WO2023185795A1 WO 2023185795 A1 WO2023185795 A1 WO 2023185795A1 CN 2023084270 W CN2023084270 W CN 2023084270W WO 2023185795 A1 WO2023185795 A1 WO 2023185795A1
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fluorescence detection
detection reagent
reagent
acid
glucose
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French (fr)
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谢槟
陈美容
刘磊
陈巍月
王文
赵楠楠
陈方
孙雷
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深圳市真迈生物科技有限公司
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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
    • 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

Definitions

  • the invention relates to the technical field of fluorescence detection, and in particular to a fluorescence detection reagent and its preparation method and application.
  • biomolecules labeled with photoluminescent molecules based on optical systems are often used in scientific research and diagnostic applications. Taking molecular diagnosis as an example, fluorescent molecules are usually used to modify nucleotides and/or oligonucleotides to detect and characterize the structure of nucleic acids.
  • nucleotide molecules with inhibitory groups and fluorescent labels are excited by a light source of a specific wavelength and generate fluorescence after being bound to the extending chain.
  • the signal is optically detected by the imaging system.
  • biochemical signals are converted into electronic signals and nucleic acid sequence determination is achieved based on the processing of these electronic signals.
  • single-molecule sequencing does not involve the amplification of the nucleic acid molecule to be tested, that is, it does not involve the amplification of the detection signal.
  • a nucleic acid molecule to be tested usually only carries one or a few fluorescent molecules. Therefore, it is understandable that For single-molecule sequencing, the detection of each nucleic acid molecule to be tested relies on the imaging system to detect the signal of a single fluorescent molecule. That is to say, it is recognized that obtaining high signal-to-noise ratio images is the key to achieving single-molecule sequencing.
  • Difficulties in other words, in the process of collecting or detecting single-molecule fluorescence signals, the intensity and stability of single-molecule fluorescence signals directly affect the accuracy and apparent read length of sequencing, and directly affect whether single-molecule sequencing can be realized .
  • nucleic acid molecule/fluorescent molecule it is often necessary to place the nucleic acid molecule/fluorescent molecule to be tested in a specific solution environment to maintain the stability of the fluorescence signal, enhance the fluorescence signal, and/or reduce the impact of photochemical reactions on the solid surface, solid phase Destruction of substances such as DNA strands, nucleotides (bases) and enzymes on the surface that participate in or catalyze reactions.
  • the so-called solution environment is a reagent or formula that is conducive to the imaging collection of fluorescence signals and is suitable for the detection of nucleic acid molecules to be tested, especially single molecule detection, including the preparation method of the reagent or formula, which needs to be provided or improved.
  • embodiments of the present invention provide A method of incorporating labeled nucleotides, a fluorescent detection reagent and its application, and a method of preparing the fluorescent detection reagent.
  • a sequencing run usually includes multiple rounds of reactions/multiple repeated reactions, and each round of reactions includes optically detectable Labeling, such as the introduction of fluorescent labels, excitation of fluorescent signals, signal acquisition and imaging, and removal of fluorescent labels.
  • the inventor believes that in the process of single-molecule sequencing based on imaging the surface of a solid-phase substrate, preferably, the solution system (sometimes also called "imaging fluid") in which the fluorescent molecules are placed should be able to enhance the fluorescence
  • the intensity of the signal such as inhibiting the ISC process, reducing the probability of quenching, avoiding or reducing light-induced damage, etc., to improve the fluorescence quantum efficiency of fluorescent molecules and extend the fluorescence lifetime to maintain the intensity (brightness) and stability of the fluorescence signal during detection .
  • the imaging fluid should contain an oxygen removal system; this is because oxygen molecules can participate in the photochemical process and cause quenching of fluorescent molecules.
  • oxygen molecules can directly react with fluorescent molecules in the excited state, causing quenching of fluorescent molecules.
  • Free radicals can also be generated to cause quenching of fluorescent molecules (Lakowicz JR, Principles of fluorescence spectroscopy, Springer science & business media; 2013 April 17; J. Vogelsang et al., A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes, AngewandteChemie International Edition, vol.
  • the oxygen removal system contained in the imaging fluid should be an effective oxygen removal system. It is expected that the solution containing an effective oxygen removal system can effectively reduce the impact of oxygen molecules on fluorescent molecules and biological macromolecules on the one hand, and can also reduce the impact of oxygen molecules on fluorescent molecules and biological macromolecules on the other.
  • the imaging fluid should contain a redox system, and the components contained in the so-called redox system include the components of the so-called oxygen removal system, intersect with the components of the so-called oxygen removal system, or are independent of the so-called oxygen removal system. deaeration system.
  • each reagent independently can effectively eliminate triplet fluorescent molecules or can restore triplet fluorescent molecules to the ground state to improve the stability of fluorescent dyes (Holzmeister P et al., Geminate recombination as a photoprotection mechanism for fluorescent dyes, Angew Chem Int.
  • solutions containing so-called oxygen scavenging systems and/or redox systems are dependent on appropriate buffer systems during preparation, storage and use, including pH value, buffering capacity, solution ionic strength, solution polarity, etc. It affects the performance of functional components and compositions, and may also directly affect the quantum efficiency of fluorescent molecules.
  • the inventor first set or prepared the same detection conditions, detection environment and detection objects, and the solution system in which the fluorescent molecules were placed during imaging disclosed in the above-mentioned published documents US 7,282,337 or US 7,666,593 was a solution system containing 30% acetonitrile.
  • the above formula is "134 ⁇ l HEPES/NaCl, 24 ⁇ l 100mM water-soluble vitamin E Trolox (configured using 2-(N-morpholine)ethanesulfonic acid, that is, MES buffer system, pH 6.1), 10 ⁇ l triethylenediamine DABCO (configured using MES, pH6.1), 8 ⁇ l 2M glucose, 20 ⁇ l NaI (50mM, configured with water) and 4 ⁇ l glucose oxidase "glucose oxidase” were tested (hereinafter referred to as the "basic formula"); specifically, the same optical imaging system was used Including the same light intensity and exposure time, multiple imaging of multiple views of a batch of solid-phase substrates with nucleic acid molecules to be tested attached to the surface obtained from the same processing method, including optimizing the redox system components Trolox and/or or the concentration of DABCO.
  • the results are basically as disclosed in the literature.
  • the application value of the obtained sequencing results is low, for example, the read length is short and the error rate is high; for example, after changing the fluorescent dye Atto647N on the reaction substrate nucleotide to Atto532, the formula Instead of enhancing the brightness of Att532, it reduces its brightness and stability.
  • the inventor when conducting component concentration optimization tests on the above-mentioned basic formula, the inventor also found that as the sequencing progressed, the fluorescence signal collected each time attenuated significantly. The inventor speculated that this was probably due to the solution being exposed to air. In the solution, O 2 in the air continuously reacts with the oxygen removal system in the solution to produce substances that make the solution acidic. If this continues, exceeding the buffering capacity of the buffer system causes the pH value of the solution to decrease, which significantly affects the intensity of the fluorescence signal. Therefore, the inventors believe that the buffering capacity of the buffer solution and the substance or combination of substances that affect the pH are factors that must be measured in formula optimization.
  • the inventor evaluated the chemical properties of the components NaI and DABCO in the basic formula and their influence/function in the detection solution system.
  • the main role of NaI should be as a catalyst to achieve the decomposition of H 2 O 2
  • DABCO is a commonly used quencher of singlet O and is used in fluorescence detection.
  • the basic formula that does not contain the specified concentration of DABCO is used as the "new basic formula" for formula optimization, that is, the above-listed oxidizing agents or reducing agents or their analogs/derivatives are used independently or based on function, redox Combine the capabilities and requirements for the reaction environment, add it to the new basic formula, and consider whether the solution system containing specific substances or combinations of substances is suitable for the mixed use of different water-soluble or electrical fluorescent dyes (can make the system Fluorescent dyes all have a higher signal-to-noise ratio, increase the stability of fluorescent dyes, etc.), whether they can reduce the degradation of nucleic acids, whether they have higher stability, etc., in order to obtain a basic formula that is significantly better than the basic formula or a new basic formula. Recipes for sequencing results.
  • the inventor developed the desired fluorescence detection solution system based on a new basic formula.
  • the invention provides a method for incorporating labeled nucleotides, the method comprising: (a) providing a hybridization complex substance, the so-called hybrid complex is a hybrid of a primer and a template molecule, the primer is configured to hybridize with the 3' end of the template molecule, and the template molecule is a single-stranded nucleic acid molecule; (b) combine polymerase, nucleotide analogs and The hybridization complex is placed under conditions suitable for the polymerization reaction, and an extension product is obtained by binding the nucleotide analogue to the hybridization complex.
  • the nucleotide analogue includes a connected sugar unit, a base, and a cleavable blocking group.
  • the so-called fluorescent detection reagent includes an enzymatic oxygen removal system and a variety of reducing agents, and does not include triethylenediamine (DABCO); (d) ) irradiating at least a portion of the hybridization complex and collecting at least a portion of the signal from the fluorescent label in the presence of a fluorescent detection reagent; and (e) replacing the fluorescent detection reagent with a cleavage reagent to cleave the cleavable blocking group on the extension product
  • the cleavage reagent is used to cleave the cleavable blocking group and fluorescent label of the nucleotide analogue.
  • the method includes the step of replacing the solution system of the extension step with a specific solution system (referred to here as a fluorescence detection reagent).
  • a fluorescence detection reagent referred to here as a fluorescence detection reagent.
  • the method including this step can place the fluorescent label on the template molecule/hybridization complex without affecting it.
  • a solution system that is conducive to its stable and effective luminescence, it is conducive to accurate detection of the signal from the fluorescent label. Therefore, it is conducive to accurate identification of the type of nucleotide incorporated into the template molecule based on the detected signal.
  • the solution system in which the fluorescent label is placed includes an enzymatic oxygen removal system and a variety of reducing agents and does not include DABCO.
  • This solution system can quickly and effectively remove oxygen, quench triplet fluorescent molecules by transferring electrons, etc., and avoid The reaction between triplet fluorescent molecules and oxygen molecules helps the fluorescent molecules return to the ground state, thus providing an effective and stable detection environment/imaging environment; and, the existence of this solution system can make the fluorescence detection process
  • the fluorescence signal is stable, nucleic acid damage is reduced or suppressed, and the quenching time of fluorescent molecules is extended, which is beneficial to increasing sequencing read length and improving sequencing quality.
  • the present invention also provides a composition or reagent formulation, also known as a fluorescence detection reagent, which composition or reagent method includes an enzymatic oxygen removal system and a plurality of reducing agents, and does not include DABCO.
  • a composition or reagent formulation also known as a fluorescence detection reagent
  • the composition was made by the inventor based on the above unexpected discovery and is suitable for applications involving the detection of fluorescence signals, especially applications involving the detection of single-molecule fluorescence signals.
  • composition or reagent formula in detecting fluorescence, especially in detecting single-molecule fluorescence signals, is also provided.
  • Figure 1 shows the fluorescence quenching time data of each experimental group and control group in Example 10;
  • Figure 2 shows the sequencing read length data of each experimental group and control group in Example 10;
  • Figure 3 is the CallRatio data of Experimental Group 1 and Experimental Group 2 in Example 12;
  • FIG. 4 shows the apparent read length data of each experimental group in Example 14
  • Figure 5 shows the changes in pH value and dissolved oxygen value DO over time after opening the bottle of the fluorescence detection reagent in the control group in Example 16 (left picture), and the imaging quality picture after 45 hours (right picture);
  • Figure 6 shows the changes in pH value and dissolved oxygen value DO over time after opening the bottle of the fluorescence detection reagent of Experiment Group 1 in Example 16 (left picture), and the imaging quality picture after 45 hours (right picture);
  • Figure 7 shows the changes in pH value and dissolved oxygen value DO over time after opening the bottle of the fluorescence detection reagent of Experiment Group 2 in Example 16 (left picture), and the imaging quality picture after 45 hours (right picture);
  • Figure 8 shows the changes in pH value and dissolved oxygen value DO over time after opening the bottle of the fluorescence detection reagent of Experiment Group 3 in Example 16 (left picture), and the imaging quality picture after 45 hours (right picture);
  • Figure 9 is the quenching curve of each experimental group in which different concentrations of ascorbic acid were added in Example 18;
  • Figure 10 is the quenching time data of each experimental group in which different concentrations of ascorbic acid were added in Example 18;
  • Figure 11 is the sequencing read length distribution data of the experimental groups added with 0mM and 20mM ascorbic acid respectively in Example 18;
  • Figure 12 is the read length distribution data of each experimental group in which different concentrations of ascorbic acid were added in Example 18;
  • Figure 13 is a comparison of the imaging quality of experimental group 1 (right picture) and control group (left picture) in Example 22;
  • Figure 14 is the fluorescence intensity data corresponding to the fluorescence detection reagents under different pH conditions in Example 28;
  • Figure 15 is the apparent sequence read length corresponding to the fluorescence detection reagent under different pH conditions in Example 28;
  • Figure 16 is the quenching curve corresponding to the fluorescence detection reagent added with different concentrations of BME in Example 30;
  • Figure 17 shows the quenching time corresponding to the fluorescence detection reagents added with different concentrations of BME in Example 30;
  • Figure 18 is the quenching curve corresponding to the fluorescence detection reagent added with different concentrations of DTT in Example 30;
  • Figure 19 is the quenching time corresponding to the fluorescence detection reagent added with different concentrations of DTT in Example 30;
  • Figure 20 shows the quenching time corresponding to the fluorescence detection reagents adding different concentrations of AA and MV under the same ratio conditions in Example 30;
  • Figure 21 shows the quenching time corresponding to the fluorescence detection reagents added with different proportions of AA and MV in Example 30;
  • Figure 22 shows the quenching time corresponding to the fluorescence detection reagents added with different proportions of AA and MV in Example 30;
  • Figure 23 is the sequencing read length distribution corresponding to the fluorescence detection reagents with or without the addition of AA and MV in Example 30;
  • Figure 24 shows the fluorescence brightness corresponding to the fluorescence detection reagents with or without the addition of AA and MV in Example 30;
  • Figure 25 shows the mapping results corresponding to the fluorescence detection reagents with or without the addition of AA and MV in Example 30.
  • nucleotide refers to the four natural nucleotides (such as dATP, dCTP, dGTP and dTTP or ATP, CTP, GTP and UTP) or their derivatives, sometimes also directly by the base they contain. Represented by base (A, T or U, C and G). Those of ordinary skill in the art will know the nucleotides or bases referred to by the expressions shown from the context.
  • sequence determination is sequence determination, which is the same as “nucleic acid sequencing” or “gene sequencing” and refers to the determination of the base order in the nucleic acid sequence; including sequencing by synthesis (sequencing by synthesis, SBS) and/or ligation Sequencing (sequencing while ligation, SBL); including DNA sequencing and/or RNA sequencing; including long fragment sequencing and/or short fragment sequencing.
  • long fragments and short fragments are relative, such as longer than 1Kb, 2Kb, 5Kb or Nucleic acid molecules of 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; including paired-end sequencing, single-end sequencing and/or paired-end sequencing, etc., the so-called paired-end sequencing or paired-end sequencing can refer to The readout of any two non-completely overlapping segments or portions of the same nucleic acid molecule.
  • sequencing includes the process of binding nucleotides (including nucleotide analogs) to templates and collecting corresponding reaction signals.
  • nucleotides including nucleotide analogs
  • the order of multiple nucleotides/bases on the template is generally determined through multiple rounds of sequencing.
  • a cycle of sequencing also called a sequencing round, can be defined as a base extension of four nucleotides/bases. In other words, it can be defined as the process of completing the determination of the base type at any specified position on the template.
  • one round of sequencing may include the process of binding four nucleotides to a so-called template at a time and collecting the corresponding reaction signals; for platforms that implement sequencing based on polymerization reactions, the reaction The system includes reaction substrate nucleotides, polymerase and template, so that a preset sequence (sequencing primer) is combined with the template.
  • the added reaction substrate polymerizes Under the catalysis of enzyme, it is controllably connected to the 3' end of the sequencing primer to achieve base pairing with the corresponding position of the template; usually, a round of sequencing can include one or more base extensions (repeat), for example, four Each nucleotide is added to the reaction system in sequence, Base extension and corresponding reaction signal collection are performed separately.
  • One round of sequencing includes four base extensions and four signal collections; for another example, four nucleotides are added to the reaction system in any combination, such as a combination of two or one. Three combinations, two combinations perform base extension and corresponding reaction signal collection respectively.
