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WO2019075711A1 - Procédé d'élimination de résidu d'agent de régénération - Google Patents

Procédé d'élimination de résidu d'agent de régénération Download PDF

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Publication number
WO2019075711A1
WO2019075711A1 PCT/CN2017/106972 CN2017106972W WO2019075711A1 WO 2019075711 A1 WO2019075711 A1 WO 2019075711A1 CN 2017106972 W CN2017106972 W CN 2017106972W WO 2019075711 A1 WO2019075711 A1 WO 2019075711A1
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Prior art keywords
sequencing
solid support
group
reagent
optionally substituted
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PCT/CN2017/106972
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English (en)
Chinese (zh)
Inventor
谢槟
徐讯
杨晋
徐崇钧
刘二凯
陈奥
章文蔚
德马纳克•斯内扎娜
卡洛•马修
拉哈戈泊尔•田西
马可心
赵芳
李计广
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MGI Tech Co Ltd
Complete Genomics Inc
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MGI Tech Co Ltd
Complete Genomics Inc
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Priority to CN201780094653.2A priority Critical patent/CN111051533B/zh
Priority to PCT/CN2017/106972 priority patent/WO2019075711A1/fr
Publication of WO2019075711A1 publication Critical patent/WO2019075711A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • the invention relates to the field of nucleic acid sequencing.
  • the present invention relates to a method of eliminating residual reagents of regenerating reagents, comprising: a. providing a solid support on which a nucleic acid molecule is immobilized, b. treating the solid support with a regenerating reagent, the regenerating reagent comprising a reducing agent, And c.
  • step b treating the solid support with a wash solution to which a compound having a disulfide bond structure is added, wherein prior to step b, the solid support is optionally treated with one or more reaction solutions containing a sequencing reagent Generating a signal representative of the nucleotide sequence of the nucleic acid molecule on a solid support, and wherein the regeneration reagent can eliminate the signal on the solid support, wherein after the treatment of step c, optionally further The solid support is treated with one or more reaction solutions containing sequencing reagents.
  • the second generation of sequencing technology is based on the development of the first generation of Sanger sequencing technology, with low cost, high throughput, automation and other features, greatly promoting the development of the gene sequencing industry.
  • Second-generation sequencing technology has been widely used in whole-genome sequencing, transcriptome sequencing, and metagenomic sequencing. It is a powerful tool for analyzing the evolution and classification of organisms, studying cancer-related genes such as autism, and conducting in vitro diagnostics. It has promoted people's understanding of life sciences and promoted the development of health industry.
  • SBS Sequencing-by-Synthesis
  • cPAS Joint Probe Anchor Polymerization
  • SBL ligation sequencing
  • the regenerated reagent will remain on the surface or reagent of the solid support after resection of the detectable label with the regenerating reagent during the sequencing process.
  • the subsequent sequencing reagents are polluted, so that the quality of the sequencing data is degraded, which affects the sequencing accuracy.
  • high-salt solutions are used to clean the chips or tubes, high salt solutions require a large amount of high salt due to dilution and by reducing the physical binding of the regenerating reagent to the chip, nucleic acid or tubing to reduce the residual amount of regenerating reagents.
  • the solution, and only to minimize the residual of the regenerating reagent in the sequencing reagent is difficult to completely remove the regenerating reagent, resulting in reduced sequencing data quality and sequencing accuracy.
  • the present invention solves the above problems by using a washing solution containing a compound having a disulfide bond structure.
  • the invention provides a method of eliminating regenerative reagent residues comprising: a. providing a solid support having nucleic acid molecules immobilized thereon, b. treating the solid support with a regeneration reagent, the regenerating reagent comprising reducing And c. treating the solid support with a washing solution to which a compound having a disulfide bond structure is added.
  • the solid support is free or substantially free of regenerating reagent after the treatment of step c.
  • the solid support is free or substantially free of reducing agent contained in the regenerating agent.
  • the solid support is optionally treated with one or more reaction solutions containing a sequencing reagent to produce a nucleotide sequence representing the nucleic acid molecule on a solid support
  • the signal and wherein the regeneration reagent can eliminate the signal on the solid support.
  • the solid support is optionally also treated with one or more reaction solutions containing sequencing reagents.
  • the solid support does not contain or substantially does not contain regeneration.
  • Reagent preferably, the solid support is free or substantially free of reducing agent contained in the regenerating agent.
  • substantially free of regenerating agent means that the amount of regenerating agent (or preferably, the reducing agent in the regenerating agent) contained on the solid support is sufficiently small that the solid support and the sequencing reagent are included.
  • reaction solution When the reaction solution is contacted, it does not cause significant contamination of the reaction solution with respect to the washing solution to which the compound containing the disulfide bond structure is not added, with respect to the regenerating agent (or preferably, the reducing agent in the regenerating agent).
  • the solid support After treating a solid support which has been treated with a regenerating agent using a washing solution to which a compound having a disulfide bond structure is added, the solid support is subsequently treated with a reaction solution containing a sequencing reaction reagent.
  • the concentration of the regenerating agent (or preferably, the reducing agent in the regenerating agent) in the reaction solution is not more than about 10%, not more than about 5%, not more than about 2%, not more than about 1%, Not more than about 0.5%, no more than about 0.2%, no more than about 0.1%, no more than about 0.05%, no more than about 0.02%, no more than about 0.001%, or even lower.
  • treating a solid support includes any means of contacting a solid support with a reagent or solution, including but not limited to flowing a reagent or solution through a solid support or soaking a solid support in an agent or solution.
  • the means for flowing the reagent or solution through the solid support includes, for example, transporting the reagent or solution to the solid support using an inlet flow channel in fluid communication with the solid support and using the outlet flow channel in fluid communication with the solid support to remove the reagent or solution from the reagent or solution Solid support discharge (described in International Application Publication WO 91/06678) a kind of treatment).
  • Soaking the solid support in the reagent or solution includes any means of immersing the solid support partially or completely in the reagent or solution.
  • the solid support may be partially or fully immersed in the reagent or solution, for example, in a vertical, oblique or horizontal manner, preferably all nucleic acid molecules immobilized on the solid support are contacted with reagents in the reaction vessel.
  • nucleic acid may be used interchangeably with “nucleic acid molecule", "polynucleotide”, which may be any type of nucleic acid, eg, the nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or An analog of DNA or RNA made from a nucleotide analog. Nucleic acids can be single stranded, double stranded or contain both single stranded and double stranded sequences.
  • the nucleic acid molecule may be derived from a double-stranded DNA (dsDNA) form (eg, genomic DNA, PCR, and amplification products, etc.), or may be derived from a single-stranded form such as DNA (ssDNA) or RNA and which may be converted to the dsDNA form, and vice versa.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded form
  • RNA RNA
  • the sequenced nucleic acid can be in the form of a single molecule (which can be a natural molecule, a modified molecule such as a labeled molecule, or a nucleic acid comprising a nucleotide analog), a concatamer of a sequence, etc.
