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WO2024164823A1 - Analogue nucléotidique et son utilisation dans le séquençage - Google Patents

Analogue nucléotidique et son utilisation dans le séquençage Download PDF

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WO2024164823A1
WO2024164823A1 PCT/CN2024/073078 CN2024073078W WO2024164823A1 WO 2024164823 A1 WO2024164823 A1 WO 2024164823A1 CN 2024073078 W CN2024073078 W CN 2024073078W WO 2024164823 A1 WO2024164823 A1 WO 2024164823A1
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unsubstituted
substituted
sequencing
nucleotide
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徐杰成
刘二凯
陈德遐
王谷丰
赵陆洋
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Shenzhen Salus Biomed Co Ltd
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Shenzhen Salus Biomed Co Ltd
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    • 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
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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    • C09K2211/1018—Heterocyclic compounds
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present application relates to the field of nucleotide technology, and in particular to nucleotide analogs and their application in sequencing.
  • the nucleotide analogs with blocking groups will terminate the next extension of the primer until the deblocking reagent is added, and the next biochemical reaction can be carried out.
  • This cycle is repeated many times, and the special markers carried by it are detected in each cycle, and the sequence of the target nucleic acid is analyzed.
  • the nucleotide analog obtained by introducing an azidomethyl group as a blocking group to protect the 3′-OH of the nucleotide will block the incorporation of the next base during the extension process of the target nucleic acid.
  • a trivalent phosphorus reagent is added to remove the azidomethyl group, and then the next round of biochemical reaction is carried out. This cycle is repeated many times, and detection is performed in each cycle to obtain the nucleic acid sequence.
  • phasing refers to the process of sequencing while synthesizing, in which a part of the DNA chain cannot be completely incorporated into the cluster by the polymerase in the next round of sequencing cycle due to incomplete removal of the 3′ terminator and the fluorophore.
  • Pre-phasing refers to the incorporation of nucleotide analogs in the absence of an effective 3′ terminator, and the incorporation process is performed in advance for one cycle. Taking the fluorescently labeled nucleotides protected by azidomethyl as an example, sodium azide is required during the synthesis of the azide group, and sodium azide is explosive.
  • organic azide compounds As an organic azide compound, its thermal stability is poor at the sequencing biochemical reaction temperature, and the azide methyl group is easily detached to expose the 3′-OH, causing the next round of biochemical reaction in sequencing, resulting in a high Pre-phasing value.
  • organic azide compounds are easily affected by reducing agents during storage and transfer; and the removal of the azide methyl group requires the use of reducing agents such as easily oxidized trivalent phosphorus reagents.
  • the excision time and excision efficiency affect the next biochemical reaction. Incomplete excision will lead to too high phasing values, and too long excision reaction time will lead to Too low efficiency will affect the biochemical reaction time of sequencing, increase sequencing costs, etc. Therefore, it is necessary to provide a nucleotide analog for sequencing, which can reduce the reaction time and phasing value in the sequencing process, and ultimately reduce the error rate and improve the accuracy.
  • the present application aims to solve at least one of the technical problems existing in the prior art.
  • the present application proposes a nucleotide analog and its application in sequencing, which can be quickly removed as a replacement for the reversible terminator in sequencing to expose 3′-OH, reduce the reaction time in the sequencing process, reduce the phasing value in the sequencing process, and ultimately reduce the error rate and improve the accuracy.
  • nucleotide analogue having a structural formula as shown in formula (I) is provided:
  • R 1 includes an optional orthogonal breaking group
  • R 2 includes a base
  • R 3 includes any one of a hydroxyl group and a phosphate group
  • R 4 includes any one of hydrogen and OR 5 , and R 5 is an optional orthogonal breaking group;
  • At least one of R 1 , R 2 , R 3 , and R 4 is also linked to a detectable label.
  • the compounds provided in the examples of the present application introduce an orthogonal cleavage group on the nucleotide.