  • One round of sequencing includes two base extensions and four signal collections; for another example, four nucleotides are added to the reaction system at the same time for base extension. Base extension and reaction signal collection, one round of sequencing includes one base extension and four signal collections.
  • Sequencing can be performed through a sequencing platform, which can be selected but is not limited to Illumina's Hiseq/Miseq/Nextseq/Novaseq sequencing platform, Thermo Fisher/Life Technologies' Ion Torrent platform, BGI's BGISEQ and MGISEQ/DNBSEQ platforms, and single Molecular sequencing platform; the sequencing method can choose single-end sequencing or paired-end sequencing; the sequencing results/data obtained are the fragments read out, which are called reads, and the length of the reads is called the read length.
  • a sequencing platform which can be selected but is not limited to Illumina's Hiseq/Miseq/Nextseq/Novaseq sequencing platform, Thermo Fisher/Life Technologies' Ion Torrent platform, BGI's BGISEQ and MGISEQ/DNBSEQ platforms, and single Molecular sequencing platform; the sequencing method can choose single-end sequencing or paired-end sequencing; the sequencing results/data obtained are the fragments read out, which are called reads, and the length
  • the so-called single molecule detection includes single molecule sequencing, which is a detection that does not involve amplification of the signal to be tested, such as a sequencing platform that does not involve amplification of the nucleic acid molecule to be tested.
  • the molecule to be tested exists in the physical form of a single molecule or a few molecules. , reflected in the signal collection results such as images, is a weak, relatively unstable signal that is easily disturbed/submerged.
  • the so-called “single molecule” refers to one or a few molecules, generally no more than 10, such as 1, 2, 3 or 5 molecules.
  • solid substrate can be any solid support that can be used to immobilize nucleic acid sequences, such as nylon membranes, glass sheets, plastics, silicon wafers, magnetic beads, etc.; sometimes also called reactors, chips or flow pool.
  • a method for incorporating labeled nucleotides including the following steps: (a) providing a hybridization complex, which is a hybrid of a primer and a template molecule, and the primer is configured to The 3' end of the molecule is hybridized, and the template molecule is a single-stranded nucleic acid molecule; (b) placing the polymerase, nucleotide analog and hybridization complex under conditions suitable for the polymerization reaction, by binding the nucleotide analog to the hybrid The complex is used to obtain an extension product, and the nucleotide analog includes connected sugar units, bases, cleavable blocking groups and fluorescent labels; (c) replace the solution system of (b) with a fluorescent detection reagent, and the fluorescent detection reagent including an enzymatic oxygen removal system and a plurality of reducing agents and excluding triethylenediamine; (d) in the presence of a fluorescent detection reagent, irradiating at least a portion of the hybridization
  • Nucleotides used in SBS sequencing generally include the use of modified nucleotides, which are also often called "nucleotide analogs". Nucleotide analogues used in SBS sequencing are usually also called terminators. In the current development stage, reversible terminators are usually used, which are generally called reversible terminators. In addition to containing nucleotides, reversible terminators generally It contains a phosphate group, a pentose sugar and a base connected in sequence, and usually also contains a removable blocking group and a removable detectable label. The removable blocking group can reversibly prevent subsequent reversible terminators or cores. The nucleotide is incorporated/binded to the next position on the template, and the detectable label enables the reversible terminator to generate a signal that can be detected upon incorporation/binding into the current position of the template.
  • the reported reversible terminators can be divided into three categories: First, the blocking group is located at the 3'-OH of the pentose, that is, the 3'-O-blocking group of the pentose, so that A phosphodiester bond cannot be formed, and the blocking group is, for example, azide; the second is that the blocking group is located on the phosphate side, making it impossible to form a phosphodiester bond; the third is that the blocking group is located on the base side of the nucleotide, In a solution environment, the formation of phosphodiester bonds is blocked based on charge and/or steric hindrance to achieve polymerization blocking. This type of reversible terminator is also often called a virtual terminator.
  • the nucleotide analogs include four nucleotide analogs: dATP, dUTP or dTTP, dCTP and dGTP. Two of the four nucleotide analogs carry fluorescent labels There is a fluorescent label Y, and the fluorescent label X and the fluorescent label Y are two fluorescent labels with different emission wavelengths.
  • the polymerization reaction in (b) includes two nuclei with different fluorescent labels among the four nucleotide analogs. Glycoside analogues.
  • the nucleotide analogs include four nucleotide analogs of dATP, dUTP or dTTP, dCTP and dGTP, and the four nucleotide analogs carry fluorescent labels with four different emission wavelengths, (b) The polymerization reaction in contains four nucleotide analogs.
  • the blocking group and the fluorescent label on the nucleotide analog are located on the same side of the base. More specifically, in one example, the 3' position of the sugar unit on the nucleotide analog is -OH, that is, the 3' position of the pentose sugar is in its natural state.
  • This nucleotide analog can block the incorporation/binding of nucleotides into the next position of the template through non-physical blocking methods such as steric hindrance and/or charge effects of the molecule.
  • the specific structure is For example, see the content disclosed in WO2019105421A1.
  • the blocking group and the fluorescent label on the nucleotide analog are located on different sides of the base. More specifically, the blocking group on the nucleotide analog is located at the 3' position of the sugar unit, that is, the pentose 3'-OH of the nucleotide analog is modified into a 3'-O-blocking group , the specific structure can be found in the content disclosed in US7057026B2, for example.
  • the above-mentioned method for incorporating labeled nucleotides further includes the following steps: (f) performing (b)-(e) at least once, and the specific number of times can be determined as needed.
  • (b)-(e) is performed, that is, a base extension or a round of sequencing reactions is performed for no less than 20, 30, 50, 100 or 150 times, etc., to detect and read a sequence of a certain length. (reads, reads) to meet the requirements of application testing for various purposes.
  • the hybridization complex is connected to the substrate surface, and (d) includes irradiating the substrate surface with light of a specific wavelength to excite a fluorescent label on the substrate surface to emit fluorescence, and collecting at least a portion of the fluorescence from the fluorescent label.
  • a specific wavelength to excite a fluorescent label on the substrate surface to emit fluorescence
  • collecting at least a portion of the fluorescence from the fluorescent label can be understood that the above-mentioned specific wavelengths can be adjusted according to the excitation wavelengths of different fluorescent labels.
  • the wavelength range used in common fluorescence signal imaging detection is 500nm-700nm.
  • Optional fluorescent dyes or dye combinations suitable for this excitation wavelength range can choose Cy3, Alexafluor 532, HEX, Atto 532, ROX, Alexafluorosis 630, Cy5, Atto647N, BODIPY650, Cy 5.5, IF700 and Alex680.
  • ATTO647N has an excitation wavelength of 646nm and an emission wavelength of 664nm
  • ATTO532 has an excitation wavelength of 532nm and an emission wavelength of 552nm
  • CY5 has an excitation wavelength of 651nm and an emission wavelength of 670nm
  • IF700 has an excitation wavelength of 690nm and an emission The wavelength is 713nm
  • the excitation wavelength of ROX is 578nm and the emission wavelength is 604nm
  • the excitation wavelength of Alexa Fluor532 is 534nm and the emission wavelength is 553nm.
  • the fluorescent label includes at least one of the combination ROX, ATTO532 and Alexa fluor532, and at least one of the combination CY5, IF700 and ATTO647N. That is, a base extension or sequencing round involves collecting signals from two or more fluorescent labels.
  • the fluorescent label is selected from a combination of fluorescent dyes with excitation wavelengths around 550 nm and around 660 nm.
  • fluorescent markers include ATTO532 and ATTO647N.
  • the enzymatic oxygen scavenging system is selected from combination I, combination II, or combination III, combination I includes glucose and glucose oxidase, combination II includes glucose, glucose oxidase, and catalase, and combination III includes protozoa Theophylline and protocatechuic acid 3,4-dioxygenase.
  • the detection solution containing any of the enzymatic oxygen removal systems can effectively remove oxygen in the solution and reduce the impact of singlet oxygen on the luminescence of fluorescent dyes.
  • the reducing agent is selected from at least two of ascorbic acid, gallic acid, analogs or derivatives of gallic acid, cyanuric acid, and water-soluble vitamin E or derivatives of water-soluble vitamin E.
  • the so-called analogs or derivatives of gallic acid are, for example, lower alkyl esters of gallic acid, such as methyl gallate, ethyl gallate, propyl gallate or combinations thereof.
  • the signal from the fluorescent label is collected by a camera to obtain an image; understandably, at least part of the signal from the fluorescent label appears as a bright spot on the image.
  • the so-called “bright spots” (dots, spots or peaks) on the image also known as “bright spots” or “light spots”, refer to the positions on the image where the signal is relatively strong. For example, the signal at this position is stronger than the surrounding ones. Appears as a relatively bright spot or point on the image, and a bright spot occupies one or more pixels.
  • the signal corresponding to the bright spot may come from target molecules or non-target substances. Detection of "bright spots” involves detection of optical signals from target molecules such as extended bases or clusters of bases.
  • the fluorescent detection reagent includes combination I, ascorbic acid and gallic acid or an analog or derivative of gallic acid.
  • Combining ascorbic acid and gallic acid can improve the detected fluorescence signal intensity and imaging quality score (image score/imagescore), make the imaging quality more stable during the signal acquisition process, and reduce the decline in imaging quality during the signal acquisition process. , therefore, better detection results can be obtained.
  • the fluorescence detection reagent of this embodiment uses glucose and glucose oxidase as the oxygen removal system, which has a fast oxygen removal speed, and is combined with ascorbic acid as a reducing agent to quench the triplet fluorescent dye by transferring electrons to avoid its interaction with
  • the reaction of oxygen molecules helps the fluorescent dye return to the ground state, thus forming an effective and stable detection solution system that can be stable during the fluorescence detection process.
  • Fluorescent signals reduce or inhibit nucleic acid damage, extend the quenching time of fluorescent molecules, thereby increasing sequencing read length and improving sequencing quality.
  • Imaging quality refers to passing in the fluorescence detection reagent, observing the image, and conducting preliminary analysis such as statistical brightness and image score value through a small program. This embodiment does not limit the evaluation method of imaging quality (image quality). Expectations can be established based on a priori data or based on theory, giving relatively high scores to those that meet or are closer to expectations, and giving relatively low scores to those that do not meet or are far from expectations. For example, the identification or detection of bright spots on an image and the evaluation of image quality can be performed with reference to the method disclosed in CN112285070A.
  • the fluorescent detection reagent includes 50mM ⁇ 300mM glucose, 2U/mL ⁇ 20U/mL glucose oxidase, 1mM ⁇ 200mM ascorbic acid, and 1mM ⁇ 20mM gallic acid or analogs or derivatives of gallic acid.
  • the fluorescence detection reagent includes 80mM ⁇ 150mM glucose, 8U/mL ⁇ 12U/mL glucose oxidase, 1mM ⁇ 50mM ascorbic acid, and 1mM ⁇ 10mM gallic acid or gallic acid analogs or derivatives.
  • the fluorescence detection reagent also includes water-soluble vitamin E (Trolox) or its derivatives, or a combination of water-soluble vitamin E or its derivatives and quinone derivatives.
  • Trolox water-soluble vitamin E
  • Tests have found that water-soluble vitamin E or its derivatives have a protective effect on fluorescent groups, but freshly prepared fluorescence detection reagents added with water-soluble vitamin E or its derivatives have poor imaging performance for fluorescent dyes such as Atto532, and after Stored fluorescent detection reagents do not have this problem.
  • the inventor speculates that this may be because the protective effect of water-soluble vitamin E or its derivatives on the fluorescent group requires the combined action of reduced Trolox and its oxidized state TX-quinone (TQ). Therefore, it is speculated that Trolox in the newly prepared fluorescence detection reagents is mostly in a reduced state and lacks TQ, which cannot create good conditions for the excitation of fluorescent dyes.
  • the inventor adjusted the method of preparing a solution containing Trolox or its derivatives, for example, increasing UV irradiation or increasing the standing time in a certain air; for another example, using a combination of Trolox and quinone compounds, And adjust the ratio of trolox or its derivatives to quinone compounds to enhance the brightness of the dye in the detection solution, control the stability of the detection solution system, and enhance the realization of the function/effect of the detection solution.
  • a certain proportion of quinone derivatives such as p-benzoquinone is added to freshly prepared fluorescence detection reagents containing Trolox or its derivatives, so that p-benzoquinone can simulate the oxidized state of Trolox, thereby enhancing fluorescence. group signal intensity, thereby helping to improve imaging quality, sequencing read length, and reduce error rates.
  • the fluorescence detection reagent also includes water-soluble vitamin E and p-benzoquinone.
  • the concentration of water-soluble vitamin E is 6mM-12mM, and the concentration of p-benzoquinone is 0.36mM-0.96mM.
  • the fluorescent detection reagent further includes cyanuric acid.
  • cyanuric acid free in solution, can react with oxygen free radicals prior to the molecules to be measured, such as nucleic acids on the surface of the chip, and can consume the oxygen free radicals generated after laser irradiation, weakening or avoiding damage to the nucleic acid chain. This can reduce base misidentifications.
  • the mass percentage of cyanuric acid is 0.0001% to 0.001%.
  • the mass percentage of cyanuric acid is 0.0003% to 0.0009%.
  • the fluorescence detection reagent further includes at least one of adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, and thymidine monophosphate.
  • adenosine monophosphate cytidine monophosphate
  • guanosine monophosphate uridine monophosphate
  • thymidine monophosphate adenosine monophosphate
  • these components are free in the solution independently or in combination, and can react with oxygen free radicals in preference to the molecules to be measured, and can consume the oxygen free radicals generated after laser irradiation, weakening or avoiding Damage to nucleic acid strands helps reduce sequencing error rates.
  • the fluorescence detection reagent also includes 1 ⁇ M ⁇ 50 ⁇ M 5'-adenylate and 1 ⁇ M ⁇ 50 ⁇ M guanosine-5'-monophosphate. In this way, images with better quality can be obtained, and sequencing results with better quality can be obtained based on these images.
  • the reagent components in the fluorescence detection reagent include Tris (trishydroxymethylaminomethane) buffer system or HEPES buffer system, and the pH of the fluorescence detection reagent is 6.5-8.5.
  • Tris trishydroxymethylaminomethane
  • HEPES buffer system HEPES buffer system
  • the pH of the fluorescence detection reagent is 6.5-8.5.
  • a certain concentration of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid can maintain the pH above 7 for a long time, thereby improving the stability of the fluorescence detection reagent.
  • Tris trishydroxymethylaminomethane
  • the fluorescence detection reagent also includes acetonitrile (ACN), and the volume percentage of acetonitrile is 5% to 40%, preferably 18% ⁇ 35%.
  • ACN acetonitrile
  • the volume percentage of acetonitrile is 5% to 40%, preferably 18% ⁇ 35%.
  • Trolox was dissolved in acetonitrile and left in the air for more than 10 hours before adding other components to prepare a p-benzoquinone-free fluorescence detection solution I.
  • the preparation contains the same concentration of Trolox, 0.36mM to 0.96mM, such as 0.52mM of p-benzoquinone, and other components at the same concentration as I without placing it in the air to obtain the fluorescence detection solution II.
  • Fluorescence detection solutions I and II were both used for sequencing, and the results showed that both formulations were able to obtain better sequencing results and were equally effective.
  • the formulas in some of the above embodiments can enhance the signals of some of the fluorescent dyes, while at the same time weakening the signals of other fluorescent dyes.
  • SBS sequencing of multiple fluorescent dyes can be effectively realized. Relatively speaking, it is basically possible to obtain read lengths with application value and the error rate is not high.
  • the fluorescence detection reagent includes the following components: 50mM ⁇ 200mM glucose, 2U/mL ⁇ 20U/mL glucose oxidase, 1mM ⁇ 200mM ascorbic acid, 1mM ⁇ 10mM gallic acid, 6mM ⁇ 12mM water-soluble vitamin E, 0.1 mM ⁇ 1.0mM p-benzoquinone, 0.0001wt% ⁇ 0.001wt% cyanuric acid, 1mM ⁇ 20 ⁇ M 5'-adenylate, 1 ⁇ M ⁇ 20 ⁇ M guanosine-5'-monophosphate, 100mM ⁇ 300mM trishydroxymethylamino acid Methane and 10v/v% ⁇ 40v/v% acetonitrile.
  • the fluorescence detection reagent includes the following components: 80mM ⁇ 150mM glucose, 8U/mL ⁇ 12U/mL glucose oxidase, 1mM ⁇ 50mM ascorbic acid, 1mM ⁇ 10mM gallic acid, 6mM ⁇ 12mM water-soluble vitamins E. 0.36mM ⁇ 0.96mM p-benzoquinone, 0.0003wt% ⁇ 0.0009wt% cyanuric acid, 4mM ⁇ 12 ⁇ M 5'-adenylate, 4 ⁇ M ⁇ 12 ⁇ M guanosine-5'-monophosphate, 100mM ⁇ 300mM trihydroxy Methyl aminomethane and 18v/v% ⁇ 35v/v% acetonitrile.
  • the volume percentage of acetonitrile is 28% to 35% to obtain high-quality detection results.
  • the fluorescence detection reagent includes the following components: 80mM ⁇ 150mM glucose, 8U/mL ⁇ 12U/mL glucose oxidase, 1mM ⁇ 50mM ascorbic acid, 1mM ⁇ 15mM gallic acid or ethyl gallate or propyl gallate , 6mM ⁇ 15mM water-soluble vitamin E, 0.36mM ⁇ 0.96mM p-benzoquinone, 0.0003wt% ⁇ 0.0009wt% cyanuric acid, 4mM ⁇ 12 ⁇ M 5'-adenylate, 4 ⁇ M ⁇ 12 ⁇ M guanosine-5'-mono Phosphoric acid, 100mM ⁇ 300mM trishydroxymethylaminomethane and 18v/v% ⁇ 35v/v% acetonitrile.
  • the reaction time usually lasts for several hours, for example, more than 12 hours.
  • the solution reagents are mixed in advance and then used for sequencing. As the sequencing proceeds, the performance of the mixed reagents exposed to the air will change. It will gradually decay, making it difficult to meet the sequencing requirements.
  • a premixed kit since its components contain a large amount of reducing substances, they are gradually oxidized during the storage process. Finally, the reducing components in the components are fully or partially oxidized, making it difficult to use the kit after storage for a certain period of time. It can also exert its performance in detection.
  • the fluorescent detection reagent also includes liquid paraffin and/or silicone oil. Sealing and storing with liquid paraffin or silicone oil can ensure the stability of the fluorescence detection reagent for a longer period of time. Moreover, it is convenient for reagent transportation.
  • inert substances such as silicone oil or liquid paraffin are added, for example, 8-20 v/v%. Since the density of any two substances is less than the density of the aqueous solution, the substance(s) can separate the air and the solution system, thus preventing or reducing the oxygen in the air from entering the solution and consuming it.