  • nucleic acid molecule can be amplified (eg, amplified as a concatamer, amplified into multiple individual molecules having the same or similar sequences, etc.), and/or can be in any other form.
  • the exact sequence of the nucleic acid molecule can be known or unknown.
  • nucleic acids genes or gene fragments (eg, probes, primers, EST or SAGE tags), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, Ribosome RNA, ribozyme, cDNA, nucleic acid library, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, any of the above sequences Nucleic acid probes, primers or amplified copies.
  • genes or gene fragments eg, probes, primers, EST or SAGE tags
  • genomic DNA genomic DNA fragments, exons, introns
  • messenger RNA messenger RNA
  • transfer RNA Ribosome RNA
  • Ribozyme Ribosome RNA
  • cDNA nucleic acid library
  • recombinant polynucleotide synthetic polynucleotide,
  • Nucleic acids can include nucleotides or nucleotide analogs. Nucleotides typically contain a sugar, a nucleobase, and at least one phosphate group. Nucleotides can be abasic (ie, lacking nucleobases). Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleosides Acids and mixtures thereof.
  • nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), and thorax.
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • TMP thymidine monophosphate
  • TDP thymidine diphosphate
  • thorax examples include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), and thorax.
  • Nucleotide analogs comprising modified nucleobases can also be used in the methods described herein.
  • Exemplary modified nucleobases that may be included in a polynucleotide, whether having a native backbone or a similar structure include, for example, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 2-amino Indole, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine , 2-thiouracil, 2-thiothymidine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6 - azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine,
  • Nucleic acid molecules that are sequenced in particular embodiments of the invention can be of any length.
  • exemplary lengths of useful nucleic acids include, for example, at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 1,000. , 5,000 or 10,000, 100,000 nucleotides or longer.
  • the length may be no longer than 1,000,000, 100,000, 10,000, 1,000, 100 nucleotides or less.
  • the length of the nucleic acid molecule can also include all integers between the above exemplary numbers.
  • the sequenced nucleic acid can be, for example, within the scope of short polynucleotides, fragments, cDNA, genes, and genomic fragments.
  • Nucleic acid molecules that are sequenced in particular embodiments of the invention may be in any number, for example, may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70 , 80, 90 or 100 or more identical or different nucleic acid molecules. Is the number of sequencing a nucleic acid molecule may also be, for example 200,300,400,500,1000,5000,10000,50000,1x10 5, 2x10 5, 3x10 5 , 4x10 5, 5x10 5, 6x10 5, 7x10 5, 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 or 1x10 7 or more identical or different nucleic acid molecules. The number of nucleic acid molecules that are sequenced may also include all integers between the above exemplary numbers.
  • Nucleic acids can be obtained from any source.
  • a nucleic acid can be prepared from a nucleic acid molecule obtained from an organism, or from a population of nucleic acid molecules obtained from a natural source comprising one or more organisms.
  • Sources of nucleic acid molecules include, but are not limited to, organelles, cells, tissues, organs, or organisms.
  • Cells that can be used as a source of nucleic acid molecules can Prokaryotic (eg bacteria); or eukaryotic, such as fungi (eg yeast), plants, protozoa and other parasites, and animals (including insects, nematodes), and mammals (eg, rats, mice, Monkeys, non-human primates, and humans); or nucleic acid molecules can be derived from viruses.
  • Prokaryotic eg bacteria
  • eukaryotic such as fungi (eg yeast), plants, protozoa and other parasites, and animals (including insects, nematodes), and mammals (eg, rats, mice, Monkeys, non-human primates, and humans); or nucleic acid molecules can be derived from viruses.
  • the nucleic acid can be obtained from a particular biological source.
  • the nucleic acid is a human nucleic acid obtained from a human, for example, a sample of human tissue.
  • the nucleic acid is a human mitochondrial nucleic acid.
  • the nucleic acid can be obtained from a metagenomic sample.
  • the nucleic acid can be obtained from an environmental source that no longer contains living organisms.
  • the term "immobilized" when used in reference to a nucleic acid means attached directly or indirectly to a solid support via a covalent bond or a non-covalent bond.
  • covalent attachment can be used, but typically only required is nucleic acid retention under conditions where it is desired to use a solid support (eg, in applications requiring nucleic acid amplification and/or sequencing) Fixed or attached to a solid support.
  • the oligonucleotide to be used as a capture primer or amplification primer is immobilized such that the 3' end is available for enzymatic extension and at least a portion of the primer sequence is capable of hybridizing to the complementary nucleic acid sequence.
  • Immobilization can occur via hybridization to surface-attached oligonucleotides, in which case the immobilized oligonucleotide or polynucleotide can be in the 3'-5' direction.
  • Another way of non-covalent attachment may be to bind the nucleic acid binding protein to the solid support by amination modification and to capture the nucleic acid molecule by the nucleic acid binding protein.
  • immobilization can occur by other means than base pair hybridization, such as the covalent attachment described above.
  • Non-limiting examples of nucleic acid attachment to solid support include nucleic acid hybridization, biotin streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, via hydrogel or other porous polymer. Physical limitations, etc.
  • Various exemplary methods for immobilizing nucleic acids on a solid support can be found, for example, in G. Steinberg-Tatman et al, Bioconjugate Chemistry 2006, 17, 841-848; Xu x. et al.
  • solid support means any insoluble substrate or matrix to which a nucleic acid can be attached, such as, for example, latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polypropylene. Amide gel, gold surface, glass surface, chip and silicon wafer.
  • the surface of the solid support can be any desired shape including, for example, planar, spherical or porous suitable for a particular application.
  • solid support The object can be a flat glass surface.
  • a solid support can be mounted inside the flow cell to allow interaction with solutions of multiple reagents. The solid support can also be moved to contact the solution in a plurality of reaction vessels, respectively.
  • the solid support can comprise an inert substrate or matrix that has been chemically functionalized, for example by applying a layer or coating of an intermediate material that allows covalent attachment to the multicore A reactive group of a glycoside.
  • the intermediate material can be attached directly or indirectly to the solid support via a covalent bond or a non-covalent bond.
  • a support can include a polyacrylamide hydrogel layer on an inert substrate such as glass.
  • the polynucleotide can be directly covalently attached to an intermediate layer (eg, a hydrogel), but the intermediate layer itself can be non-covalently attached to other substrates or substrates (eg, glass substrates) layers.
  • the term “signal” includes any signal that can be detected, including but not limited to optical signals, electrical signals, electromagnetic signals, radiated signals, and the like.