  • the orthogonal cleavage group as a protecting group can be quickly removed through an inverse electron demand Diels-Alder (IEDDA) reaction, thereby exposing 3′-OH for the next round of biochemical reaction, greatly shortening the reaction time in the sequencing process, reducing the phasing value, and ultimately reducing the error rate in the sequencing process and improving the accuracy of sequencing.
  • IEDDA inverse electron demand Diels-Alder
  • the orthogonal cleavage group refers to a group based on the bioorthogonal cleavage (click to release) principle, which has bioorthogonality and controllable bond cleavage properties.
  • the orthogonal cleavage group is a dienophile group.
  • the dienophile group as a linker for orthogonal cleavage, an inverse electron demand Diels-Alder (IEDDA) click reaction occurs, so that the orthogonal cleavage group as a protecting group can be quickly released from the 3′-OH position, achieving a shorter excision time and higher excision efficiency, and reducing the time required for sequencing.
  • IEDDA inverse electron demand Diels-Alder
  • the orthogonal breaking group is -LR 6 ;
  • L is selected from non-existent, substituted or unsubstituted C1-C10 alkylene, substituted or unsubstituted C1-C10 heteroalkylene any one of substituted or unsubstituted C1-C10 cycloalkylene, substituted or unsubstituted C1-C10 heterocycloalkylene, substituted or unsubstituted C6-C10 arylene and substituted or unsubstituted C6-C10 heteroarylene;
  • R6 is selected from a substituted or unsubstituted trans-cyclooctene group, a substituted or unsubstituted norbornene group, a substituted or unsubstituted benzonorbornene group, a substituted or unsubstituted vinyl group, a substituted or unsubstituted cyano group, and a substituted or unsubstituted azidophenyl group.
  • the substituent of the group in the above L or R6 can be selected from at least one of halogen, haloalkyl, cyano, amino, nitro, carboxyl, thiol, sulfonic acid, acyl, amide, sulfonyl, carbonyl, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted alkoxy, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted heteroaryl.
  • heteroatoms in the heteroalkyl, heteroalkylene, heteroaryl, heteroarylene, heterocycloalkyl, and heterocycloalkylene include but are not limited to atoms such as O, N, P, S, and Si, and the number of heteroatoms can be at least one.
  • L is selected from any one of absent, substituted or unsubstituted C1-C8 alkylene, substituted or unsubstituted C1-C8 heteroalkylene, substituted or unsubstituted C1-C8 cycloalkylene, substituted or unsubstituted C1-C8 heterocycloalkylene, substituted or unsubstituted C6-C8 arylene and substituted or unsubstituted C6-C8 heteroarylene.
  • L is selected from any one of absent, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C1-C6 cycloalkylene, substituted or unsubstituted C1-C6 heterocycloalkylene, substituted or unsubstituted C6-C8 arylene and substituted or unsubstituted C6-C8 heteroarylene.
  • L is selected from any one of absent, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C1-C6 cycloalkylene, substituted or unsubstituted C1-C6 heterocycloalkylene, substituted or unsubstituted phenylene and substituted or unsubstituted heterophenylene.
  • L is selected from any one of absent, substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 heteroalkylene, substituted or unsubstituted C1-C4 cycloalkylene, and substituted or unsubstituted C1-C4 heterocycloalkylene.
  • L is selected from any one of absent, substituted or unsubstituted C1-C2 alkylene, substituted or unsubstituted C1-C2 heteroalkylene, substituted or unsubstituted C1-C2 cycloalkylene, and substituted or unsubstituted C1-C2 heterocycloalkylene.
  • R 6 is a substituted or unsubstituted trans-cyclooctene group.
  • the orthogonal breaking group is selected from any one of the following structures:
  • L 1 , L 2 , and L 3 are each independently absent, an ester group, an ether group, or a C1-C6 alkylene group;
  • R6 is hydrogen or a boronic acid group.