  • the oxygen scavenging reagent in the solution causes changes in reducing substances and/or undesirable reactions with substances or intermediate products in the solution, changes the pH of the solution and/or affects the performance of the solution system, etc. Therefore, the stability of the detection solution during use can be increased and the shelf life of the detection solution can be extended.
  • the irradiation time is 10-100 milliseconds, preferably, the irradiation time is 50-100 milliseconds.
  • the light intensity for irradiating ATTO532 is 30-40 milliwatts, and the light intensity for irradiating ATTO647N is 60-80 milliwatts; and/or, the duration of irradiating ATTO532 or ATTO647N in one field of view is is 50-500 milliseconds.
  • the duration of irradiating ATTO532 or ATTO647N in one field of view is 50-100 milliseconds.
  • the fluorescence detection reagent according to one embodiment of the present invention includes an enzymatic oxygen removal system and a variety of reducing agents, and does not include triethylenediamine.
  • the enzymatic oxygen scavenging system is selected from combination I, combination II, or combination III, combination I includes glucose and glucose oxidase, combination II includes glucose, glucose oxidase, and catalase, and combination III includes protozoa Theophylline and protocatechuic acid 3,4-dioxygenase.
  • the plurality of reducing agents are selected from at least two of ascorbic acid, gallic acid, analogs or derivatives of gallic acid, cyanuric acid, and water-soluble vitamin E.
  • the fluorescent detection reagent includes combination I, ascorbic acid and gallic acid or an analog or derivative of gallic acid.
  • the fluorescence detection reagent of this embodiment uses glucose and glucose oxidase as the oxygen removal system, which removes oxygen quickly. It is combined with ascorbic acid as a reducing agent to quench the triplet fluorescent dye by transferring electrons to avoid its reaction with oxygen molecules. , helps the fluorescent dye return to the ground state, thereby forming an effective and stable detection solution system, which can stabilize the fluorescence signal, reduce or inhibit nucleic acid damage, prolong the quenching time of fluorescent molecules, and thereby increase Sequencing read length improves sequencing quality.
  • Imaging quality refers to the introduction of fluorescent detection reagents, observation of single molecule images, and preliminary analysis through small programs such as statistical brightness and image score values.
  • the fluorescence detection reagent includes 50mM to 200mM glucose, 2U/mL to 20U/mL glucose oxidase, 1mM to 200mM ascorbic acid, and 1mM to 10mM gallic acid or gallic acid analogs or derivatives.
  • the fluorescence detection reagent includes 80mM to 150mM glucose, 8U/mL to 12U/mL glucose oxidase, 1mM to 50mM ascorbic acid, and 1mM to 10mM gallic acid or gallic acid analogs or derivatives.
  • the fluorescent detection reagent also includes water-soluble vitamin E (Trolox) and p-benzoquinone.
  • water-soluble vitamin E has a protective effect on fluorescent groups, but freshly prepared fluorescence detection reagents added with water-soluble vitamin E have poor imaging performance for fluorescent dyes such as Atto532, while stored fluorescence detection reagents do not have this question.
  • the protective effect of water-soluble vitamin E on the fluorescent group requires the combined action of reduced Trolox and its oxidized state TX-quinone (TQ). Therefore, it is speculated that Trolox in the newly prepared fluorescence detection reagents is mostly in a reduced state and lacks TQ, which cannot create good conditions for the excitation of fluorescent dyes.
  • the concentration of water-soluble vitamin E is 6mM ⁇ 12mM, and the concentration of p-benzoquinone is 0.1mM ⁇ 1mM; preferably, the concentration of p-benzoquinone is 0.36mM ⁇ 0.96mM.
  • the fluorescent detection reagent further includes cyanuric acid.
  • cyanuric acid is free in the solution and reacts with oxygen free radicals prior to the molecules to be measured, such as nucleic acids on the surface of the chip. It can consume the oxygen free radicals generated after laser irradiation, weaken or avoid damage to the nucleic acid chain, thereby reducing Misidentification of nucleic acids in sequencing technologies.
  • the mass percentage of cyanuric acid is 0.0001% to 0.001%; preferably, the mass percentage of cyanuric acid is 0.0003% to 0.0009%.
  • the fluorescent detection reagent further includes adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, uridine monophosphate, and At least one kind of thymidine monophosphate. Similar to cyanuric acid, the inventors speculate that these components are free in the solution and react with oxygen free radicals prior to the molecules to be measured. They can consume the oxygen free radicals generated after laser irradiation and weaken or avoid damage to the nucleic acid chain. Helps reduce sequencing error rates.
  • the fluorescent detection reagent also includes 5'-adenylate and guanosine-5'-monophosphate.
  • the fluorescence detection reagent also includes 1mM ⁇ 20 ⁇ M 5'-adenylate and 1 ⁇ M ⁇ 20 ⁇ M guanosine-5'-monophosphate.
  • the fluorescence detection reagent also includes 4mM ⁇ 12 ⁇ M 5'-adenylate and 4 ⁇ M ⁇ 12 ⁇ M guanosine-5'-monophosphate.
  • the fluorescence detection reagent uses a Tris (trihydroxymethylaminomethane) buffer system or a HEPES buffer system, and the pH value of the fluorescence detection reagent is 6.5-8.5. Preferably, the pH value of the fluorescence detection reagent is 8.5.
  • the concentration of trishydroxymethylaminomethane or HEPES is 80mM ⁇ 120mM.
  • glucose oxidase and glucose were used as the oxygen removal system to determine that the oxidation of glucose into gluconic acid was the cause of the pH decrease.
  • glucose was exhausted and dissolved oxygen increased.
  • a certain concentration of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) can maintain the pH above 7 for a long time, thereby improving the stability of the fluorescence detection reagent.
  • Tris trishydroxymethylaminomethane
  • the fluorescence detection reagent further includes acetonitrile, and the volume percentage of acetonitrile is 10% to 40%; preferably, the volume percentage of acetonitrile is 18% to 35%; more preferably, the volume percentage of acetonitrile is 28%. ⁇ 35%.
  • the combination of Tris and ACN performs better in terms of fluorescence signal intensity, imaging quality, and error rate.
  • the fluorescence detection reagent includes the following components: 50mM ⁇ 200mM glucose, 2U/mL ⁇ 20U/mL glucose oxidase, 1mM ⁇ 200mM ascorbic acid, 1mM ⁇ 10mM gallic acid, 6mM ⁇ 12mM water-soluble vitamin E, 0.1 mM ⁇ 1.0mM p-benzoquinone, 0.0001wt% ⁇ 0.001wt% cyanuric acid, 1mM ⁇ 20 ⁇ M 5'-adenylate, 1 ⁇ M ⁇ 20 ⁇ M guanosine-5'-monophosphate, 100mM ⁇ 300mM trishydroxymethylamino acid Methane and 18v/v% ⁇ 35v/v% acetonitrile.
  • the fluorescence detection reagent also includes a monovalent soluble salt such as sodium chloride, combined with a buffer component.
  • the fluorescence detection reagent includes the following components: 80mM ⁇ 150mM glucose, 8U/mL ⁇ 12U/mL glucose oxidase, 1mM ⁇ 50mM ascorbic acid, 1mM ⁇ 10mM gallic acid, 6mM ⁇ 12mM water-soluble vitamin E, 0.36mM ⁇ 0.96mM p-benzoquinone, 0.0003wt% ⁇ 0.0009wt% cyanuric acid, 4mM ⁇ 12 ⁇ M 5'-adenylate, 4 ⁇ M ⁇ 12 ⁇ M guanosine-5'-monophosphate, 100mM ⁇ 300mM trihydroxymethyl Aminomethane and 18v/v% ⁇ 35v/v% acetonitrile.
  • a method for preparing the above-mentioned fluorescence detection reagent according to an embodiment of the present invention includes the following steps: before encapsulating the fluorescence detection reagent, add liquid paraffin and/or silicone oil.
  • a kit according to an embodiment of the present invention includes the above-mentioned fluorescence detection reagent and the above-mentioned nucleotide analogue. It can be understood that the above-mentioned fluorescence detection reagents can be used not only for sequencing, but also for other methods or products that require fluorescence detection.
  • FIG. 1 shows the detection data of different enzymatic deoxygenation systems.
  • an oxygen removal system using glucose and glucose oxidase as fluorescence detection reagents can prolong the quenching time of fluorescent molecules.
  • HEPES easily generates hydrogen peroxide under light, which may accelerate the quenching of fluorescent molecules and increase the photochemical reaction of biological macromolecules. Therefore, in principle, it is more reasonable to replace HEPES with trishydroxymethylaminomethane (Tris).
  • Tris trishydroxymethylaminomethane
  • Figure 2 shows that there is no significant difference in the sequencing read length between experimental group 1 and experimental group 2.
  • Figure 1 shows that the average quenching time of experimental group 2 is 99s. Under the extreme exposure time of 0.5s, the fluorescence detection reagent using Tris instead of HEPES will have 0.6% of fluorescent molecules quenched, and the quenching ratio is small.
  • the fluorescence quantum efficiency of fluorescent molecules is also affected by the polarity of the solvent. Therefore, different proportions of acetonitrile may cause different fluorescence quantum efficiencies, thereby presenting different fluorescence signal intensities. As shown in the table below, this embodiment adjusts the final concentration of acetonitrile in the fluorescence detection reagent, and compares the effects of different concentrations of acetonitrile on sequencing read length, imaging quality, and sequencing error rate.
  • the sequencing involves two fluorescent dyes, Atto532 and Atto647N.
  • Figure 9 shows the quenching curve of Atto532 after adding different concentrations of AA. It can be seen from the curve that as the concentration of AA increases, the quenching curve decreases more slowly. The quenching curve of 50mM AA decreases the slowest. Combined with Figure 10 It can be seen from the quenching time that the quenching time of the control group without adding AA is within 5s, but after adding AA, the quenching time is extended. As the AA concentration increases, the quenching time of the 50mM AA group reaches 30s, that is, AA helps Extend the quenching time of Atto532. Moreover, as the concentration of AA increases, the sequencing read length increases.
  • Figure 11 shows the distribution of sequencing read lengths in the 20mM AA group and the control group (0mM AA). From the read length peak, after adding 20mM AA, the main peak is higher, and the short video segment reduction.
  • Figure 12 shows the read length distribution of the groups adding different concentrations of ascorbic acid. It can be seen that the 40mM AA group and the 50mM AA group are better concentration ranges. The read length increases by 1bp, the main peak is more prominent, and the small fragments are significantly reduced. From the above studies, it can be seen that as the concentration of ascorbic acid continues to increase, the stability of the system can be further increased. The above results prove that adding AA can help prolong the quenching time of fluorescent molecules and increase the sequencing read length.
  • the optimal concentration range is 40mM to 50mM.
  • the fluorescence detection reagents of the experimental group and the control group were prepared separately. The difference between the two was that the experimental group added ascorbic acid and gallic acid at the same time, while the control group added ascorbic acid without adding gallic acid (GA).
  • the fluorescence detection reagents of the experimental group and the control group were used for sequencing, and data such as fluorescence signal intensity and imaging quality scores were compared.
  • the imaging quality of Atto532 using the fluorescence detection reagent of experimental group 1 is significantly better than that of the fluorescence detection reagent of the control group (left picture).
  • 6-12mM water-soluble vitamin E combined with 0.36-0.96mM p-benzoquinone can obtain better imaging quality, read length and relatively low
  • 6mM water-soluble vitamin E combined with 0.96mM p-benzoquinone is the optimal condition. Therefore, experiments have proven that the combination of water-soluble vitamin E and p-benzoquinone can help improve imaging quality, sequencing read length and reduce error rates.
  • the optimal concentration range is 6-12mM water-soluble vitamin E combined with 0.36-0.96mM p-benzoquinone. .
  • each group of fluorescence detection reagents Prepare each group of fluorescence detection reagents according to the formula shown in the table below. The difference is that each group adds different proportions of cyanuric acid or does not add cyanuric acid. Sequencing was performed using the fluorescent detection reagents of each group, and the error rate data were compared.
  • the Atto647N-labeled G terminator is dissolved in Tris solutions with different pH levels, and the different solutions are placed in quartz cavities, and a 640 nm laser with constant light intensity is used for excitation and fluorescence intensity is measured.
  • a 640 nm laser with constant light intensity is used for excitation and fluorescence intensity is measured.
  • the higher the pH value the higher the fluorescence intensity of Atto647N.
  • the apparent sequencing read length corresponding to the fluorescence detection reagent after raising the pH and without raising the pH is compared.
  • the sequencing read length corresponding to the fluorescence detection reagent at pH 8.5 has a slight improvement, and the advantage is not obvious.
  • the oxygen removal system will cause the pH of the solution to further drop during the oxygen removal process, thereby affecting the oxygen removal rate, it is recommended to increase the pH of the fluorescence detection reagent to 8.5. Therefore, experiments have proven that the fluorescence detection reagent is in the range of pH 6.5 to 8.5. The higher the pH value, the higher the fluorescence intensity of Atto647N, and it is more conducive to the stability of the oxygen removal system.
  • the quenching curve reflects the quenching situation of a base fluorescent dye. The faster the quenching curve decreases and the shorter the quenching time, the worse the stability of the fluorescent dye in the imaging system. On the contrary, the stability is better; Quenching time definition: refers to the time corresponding to the quenching of the number of spots (fluorescence signal) reduced to 50%.
  • Target sequence that reacts with the base to be tested in the first round as the target to be tested (template molecule with a known sequence);
  • the additives obtained through quenching curve screening were used to verify their impact on sequencing quality through two channel sequencing (two-color sequencing).
  • the test process base polymerization/extension reaction - photo collection under fluorescent detection reagent - shearing process , based on this cycle, 60 cycles of synthetic target sequencing or 80 cycles of biological sample sequencing are performed.
  • Substrate modified nucleotides/transformed nucleotides, also known as reversible terminators, which are nucleotides with fluorescent molecules that can inhibit other nucleotides from binding to the next position of the template to be tested
  • configuration Prepare respectively 50-150nM such as 125nM C_Atto647N (dCTP with Atto647N), 50-150nM such as 125nM T_Atto647N (dTTP with Atto647N), 100-200 such as 200nM A_Atto647N (dATP with Atto647N), 50-200nM such as 75 nM G_Atto647N (dGTP with Atto647N), 1000nM T_Atto532 (dTTP with Atto532), and 1000nM C_Atto532 (dCTP with Atto532), AT mixture mix (A_Atto647N and T_Atto532), CG mixture mix (G_Atto
  • the fluorescence detection solution used contains the components in the following two tables:
  • the target sequence has 3'-fam.
  • the single target sequence, target sequence, target, etc. referred to in this article all refer to the template; the C target in the table below refers to the preset template for the sequence of the C base (added C nucleotide analog) in the first round of reaction.
  • the target sequence is connected to the designated surface/chip, and C_Atto647N and C_Atto532 are added respectively.
  • detection including base extension reaction, pass the above fluorescent detection reagents to the designated surface and collect images on the surface.
  • the laser exposure setting of C_Atto647N is 100ms or 500ms, laser: 60mW
  • the exposure setting of C_Atto532 is 100ms, laser: 30mW; in this way, select different exposure times according to different test bases to continuously collect photos until the number of bright spots is almost quenched, and finally perform fitting analysis through origin software;
  • Figure 16 shows the changes in the quenching curve of Atto532 under different concentrations of BME. It can be seen from the curve that the quenching curves of 1mM BME, 10mM BME and 25mM BME have no significant difference from those without adding BME, while 50mM The number of starting points of BME is significantly lower than that of other experimental groups. The density of hybridization under the same concentration and conditions cannot be doubled. Combining the structure of the base linker and the characteristics of BME, it can be speculated that BME has the ability to excise the disulfide bond of the base linker. effect, resulting in a lower number of starting points. Combined with Figure 17, it can be seen that the quenching time of Atto532 does not change significantly under different concentrations of BME treatment.
  • Figure 18 shows the quenching of Atto532 under different concentrations of DTT. It can be seen from the quenching curve that compared with the control group without adding DTT, the quenching curves of 1mM and 10mM DTT decrease more slowly, but DTT and BME have similar characteristics. Function, high concentration 50mM DTT has an obvious effect on disulfide bonds, and the number of starting points is significantly lower than other test groups. For the quenching conditions of 1mM and 10mM DDT, the quenching time was calculated, as shown in Figure 19. It can be found from the figure that as the concentration of DTT increases, the quenching time increases significantly.
  • the quenching time of Atto532 at 20mM was extended to 5s; in addition, a gradient experiment was conducted on the ratio of AA and MV, and comparative analysis was conducted with the control group 20mM AA, combined with Figures 21 and 22 It can be seen from the bar graph that when low concentration of MV is added to 20mM AA, the quenching time of Atto532 is shortened. As the concentration of added MV increases, the quenching time can reach up to 10-15s, but it is still significantly lower than that of the control group 20mM AA. , that is, for Atto532, adding MV on the basis of AA is not conducive to prolonging its quenching time.
  • Figures 23 and 24 show the test results of the AA and MV combination system.
  • adding AA alone will not significantly affect the quality of single-color sequencing, while adding MV in combination will obviously increase the deletion ratio, shift the read length to the left (indicating that the read length becomes shorter), and the sequencing quality will be poor, making it impossible to continue to apply it.
  • Formula 1 100mM glucose, 10U/mL glucose oxidase, 30mM ascorbic acid, 1mM gallic acid, water-soluble vitamin E (Trolox), 0.50mM p-benzoquinone (BQ), 0.0001wt% cyanuric acid, 5 ⁇ M 5'-aden Glycolic acid, 5 ⁇ M guanosine-5'-monophosphate, 200 mM tris, and 30 v/v% acetonitrile, 15 v/v% liquid paraffin, pH 7.5.
  • Formula 2 120mM glucose, 12U/mL glucose oxidase, 5mM ascorbic acid, 15mM ethyl gallate, 6mM water-soluble vitamin E (Trolox), 0.0001wt% cyanuric acid, 10 ⁇ M 5'-adenylate, 10 ⁇ M guanosine -5'-monophosphate, 200mM Tris, 20mM NaCl and 20v/v% acetonitrile, 10v/v% liquid paraffin, pH7.5.
  • acetonitrile to dissolve water-soluble vitamin E and leave it in the air for more than 10 hours before adding other components to complete the preparation.
  • Formula 3 150mM glucose, 15U/mL glucose oxidase, 10mM ascorbic acid, 10mM propyl gallate, 5mM hydroquinone, 12mM water-soluble vitamin E (Trolox), 0.0005wt% cyanuric acid, 2 ⁇ M 5'-aden Acid, 2 ⁇ M guanosine-5'-monophosphate, 250 mM Tris and 20 v/v% acetonitrile, 10 v/v% liquid paraffin, pH 8.0.
  • the three formulas were tested under the same other conditions.
  • the quality of the results obtained by sequencing the same samples using the three formulas were all good, as shown in the read length, throughput and error rate indicators. All are similar.
  • New basic formula does not contain 10 ⁇ l triethylenediamine DABCO (configured using MES, pH 6.1), optionally contains or does not contain NaI, and is the same as the above basic formula.
  • the two formulas were tested under the same other conditions.
  • the results obtained by sequencing the same sample using the two formulas were similar to those disclosed in the patent document specification, and both showed that it was basically possible to achieve single sequencing. Molecular sequencing, but with shorter read lengths and higher error rates.