  • the term “signal representing a nucleotide sequence of a nucleic acid molecule” means that the signal carries information of the nucleotide sequence and can be decoded into the nucleotide sequence of the nucleic acid molecule being sequenced. For example, where a labeled (eg, fluorescently labeled) sequencing probe is used, the signal generated by the label carries the nucleotide sequence information of the sequencing probe, and the signal is used to know the sequenced nucleic acid molecule and the sequencing The nucleotide sequence of the position where the probe is complementary to the pair.
  • a labeled eg, fluorescently labeled
  • the signal generated by the label carries the identity information of the nucleotide, and the signal is used to know the nucleotide molecule being sequenced and the nucleotide The nucleotide sequence of the position of the complementary pair.
  • the signal is a fluorescent signal.
  • the label is a fluorescent label.
  • the manner in which fluorescent labels or signals are detected is well known in the art. For example, it can be realized by a device that detects the wavelength of fluorescence. Such devices are well known in the art.
  • such a device can be a confocal scanning microscope that scans the surface of the solid support with a laser to image the fluorophore directly on the nucleic acid molecule being sequenced.
  • each of the generated signals can be observed, for example, with a sensitive 2-D detector, such as a charge coupled detector (CCD).
  • CCD charge coupled detector
  • Other techniques such as scanning near-field optical microscopy (SNOM) can also be used, for example.
  • sequencing reagent refers to an agent that is suitable for use in any sequencing method for sequencing nucleic acid molecules on a solid support.
  • sequencing methods can include, but are not limited to, electrophoretic sequencing, synthetic sequencing (including combinatorial probe anchor polymerization sequencing), ligation sequencing, hybrid sequencing, single molecule sequencing, and real-time sequencing methods.
  • a signal eg, a fluorescent signal
  • sequencing reagent used in the method of the invention and the one or more reaction solutions containing the sequencing reagent will depend on the sequencing method employed. It is within the ability of those skilled in the art to determine the particular sequencing reagents used in the methods of the invention, as well as one or more reaction solutions containing sequencing reagents, according to sequencing methods.
  • the sequencing reagent comprises a sequencing reagent for ligation sequencing.
  • Such reagents can produce a ligation product on the sequenced nucleic acid molecule and generate a signal representative of the nucleotide sequence of the nucleic acid molecule.
  • Ligation sequencing as used herein is a variety of ligation sequencing methods well known in the art. Basically, ligation sequencing involves the hybridization and ligation of labeled (eg, fluorescently labeled) sequencing probes and anchor probes (also referred to as "primers" in SOLiD sequencing) to DNA strands.
  • the sequencing probe contains one or two fixed known sequencing bases (single base sequencing probe or double base sequencing probe) and a series of degenerate or universal bases that allow for sequencing probes and nucleic acid templates Perform complementary pairing.
  • the anchor probe sequence is complementary to an adaptor sequence on the nucleic acid template (an adaptor sequence means an oligonucleotide of known sequence in the nucleic acid template) and provides a known sequence of sites for initiation of ligation. After ligation, the template is imaged and the one or two known bases in the sequencing probe are identified. After the complete removal of the anchor probe-sequencing probe complex or after cleavage removal of the label (eg, fluorophore) and regeneration of the junction site, the next ligation sequencing cycle begins.
  • an adaptor sequence means an oligonucleotide of known sequence in the nucleic acid template
  • the sequencing reagent comprises an anchor probe, a labeled (eg, fluorescently labeled) sequencing probe, a ligase, or a mixture thereof, and one comprising a sequencing reagent
  • the one or more reaction solutions include a solution comprising an anchor probe, a labeled (eg, fluorescently labeled) sequencing probe, a ligase, or a mixture thereof, provided that the solid support and anchor probe, labeled sequencing probe
  • Each of the ligases is contacted, and wherein the signal on the solid support is produced by a labeled sequencing probe that is complementary to a nucleic acid molecule on a solid support, the labeled sequencing probe
  • An anchor probe that is complementary to the same nucleic acid molecule is ligated via a ligase.
  • the sequencing reagent may not comprise an anchor probe and the solid support and the anchor probe (also referred to as "in SOLiD sequencing") prior to treatment of the solid support with one or more reaction solutions containing the sequencing reagent
  • the primer contacts such that the anchor probe hybridizes to the nucleic acid molecule on the solid support.
  • the one or more reaction solutions containing the sequencing reagent can include only one reaction solution comprising a ligase and a labeled sequencing probe, optionally, Also included is an anchor probe; or one or more reaction solutions containing a sequencing reagent may also include two reaction solutions, one of which contains an anchor probe and the other of which contains a labeled sequencing probe , And at least one of the two reaction solutions comprises a ligase, in which case sequencing of the nucleic acid molecule comprises immersing the solid support in a reaction solution comprising an anchor probe, washing, and then immersing in the inclusion of the labeled The reaction solution of the sequencing probe is washed and washed.
  • the one or more reaction solutions containing the sequencing reagent may further comprise three reaction solutions comprising an anchor probe, a labeled sequencing probe and a ligase, respectively, in which case the nucleic acid molecule is sequenced.
  • the method includes: immersing a solid support in a reaction solution containing an anchor probe, washing, then immersing in a reaction solution containing a labeled sequencing probe, washing, and then immersing in a reaction solution containing a ligase, washing.
  • the one or more reaction solutions containing the sequencing reagent can include both a ligase, a first anchor probe, a second anchor probe, and labeled sequencing. Only one reaction solution of the probe, or a plurality of reaction solutions containing a ligase, a first anchor probe, a second anchor probe, a labeled sequencing probe, or a mixture thereof, respectively.
  • ligation sequencing in particular with regard to anchoring probes, sequencing probes, see, for example, WO2013066975, U.S. Patent Nos. 60/992,485, 61/026,337, 61/035,914, and 61/061,134.
  • ligation sequencing includes combined probe anchor ligation (cPAL) sequencing (see document WO2013066975).
  • ligase refers to a nucleic acid modifying enzyme that catalyzes the intramolecular formation and intermolecular formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of a nucleic acid strand.
  • Ligase can be obtained from recombinant or natural sources.
  • One or more low temperature (eg, room temperature or lower) ligases eg, T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, and/or E. coli DNA ligase
  • the ligase may also be a thermostable ligase.
  • a thermostable ligase from a thermophilic organism can be used.
  • thermostable DNA ligases include, but are not limited to, Tth DNA ligase (from Thermus thermophilus, available from, for example, Eurogentec and GeneCraft); Pfu DNA ligase (from intense Hyperthermophilic ligase of Pyrococcus furiosus; Taq ligase (from Thermus aquaticus), and any other suitable thermostable ligase, or any combination thereof.