  • L 1 is selected from any one of absence, ester group, and ether group.
  • L2 is absent.
  • L 3 is any one of C1 to C6 alkylene. In some embodiments, L 3 is any one of C1 to C3 alkylene.
  • the orthogonal breaking group is selected from any one of the following:
  • the phosphate group in R 3 can be a monophosphate or a polyphosphate group (eg, 2 or more).
  • the second aspect of the present application further provides the use of the above nucleotide analogs in the synthesis or sequencing of nucleic acids.
  • the synthesis of nucleic acids includes any one of enzymatic synthesis and chemical synthesis.
  • the synthesis of nucleic acid includes any one of enzymatic synthesis and chemical synthesis of DNA.
  • the enzymatic synthesis of nucleic acids refers to the use of the aforementioned compounds as synthesis substrates, a system including a DNA template and an enzyme to complete the synthesis within a set reaction program including temperature conditions and reaction time.
  • the enzyme in the system includes a DNA polymerase, and in some embodiments, it can be an enzyme that can catalyze a template-dependent DNA polymerization reaction, such as Taq DNA polymerase, Pfu DNA polymerase, etc.
  • chemical synthesis of nucleic acid refers to using the aforementioned compounds as synthetic raw materials, and completing the synthesis process by protecting the non-reactive groups before the reaction, activating the reactive groups, coupling, and deprotecting after the reaction.
  • chemical synthesis of nucleic acid includes at least one of the phosphodiester bond method, the phosphotriester bond method, and the phosphite amide method.
  • chemical synthesis of nucleic acid is solid phase synthesis, that is, the synthesis reaction is carried out on a solid phase carrier.
  • the nucleic acid comprises more than 2 nucleotides, further more than 10, 20, 30, 50, 100, 200, 500, 1000 nucleotides.
  • the third aspect of the present application provides a sequencing method, comprising the following steps:
  • nucleotide analog includes four different tags according to different base types;
  • the cleavage agent includes a tetrazine compound.
  • the tetrazine compound has a general formula as shown in formula (II):
  • R 7 and R 8 are independently selected from hydrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 heteroalkyl, substituted or unsubstituted C1-C10 cycloalkyl, substituted or unsubstituted C1-C10 heterocycloalkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C1-C10 heteroaryl.
  • R 7 and R 8 are not hydrogen at the same time.
  • the substituents of the C1-C10 alkyl, C1-C10 heteroalkyl, C1-C10 cycloalkyl, C1-C10 heterocycloalkyl, C6-C10 aryl and C1-C10 heteroaryl are selected from at least one of amino and carboxyl groups.
  • R7 is hydrogen
  • R8 is selected from any one of C1 ⁇ C10 alkyl substituted by amino and/or carboxyl, C1 ⁇ C10 heteroalkyl substituted by amino and/or carboxyl, C1 ⁇ C10 cycloalkyl substituted by amino and/or carboxyl, C1 ⁇ C10 heterocycloalkyl substituted by amino and/or carboxyl, C6 ⁇ C10 aryl substituted by amino and/or carboxyl, and C1 ⁇ C10 heteroaryl substituted by amino and/or carboxyl.
  • the detectable label is a fluorescent group.
  • fluorescent groups include but are not limited to FAM, HEX, TAMRA, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas, ROX, JOE, R6G, EDANS, IFluor, Alexa Fluor, FITC, etc.
  • the nucleotide analogs used for sequencing generally have four different bases.
  • the nucleotide analogs of different base types have different detectable labels.
  • the detectable label is a fluorescent group
  • the emission wavelengths of the fluorescent groups of the nucleotide analogs of different base types are significantly different.
  • the detectable label can also be other labels known in the art that can distinguish different base types by at least one method in physics, chemistry, and biology.