Abstract

本发明公开了一种提供一种荧光检测试剂、利用该试剂掺入带标记的核苷酸的方法以及试剂盒或混合物体系。该荧光检测试剂包含酶促除氧系统和多种还原剂、并且不包含DABCO。该组合物适于涉及检测荧光信号的应用情景，特别是包含单分子荧光信号检测的应用

Description

荧光检测试剂及其制备方法及应用 技术领域
本发明涉及荧光检测技术领域，特别是涉及一种荧光检测试剂及其制备方法及应用。
背景技术
本部分中讨论的主题不应仅因为在本部分中有提及就被认为是现有技术。类似地，在本部分中提及的或与作为背景技术提供的主题相关联的技术问题不应被认为先前在现有技术中已被认识到。本部分中的主题仅表示不同的方法，这些方法本身也可对应于权利要求书的技术方案的具体实施方式。
基于光学系统检测带有光致发光分子标记的生物分子在科学研究和诊断应用中常常被使用。以分子诊断为例，荧光分子通常用来修饰核苷酸和/或寡核苷酸等来实现检测和表征核酸的结构。
例如，在边合成边测序(sequencing by synthesis，SBS)技术中，具有抑制基团和荧光标记的核苷酸分子在结合到延伸链之后，被特定波长的光源激发并产生荧光，该信号被光学成像系统所探测到。通过不断重复该步骤，转化生化信号为电子信号并基于该些电子信号的处理实现核酸的序列测定。
然而，在多次重复的信号采集过程中，一般信号会变弱，甚至会出现在原有信号位置无法再收集到信号的情况。导致该情况的原因有多种可能，例如：扩增测序反应的失相，随测序的进行反应效率下降，光信号强度会减弱(请参见WO2006/064199以及LakowiczJR,Principles of fluorescence spectroscopy,3rd edition,Springer,2006.)。
而对于基于SBS的单分子测序，特别是基于对固相表面的待测核酸分子进行成像以实现单分子测序的平台，该现象的影响尤为严重。一般地，单分子测序不涉及扩增待测核酸分子，亦即，不涉及检测信号的放大，而且，一个待测核酸分子通常只带有一个或少数几个荧光分子，因此，可以理解地，对于单分子测序，对每个待测核酸分子的检测都依赖于成像系统对其所带有的单个荧光分子的信号的检测，亦即公认的，获取高信噪比的图像是实现单分子测序的难点；换句话说，在对单分子荧光信号进行采集或检测的过程中，单分子荧光信号的强度和稳定性直接影响测序的准确性和表观读长，直接影响单分子测序能否实现。在此，需要考虑激光照射下可能出现的一系列的生化现象。其中包括了激光照射对DNA分子的损伤、标记的具有荧光分子的荧光亮度、荧光稳定性。可以理解地，当荧光基团被激发进入到激发的单线态(S1)，可以通过释放光子的形式(即荧光)回到基态(S0)；也可以通过其他途径例如，通过系间窜越(intersystem crossing，ISC)的方式改变状态，进入到激发的三线态(T1)，而三线态回到基态的方式的时间(lifetime)较长，这会导致亮度下降。而且，在光照下产生的光致损伤(photo-induced damage)等也会导致亮度下降，甚至荧光信号难以被采集到。此外，处于S1以及T1状态的分子具有更高的反应活性，可以和溶液里的自由基其他分子反应，从而导致光漂白。同时，光诱导的情况下也可能会使核酸产生断裂/降解，导致待测的DNA链受到破坏，进而难以进行测序。
因而，在检测荧光分子的过程中，经常需要使待测核酸分子/荧光分子置于特定溶液环境中，以维持荧光信号的稳定、增强荧光信号和/或降低光化学反应对固相表面、固相表面上的DNA链、核苷酸(碱基)和酶等参与或催化反应的物质的破坏。
所称的溶液环境，亦即利于荧光信号的成像采集且适于待测核酸分子检测，特别是单分子检测的试剂或配方包括该试剂或配方的制备方法，有待提供或改进。
申请内容
为至少一定程度地解决上述技术问题至少之一或者提供一种实用的商业手段，本发明的实施方式提供 一种掺入带标记的核苷酸的方法、一种荧光检测试剂及其应用以及一种制备该荧光检测试剂的方法。
基于SBS原理，且通过成像采集光学信号、处理和转化图像信号为碱基/核苷酸信息实现测序的平台，一次测序运行通常包含多轮反应/多次重复反应，每轮反应包括光学可检测标记如荧光标记的引入、荧光信号的激发、信号的采集成像和荧光标记的去除。
可以理解地，至少由于每个生化反应都无法百分百的进行，多轮反应后，反应条件/环境会越来越不利于后续反应/检测的进行，表现为采集到的相应视野的图像的信噪比越来越低、图像上的信号与待测核酸分子的对应关系的可信度越来越低。
虽然无法明确影响上述问题的诸多因素、也难于把握因素之间的关联关系，但发明人认为，若能配置出利于荧光发光和荧光信号检测的溶液环境，应该能明显地改善上述问题。
为此，发明人基于自身查到的认为与在在开发平台采用的技术最接近的文献(US7,282,337和US7,666,593)公开的内容包括所采用的溶液环境、说明和示例的测试结果，结合自身对荧光发光原理和已报道的影响荧光发光的因素的理解以及对氧化或还原反应或物质的认识，以及自己多次对比试验测试中的发现，总结归纳如下并且作出该溶液体系。
具体地，发明人认为，在基于对固相基底表面进行成像实现单分子测序的过程中，较佳地，荧光分子所置于的溶液体系(有时也称为“成像液”)应能提升荧光信号的强度，例如抑制ISC过程、降低淬灭的概率、避免或减少光致损伤等，以提升荧光分子的荧光量子效率以及延长荧光寿命，以维持检测中荧光信号的强度(亮度)和稳定性。
而且，较佳地，成像液应包含除氧系统；这是由于氧分子能够参与到光化学过程中造成荧光分子淬灭，例如，氧分子可直接与激发态的荧光分子反应导致荧光分子淬灭，也可产生自由基导致荧光分子淬灭(Lakowicz JR,Principles of fluorescence spectroscopy,Springer science&business media；2013 Apr 17；J.Vogelsang等,A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes,AngewandteChemie International Edition,vol.47,no.29,pp.5465-5469,2008.)，同时地，也能够通过光化学反应对DNA中的鸟嘌呤(Helmut Sies等,Singlet oxygen induced DNA damage,Mutation Research/DNAging,1992；H.Piwoński等,Optimal oxygen concentration for the detection of single indocarbocyanine molecules in a polymeric matrix,Chemical Physics Letters,2005,405(4-6):352-356.)和蛋白中的色氨酸、半胱氨酸以及组氨酸等氨基酸残基(Davies M J,Reactive species formed on proteins exposed to singlet oxygen,Photochemical&Photobiological ences Official Journal of the European Photochemistry Association&the European Society for Photobiology,2004,3(1):17-25.)具有明显作用，导致测序反应中的DNA链、碱基以及酶等重要原料的损伤，从而导致碱基的错误识别。
而且，该成像液包含的除氧系统应是有效的除氧系统，期望包含有效的除氧系统的溶液一方面能有效减少氧分子对荧光分子和生物大分子的影响，另一方面也能够降低三线态(三重态)荧光分子的比例(C.Steinhauer等,Superresolution microscopy on the basis of engineered dark states,Journal of the American Chemical Society,vol.130,pp.16840-16841,Dec.2008；R.Zondervan等,Photoblinking of rhodamine 6g in poly(vinyl alcohol):Radical dark state formed through the triplet,The Journal of Physical Chemistry A,vol.107,no.35,pp.6770-6776,2003；T.Basche等,Direct spectroscopic observation of quantum jumps of a single molecule,Nature,vol.373,pp.132-134,Jan.1995；T.Ha等,Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging,Annual review of physical chemistry,vol.63,pp.595-617,2012.)，这主要是因为大量进入三线态的荧光分子会降低荧光量子效率并且增加淬灭概率。
再者，该成像液应包含氧化还原体系，所称的氧化还原体系包含的组分包括所称的除氧体系的组分、与所称的除氧体系的组分有交叉或者独立于所称的除氧体系。包括教科书在内的大量公开文献披露了多种氧化剂和还原剂，例如，甲基紫精(MV)、对硝基苄醇、水溶性维生素E氧化醌(TXQ)、抗坏血酸(AA)、没食子酸丙酯(nPG)、水溶性维生素E(Trolox)、巯基乙醇(BME)、二硫苏糖醇(DTT)、巯基乙胺(MEA) 等等，各试剂独立地能够有效地消除三线态荧光分子或者能够使三线态荧光分子恢复到基态利于提高荧光染料的稳定性(Holzmeister P等,Geminate recombination as a photoprotection mechanism for fluorescent dyes,Angew Chem Int Ed Engl,2014,53(22):5685-5688；Vogelsang J等,A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes,AngewandteChemie,2010,47(29):5261-5261；Aitken等,An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments,Biophysical Journal,2008,94(5):1826-1835.)。
此外，包含所称的除氧体系和/或氧化还原体系的溶液在配制、存储和使用过程中均依赖于合适的缓冲体系，包括pH值、缓冲能力、溶液离子强度、溶液极性等均可能对功能组分、组合物的性能产生影响、也可能直接影响荧光分子的量子效率。
综合以上，基于本领域常规认识、文献报道、猜测和试验测试验证发明人发现和总结出了上述信息包括明确了一些认为需要关注的因素和方向，但鉴于不知试剂/组分之间的关联关系、是否存在其它影响因素以及影响因素之间的关联关系等，能否作出一个满足上述期望、有效而稳定的荧光检测溶液，难以预测。
为此，首先，发明人设置或配制了相同的检测条件、检测环境和检测对象，对上述公开文献US 7,282,337或US 7,666,593中披露的成像时荧光分子置于的溶液体系即包含30％乙腈的下述配方“134μl HEPES/NaCl，24μl 100mM水溶性维生素E Trolox(利用2-(N-吗啉)乙磺酸即MES缓冲体系配置，pH6.1)，10μl三乙烯二胺DABCO(利用MES配置，pH6.1)，8μl 2M葡萄糖glucose，20μl NaI(50mM，利用水配置)以及4μl葡萄糖氧化酶glucose oxidase”进行测试(以下简称该配方为“基础配方”)；具体地，采用相同的光学成像系统包括相同光强和曝光时间，对获自相同的处理方式的一批表面连接有待测核酸分子的固相基底的多个视野进行多次成像，包括优化其中的氧化还原体系组分Trolox和/或DABCO的浓度。结果基本如文献所披露的，获得的测序结果的应用价值较低，例如，读长较短、错误率较高；例如，改变反应底物核苷酸上的荧光染料Atto647N为Atto532后，该配方非但不能增强Att532的亮度、反而降低了其亮度和稳定性。
而且，在对上述基础配方进行组分浓度优化试验时发明人还发现，随着测序的进行，每次所采集的荧光信号的衰减较明显，发明人猜测，这很可能是由于溶液暴露于空气中，空气中的O2不断与溶液中的除氧系统反应而产生了使溶液变酸的物质，持续如此，超过缓冲体系的缓冲能力导致溶液pH值降低，以致明显影响荧光信号的强度。因此，发明人认为缓冲溶液的缓冲能力以及影响pH的物质或物质的组合在配方优化中是必需衡量的因素。
进一步地，发明人对基础配方中的组分NaI和DABCO的化学性能及其在检测溶液体系的影响/作用进行了评估。在该基础配方中，NaI的主要作用应该是作为催化剂来实现H2O2的分解，而DABCO则是一个常用的单线态O的淬灭剂、被运用到荧光检测中。试验测试发现：(1)NaI表现为较不稳定、较容易被氧化，配制后在分装保存过程中容易形成沉淀；而且，意外地，发明人发现将其从该配方中去除，并未影响配方的工作效用，换句话说，NaI的存在与否似乎不影响或不明显影响该基础配方的功能和效果的实现；(2)更意外地，发明人还发现，利用不包含指定浓度的DABCO的基础配方进行荧光成像获得的测序结果，与利用完整基础配方的测序结果基本没有差别。
并且，以不包含指定浓度的DABCO的基础配方作为“新的基础配方”进行配方优化，亦即，将上述所列的氧化剂或还原剂或其类似物/衍生物独立地或者基于功能、氧化还原能力和对反应环境的要求进行组合，增加到新的基础配方中，并且考量包含特定物质或物质组合的溶液体系是否适配不同水溶性或电性的荧光染料的混合使用(能使得体系内的荧光染料都具有较高的信噪比、增加荧光染料的稳定性等)、能否减少核酸的降解、是否具有更高的稳定性等，以求获得能够显著优于基础配方或新的基础配方的测序结果的配方。
基于上述意外发现和多次试验调整优化，发明人基于新的基础配方开发期望的荧光检测溶液体系。
在某个实施方式中，本发明提供了一种掺入带标记的核苷酸的方法，该方法包括：(a)提供杂交复合 物，所称的杂交复合物为引物和模板分子的杂交体，引物配置为与模板分子的3'末端杂交，模板分子为单链核酸分子；(b)将聚合酶、核苷酸类似物和杂交复合物置于适于聚合反应的条件下，通过使核苷酸类似物结合至杂交复合物以获得延伸产物，核苷酸类似物包括连接的糖单元、碱基、可切割的阻断基团和荧光标记；(c)用荧光检测试剂替换(b)的溶液体系，所称的荧光检测试剂包括酶促除氧系统和多种还原剂、并且不包括三乙烯二胺(DABCO)；(d)在荧光检测试剂存在的情况下，照射至少一部分杂交复合物并采集至少一部分来自荧光标记的信号；以及(e)用切割试剂替换荧光检测试剂，以切除延伸产物上的可切割的阻断基团和荧光标记，切割试剂用于切除核苷酸类似物的可切割的阻断基团和荧光标记。
该方法包括替换延伸步骤的溶液体系为特定的溶液体系(这里所称的荧光检测试剂)的步骤，包含该步骤的方法能够在不影响模板分子/杂交复合物的情况下，使荧光标记置于有利于其稳定且有效发光的溶液体系中，有利于对来自荧光标记的信号实现准确检测，因此，有利于基于检测的信号准确识别掺入模板分子的核苷酸的类型。
该方法适用于基于荧光成像检测实现边合成边测序的平台，特别是基于SBS的单分子测序平台。具体地，荧光标记所置于的溶液体系包括酶促除氧系统和多种还原剂并且不包括DABCO，该溶液体系能够快速有效除氧、通过转移电子等的方式淬灭三线态荧光分子，避免三线态荧光分子与氧分子的反应，有助于荧光分子恢复到基态，从而很好地提供了一个有效而稳定的检测环境/成像环境；并且，该溶液体系的存在能使得在荧光检测过程中荧光信号稳定、核酸损伤减少或被抑制、荧光分子的淬灭时间延长，进而利于增加测序读长和提高测序质量。
在另一实施方式中，本发明还提供了一种组合物或试剂配方，也称为荧光检测试剂，该组合物或试剂方法包含酶促除氧系统和多种还原剂、并且不包含DABCO。该组合物是发明人基于上述意外发现作出的，适于涉及检测荧光信号的应用，特别是包含单分子荧光信号检测的应用。
在本发明的又一实施方式中，还提供上述组合物或试剂配方在检测荧光中的用途，特别是在检测单分子荧光信号中的用途。
本发明实施方式的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明实施方式的实践了解到。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
其中：
图1为实施例10中各实验组和对照组的荧光淬灭时间数据；
图2为实施例10中各实验组和对照组的测序读长数据；
图3为实施例12中实验组1和实验组2的CallRatio数据；
图4为实施例14中各实验组的表观读长数据；
图5为实施例16中对照组荧光检测试剂开瓶后的pH值与溶氧值DO随时间的变化(左图)，以及45小时后的成像质量图(右图)；
图6为实施例16中实验组1荧光检测试剂开瓶后的pH值与溶氧值DO随时间的变化(左图)，以及45小时后的成像质量图(右图)；
图7为实施例16中实验组2荧光检测试剂开瓶后的pH值与溶氧值DO随时间的变化(左图)，以及45小时后的成像质量图(右图)；
图8为实施例16中实验组3荧光检测试剂开瓶后的pH值与溶氧值DO随时间的变化(左图)，以及45小时后的成像质量图(右图)；
图9为实施例18中分别添加了不同浓度的抗坏血酸的各实验组的淬灭曲线；
图10为实施例18中分别添加了不同浓度的抗坏血酸的各实验组的淬灭时间数据；
图11为实施例18中分别添加了0mM和20mM抗坏血酸的实验组的测序读长分布数据；
图12为实施例18中分别添加了不同浓度的抗坏血酸的各实验组的读长分布数据；
图13为实施例22中实验组1(右图)和对照组(左图)的成像质量对比；
图14为实施例28中不同pH条件下的荧光检测试剂对应的荧光强度数据；
图15为实施例28中不同pH条件下的荧光检测试剂对应的表观测序读长；
图16为实施例30中添加不同浓度BME的荧光检测试剂对应的淬灭曲线；
图17为实施例30中添加不同浓度BME的荧光检测试剂对应的淬灭时间；
图18为实施例30中添加不同浓度DTT的荧光检测试剂对应的淬灭曲线；
图19为实施例30中添加不同浓度DTT的荧光检测试剂对应的淬灭时间；
图20为实施例30中相同比例条件下添加不同浓度AA和MV的荧光检测试剂对应的淬灭时间；
图21为实施例30中添加不同比例AA和MV的荧光检测试剂对应的淬灭时间；
图22为实施例30中添加不同比例AA和MV的荧光检测试剂对应的淬灭时间；
图23为实施例30中添加或不添加AA和MV的荧光检测试剂对应的测序读长分布；
图24为实施例30中添加或不添加AA和MV的荧光检测试剂对应的荧光亮度；
图25为实施例30中添加或不添加AA和MV的荧光检测试剂对应的mapping结果。
具体实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
除非另有定义，本文所使用的所有的技术和科学术语与本领域技术人员通常理解的含义相同。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
在本文中，除非另有说明，核苷酸指四种天然核苷酸(如dATP、dCTP、dGTP和dTTP或者ATP、CTP、GTP和UTP)或其衍生物，有时也直接以其包含的碱基(A、T或U、C和G)表示。本领域普通技术人员根据上下文记载可以知晓所示表达方式所指代的核苷酸或碱基。
在本文中，所称的“测序”为序列测定，同“核酸测序”或“基因测序”，指核酸序列中碱基次序的测定；包括合成测序(边合成边测序，SBS)和/或连接测序(边连接边测序，SBL)；包括DNA测序和/或RNA测序；包括长片段测序和/或短片段测序，所称的长片段和短片段是相对的，如长于1Kb、2Kb、5Kb或者10Kb的核酸分子可称为长片段，短于1Kb或者800bp的可称为短片段；包括双末端测序、单末端测序和/或配对末端测序等，所称的双末端测序或者配对末端测序可以指同一核酸分子的不完全重叠的任意两段或两个部分的读出。
所称的测序包括使核苷酸(包括核苷酸类似物)结合到模板并采集相应的反应信号的过程。在一些使核苷酸结合到模板和采集相应的反应信号非同步/实时进行的测序平台中，一般是通过多轮测序来实现模板上的多个核苷酸/碱基的次序的测定，一轮测序(cycle)也称为测序轮，可定义为四种核苷酸/碱基的一次碱基延伸，换个说法，可定义为完成模板上任意一个指定位置的碱基类型的测定过程。
对于基于控制聚合或连接反应实现测序的测序平台，一轮测序可包括实现一次四种核苷酸结合到所称的模板并采集相应的反应信号的过程；对于基于聚合反应实现测序的平台，反应体系包括反应底物核苷酸、聚合酶和模板，使模板上结合有一段预设序列(测序引物)，基于碱基配对原则和聚合反应原理，加入的反应底物(核苷酸)在聚合酶的催化下，可控地连接到测序引物的3'末端、实现与模板的相应位置碱基的配对；通常地，一轮测序可包括一次或多次碱基延伸(repeat)，例如，四种核苷酸依次加入到反应体系中， 分别进行碱基延伸和相应的反应信号的采集，一轮测序包括四次碱基延伸、四次信号采集；又例如，四种核苷酸任意组合加入到反应体系中，例如两两组合或者一三组合，两个组合分别进行碱基延伸和相应的反应信号的采集，一轮测序包括两次碱基延伸、四次信号采集；再例如，四种核苷酸同时加入到反应体系中进行碱基延伸和反应信号的采集，一轮测序包括一次碱基延伸和四次信号采集。
测序可以通过测序平台进行，测序平台可选择但不限于Illumina公司的Hiseq/Miseq/Nextseq/Novaseq测序平台、Thermo Fisher/Life Technologies公司的Ion Torrent平台、华大基因的BGISEQ和MGISEQ/DNBSEQ平台以及单分子测序平台；测序方式可以选择单端测序，也可以选择双末端测序；获得的测序结果/数据即测读出来的片段，称为读段(reads)，读段的长度称为读长。
所称的单分子检测包括单分子测序，为不涉及放大待测信号的检测，例如不涉及对待测核酸分子进行扩增的测序平台，待测分子以单个分子或少数几个分子的物理形式存在，体现在信号采集结果例如图像上，是微弱的、易被干扰/淹没的相对不稳定的信号。所称的“单分子”指一个或少数几个分子，一般不超过10个，例如1个、2个、3个或5个分子。
在本文中，所称的“固体基底”可以是任何可用于可用于固定核酸序列的固体支持物，例如尼龙膜、玻璃片、塑料、硅片、磁珠等；有时也称为反应器、芯片或流动池。
根据本发明的实施方式，提供一种掺入带标记的核苷酸的方法，包括以下步骤：(a)提供杂交复合物，杂交复合物为引物和模板分子的杂交体，引物配置为与模板分子的3'末端杂交，模板分子为单链核酸分子；(b)将聚合酶、核苷酸类似物和杂交复合物置于适于聚合反应的条件下，通过使核苷酸类似物结合至杂交复合物以获得延伸产物，核苷酸类似物包括连接的糖单元、碱基、可切割的阻断基团和荧光标记；(c)用荧光检测试剂替换(b)的溶液体系，荧光检测试剂包括酶促除氧系统和多种还原剂并且不包括三乙烯二胺；(d)在荧光检测试剂存在的情况下，照射至少一部分杂交复合物并采集至少一部分来自荧光标记的信号；(e)用切割试剂替换荧光检测试剂，以切除延伸产物上的可切割的阻断基团和荧光标记，切割试剂用于切除核苷酸类似物的可切割的阻断基团和荧光标记。除非有另外说明，这里的带标记的核苷酸和核苷酸类似物等同。