  • the sequencing reagent comprises a sequencing reagent for sequencing by synthesis.
  • a sequencing reagent for sequencing by synthesis Such an agent may be a reagent that performs polymerization by using the sequenced nucleic acid molecule as a template and generates a signal representing a nucleotide sequence of the nucleic acid molecule,
  • Synthetic sequencing as used herein is a variety of synthetic sequencing methods well known in the art. Basically, synthetic sequencing involves first hybridizing the sequenced nucleic acid molecule to a sequencing primer, followed by polymerization of the labeled nucleic acid molecule as a template at the 3' end of the sequencing primer (eg, fluorescent labeling) in the presence of a polymerase. Nucleotide. After polymerization, the labeled nucleotide is identified by detecting the label. After removal of the label (eg, fluorophore) from the labeled nucleotide, the next polymerization sequencing cycle begins.
  • a sequencing primer e.g, fluorescent labeling
  • the sequenced nucleic acid molecule is immobilized on a solid support by hybridization to a sequencing primer immobilized on a solid support.
  • the solid support is contacted with a sequencing primer prior to treatment of the solid support on which the nucleic acid molecule is immobilized using one or more reaction solutions containing the sequencing reagent, such that the sequencing primer hybridizes to A nucleic acid molecule on the solid support.
  • the nucleic acid molecules on the solid support are sequenced using the BGISEQ-500 sequencing platform.
  • the sequencing reagent comprises a polymerase, a labeled (eg, fluorescently labeled) nucleotide, or a mixture thereof, and one or more reactions comprising a sequencing reagent
  • the solution includes a solution comprising a polymerase, a labeled (eg, fluorescently labeled) nucleotide, or a mixture thereof, provided that the solid support contacts each of the polymerase, the labeled nucleotide, and wherein the solid
  • the signal on the support is produced by a labeled nucleotide that is complementary bound to a nucleic acid molecule on a solid support, the labeled nucleotide being polymerized via a polymerase with a nucleic acid molecule on a solid support as a template to The 3' end of the primer was sequenced.
  • the regeneration reagent comprises an agent for removing the label from the labeled (eg, fluorescently labeled) nucleotide.
  • the solid support is optionally treated with a reaction solution comprising a polymerase and a labeled (e.g., fluorescently labeled) nucleotide.
  • the sequencing reagent comprises a reagent in a sequencing kit for the BGISEQ-500 sequencing platform, such as the BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizhi Technology Co., Ltd., article number Sequencing reagent in PF-UM-PEV30).
  • a sequencing kit for the BGISEQ-500 sequencing platform such as the BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizhi Technology Co., Ltd., article number Sequencing reagent in PF-UM-PEV30).
  • Labeled (e.g., fluorescently labeled) nucleotides suitable for use in synthetic sequencing and reagents for removing labels from labeled (e.g., fluorescently labeled) nucleotides are well known in the art, such nucleotides and reagents Non-limiting examples can be found in, for example, the labeled nucleotides disclosed in WO04018497, WO04018493, U.S. Patent No. 7,427, 673, and U.S. Pat. Nucleotide removal of labeled reagents.
  • the labeled nucleotide may further comprise a 3' blocking group.
  • the 3' blocking group prevents the incorporation of other nucleotides when the labeled nucleotide is polymerized onto the growing nucleotide strand.
  • 3' blocking The group is removed along with the label.
  • Suitable 3' blocking groups and reagents for removing 3' blocking groups from nucleotides are well known in the art, and non-limiting examples of such 3' blocking groups and reagents can be found, for example, in Greene. & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Guillier; Metzker et al.
  • the label on the labeled nucleotide itself can be used as a 3' blocking group.
  • a label may be of a size or structure sufficient to function to block the incorporation of other nucleotides into the polynucleotide strand. The blockage may be due to steric hindrance or may be due to a combination of size, charge and structure.
  • the labeling and blocking groups on the labeled nucleotides can be different, but preferably the labeling and blocking groups can be removed from the nucleotides in the same manner.
  • the regenerative reagents described herein comprise an agent that removes both the label and the 3' blocking group from the labeled nucleotide.
  • the regenerative reagents described herein can include an agent that can remove the label from the labeled nucleotide and an agent that can remove the 3' blocking group from the labeled nucleotide.
  • polymerase refers to an enzyme that synthesizes a nucleic acid strand or polymer, including DNA polymerases and RNA polymerases.
  • the polymerase used herein is a DNA polymerase. May be used is one polymerase Sequenase TM (7 DNA polymerase from bacteriophage enzyme, which sequence is modified to improve its properties - see Tabor and Richarson, Proc.Nat.Acad.Sci.USA, 84: 4767-4771 (1987), available from, for example, United States Biochemical Corporation, Cleveland, Ohio).
  • TM Sequenase
  • Other polymerases that can be used in place of Sequenase (TM) include, but are not limited to, the Klenow fragment of DNA polymerase I, AMV reverse transcriptase, and Taq polymerase. Further description of the polymerase can also be found in WO05024010 and WO06120433, the entire contents of which are hereby incorporated by reference.
  • the polymerization conditions generally used are the polymerization conditions of these enzymes known in the art.
  • the polymerization conditions include a temperature in the range of from about room temperature to about 45 °C; and pH 7 buffer to 8, preferably pH 7.3 to 7.7; an enzyme concentration of from about 0.01 units / [mu] l to about 1 unit / The microliter is for a period of from about 1 to about 20 minutes, preferably from 1 to 5 minutes.
  • Typical for Sequenase TM buffer consisting of: 0.040M Tris HCI (pH7.5) 0.050M sodium chloride, 0.010M magnesium chloride, 0.010M dithiothreitol.
  • these typical conditions include a temperature in the range of from about 10 ° C to about 45 ° C, preferably from about 15 ° C to about 40 ° C; a buffer of pH 6.8 to 7.4, preferably pH 7.0 To 7.4; the enzyme concentration is from about 0.01 unit/microliter to about 1 unit/microliter, preferably from about 0.02 to about 0.15 unit/microliter, and the reaction time is from about 1 to about 40 minutes.
  • a typical buffer for the Klenow fragment of DNA polymerase I consists of 0.05 M trishydroxymethylammonium chloride, pH 7.5 0.05 M magnesium chloride, 0.05 M sodium chloride, 0.010 M dithiothreitol.
  • complementary refers to hybridization or base pairing or duplex formation between nucleotides or nucleic acids. If a nucleotide of one nucleic acid at a given position is capable of forming a hydrogen bond with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to each other at that position.
  • Complementary nucleotides are typically A and T (or A and U) or C and G.
  • RNA or DNA molecules are said to be substantially complementary.
  • hybridization refers to a sufficient hydrogen bonding between complementary nucleotide or nucleotide bases, which may be, for example, Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding, Stable and specific binding occurs between nucleic acid strands.