  • the cleavage reaction uses pyridazine elimination triggered by IEDDA reaction, and tetrazine reacts with a dienophilic group (such as trans-cyclooctenyl) to form a dihydropyridazine intermediate, which can spontaneously lead to the release of the group at the allylic position through 1,4-elimination.
  • dienophilic groups especially trans-cyclooctenyl, are more suitable for the production of pyridazine.
  • the group has better stability.
  • a method for synthesizing nucleic acid comprises using the aforementioned nucleotide analogs as synthetic raw materials.
  • the synthesis method is a chemical synthesis method of nucleic acid or an enzymatic synthesis method of nucleic acid.
  • the fourth aspect of the present application also provides a composition comprising the aforementioned nucleotide analogs.
  • the composition includes sequencing reagents.
  • the composition includes a polymerization reagent.
  • the composition includes a polymerization reaction reagent and a cleavage reaction reagent.
  • the polymerization reaction reagent includes the aforementioned compound, and the cleavage reaction reagent includes a cleavage reagent.
  • the composition includes a polymerization reaction reagent and a cleavage reaction reagent.
  • the polymerization reaction reagent includes an orthogonal cleavage group selected from Any one of the aforementioned compounds, wherein the cleavage reaction reagent includes a tetrazine compound.
  • the polymerization reaction reagent further includes a polymerase.
  • “several” means more than one
  • “more” means more than two
  • “greater than”, “less than”, “exceed” etc. are understood as not including the number itself
  • “above”, “below”, “within” etc. are understood as including the number itself
  • “about” means within ⁇ 20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. If there is a description of first or second, it is only for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.
  • a nucleoside (Compound 1) is reacted with tert-butyldimethylsilyl chloride (TBSCl) and imidazole in N,N-dimethylformamide (DMF) at room temperature for 24 hours to add a TBS protecting group to the 5′-OH to obtain Compound 2.
  • TBSCl tert-butyldimethylsilyl chloride
  • DMF N,N-dimethylformamide
  • triphenylphosphine (PPh 3 ) and trans-cyclooctene-2′-OH were weighed into a round-bottom flask, tetrahydrofuran (THF) was added as a solvent and stirred to dissolve, diethyl azodicarboxylate (DEAD) was slowly added dropwise over 5 min, and then compound 2 dissolved in tetrahydrofuran was slowly added to the above mixture, and the reaction was monitored by TLC. After compound 2 was completely reacted, the reaction solvent was removed by a rotary evaporator, and the intermediate compound 3 was purified by normal silica gel chromatography to obtain a white solid.
  • THF tetrahydrofuran
  • DEAD diethyl azodicarboxylate
  • This embodiment provides a tetrazine compound 11 for excision reaction, and its synthesis route is as follows:
  • n-boc-b-cyano-l-alanine (compound 10) (600 mg, 2.8 mmol), acetic acid formamidinium salt (1.46 g, 14 mmol), Zn(OTf) 2 (255 mg, 0.7 mmol), dioxane (8.4 mL, 98 mmol) and hydrazine monohydrate (6.8 mL, 140 mmol) were added to a round-bottom flask and mixed evenly, and the mixture was immediately sealed. Stirred at 30°C for 72 hours, and then poured into 50 mL of ice water. After adding NaNO 2 (3.9 g, 56 mmol), the solution was acidified with 2N HCI aqueous solution (60 mL).
  • This embodiment provides a tetrazine compound 13 for excision reaction, and its synthesis route is as follows:
  • This example provides a nucleotide analogue, the synthesis route of which is different from that of Example 1 in that 2 ⁇ 3 is replaced by the following 2 ⁇ 14 ⁇ 15, and then compound 15 is used to replace compound 3 in steps (3) to (4) of Example 1:
  • This example provides a nucleotide analogue, the synthesis route of which is different from that of Example 1 in that 2 ⁇ 3 is replaced by 2 ⁇ 16 ⁇ 17 ⁇ 18 ⁇ 19 as follows, and then compound 19 is used to replace compound 3 in steps (3) to (4) of Example 1:
  • This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • nucleotide analogs dATP, dCTP, dGTP and dTTP include nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • dCTP nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • the difference between it and the nucleotide analog in Example 1 is that the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:
  • dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.