应用于SBS测序的核苷酸一般都包括使用改造的核苷酸，改造的核苷酸通常也称为“核苷酸类似物”。应用于SBS测序的核苷酸类似物通常也称为终止子，在当前发展阶段使用的通常是可逆的终止子，亦即一般所称的可逆终止子，可逆终止子除了包含核苷酸一般都包含的依次连接的磷酸基团、戊糖和碱基，通常还包含可切除的阻断基团以及可切除的可检测标记，可切除的阻断基团能够可逆地阻止后续可逆终止子或核苷酸掺入/结合到模板的下一个位置，可检测标记使得可逆终止子掺入/结合到模板的当前位置后能够产生信号以被检测到。
基于阻断基团的连接位置，有报道的可逆终止子可分为三类：一是阻断基团位于戊糖的3'-OH，即戊糖3'-O-阻断基团，使无法形成磷酸二酯键，阻断基团例如为叠氮；二是阻断基团位于磷酸侧，使无法形成磷酸二酯键；三是阻断基团位于核苷酸的碱基侧，在溶液环境中基于电荷和/或空间位阻阻挡磷酸二酯键的形成以实现聚合阻断，该种可逆终止子也常称为虚拟终止子。
在一个具体示例中，核苷酸类似物包括dATP、dUTP或dTTP、dCTP和dGTP四种核苷酸类似物，四种核苷酸类似物中的两种带有荧光标记X、另外两种带有荧光标记Y，荧光标记X和荧光标记Y为具有不同发射波长的两种荧光标记，(b)中的聚合反应包含该四种核苷酸类似物中的带有不同荧光标记的两种核苷酸类似物。
在一个具体示例中，核苷酸类似物包括dATP、dUTP或dTTP、dCTP和dGTP四种核苷酸类似物，四种核苷酸类似物带有四种不同发射波长的荧光标记，(b)中的聚合反应包含四种核苷酸类似物。
在一个具体示例中，核苷酸类似物上的阻断基团和荧光标记位于碱基的相同侧。更具体地，在一个示例中，核苷酸类似物上的糖单元的3'位为-OH，即戊糖的3'位为天然状态。该种核苷酸类似物能够通过分子的空间位阻和/或电荷的作用等非物理阻断的方式阻断核苷酸掺入/结合到模板的下一个位置，具体结构 例如可参见WO2019105421A1公开的内容。
在一个具体示例中，核苷酸类似物上的阻断基团和荧光标记位于碱基的不同侧。更具体地，核苷酸类似物上的阻断基团位于糖单元的3'位，亦即，核苷酸类似物的戊糖3'-OH被改造成3'-O-阻断基团，具体结构例如可参见US7057026B2公开的内容。
在一个具体示例中，上述掺入带标记的核苷酸的方法还包括以下步骤：(f)进行(b)-(e)至少一次，具体次数可根据需要确定。通常地，进行(b)-(e)即进行一次碱基延伸或一轮测序反应不少于20个、30个、50个、100个或150个等，以测读得具有一定长度的序列(读段，reads)，以达到各类目的的应用检测的要求。
在一个具体示例中，杂交复合物连接于基底表面，(d)包括利用特定波长的光照射基底表面以激发基底表面上的荧光标记发出荧光，以及采集至少一部分来自荧光标记的荧光。可以理解，上述特定波长可根据不同荧光标记的激发波长而进行调整。例如，常见的荧光信号成像检测所使用的波长范围为500nm-700nm，可选的且适合该激发波长范围的荧光染料或染料组合可选择Cy3，Alexafluor 532，HEX，Atto 532，ROX，Alexafluorosis 630，Cy5，Atto647N，BODIPY650，Cy 5.5，IF700和Alex680。更具体地，例如，ATTO647N的激发波长为646nm，发射波长为664nm；ATTO532的激发波长为532nm，发射波长为552nm；CY5的激发波长为651nm，发射波长为670nm；IF700的激发波长为690nm，发射波长为713nm；ROX的激发波长为578nm，发射波长为604nm；Alexa Fluor532的激发波长为534nm，发射波长为553nm。
在一个具体示例中，荧光标记包括组合ROX、ATTO532和Alexa fluor532中的至少一种，以及组合CY5、IF700和ATTO647N中的至少一种。亦即，一次碱基延伸或者一轮测序涉及采集来自两种或多种荧光标记的信号。
在一个具体示例中，荧光标记选自激发波长为550nm附近以及660nm附近的多种荧光染料的组合。例如，荧光标记包括ATTO532和ATTO647N。
在一个具体示例中，酶促除氧系统选自组合I、组合II或组合III，组合I包括葡萄糖和葡萄糖氧化酶，组合II包括葡萄糖、葡萄糖氧化酶和过氧化氢酶，组合III包括原儿茶酸和原儿茶酸3,4-双加氧酶。包含任一该酶促除氧体系的检测溶液均可以有效去除溶液中的氧气，可以降低单线态氧对荧光染料发光的影响。
在一个具体示例中，还原剂选自抗坏血酸、没食子酸、没食子酸的类似物或衍生物、三聚氰酸和水溶性维生素E或水溶性维生素E的衍生物中的至少两种。所称的没食子酸的类似物或衍生物例如没食子酸的低级烷酯，如没食子酸甲酯、没食子酸乙酯、没食子酸丙酯或其组合。如此，通过多种还原几的组合使用，可以对具有不同特性的荧光染料产生作用。同时，可以保护试剂组分中的其他还原剂和/或增强核酸分子的稳定性。
在某些示例中，在荧光信号检测时，通过相机采集来自荧光标记的信号，获得图像；可以理解地，至少一部分来自荧光标记的信号在图像上表现为亮斑。
所称的图像上的“亮斑”(dots，spots或者peaks)，也称为“亮点”或“光点”，指图像上的信号相对强的位置，例如该位置的信号较周围的强，在图像上表现为相对亮的一个斑或点，一个亮斑占有一个或多个像素。亮斑对应的信号可能来自目标分子，也可能来自非目标物质。对“亮斑”的检测包括对目标分子如延伸碱基或碱基簇的光学信号的检测。
在一个具体示例中，荧光检测试剂包括组合I、抗坏血酸和没食子酸或没食子酸的类似物或衍生物。将抗坏血酸与没食子酸进行组合，能够提升检测到的荧光信号强度和成像质量打分(图像分值/imagescore)，并且使得信号采集过程中成像质量更稳定，减小信号采集过程中成像质量的下降幅度，因此，能够获得较佳的检测结果。发明人猜测，本实施例的荧光检测试剂以葡萄糖和葡萄糖氧化酶为除氧系统，除氧速度较快，并结合抗坏血酸作为还原剂，通过转移电子的方法淬灭三重态荧光染料，避免其与氧分子的反应，有助于荧光染料恢复到基态，从而很好地形成一个有效而稳定的检测溶液系统，在荧光检测过程中能够稳定 荧光信号、减少或抑制核酸损伤，延长荧光分子的淬灭时间，进而增加测序读长，提高测序质量。
成像质量是指通入荧光检测试剂，观察图像，通过小程序统计亮度、图像score值等初步分析。本实施方式对成像质量(图像质量)的评估方式不作限定。可基于先验数据或者基于理论建立期望，对满足或越接近期望的赋予相对高的分值，对不满足或远离期望则赋予相对低的分值。例如，关于图像上亮斑的识别或检测以及图像质量的评估例如可参见CN112285070A公开的方法进行。
在一个具体示例中，荧光检测试剂包括50mM～300mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸以及1mM～20mM没食子酸或没食子酸的类似物或衍生物。
较佳地，在一个具体示例中，荧光检测试剂包括80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸以及1mM～10mM没食子酸或没食子酸的类似物或衍生物。
在一个示例中，荧光检测试剂还包括水溶性维生素E(Trolox)或其衍生物，或水溶性维生素E或其衍生物与醌类衍生物的组合。测试发现，水溶性维生素E或其衍生物对荧光基团具有保护作用，但新鲜配制的添加有水溶性维生素E或其衍生物的荧光检测试剂对于荧光染料如Atto532的成像性能较差，而经过存放的荧光检测试剂就没有这个问题。发明人猜测，这可能是由于水溶性维生素E或其衍生物对荧光基团的保护作用需要还原态Trolox及其氧化态TX-quinone(TQ)共同作用。因此，推测新配制荧光检测试剂中Trolox多为还原态，缺少TQ，无法为荧光染料的激发创造良好的条件。
基于以上测试发现和推测，发明人调整配置包含Trolox或其衍生物的溶液的方法，例如，增加紫外照射或增加一定空气中的静置时间；又例如，采用通过Trolox与醌类化合物的组合，并调整trolox或其衍生物与醌类化合物的比例等，以增强染料该在检测溶液中的亮度、对检测溶液体系稳定性的控制，增强该检测溶液的功能/作用的实现。
在一个具体示例中，在新鲜配制的包含Trolox或其衍生物的荧光检测试剂中加入一定比例的醌类衍生物如对苯醌，让对苯醌来模拟氧化态的Trolox，能够达到了增强荧光基团信号强度的目的，进而助于提高成像质量、测序读长和降低错误率。
较佳地，在一个示例中，荧光检测试剂还包括水溶性维生素E和对苯醌。
在一个具体示例中，水溶性维生素E的浓度为6mM～12mM，对苯醌的浓度为0.36mM～0.96mM。
在一个具体示例中，荧光检测试剂还包括三聚氰酸。如此，能够获得质量较佳的图像。发明人猜测，三聚氰酸游离在溶液中，能够优先于待测分子如芯片表面的核酸与氧自由基反应，能消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，从而可减少碱基的错误识别。
在一个具体示例中，三聚氰酸的质量百分比为0.0001％～0.001％。
较佳地，在一个示例中，三聚氰酸的质量百分比为0.0003％～0.0009％。
在一个具体示例中，荧光检测试剂还包括单磷酸腺苷、单磷酸胞苷、单磷酸鸟苷、单磷酸尿苷和胸苷单磷酸中的至少一种。发明人猜测，与三聚氰酸类似，这些组分独立或组合地游离在溶液中，能够优先于待测分子与氧自由基反应，能消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，有助于降低测序的错误率。
在一个具体示例中，荧光检测试剂还包括1μM～50μM 5'-腺苷酸和1μM～50μM鸟苷-5'-单磷酸。如此，能够获得质量较佳的图像，以基于该些图像获得质量较佳的测序结果。
在一个具体示例中，荧光检测试剂中的试剂组分包含了Tris(三羟甲基氨基甲烷)缓冲体系或HEPES缓冲体系，荧光检测试剂的pH为6.5-8.5。通过在不同时间对荧光检测试剂的理化性质进行检测，发现其pH值会逐渐降低，溶氧值会逐渐升高。根据荧光检测试剂将葡萄糖氧化酶和葡萄糖作为除氧体系，确定葡萄糖氧化成葡萄糖酸为pH降低的原因，到测序后期葡萄糖消耗殆尽，溶解氧增加。通过一定浓度的4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES)可以较长时间地维持pH在7以上，从而提高荧光检测试剂的稳定性。另外，进一步发现采用三羟甲基氨基甲烷(Tris)代替HEPES更有助于提高荧光检测试剂的稳定性。
在一个具体示例中，荧光检测试剂还包括乙腈(ACN)，乙腈的体积百分比为5％～40％，较佳地为 18％～35％。测试中发现，在不含乙腈的溶液体系中，部分测试的还原性物质的溶解性较差；例如，水溶性维生素E或一些醌类衍生物在不含乙腈的溶液中的溶解较困难。通过添加10％以上的乙腈，可以增强该些功能物质在溶液中的溶解。
测试发现，Tris和ACN组合使用，所采集得的图像在荧光信号强度和成像质量以及检测结果的错误率方面都表现更优。
具体地，在一个示例中，Trolox在用乙腈溶解后，在空气中放置超过10h再加入其他组分配制出不含对苯醌的荧光检测溶液I。在另一个示例中，配制包含相同浓度的Trolox、0.36mM～0.96mM例如0.52mM的对苯醌以及与I相同浓度的其他组分并且不需在空气中放置，以获得荧光检测溶液II。荧光检测溶液I和II均用于测序，结果显示，该两配方均能够获得较好的测序结果并且效果相当。
在某些测试中发现，例如反应体系中包含多种荧光染料的测序，上述部分实施例中的配方能增强其中的部分荧光染料的信号、而同时又减弱了另一部分荧光染料的信号。但从各示例的测序数据来看，均能有效的实现多荧光染料的SBS测序，相对来说，基本能获得有应用价值的读长且错误率也不高。
在一个具体示例中，荧光检测试剂包括以下组分：50mM～200mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、1mM～10mM没食子酸、6mM～12mM水溶性维生素E、0.1mM～1.0mM对苯醌、0.0001wt％～0.001wt％三聚氰酸、1mM～20μM 5'-腺苷酸、1μM～20μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和10v/v％～40v/v％乙腈。
较佳地，在一个示例中，荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸、1mM～10mM没食子酸、6mM～12mM水溶性维生素E、0.36mM～0.96mM对苯醌、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
更佳地，使其中的乙腈的体积百分比为28％～35％更是能够获得高质量的检测结果。
在一些较佳示例中，荧光检测试剂包括以下组分80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸、1mM～15mM没食子酸或没食子酸乙酯或没食子酸丙酯、6mM～15mM水溶性维生素E、0.36mM～0.96mM对苯醌、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
或者，包括以下组分80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、10mM～100mM抗坏血酸、10mM～15mM没食子酸乙酯、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
或者，包括以下组分80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、50mM～150mM抗坏血酸、10mM～15mM没食子酸丙酯、3～7mM对苯二酚、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
分别应用于核酸测序，特别是涉及多种荧光染料检测的测序中，发现该些配方均能获得较好的测序结果、并且各自获得的测序结果的质量相当。
可以理解地，在一个边合成边测序的测序过程，反应时间通常持续数小时例如12h以上，将该各溶液试剂提前混合之后用于测序，随着测序的进行，暴露在空气中的混合试剂性能会逐渐衰减，导致难以满足测序的要求。而且，作为预混合试剂盒，由于其组分中含有大量还原性物质，在保存过程中逐渐被氧化，最后组分内还原成分被全部或部分氧化，使得该试剂盒难以在一定时间存储后用于检测还能发挥其性能。
在一些示例中，荧光检测试剂还包括液体石蜡和/或硅油。用液体石蜡或硅油封闭保存，能够更长时间地保证荧光检测试剂的稳定性。而且，利于试剂运输。
在一个具体示例中，加入硅油或液体石蜡等惰性物质，例如8-20v/v％。鉴于任意这两物质的密度小于水溶液的密度，该(些)物质能隔开空气与溶液体系，因此能避免或减少空气中的氧气进入溶液消耗其 中的除氧试剂、造成还原性物质的变化和/或与溶液中的物质或中间产物发生不期望的反应，改变溶液的pH和/或影响溶液体系性能的发挥等。因此，能够增加检测溶液在使用时的稳定性、增长检测溶液的保时效期。
测试发现，上述示例的三个较佳荧光检测试剂配方，在配制完成后的12小时之内添加10v/v％的硅油，暴露在空气中48h再应用于测序，性能不减，仍旧能获得较好的测序结果。而且，效期试验结果证明，包含一定量的硅油或液体石蜡的荧光检测试剂的有效期可达6个月。可见，该配方可批量生产，具有较高的工业实用性。
在一个具体示例中，照射的时间为10-100毫秒，较佳地，照射的时间为50-100毫秒。
在一个具体示例中，(d)步骤中，照射ATTO532的光强为30-40毫瓦，照射ATTO647N的光强为60-80毫瓦；和/或，照射一个视野中的ATTO532或ATTO647N的时长为50-500毫秒。较佳地，照射一个视野中的ATTO532或ATTO647N的时长为50-100毫秒。
本发明一实施例的荧光检测试剂，包含酶促除氧系统和多种还原剂、并且不包括三乙烯二胺。
在一个具体示例中，酶促除氧系统选自组合I、组合II或组合III，组合I包括葡萄糖和葡萄糖氧化酶，组合II包括葡萄糖、葡萄糖氧化酶和过氧化氢酶，组合III包括原儿茶酸和原儿茶酸3,4-双加氧酶。
在一个具体示例中，多种还原剂选自抗环血酸、没食子酸、没食子酸的类似物或衍生物、三聚氰酸和水溶性维生素E中的至少两种。
在一个具体示例中，荧光检测试剂包括组合I、抗坏血酸和没食子酸或没食子酸的类似物或衍生物。本实施例的荧光检测试剂以葡萄糖和葡萄糖氧化酶为除氧系统，除氧速度较快，并结合抗坏血酸作为还原剂，通过转移电子的方法淬灭三重态荧光染料，避免其与氧分子的反应，有助于荧光染料恢复到基态，从而很好地形成一个有效而稳定的检测溶液系统，在荧光检测过程中能够稳定荧光信号、减少或抑制核酸损伤，延长荧光分子的淬灭时间，进而增加测序读长，提高测序质量。将抗坏血酸与没食子酸进行组合，能够提升检测到的荧光信号强度和成像质量打分，并且使得信号采集过程中成像质量更稳定，减小信号采集过程中成像质量的下降幅度。成像质量是指通入荧光检测试剂，观察单分子图像，通过小程序统计亮度、图像score值等初步分析。
在一个具体示例中，荧光检测试剂包括50mM～200mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸以及1mM～10mM没食子酸或没食子酸的类似物或衍生物。
较佳地，在一个示例中，荧光检测试剂包括80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸以及1mM～10mM没食子酸或没食子酸的类似物或衍生物。
在一个具体示例中，荧光检测试剂还包括水溶性维生素E(Trolox)和对苯醌。研究发现，水溶性维生素E对荧光基团具有保护作用，但新鲜配制的添加有水溶性维生素E的荧光检测试剂对于荧光染料如Atto532的成像性能较差，而经过存放的荧光检测试剂就没有这个问题。可能是由于水溶性维生素E对荧光基团的保护作用需要还原态Trolox及其氧化态TX-quinone(TQ)共同作用。因此推测新配制荧光检测试剂中Trolox多为还原态，缺少TQ，无法为荧光染料的激发创造良好的条件。基于以上推测，尝试在新鲜配制的荧光检测试剂中加入一定比例的醌类的衍生物，例如，对本醌，让对苯醌来模拟氧化态的Trolox，从而达到了增强荧光基团信号强度的目的，有助于提高成像质量、测序读长和降低错误率。
可选地，水溶性维生素E的浓度为6mM～12mM，对苯醌的浓度为0.1mM～1mM；较佳地，其中的对苯醌的浓度为0.36mM～0.96mM。
在一个具体示例中，荧光检测试剂还包括三聚氰酸。如此，三聚氰酸游离在溶液中，优先于待测分子如芯片表面的核酸与氧自由基反应，能消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，从而可减少测序技术中核酸的错误识别。在某些示例中，三聚氰酸的质量百分比为0.0001％～0.001％；较佳地，三聚氰酸的质量百分比为0.0003％～0.0009％。
在一个具体示例中，所述荧光检测试剂还包括单磷酸腺苷、单磷酸胞苷、单磷酸鸟苷、单磷酸尿苷和 胸苷单磷酸中的至少一种。与三聚氰酸类似，发明人猜测，这些组分游离在溶液中，优先于待测分子与氧自由基反应，能消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，有助于降低测序的错误率。
在一个具体示例中，荧光检测试剂还包括5'-腺苷酸和鸟苷-5'-单磷酸。可选地，荧光检测试剂还包括1mM～20μM 5'-腺苷酸和1μM～20μM鸟苷-5'-单磷酸。较佳地，荧光检测试剂还包括4mM～12μM 5'-腺苷酸和4μM～12μM鸟苷-5'-单磷酸。
在一个具体示例中，荧光检测试剂采用Tris(三羟甲基氨基甲烷)缓冲体系或HEPES缓冲体系，荧光检测试剂的pH为6.5-8.5，较佳地，荧光检测试剂的pH值为8.5。可选地，三羟甲基氨基甲烷或HEPES的浓度为80mM～120mM。在测序中发现，随着测序的进行，成像质量会逐渐变差，严重影响了测序后期的数据质量。通过在不同时间对荧光检测试剂的理化性质进行检测，发现其pH值会逐渐降低，溶氧值会逐渐升高。根据荧光检测试剂将葡萄糖氧化酶和葡萄糖作为除氧体系，确定葡萄糖氧化成葡萄糖酸为pH降低的原因，到测序后期葡萄糖消耗殆尽，溶解氧增加。通过一定浓度的4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES)可以较长时间地维持pH在7以上，从而提高荧光检测试剂的稳定性。另外，进一步发现采用三羟甲基氨基甲烷(Tris)代替HEPES更有助于提高荧光检测试剂的稳定性。
在一个具体示例中，荧光检测试剂还包括乙腈，乙腈的体积百分比为10％～40％；较佳地，乙腈的体积百分比为18％～35％；更佳地，乙腈的体积百分比为28％～35％。将Tris和ACN组合使用，在荧光信号强度、成像质量、错误率方面都表现更优。
在一个具体示例中，荧光检测试剂包括以下组分：50mM～200mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、1mM～10mM没食子酸、6mM～12mM水溶性维生素E、0.1mM～1.0mM对苯醌、0.0001wt％～0.001wt％三聚氰酸、1mM～20μM 5'-腺苷酸、1μM～20μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。可选地，荧光检测试剂还包括一价可溶性盐如氯化钠，与缓冲组分配合。
在一个较佳示例中，荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸、1mM～10mM没食子酸、6mM～12mM水溶性维生素E、0.36mM～0.96mM对苯醌、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
本发明一实施例的制备上述荧光检测试剂的方法，包括以下步骤：在封装荧光检测试剂之前，加入液体石蜡和/或硅油。
本发明一实施例的试剂盒，其包括上述荧光检测试剂和上述核苷酸类似物。可以理解，上述荧光检测试剂不仅可用于测序，也可以用于其他需要进行荧光检测的方法或产品中。
以下通过具体实施例对本发明做详细的阐述，应当理解，实施例仅是示例性的。实施例中涉及的材料、试剂以及序列等，如无特殊说明，可自行制备、合成或者通过市售途径获取。
实施例10
按照下表所示配方分别配制各实验组和对照组的荧光检测试剂，实验组1与实验组3的区别在于酶促除氧体系不同，实验组1为葡萄糖和葡萄糖氧化酶，实验组3为原儿茶酸-3,4-二加氧酶(PCD)与3,4-二羟基苯甲酸(原儿茶酸/PCA)，实验组2相比于实验组1的区别在于缓冲体系将HEPES替换为Tris。分别使用各实验组的荧光检测试剂进行测序，并比较用Atto647N标记的终止子在特定激光工作强度下的荧光淬灭时间。