  • Hybridization capacity is determined according to stringent conditions, including appropriate buffer concentrations and temperatures, which allow for specific hybridization to a target nucleic acid having a fully or partially complementary region. Therefore, not all nucleotides of a nucleic acid need to be complementary.
  • a nucleic acid strand is "substantially complementary" when the nucleic acid strand hybridizes to all, part or overlapping regions of the target nucleic acid.
  • the term "regeneration agent” refers to an agent that, upon sequencing a nucleic acid molecule on a solid support, is capable of eliminating sequencing signals (eg, fluorescent signals) on a solid support to enable next round of sequencing.
  • the particular regenerating reagent used in the methods of the invention will depend on the sequencing method employed. Determining the specific regenerative reagents used in the methods of the invention according to sequencing methods is within the skill of those skilled in the art. Inside.
  • a regenerating reagent in ligation sequencing, includes an agent that eliminates a signal representing a nucleotide sequence of the nucleic acid molecule from the sequenced nucleic acid molecule and enables initiation of the next ligation sequencing reaction, preferably such a reagent It is a reducing agent.
  • the regenerating reagent comprises an agent that removes a label (eg, a fluorescent label) from a labeled (eg, fluorescently labeled) sequencing probe, preferably such reagent is a reducing agent.
  • the regenerating reagent comprises reagents for eliminating a signal representative of the nucleotide sequence of the nucleic acid molecule from the sequenced nucleic acid molecule and enabling initiation of the next polymerization sequencing reaction, preferably, Such an agent is a reducing agent.
  • the regenerating reagent comprises an agent that removes a label (eg, a fluorescent label) from a labeled (eg, fluorescently labeled) nucleotide, preferably such reagent is a reducing agent.
  • the reagent that removes the label (eg, fluorescent label) from the labeled (eg, fluorescently labeled) sequencing probe or nucleotide can be a reducing agent, such as by linking the label with a functional group that is susceptible to reductive cleavage. In the case of nucleotides.
  • a reducing agent examples include, but are not limited to, a metal catalyst such as a nickel catalyst, a platinum catalyst, a palladium catalyst, an alcohol compound, a phenol compound, an aldehyde compound, an olefin compound, an amine compound, a ketone compound, a phosphorus-containing compound, Peroxides, thiol compounds such as ⁇ -mercaptoethanol, dithiothreitol, phosphines (eg, tris(hydroxymethyl)phosphine (THP), tris(2-carboxyethyl)phosphine, etc.) (TCEP), etc.
  • a metal catalyst such as a nickel catalyst, a platinum catalyst, a palladium catalyst
  • an alcohol compound such as a nickel catalyst, a platinum catalyst, a palladium catalyst
  • an alcohol compound such as a phenol compound, an aldehyde compound, an olefin compound, an amine compound, a ketone compound, a
  • the reducing agent is any suitable reducing agent capable of reducing disulfide bonds, including but not limited to thiol compounds such as ⁇ -mercaptoethanol, dithiothreitol, phosphines (eg, tris(hydroxymethyl)phosphine (THP) ), tris(2-carboxyethyl)phosphine, etc.) (TCEP) and the like.
  • thiol compounds such as ⁇ -mercaptoethanol, dithiothreitol, phosphines (eg, tris(hydroxymethyl)phosphine (THP) ), tris(2-carboxyethyl)phosphine, etc.) (TCEP) and the like.
  • the regenerant containing reductant used in embodiments of the invention should be capable of removing labels (e.g., fluorescent labels) from labeled (e.g., fluorescently labeled) sequencing probes or nucleotides without disrupting sequencing.
  • labels e.g., fluorescent labels
  • the regenerating reagent comprises a regenerative reagent for use in a sequencing kit for the BGISEQ-500 sequencing platform, such as the BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhicheng Technology Co., Ltd., Regeneration reagent in item number PF-UM-PEV30).
  • a sequencing kit for the BGISEQ-500 sequencing platform such as the BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhicheng Technology Co., Ltd., Regeneration reagent in item number PF-UM-PEV30).
  • the invention relates to a method of eliminating regenerative reagent residues comprising: a. providing a solid support on which a nucleic acid molecule is immobilized, b. using a sequencing kit for the BGISEQ-500 sequencing platform
  • the regenerative reagent such as the BGISEQ-500 high-throughput sequencing kit (SE50 V3.0, Shenzhen Huada Zhizhi Technology Co., Ltd., Cat. No. PF-UM-PEV30), treats the solid support, the regenerating reagent Containing a reducing agent, and c. treating the solid with a washing solution to which a compound having a disulfide bond structure is added Support.
  • the solid support is free or substantially free of regenerating reagent after the treatment of step c.
  • a sequencing reagent containing a sequencing kit for the BGISEQ-500 sequencing platform eg, BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0, Shenzhen Hua) is optionally used.
  • the solid support is treated with one or more reaction solutions of a sequencing reagent in Dazhicheng Technology Co., Ltd., No.
  • PF-UM-PEV30 to generate a nucleotide sequence representing the nucleic acid molecule on a solid support a signal (eg, a fluorescent signal), and wherein the regeneration reagent can eliminate the signal (eg, a fluorescent signal) on the solid support.
  • a sequencing reagent in a sequencing kit for the BGI-SEQ500 sequencing platform eg, BGISEQ-500 High Throughput Sequencing Kit (SE50 V3.0
  • SE50 V3.0 the sequencing kit for the BGI-SEQ500 sequencing platform
  • the solid support is treated with one or more reaction solutions of Shenzhen Huada Zhixing Technology Co., Ltd., the sequencing reagent in PF-UM-PEV30).
  • the invention also relates to a kit for sequencing, the kit comprising a sequencing reagent, a washing reagent, a regenerating reagent comprising a reducing agent, and a washing solution to which a compound containing a disulfide bond structure is added, as described herein,
  • the sequencing reagent can generate a signal (eg, a fluorescent signal) representing a nucleotide sequence of the nucleic acid molecule on a solid support to which the nucleic acid molecule is immobilized, and wherein the regeneration reagent can eliminate the solid support The signal.
  • the sequencing reagent comprises a sequencing reagent for ligation sequencing or synthetic sequencing.
  • a wash reagent is any solution that is capable of washing away a substance on its solid support that is non-specifically bound thereto and does not adversely affect subsequent reactions.
  • the wash reagent contains a buffer, such as an organic salt, to maintain a stable pH of from about pH 6 to pH 9, and may also contain monovalent or divalent cations to remove non-specifically bound molecules from the solid support.
  • Exemplary washing reagents can include, for example, 100 mM Tris-HCl buffer at pH 6.5, TE buffer (Tris-HCl pH 8, 10 mM and EDTA, 1 mM), and the like.