当溶液内添加除氧体系后，体系内的溶氧含量经过溶氧仪的测试都可以确保无氧气检出。但不同体系在实际使用时的淬灭时间却有差异。图1显示不同酶促除氧体系的检测数据，葡萄糖和葡萄糖氧化酶除氧体系下Atto647N的平均荧光淬灭时间为278s，而PCD-PCA除氧体系下平均荧光淬灭时间为11s，实验重复次数N＝9。在葡萄糖和葡萄糖氧化酶除氧体系下，Atto647N的荧光淬灭时间更长。若曝光时间为0.5s，葡萄糖和葡萄糖氧化酶除氧体系下将有0.2％的荧光分子发生淬灭，而PCD-PCA除氧体系将有4.5％的荧光分子发生淬灭。因此，采用葡萄糖和葡萄糖氧化酶作为荧光检测试剂的除氧系统可以延长荧光分子的淬灭时间。
此外，HEPES在光照下易产生过氧化氢，从而可能加快荧光分子的淬灭以及增加生物大分子的光化学反应，因此原理上将HEPES替换成三羟甲基氨基甲烷(Tris)更合理，我们同样对比了两种不同缓冲体系下的表观测序读长。图2显示实验组1和实验组2的测序读长没有明显差异，图1显示实验组2平均淬灭时间为99s。在极端的0.5s曝光时间下，使用Tris替代HEPES的荧光检测试剂将有0.6％的荧光分子发生淬灭，淬灭比例较小。
实施例12
为了简化配方，我们用100mM Tris替换前述配方中的4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES)、2-(N-吗啉)乙磺酸(MES)、三羟甲基氨基甲烷(Tris)组合缓冲体系，如图3所示，随着测序进行，100mM Tris的CallRatio(CallRatio表示能够识别出的碱基的比例，能够一定程度上反应成像液的测序性能)逐渐好于77mM HEPES+25mM MES+3mM Tris的组合缓冲体系，尤其在测序后期表现更为明显，说明100mM Tris更有助于提高荧光检测试剂性能的稳定性。
另外，还对比了1,3-双((三羟甲基)甲基氨基)丙烷(Bis-tris propane)、4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES)、三羟甲基氨基甲烷(Tris)和乙腈(ACN)组合并结合石蜡或硅油保存、磷酸缓冲盐溶液(PBS)，其中，Tris和乙腈组合在荧光信号强度、成像质量、错误率方面都表现更优。

实施例14
荧光分子的荧光量子效率还受溶剂极性的影响，因而不同比例的乙腈可能造成荧光量子效率的不同，进而呈现不同的荧光信号强度。如下表所示，本实施例调整荧光检测试剂中乙腈的终浓度，对比不同浓度乙腈对测序读长、成像质量以及测序错误率的影响。这里的测序涉及Atto532和Atto647N两种荧光染料。
结果如图4所示，荧光检测试剂中不同的乙腈浓度并未造成明显的表观读长差异，而如下表所示，荧光检测试剂中不同的乙腈浓度检测到的信号强度不同，成像质量的打分及错误率不同。
因此，实验证明乙腈在10％～40％浓度范围内特别是28-35％范围内均可以获得较好的测序读长，其中，含有30％乙腈的荧光检测试剂的信号强度、成像质量打分和错误率的表现较优。
实施例16
如图5所示，在测序中发现，随着测序的进行，成像质量会逐渐变差，严重影响了测序后期的数据质量。通过在不同时间对荧光检测试剂的理化性质进行检测，发现其pH值会逐渐降低，溶氧值会逐渐升高。根据葡萄糖氧化酶和葡萄糖作为除氧体系，确定葡萄糖氧化成葡萄糖酸为pH降低的原因。到测序后期，葡萄糖消耗殆尽，溶解氧增加。
因此进行如下实验来增加荧光检测试剂的稳定性，配制25mL成像液在试剂盒中4度放置40小时，在不同的时间点测量溶液的pH和溶氧，变量为葡萄糖浓度和HEPES(pH8.8)的浓度，具体如下表所示。

结果如图5～8所示，增加HEPES(pH8.8)的浓度可以在40小时内维持pH在7以上，增加葡萄糖浓度可以减慢荧光检测试剂中溶解氧上升的速度。因此，HEPES浓度(pH8.8)和葡萄糖浓度的范围在80～150mM对荧光检测试剂有更佳的稳定作用。
实施例18
按照下表所示配方分别配制各组的荧光检测试剂，区别在于，各组分别添加了不同浓度的抗坏血酸(AA)或未添加抗坏血酸。分别使用各组的荧光检测试剂进行测序，并比较用Atto532标记的终止子在激光工作强度下的荧光淬灭时间。
图9展示了添加不同浓度AA后，Atto532的淬灭曲线，从曲线图可知，随着AA浓度的增加，淬灭曲线下降更缓慢，50mM AA的淬灭曲线下降幅度最慢，结合图10的淬灭时间可知，未添加AA的对照组，淬灭时间在5s以内，而添加AA后，淬灭时间延长，随着AA浓度增加，50mM AA组的淬灭时间达到30s，即AA有助于延长Atto532的淬灭时间。而且随着AA浓度升高测序读长增长，如图11展示了20mM AA组和对照组(0mM AA)测序读长的分布，从读长峰来看，添加20mM AA后，主峰更高，短片段减少。图12展示了添加不同浓度抗坏血酸组的读长分布，可见40mM AA组和50mM AA组为较好的浓度范围，读长都增加1bp，主峰更突出，小片段显著减少。从上述研究可以看到当抗坏血酸浓度不断提升可以进一步增加体系的稳定性。通过上述结果可证，添加AA有助于延长荧光分子的淬灭时间，增加测序读长，40mM～50mM为较优的浓度范围。
在其它试验测试中发现，AA在溶液中的浓度达到100-200mM也可以进一步降低淬灭时间(数据未在此处展示)。
实施例20
分别配制实验组和对照组的荧光检测试剂，二者区别在于，实验组同时添加了抗坏血酸和没食子酸，而对照组添加抗坏血酸未添加没食子酸(GA)。分别使用实验组和对照组的荧光检测试剂进行测序，并比较荧光信号强度和成像质量打分等数据。

AA的梯度测试显示随着还原剂AA浓度增加，Atto532的淬灭时间明显增加，但是荧光信号强度随着AA浓度增加有下降趋势。而在AA的基础上，结合GA进行组合测试，结果如下表所示。结果显示，抗坏血酸组合没食子酸能够提升检测到的荧光信号强度和成像质量打分，并且使得信号采集过程中成像质量更稳定，减小信号采集过程中成像质量的下降幅度。因此，有抗坏血酸存在的情况下，添加没食子酸能够增强成像质量的稳定性。
实施例22
按照下表所示配方分别配制各实验组和对照组的荧光检测试剂，二者区别在于，各实验组同时添加了不同浓度的水溶性维生素E和对苯醌，而对照组仅添加水溶性维生素E未添加对苯醌。分别使用各实验组和对照组的荧光检测试剂进行测序，并比较成像质量、读长和错误率等数据。
如图13所示，实验组1(右图)的荧光检测试剂对Atto532的成像质量显著优于对照组(左图)的荧光检测试剂。再如下表所示，从成像质量打分、读长和错误率综合评价来看，6～12mM水溶性维生素E组合0.36-0.96mM对苯醌能够获得较好的成像质量、读长和相对较低的错误率，6mM水溶性维生素E组合0.96mM对苯醌为最优条件。因此，实验证明水溶性维生素E与对苯醌组合加入有助于提高成像质量、测序读长和降低错误率，6～12mM水溶性维生素E组合0.36～0.96mM对苯醌为较优的浓度范围。
实施例24
按照下表所示配方分别配制各组的荧光检测试剂，区别在于各组分别添加了不同比例的三聚氰酸或未添加三聚氰酸。分别使用各组的荧光检测试剂进行测序，并比较错误率数据。
结果如下表所示，在荧光检测试剂中添加不同比例的三聚氰酸，有助于降低测序的错误率。因此，实验证明添加三聚氰酸能够明显降低错误率，0.0001％～0.001％浓度范围均可以达到较好效果。发明人猜测，三聚氰酸游离在溶液中，能够优先于芯片表面的核酸与氧自由基反应，消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，从而减少测序技术中核酸的错误识别。
实施例26
按照下表所示配方分别配制各实验组和对照组的荧光检测试剂，二者区别在于，各实验组分别添加了不同浓度的5'-腺苷酸(AMP)和鸟苷-5'-单磷酸(GMP)，而对照组未添加5'-腺苷酸(AMP)和鸟苷-5'-单磷酸(GMP)。分别使用各实验组和对照组的荧光检测试剂进行测序，并比较错误率数据。

结果如下表所示，在荧光检测试剂中添加不同浓度的单磷酸，有助于降低测序的错误率。因此，实验证明添加5'-腺苷酸(AMP)和鸟苷-5'-单磷酸(GMP)组合，能够降低错误率，1μM～20μM效果显著。发明人猜测，单磷酸游离在溶液中，能够优先于芯片表面的核酸与氧自由基反应，消耗掉激光照射后生成的氧自由基，减弱或避免核酸链受损，从而减少碱基错误识别。换句话说，添加不具有反应活性的核苷酸类似物，可以作为牺牲或竞争试剂，用来替代或保护目标反应底物核苷酸。
实施例28
由于酸碱度能影响荧光分子的电子转移，因而不同的pH值可能造成荧光分子的荧光量子效率不同，进而造成荧光亮度不同。本实施例将Atto647N标记的G终止子溶于不同酸碱度的Tris溶液中，并将不同溶液分别置于石英腔中，使用640nm、光强恒定的激光进行激发并测试荧光强度。如图14所示，在pH6.5～8.5范围内，pH值越高，Atto647N的荧光强度越高。通过测序对比升高pH后和未升高pH的荧光检测试剂对应的表观测序读长，如图15所示，pH8.5的荧光检测试剂对应的测序读长有微弱的提升，优势不明显。但考虑到除氧体系在除氧过程中会导致溶液pH进一步下降，进而影响除氧速率，因而推荐提升荧光检测试剂的pH至8.5。因此，通过实验证明荧光检测试剂在pH6.5～8.5范围内，pH值越高，Atto647N的荧光强度越高，且更利于除氧体系的稳定。
实施例30
本实施例进一步对其他添加剂对荧光检测试剂的性能影响进行检测，具体步骤如下。
1.淬灭曲线
淬灭曲线反映了一个碱基荧光染料淬灭的情况，淬灭曲线下降越快，淬灭时间越短，那么荧光染料在成像体系中的稳定性就越差，反之，则稳定性较好；淬灭时间定义：指点数(荧光信号)淬灭降低到50％所对应的时间。
1.1.靶序列的选择原则
选取一条首轮反应待测试碱基的靶序列作为待测试的靶(序列已知的模板分子)；
1.2.数据分析的原则
A.统计每个图片的亮点数目(荧光信号在图像上表现为亮点或亮斑)，例如可选取固定大小的图像区域1024*1024或512*512进行统计；
B.分析点数随着时间变化的散点图，通过origin软件拟合分析统计淬灭时间。
1.3.测试流程
假设待测试的碱基为C_Atto532(带有Atto532的dCTP)，通过选取首轮靶序列为G的单靶进行测试：按照的通常的单分子边合成边测序流程加入C_Atto532碱基，保证激光强度不变的情况，选择一个固定区域，每隔100ms采集一张照片，直到所有荧光信号完全淬灭；
2.长测序验证
经过淬灭曲线筛选获得的添加剂，通过双通道测序(two channel，亦即双色测序)验证其对测序质量的影响，测试流程：碱基聚合/延伸反应—荧光检测试剂下采集照片—剪切处理，以此循坏，进行60轮(cycles)合成靶测序或80cycles生物样本测序。
3.具体测试
底物(修饰的核苷酸/改造的核苷酸，也称为可逆终止子，为带荧光分子的、可抑制其它核苷酸结合到待测模板的下一个位置的核苷酸)配置：分别配制50-150nM例如125nM C_Atto647N(带有Atto647N的dCTP)，50-150nM例如125nM T_Atto647N(带有Atto647N的dTTP)，100-200例如200nM A_Atto647N(带有Atto647N的dATP)，50-200nM例如75nM G_Atto647N(带有Atto647N的dGTP)，1000nM T_Atto532(带有Atto532的dTTP)，以及1000nM C_Atto532(带有Atto532的dCTP)，AT混合物mix(A_Atto647N和T_Atto532)，CG混合物mix(G_Atto647N和C_Atto532)1mL，具体配制如下表，具体测试体积根据实验情况进行调整。
利用的荧光检测溶液包含下面两表的组分：

选择首轮反应C碱基的单靶序列(首轮反应碱基的结合位置如表中带下划线的碱基)，靶序列带有3'-fam，具体序列可参考下表。本文所称的单靶序列、靶序列、靶等均指模板；如下表中的C靶指首轮反应C碱基(加入C核苷酸类似物)序列预设的模板。
通过与表面上的探针杂交，使靶序列连接到指定表面/芯片，分别加入C_Atto647N和C_Atto532进行 检测，包括碱基延伸反应之后，通入上述各荧光检测试剂至指定表面后对该表面进行图像采集，其中C_Atto647N的激光曝光设置为100ms或500ms、激光：60mW，C_Atto532曝光设置为100ms、激光：30mW；如此，根据测试碱基的不同选择不同的曝光时间进行连续采集照片，直到亮点数几乎淬灭完，最后通过origin软件进行拟合分析；
结果：如图16所示为不同浓度BME处理下，Atto532的淬灭曲线变化，从曲线图可知，1mM BME、10mM BME以及25mM BME的淬灭曲线图和不添加BME的无明显差异，而50mM BME起始点数明显低于其他实验组，相同浓度相同条件下杂交的密度不可能出现一倍的差异，结合碱基linker的结构以及BME的特性，可以推测BME对碱基linker二硫键具有切除效果，从而导致起始点数较低的现象，结合图17可知，不同浓度BME处理下，Atto532的淬灭时间无明显变化。
图18所示为不同浓度DTT条件下，Atto532的淬灭情况，从淬灭曲线图可知，相对于不添加DTT对照组，1mM、10mM DTT的淬灭曲线下降更缓慢，但是DTT与BME具有近似功能，高浓度50mM DTT对于二硫键具有明显切除效果，起始点数明显低于其他测试组。针对1mM以及10mM DDT淬灭情况，统计了淬灭时间，如图19所示。从图可以发现，随着DTT浓度增加，淬灭时间明显增加，10mM DTT处理下，淬灭时间延长了一倍，即DTT对于提高Atto532的淬灭时间有明显作用，但是由于对碱基linker具有切除效果，因此，难以应用。针对AA测试情况，尝试了AA和MV的组合体系，图20相同比例条件下(AA:MV＝1：1)，随着AA和MV浓度增加，淬灭时间相对无添加的对照组有一定提升，Atto532在20mM(AA:MV＝1：1)的淬灭时间延长到5s；此外，针对AA和MV的比例进行了梯度实验，通过和对照组20mM AA进行比较分析，结合图21及22两个柱形图可知，20mM AA情况下添加低浓度的MV，Atto532的淬灭时间缩短，随着添加MV浓度的增加，淬灭时间最高可达到10～15s，但是仍然明显低于对照组20mM AA，即对于Atto532而言，在AA的基础上，添加MV不利于延长其淬灭时间。
图23和24展示了AA和MV组合体系的测试情况，读长分布图23显示，添加了20mM AA和20mM MV(AA:MV＝1:1)后，读长主峰左移，测序读长变短，且单分子荧光亮度只有300左右，明显低于对照组，导致deletion比例(无法识别出碱基的比例)较高，如图25的比对结果(mapping结果)，且比对上的比例(mapped rate)偏低，整体测序质量较差。因此，单独添加AA不会明显影响单色测序的质量，而组合添加MV明显会导致deletion比例升高，读长左移(表示读长变短)，测序质量较差，无法继续应用。
实施例32
引入多种抗氧化剂后，原配方当中的抗氧化剂DABCO的效果未知，碘化钠作为GOD除氧体系的催化剂效果也未知，因此，我们尝试去除原配方中的DABCO和碘化钠进行对比测试。
结果如下表所示，DABCO和NaI去除前后各个指标表现没有差异。
实施例34
配方1：100mM葡萄糖、10U/mL葡萄糖氧化酶、30mM抗坏血酸、1mM没食子酸、水溶性维生素E(Trolox)、0.50mM对苯醌(BQ)、0.0001wt％三聚氰酸、5μM 5'-腺苷酸、5μM鸟苷-5'-单磷酸、200mM三羟甲基氨基甲烷(Tris)和30v/v％乙腈，15v/v％液体石蜡，pH7.5。
配方2：120mM葡萄糖、12U/mL葡萄糖氧化酶、5mM抗坏血酸、15mM没食子酸乙酯、6mM水溶性维生素E(Trolox)、0.0001wt％三聚氰酸、10μM 5'-腺苷酸、10μM鸟苷-5'-单磷酸、200mMTris、20mM NaCl和20v/v％乙腈，10v/v％液体石蜡，pH7.5。并且，在配制该试剂时，利用乙腈溶解水溶性维生素E，并在空气中放置超过10h之后再加入其他组分，以完成配制。
配方3：150mM葡萄糖、15U/mL葡萄糖氧化酶、10mM抗坏血酸、10mM没食子酸丙酯、5mM对苯二酚、12mM水溶性维生素E(Trolox)、0.0005wt％三聚氰酸、2μM 5'-腺苷酸、2μM鸟苷-5'-单磷酸、250mM Tris和20v/v％乙腈，10v/v％液体石蜡，pH8.0。
参照上述任一实施例，在相同的其它条件下对该三配方进行测试，利用该三配方对相同的样本进行测序获得的结果的质量均较好，表现在读长、通量和错误率指标上均相近。
实施例36
US 7,282,337或US 7,666,593披露的(原始)基础配方：134μl HEPES/NaCl，24μl 100mM水溶性维生素E Trolox(利用2-(N-吗啉)乙磺酸即MES缓冲体系配置，pH6.1)，10μl三乙烯二胺DABCO(利用MES配置，pH6.1)，8μl 2M葡萄糖glucose，20μl NaI(50mM，利用水配置)，4μl葡萄糖氧化酶glucose oxidase以及30％乙腈。
新的基础配方：不包含10μl三乙烯二胺DABCO(利用MES配置，pH6.1)，可选地包含或不包含NaI，其它同上述基础配方。
参照上述任一实施例，在相同的其它条件下对该两配方进行测试，利用该两配方对相同的样本进行测序获得的结果均类似于该专利文献说明书所披露的，均表现基本能够实现单分子测序，但读长较短且错误率较高。
在本说明书的描述中，参考术语“一个实施方式”、“一些实施方式”、“示意性实施方式”、“示例”、“某些示例”、“具体示例”或“实施例”等的描述意指结合所述实施方式或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施方式或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施方式或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施方式或示例中以合适的方式结合。
尽管上面已经示出和描述了本发明的实施方式，可以理解的是，上述实施方式是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施方式进行变化、修改、替换和变型。