  • the buffering agent can also be, for example, a buffering agent as described below.
  • a compound having a disulfide bond structure includes any compound having a bond (also referred to as a disulfide bond) between sulfur atoms in the -S-S-form in its chemical structure.
  • the compound containing a disulfide bond structure has the formula -RSS-R'-, wherein R and R' are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, Optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted Heterocyclyl, optionally substituted heteroalicyclic, optionally substituted aralkyl or optionally substituted (heteroalicyclic)alkyl.
  • R and R' are each independently selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl, Optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted cycloalkynyl, optionally substituted aryl, optionally substituted heteroaryl,
  • the compound containing a disulfide bond structure has the formula -C(R 1 ) m (R 2 ) m' -C(R 3 ) p -SSC(R 4 ) q -C(R 5 ) n (R 6 ) n' -, wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of hydrogen, oxo, hydroxy, protected hydroxy, alkoxy, aryloxy Base, acyl, decyl, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl , C-amido, N-acylamino, S-sulfonylamino, N-sulfonylamino, C-carboxyl
  • the compound containing a disulfide bond structure may be selected from, but not limited to, such as diethyl disulfide, di-n-propyl disulfide, di-n-butyl disulfide, di- Sec-butyl disulfide, di-tert-butyl disulfide, di-n-pentyl disulfide, di-tert-amyl disulfide, di-tert-hexyl disulfide, di-n-octyl Dithioether, di-tert-octyl disulfide, di-n-dodecyl disulfide, di-tert-dodecyl disulfide, di-n-stearyl disulfide, ethyl- Dialkyl disulfide compounds such as n-propyl disulfide, ethyl-tert-butyl disulfide, ethyl-tert-but
  • the compound containing a disulfide bond structure is selected from the group consisting of cystamine, cystine, 5,5'-dithiobis(2-nitrobenzoic acid), and 2-hydroxyethyl disulfide. Its derivatives.
  • the R, R', R 1 , R 2 , R 3 , R 4 , R 5 and R 6 groups represent substituents which are capable of attaching to a specified atom.
  • the R, R', R 1 , R 2 , R 3 , R 4 , R 5 and R 6 groups may be substituted or unsubstituted.
  • the R, R', R 1 , R 2 , R 3 , R 4 , R 5 and R 6 groups together with the atoms to which they are attached are capable of forming a cycloalkyl, aryl, heteroaryl or heterocyclic ring.
  • the group may be unsubstituted or substituted with one or more specified substituents.
  • the substituent may be selected from one or more of the specified substituents.
  • substituent it is meant that the specified "optionally substituted” or “substituted” group may be substituted, individually and independently, by one or more groups, which are individually and independently selected from the group consisting of Functional groups, including but not limited to: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroarylalkyl, (heteroalicyclic)alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, decyl, alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamoyl , N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-acylamino, S-sulfonyla
  • Functional groups
  • alkyl refers to a straight or branched hydrocarbon chain that includes a fully saturated (no double or triple bond) hydrocarbon group.
  • the alkyl group can have from 1 to 20 carbon atoms (when it appears herein, such as the numerical range of "1-20” refers to a range including endpoints within a given range.
  • Each integer; for example, “1-20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms, however this definition also covers The occurrence of the term "alkyl” which does not specify a range of values).
  • the alkyl group can also be a medium size alkyl group having from about 7 to about 10 carbon atoms.
  • the alkyl group may also be a lower alkyl group having 1 to 6 carbon atoms.
  • the alkyl group of the compound can be designated as "C 1 -C 4 alkyl group” or the like.
  • “C 1 -C 4 alkyl” means that one to four carbon atoms are present in the alkyl chain, ie, the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl Base, isobutyl, sec-butyl and tert-butyl.
  • Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, and hexyl.
  • the alkyl group can be substituted or unsubstituted.
  • alkenyl refers to a hydrocarbyl group containing one or more double bonds in a straight or branched hydrocarbon chain.
  • the alkenyl group can be unsubstituted or substituted.
  • alkynyl refers to a hydrocarbyl group containing one or more triple bonds in a straight or branched hydrocarbon chain.
  • An alkynyl group can be unsubstituted or substituted.
  • cycloalkyl refers to a fully saturated (no double or triple bond) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be bonded together in a fused form.
  • the cycloalkyl group may have 3 to 10 atoms in the ring. In certain embodiments, a cycloalkyl group can contain from 3 to 8 atoms in the ring.
  • a cycloalkyl group can be unsubstituted or substituted.
  • Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • aryl refers to a monocyclic or polycyclic aromatic ring system of carbon rings (all carbon) (including, for example, fused, bridged or spiro ring systems in which two carbon rings share a chemical bond, For example, one or more aromatic rings carry one or more aromatic or non-aromatic rings) having a fully delocalized pi-electron system throughout at least one ring.
  • the number of carbon atoms in the aryl group is variable.
  • the aryl group can be a C 6 -C 14 aryl group, a C 6 -C 1o aryl group, or a C 6 aryl group.
  • Examples of aryl groups include, but are not limited to, benzene, naphthalene, and chamomile rings.
  • the aryl group can be substituted or unsubstituted.
  • heterocyclyl refers to a ring system comprising at least one hetero atom (eg, O, N, S). Such systems may be unsaturated, may include partial unsaturation, or may contain some aromatic moieties, or be completely aromatic.
  • the heterocyclic group may be unsubstituted or substituted.
  • heteroaryl refers to a monocyclic or polycyclic aromatic ring system (a ring system having at least one ring that is a fully delocalized pi-electron system) that contains one or more heteroatoms, ie, carbon Elements other than, including but not limited to nitrogen, oxygen and sulfur, and comprising at least one aromatic ring.
  • the number of atoms in the ring of the heteroaryl group is variable.
  • a heteroaryl group can contain 4-14 ring atoms, 5-10 ring atoms, or 5-6 ring atoms.
  • heteroaryl includes fused ring systems in which two rings, for example at least one aromatic ring and at least one heteroaryl ring, or at least two heteroaryl rings share at least one chemical bond.
  • heteroaromatic rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, pyridazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxa Diazole, thiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole, benzimidazole, indole, oxazole, pyrazole, benzopyrazole, different Oxazole, benzisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyr
  • heteroalicyclic refers to three, four, five, six, seven, eight, nine, ten, up to 18-membered monocyclic, bicyclic, and tricyclic
  • a ring system in which a carbon atom and 1-5 heteroatoms together form the ring system.
  • the heterocycle can optionally contain one or more unsaturated bonds positioned in such a manner that there is no fully delocalized pi-electron system throughout all of the rings.
  • the heteroatoms are independently selected from the group consisting of oxygen, sulfur, and nitrogen.
  • the heterocyclic ring may further comprise one or more carbonyl or thiocarbonyl functional groups, such that the definition includes oxo systems and sulfur Generation systems such as lactams, lactones, cyclic imides, cyclic thioimides, and cyclic carbamates. When composed of two or more rings, the rings may be bonded together in a fused form. Additionally, any nitrogen in the heteroalicyclic ring can be quaternized.
  • the heteroalicyclic or heteroalicyclic group may be unsubstituted or substituted.
  • heteroalicyclic or “heteroalicyclic” groups include, but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane, 1,3-dioxolan, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxethiohexadiene, 1,3-oxathiolane, 1,3-dithiolepine, 1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4- Thiazide, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil , trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine, isox
  • aralkyl and aryl (alkyl) refer to an aryl group as a substituent attached through a lower alkylene group.
  • the lower alkylene and aryl groups of the aralkyl group may be substituted or unsubstituted. Examples include, but are not limited to, benzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.
  • heteroaryl and “heteroaryl (alkyl)” refers to a heteroaryl group attached as a substituent through a lower alkylene group.
  • the lower alkylene and heteroaryl groups of the heteroarylalkyl group may be substituted or unsubstituted. Examples include, but are not limited to, 2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl And their benzo-fused analogs.
  • hydrocarbyloxy refers to the formula -OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl or cycloalkynyl as defined above.
  • alkoxy groups are methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy And tert-butoxy.
  • the alkoxy group may be substituted or unsubstituted.
  • the C-acylamino group may be substituted or unsubstituted.
  • R and R a may independently be hydrogen, alkyl, alkenyl, alkynyl.
  • the N-acylamino group may be substituted or unsubstituted.
  • halogen atom means any of the radiation-stable atoms of the seventh column of the periodic table, such as fluorine, chlorine, bromine and iodine.
  • amine refers to a -NH 2 group, wherein one or more hydrogen may optionally be substituted with R groups.
  • R may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl or (heteroalicyclic) alkyl.
  • aldehyde refers to a -R c -C (O) H group, wherein R c may be absent, or independently selected from alkylene, alkenylene, alkynylene, cycloalkylene, alkylene Cycloalkenyl, cycloalkynylene, arylene, heteroarylene, heteroalicyclic, aralkylene or (heteroalicyclic)alkyl.
  • amino refers to a -NH 2 group.
  • hydroxy refers to an -OH group.
  • cyano refers to a "-CN” group.
  • azido refers to a -N 3 group.
  • mercapto refers to a -SH group.
  • carboxylic acid refers to -C(O)OH.
  • thiocyanate refers to a -S-C ⁇ N group.
  • amine group refers to the group -O-NH 2, wherein one or more hydrogens on -NH 2 may optionally be substituted with R groups.
  • R may independently be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclic, aralkyl or (heteroalicyclic) alkyl.
  • the term "derivative” means a similar compound that is derived from a specified compound by physical or chemical processes. Derivatives can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and are for example, by J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure (Advanced Organic Chemistry: Reactions, Mechanisms). And Structure ”)", 4th edition (New York: Wiley-Interscience, 1992).
  • a base addition salt is prepared from a compound using conventional means, including reacting one or more free hydroxyl groups of the compound with a suitable base.
  • the compound is dissolved in a polar organic solvent such as methanol or ethanol and a base is added thereto.
  • Salts suitable for the formation of cystamines of base addition salts refer to cystamine salts derived from inorganic and organic acids and bases.
  • suitable acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, k nitrate, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, Tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and oxalic acid.
  • salts derived from amino acids such as L-arginine, L-lysine.
  • suitable bases include, but are not limited to, inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.
  • a wash solution comprising a compound having a disulfide bond structure also contains a buffer.
  • the buffer is any buffer suitable for treating a fixed support to which a nucleic acid molecule is immobilized. It should be understood that the treatment of such a buffer with a solid support should not deal with the structure of the nucleic acid molecule on the solid support, the binding of the nucleic acid molecule to the solid support, and the sequencing enzyme, probe and nucleotide structure in subsequent sequencing reagents and Activity has an adverse effect. Suitable buffers can be readily determined by those skilled in the art.
  • Suitable buffering agents may contain, for example, an organic salt to maintain a stable pH of from about pH 6 to pH 9, and may also contain monovalent or divalent cations or detergents to remove non-specifically bound molecules from the solid support.
  • Exemplary buffers can include, for example, Tris-HCl buffer (eg, 100 mM Tris-HCl buffer at pH 6.5), SSC buffer (eg, 0.3 x SSC/0.1% Tween), TE buffer (eg, 10 mM Tris with pH 8). -HCl and 1 mM EDTA in TE buffer) and the like.
  • the disulfide-containing compound of the present invention is capable of undergoing a redox reaction with a reducing agent in a regenerating agent, and at the same time, such a disulfide-containing bond of the present invention
  • the compound does not cause any significant adverse effects on the sequencing of the nucleic acid, such as the nature of the sequencing reagent (eg, sequencing enzyme, labeled probe or nucleotide), the nature of the nucleic acid molecule on the solid support, and The binding of the solid support to the nucleic acid molecule immobilized thereon causes any significant adverse effects.
  • many other commonly used oxidants can adversely affect sequencing, such as disrupting the structure of dNTPs, affecting the activity of polymerases, and the like.
  • the oxidizing property of the disulfide bond-containing compound of the present invention may be derived only from a disulfide bond structure. That is, the disulfide bond-containing compound of the present invention may not contain any other oxidizing group other than -S-S-.
  • the disulfide bond-containing compound of the present invention may contain at least one disulfide bond.
  • a compound containing a disulfide bond structure may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds.
  • the compound containing a disulfide bond structure contains 1-3 disulfide bonds.
  • the compound containing a disulfide bond structure contains 1-2 disulfide bonds.
  • the compound containing a disulfide bond structure contains only one disulfide bond.
  • the amount of the disulfide-containing structure-containing compound in the solution containing the compound is sufficient to significantly reduce or even eliminate or be in fluid communication with the solid support during the sequencing process. Any amount of residual reagent regenerating on the flow channel.
  • the amount of the disulfide-containing structure-containing compound in the solution containing the compound can be, for example, at least about 0.1 mM, at least about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM, at least about 0.5 mM, At least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, at least about 0.10 mM, at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least About 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at
  • the amount of the disulfide-containing structure-containing compound in the solution containing the compound can be, for example, up to about 1 mM, up to about 2 mM, up to about 3 mM, up to about 4 mM, up to about 5 mM, up to about 6 mM, up to About 7 mM, up to about 8 mM, up to about 9 mM, up to about 10 mM, up to about 15 mM, up to about 20 mM, up to about 25 mM, up to about 30 mM, up to about 35 mM, up to about 40 mM, up to about 45 mM, up to about 50 mM, up to about 55 mM Up to about 60 mM, up to about 70 mM, up to about 80 mM, up to about 90 mM, up to about 100 mM, or a range between any two of the foregoing.