Claims (65)

  1. 一种掺入带标记的核苷酸的方法，其特征在于，包括以下步骤：
    (a)提供杂交复合物，所述杂交复合物为引物和模板分子的杂交体，所述引物配置为与所述模板分子的3'末端杂交，所述模板分子为单链核酸分子；
    (b)将聚合酶、核苷酸类似物和所述杂交复合物置于适于聚合反应的条件下，通过使所述核苷酸类似物结合至所述杂交复合物以获得延伸产物，所述核苷酸类似物包括连接的糖单元、碱基、可切割的阻断基团和荧光标记；
    (c)用荧光检测试剂替换(b)的溶液体系，所述荧光检测试剂包括酶促除氧系统和多种还原剂、并且不包括三乙烯二胺；
    (d)在所述荧光检测试剂存在的情况下，照射至少一部分所述杂交复合物并采集至少一部分来自所述荧光标记的信号；
    (e)用切割试剂替换所述荧光检测试剂，以切除所述延伸产物上的可切割的阻断基团和荧光标记，所述切割试剂用于切除所述核苷酸类似物的可切割的阻断基团和荧光标记。
  2. 根据权利要求1所述的方法，其特征在于，所述核苷酸类似物包括dATP、dUTP或dTTP、dCTP和dGTP四种核苷酸类似物，四种所述核苷酸类似物中的两种带有荧光标记X、另外两种带有荧光标记Y，所述荧光标记X和所述荧光标记Y为具有不同发射波长的两种荧光标记，(b)中的聚合反应包含该四种核苷酸类似物中的带有不同荧光标记的两种核苷酸类似物。
  3. 根据权利要求1所述的方法，其特征在于，所述核苷酸类似物包括dATP、dUTP或dTTP、dCTP和dGTP四种核苷酸类似物，四种所述核苷酸类似物带有四种不同发射波长的荧光标记，(b)中的聚合反应包含所述四种核苷酸类似物。
  4. 根据权利要求1所述的方法，其特征在于，所述核苷酸类似物上的所述阻断基团和所述荧光标记位于所述碱基的相同侧。
  5. 根据权利要求4所述的方法，其特征在于，所述核苷酸类似物上的所述糖单元的3'位为-OH。
  6. 根据权利要求1所述的方法，其特征在于，所述核苷酸类似物上的所述阻断基团和所述荧光标记位于所述碱基的不同侧。
  7. 根据权利要求6所述的方法，其特征在于，所述核苷酸类似物上的所述阻断基团位于所述糖单元的3'位。
  8. 根据权利要求1-7任一项所述的方法，其特征在于，还包括(f)进行(b)-(e)至少一次。
  9. 根据权利要求1-8任一项所述的方法，其特征在于，所述杂交复合物连接于基底表面，(d)包括利用特定波长的光照射所述基底表面以激发所述基底表面上的荧光标记发出荧光，以及采集至少一部分来自所述荧光标记的荧光。
  10. 根据权利要求9所述的方法，其特征在于，所述荧光标记包括组合ROX、ATTO532和Alexa fluor532中的至少一种，以及组合CY5、IF700和ATTO647N中的至少一种。
  11. 根据权利要求10所述的方法，其特征在于，所述荧光标记包括ATTO532和ATTO647N。
  12. 根据权利要求10或11所述的方法，其特征在于，所述酶促除氧系统选自组合I、组合II或组合III，组合I包括葡萄糖和葡萄糖氧化酶，组合II包括葡萄糖、葡萄糖氧化酶和过氧化氢酶，组合III包括原儿茶酸和原儿茶酸3,4-双加氧酶。
  13. 根据权利要求12所述的方法，其特征在于，所述还原剂选自抗环血酸、没食子酸、没食子酸的类似物或衍生物、三聚氰酸和水溶性维生素E中的至少两种。
  14. 根据权利要求13所述的方法，其特征在于，所述荧光检测试剂包括组合I、抗坏血酸和没食子酸或没食子酸的类似物或衍生物。
  15. 根据权利要求14所述的方法，其特征在于，所述荧光检测试剂包括50mM～300mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸以及1mM～20mM没食子酸或没食子酸的类似物或衍生物。
  16. 根据权利要求15所述的方法，其特征在于，所述荧光检测试剂包括80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～50mM抗坏血酸以及1mM～10mM没食子酸或没食子酸的类似物或衍生物。
  17. 根据权利要求14-16任一项所述的方法，其特征在于，所述荧光检测试剂还包括水溶性维生素E或其衍生物以及醌类化合物。
  18. 根据权利要求14-16任一项所述的方法，其特征在于，所述荧光检测试剂还包括水溶性维生素E和对苯醌。
  19. 根据权利要求18所述的方法，其特征在于，所述水溶性维生素E的浓度为6mM～12mM，所述对苯醌的浓度为0.36mM～0.96mM。
  20. 根据权利要求14-19任一项所述的方法，其特征在于，所述荧光检测试剂还包括三聚氰酸。
  21. 根据权利要求20所述的方法，其特征在于，所述三聚氰酸的质量百分比为0.0001％～0.001％。
  22. 根据权利要求20所述的方法，其特征在于，所述三聚氰酸的质量百分比为0.0003％～0.0009％。
  23. 根据权利要求14-22任一项所述的方法，其特征在于，所述荧光检测试剂还包括单磷酸腺苷、单磷酸胞苷、单磷酸鸟苷、单磷酸尿苷和胸苷单磷酸中的至少一种。
  24. 根据权利要求23所述的方法，其特征在于，所述荧光检测试剂还包括1mM～50μM 5'-腺苷酸和1μM～50μM鸟苷-5'-单磷酸。
  25. 根据权利要求23所述的方法，其特征在于，所述荧光检测试剂还包括4mM～12μM 5'-腺苷酸和4μM～12μM鸟苷-5'-单磷酸。
  26. 根据权利要求14-25任一项所述的方法，其特征在于，所述荧光检测试剂采用Tris缓冲体系，所述荧光检测试剂的pH为6.5-8.5。
  27. 根据权利要求14-26任一项所述的方法，其特征在于，所述荧光检测试剂还包括乙腈，所述乙腈的体积百分比为10％～40％。
  28. 根据权利要求14-26任一项所述的方法，其特征在于，所述荧光检测试剂还包括乙腈，所述乙腈的体积百分比为18％～35％。
  29. 根据权利要求14-26所述的方法，其特征在于，所述乙腈的体积百分比为28％～35％。
  30. 根据权利要求12-29任一项所述的方法，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、1mM～15mM没食子酸或没食子酸乙酯或没食子酸丙酯、6mM～15mM水溶性维生素E、0.36mM～0.96mM对苯醌、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  31. 根据权利要求12-29任一项所述的方法，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、10mM～15mM没食子酸乙酯、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  32. 根据权利要求12-29任一项所述的方法，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、10mM～15mM没食子酸丙酯、3～7mM对苯二酚、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  33. 根据权利要求9-32任一项所述的方法，其特征在于，所述照射的时间为10-100毫秒。
  34. 根据权利要求9-32任一项所述的方法，其特征在于，所述照射的时间为50-100毫秒。
  35. 根据权利要求11-32任一项所述的方法，其特征在于，(d)中，照射ATTO532的光强为30-40毫瓦，照射ATTO647N的光强为60-80毫瓦；和/或，照射一个视野中的ATTO532或ATTO647N的时长为50-500毫秒。
  36. 根据权利要求35所述的方法，其特征在于，照射一个视野中的ATTO532或ATTO647N的时长为50-100毫秒。
  37. 一种荧光检测试剂，其特征在于，包含酶促除氧系统和多种还原剂、并且不包括三乙烯二胺。
  38. 根据权利要求37所述的试剂，其特征在于，所述酶促除氧系统选自组合I、组合II或组合III，组合I包括葡萄糖和葡萄糖氧化酶，组合II包括葡萄糖、葡萄糖氧化酶和过氧化氢酶，组合III包括原儿茶酸和原儿茶酸3,4-双加氧酶。
  39. 根据权利要求38所述的试剂，其特征在于，所述多种还原剂选自抗环血酸、没食子酸、没食子酸的类似物或衍生物、三聚氰酸和水溶性维生素E中的至少两种。
  40. 根据权利要求39所述的试剂，其特征在于，所述荧光检测试剂包括组合I、抗坏血酸和没食子酸或没食子酸的类似物或衍生物。
  41. 根据权利要求40所述的试剂，其特征在于，所述荧光检测试剂包括50mM～300mM葡萄糖、2U/mL～20U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸以及1mM～20mM没食子酸或没食子酸的类似物或衍生物。
  42. 根据权利要求41所述的试剂，其特征在于，所述荧光检测试剂包括80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸以及1mM～10mM没食子酸或没食子酸的类似物或衍生物。
  43. 根据权利要求40-42任一项所述的试剂，其特征在于，所述荧光检测试剂还包括水溶性维生素E或其衍生物以及醌类化合物。
  44. 根据权利要求40-42任一项所述的试剂，其特征在于，所述荧光检测试剂还包括水溶性维生素E和对苯醌。
  45. 根据权利要求44所述的试剂，其特征在于，所述水溶性维生素E的浓度为6mM～12mM，所述对苯醌的浓度为0.36mM～0.96mM。
  46. 根据权利要求40-45任一项所述的试剂，其特征在于，所述荧光检测试剂还包括三聚氰酸。
  47. 根据权利要求46所述的试剂，其特征在于，所述三聚氰酸的质量百分比为0.0001％～0.001％。
  48. 根据权利要求46所述的试剂，其特征在于，所述三聚氰酸的质量百分比为0.0003％～0.0009％。
  49. 根据权利要求40-48任一项所述的试剂，其特征在于，所述荧光检测试剂还包括单磷酸腺苷、单磷酸胞苷、单磷酸鸟苷、单磷酸尿苷和胸苷单磷酸中的至少一种。
  50. 根据权利要求49所述的试剂，其特征在于，所述荧光检测试剂还包括1mM～50μM 5'-腺苷酸和1μM～50μM鸟苷-5'-单磷酸。
  51. 根据权利要求49所述的试剂，其特征在于，所述荧光检测试剂还包括4mM～12μM 5'-腺苷酸和4μM～12μM鸟苷-5'-单磷酸。
  52. 根据权利要求40-51任一项所述的试剂，其特征在于，所述荧光检测试剂采用Tris缓冲体系，所述荧光检测试剂的pH为6.5-8.5。
  53. 根据权利要求40-52任一项所述的试剂，其特征在于，所述荧光检测试剂还包括乙腈，所述乙腈的体积百分比为10％～40％。
  54. 根据权利要求40-52任一项所述的试剂，其特征在于，所述荧光检测试剂还包括乙腈，所述乙腈的体积百分比为18％～35％。
  55. 根据权利要求40-52任一项所述的试剂，其特征在于，所述乙腈的体积百分比为28％～35％。
  56. 根据权利要求38-55任一项所述的试剂，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、1mM～15mM没食子酸或没食子酸乙酯或没食子酸丙酯、6mM～15mM水溶性维生素E、0.36mM～0.96mM对苯醌、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  57. 根据权利要求38-55任一项所述的试剂，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、10mM～15mM没食子酸乙酯、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  58. 根据权利要求38-55任一项所述的试剂，其特征在于，所述荧光检测试剂包括以下组分：80mM～150mM葡萄糖、8U/mL～12U/mL葡萄糖氧化酶、1mM～200mM抗坏血酸、10mM～15mM没食子酸丙酯、3～7mM对苯二酚、6mM～12mM水溶性维生素E、0.0003wt％～0.0009wt％三聚氰酸、4mM～12μM 5'-腺苷酸、4μM～12μM鸟苷-5'-单磷酸、100mM～300mM三羟甲基氨基甲烷和18v/v％～35v/v％乙腈。
  59. 根据权利要求37-58任一项所述的试剂，其特征在于，所述荧光检测试剂还包括液体石蜡和/或硅油。
  60. 根据权利要求59所述的试剂，其特征在于，所述荧光检测试剂包括8～20v/v％的液体石蜡和/或硅油。
  61. 一种制备权利要求37-60任一项所述的试剂的方法，其特征在于，在封装所述荧光检测试剂之前，加入所述液体石蜡和/或硅油8～20v/v％的液体石蜡和/或硅油。
  62. 权利要求37-60任一项所述的试剂在检测荧光信号中的用途。
  63. 一种试剂盒，其特征在于，包括权利要求1-36任一项所述的方法中的所述荧光检测试剂和所述核苷酸类似物。
  64. 一种混合物体系，其特征在于，其包含置于溶液中的杂交复合物，所述杂交复合物为引物和模板分子的杂交体，所述溶液为权利要求37-60任一项所述试剂。
  65. 权利要求64的混合物体系，其特征在于，所述杂交复合物固定于固相基底表面。
PCT/CN2023/084270 2022-04-02 2023-03-28 荧光检测试剂及其制备方法及应用 WO2023185795A1 (zh)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030225247A1 (en) * 2002-03-12 2003-12-04 Stavrianopoulos Jannis G. Labeling reagents and labeled targets, target labeling processes and other processes for using same in nucleic acid determinations and analyses
CN1764729A (zh) * 2003-01-24 2006-04-26 人类遗传标记控股有限公司 使用嵌入核酸检测核酸中甲基化改变的分析
CN101180530A (zh) * 2005-05-20 2008-05-14 日立化成工业株式会社 生物化学物质的分析方法
CN101578517A (zh) * 2005-12-02 2009-11-11 加利福尼亚太平洋生物科学公司 分析反应中光损伤的缓解
CN102124128A (zh) * 2008-06-16 2011-07-13 Plc诊断股份有限公司 通过定相合成来进行核酸测序的系统和方法
CN112218640A (zh) * 2018-03-15 2021-01-12 哥伦比亚大学董事会 核苷酸类似物及其在核酸测序和分析中的用途
WO2021007458A1 (en) * 2019-07-09 2021-01-14 The Trustees Of Columbia University In The City Of New York Novel nucleotide analogues and methods for use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030225247A1 (en) * 2002-03-12 2003-12-04 Stavrianopoulos Jannis G. Labeling reagents and labeled targets, target labeling processes and other processes for using same in nucleic acid determinations and analyses
CN1764729A (zh) * 2003-01-24 2006-04-26 人类遗传标记控股有限公司 使用嵌入核酸检测核酸中甲基化改变的分析
CN101180530A (zh) * 2005-05-20 2008-05-14 日立化成工业株式会社 生物化学物质的分析方法
CN101578517A (zh) * 2005-12-02 2009-11-11 加利福尼亚太平洋生物科学公司 分析反应中光损伤的缓解
CN102124128A (zh) * 2008-06-16 2011-07-13 Plc诊断股份有限公司 通过定相合成来进行核酸测序的系统和方法
CN112218640A (zh) * 2018-03-15 2021-01-12 哥伦比亚大学董事会 核苷酸类似物及其在核酸测序和分析中的用途
WO2021007458A1 (en) * 2019-07-09 2021-01-14 The Trustees Of Columbia University In The City Of New York Novel nucleotide analogues and methods for use

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