  • the amount of the compound containing a disulfide bond structure in the solution containing the compound may be, for example, from about 1 mM to about 50 mM. In one embodiment, the amount of the compound containing a disulfide bond structure in the solution containing the compound may be, for example, from about 2 mM to about 20 mM. In one embodiment, the amount of the compound containing a disulfide bond structure in the solution containing the compound may be, for example, from about 5 mM to about 10 mM.
  • RunOn is used to evaluate the phenomenon of base synthesis advancement in sequencing.
  • Advance base synthesis directly leads to a decline in the quality of sequencing data.
  • RunOn detection can be performed and reported according to the software that comes with the BGISEQ-500 sequencing platform (Shenzhen Huada Zhizhi Technology Co., Ltd.) or other suitable methods. Residues of regenerated reagents typically result in higher RunOn values during sequencing.
  • the inventors have discovered that the addition of a compound containing a disulfide bond structure as described herein during the washing of the regenerating agent in the sequencing procedure can significantly reduce the RunOn value and is not detrimental
  • the effect of the sequencing reagent used in the subsequent sequencing cycle is affected.
  • other oxidants such as hydrogen peroxide, iron sulfate, and copper hydroxide severely interfere with the sequencing reaction, resulting in the inability to measure the RunOn value.
  • Figure 1 shows a comparison of RunOn values for sequencing using a wash solution with or without added cystamine.
  • Figure 2 shows a comparison of RunOn values for sequencing using a wash solution with or without added cystine.
  • Figure 3 shows a comparison of RunOn values for sequencing using a wash solution with or without addition of 5,5'-dithiobis(2-nitrobenzoic acid).
  • Figure 4 shows a comparison of RunOn values for sequencing using a wash solution with or without addition of 2-hydroxyethyl disulfide.
  • Figure 5 shows a comparison of RunOn values for sequencing using a wash solution with or without added cystamine.
  • Figure 6 shows a comparison of signal intensities for sequencing using a wash solution supplemented with the addition of hydrogen peroxide, iron sulfate or copper hydroxide or 2-hydroxyethyl disulfide.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip.
  • the prepared sequencing chip was loaded into BGISEQ-500 sequencing using the BGISEQ-500 High-throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30) according to the manufacturer's instructions.
  • the platform (Shenzhen Huada Zhizao Technology Co., Ltd.) completed the sequence determination of 30 bases and selected 1*1 of the photographed area.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip.
  • the prepared sequencing chip was loaded into BGISEQ-500 sequencing using the BGISEQ-500 High-throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30) according to the manufacturer's instructions.
  • the platform (Shenzhen Huada Zhizao Technology Co., Ltd.) completed the sequence determination of 20 bases and selected 1*1 of the photographed area.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip.
  • the prepared sequencing chip was loaded into BGISEQ-500 sequencing using the BGISEQ-500 High-throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30) according to the manufacturer's instructions.
  • the platform (Shenzhen Huada Zhizao Technology Co., Ltd.) completed the sequence determination of 40 bases and selected 1*1 of the photographed area.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip.
  • the prepared sequencing chip was loaded into BGISEQ-500 sequencing using the BGISEQ-500 High-throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30) according to the manufacturer's instructions.
  • the platform (Shenzhen Huada Zhizao Technology Co., Ltd.) completed the sequence determination of 50 bases and selected 1*1 of the photographed area.
  • MGIEasy TM DNA library preparation kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip.
  • the prepared sequencing chip was subjected to the sequence determination of 50 bases according to the following procedure, and the photographing area of 1*1 was selected:
  • the chip was immersed in the polymerization reagent for about 1 min, the chip was removed, and then immersed in the washing reagent 2 for about 1 min, the chip was removed, and then immersed in the protective reagent for about 1 min, the chip was removed, and photographed for 20-30 min to detect the representative.
  • Fluorescence signal of identity information of one base then immersing the chip in the regeneration reagent for about 1 min, removing the chip, then immersing in the washing reagent 1 for about 1 min, removing the chip; then repeating the foregoing steps on the chip to proceed to the next base Sequencing of the base; the reagents used were all from the BGISEQ-500 sequencing kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30), the temperature and other reaction conditions of each step and the photographing procedure were referenced. Instructions for the kit and standard procedures for the BGISEQ-500 sequencing platform were performed.
  • E. coli genomic DNA prepared using MGIEasy TM DNA library kit (Shenzhen Hua Taizhi Ltd. made) to a standard strain of E. coli extract as raw material DNA construct a DNA library for sequencing, loaded onto a sequencing chip on.
  • the prepared sequencing chip was loaded into BGISEQ-500 sequencing using the BGISEQ-500 High-throughput Sequencing Kit (SE50 V3.0, Shenzhen Huada Zhizao Technology Co., Ltd., item number PF-UM-PEV30) according to the manufacturer's instructions.
  • the platform (Shenzhen Huada Zhizao Technology Co., Ltd.) completed the 50-base sequence determination and selected 1*1 of the photographed area, using the elution reagent 1 in the BGISEQ-500 sequencing kit V3.0 (recycling reagent in use) 50 mM 2-hydroxyethyl disulfide was added to the reagent for washing the chip after processing the chip.
  • the fluorescence signal intensity values of the four sets of sequencing experiments were output using the program provided by the BGISEQ-500 sequencing platform, and the results obtained are shown in FIG. 6.

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  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de traitement d'un support solide fixé avec des molécules d'acide nucléique sur celui-ci, le support solide étant déjà traité par un agent de régénération et l'agent de régénération comprend un agent réducteur qui peut éliminer un groupe de blocage et une étiquette à partir d'un nucléotide marqué bloqué en position 3'. Le procédé comprend l'utilisation d'une solution de lavage à laquelle est ajouté un composé contenant une structure de liaison disulfure pour traiter le support solide. Le support solide est également facultativement traité à l'aide d'une solution de réaction contenant une polymérase, un nucléotide marqué bloqué en 3' et des amorces facultatives après le traitement avec la solution de lavage, par exemple, les amorces contiennent la séquence complémentaire d'au moins une partie de chaque molécule d'acide nucléique sur le support solide.
PCT/CN2017/106972 2017-10-20 2017-10-20 Procédé d'élimination de résidu d'agent de régénération Ceased WO2019075711A1 (fr)

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WO2024108376A1 (fr) * 2022-11-22 2024-05-30 深圳华大智造科技股份有限公司 Kit de test réactif et son application au séquençage

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