  • This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • nucleotide analogs dATP, dCTP, dGTP and dTTP include nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • dCTP nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:
  • dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.
  • This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • nucleotide analogs dATP, dCTP, dGTP and dTTP including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • dCTP nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:
  • dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.
  • This example provides a group of four nucleotide analogs with different detectable labels, including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • nucleotide analogs dATP, dCTP, dGTP and dTTP including nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • dCTP nucleotide analogs dATP, dCTP, dGTP and dTTP.
  • the base is modified with a detectable label through a linking group, specifically an optional fluorescent group Fluor, and the structural formula is as follows:
  • dATP, dGTP and dTTP are each modified with a fluorescent group via a linker group on the base, and the excitation bands of these fluorescent groups are different.
  • the proportion of nucleotides with unblocked 3′-OH was analyzed by HPLC, and the stability of the R1 protecting group in Examples 1, 2, 5, and 6 was determined according to the HPLC retention time, peak height, and peak area.
  • the R1 protecting group of the above nucleotide analogs has good thermal stability, which is 3.4 times higher than the stability of the nucleotides protected by azidomethyl under the same conditions. Therefore, the nucleotide analogs provided in the examples of the present application can ensure the smooth progress of polymerization reaction and signal acquisition in the sequencing cycle, reduce the phasing value, and facilitate the sequencing of the template with high accuracy.
  • THPP was used as a deprotection agent to perform a removal reaction on Comparative Example 1 (azidomethyl-protected nucleotide analogue).
  • Tetrazine compounds 11 and 13 prepared in Examples 5 and 6 were used as deprotection reagents to perform excision reactions on the nucleotide analogs prepared in Examples 1, 2, 5, and 6, and their respective half-retention rates were compared.
  • the principle of the tetrazine excision reaction is shown in the following route:
  • This embodiment provides a method for sequencing a human genome, and the specific process is as follows:
  • Illumina's MiSeq sequencer and its accompanying sequencing kit (MiSeq Reagent Kit v3) were used for library denaturation, library loading, and chip surface amplification to obtain DNA amplification clusters, and sequencing primers were added.
  • ACACTCTTTCCCTACACGACGCTCTTCCGATC SEQ ID No. 1.
  • a polymerization reaction solution is prepared, which contains DNA polymerase, Mg 2+ and 1 ⁇ M each of the nucleotide analogs dATP, dCTP, dGTP and dTTP in Example 7; as well as an elution buffer, a pre-wash buffer and an excision reaction solution containing compound 11.
  • the pre-wash buffer and the polymerization reaction solution are pumped in sequence to start the polymerization reaction.
  • the signal of the entire sequencing chip is collected to determine the type of base bound to the primer chain on each amplification cluster.
  • the elution buffer and the excision reaction solution are pumped in sequence to react, and then the elution buffer is pumped in for cleaning.
  • Comparative Example 2 A method for sequencing the human genome is provided, which differs from Example 12 in that the nucleotide analogs in the polymerization reaction solution are replaced by the corresponding nucleotides with azidomethyl protected 3′-OH, and at the same time, THPP is used for compound 11 in the excision reaction solution.
  • This embodiment provides a sequencing method, which is different from Embodiment 13 in that the nucleotide analogs used in the polymerization reaction solution are a group of four nucleotide analogs in Embodiment 8.
  • This embodiment provides a sequencing method, which is different from embodiment 13 in that the nucleotides used in the polymerization reaction solution are The analogs are a set of four nucleotide analogs in Example 9.
  • This embodiment provides a sequencing method, which is different from Embodiment 13 in that the nucleotide analogs used in the polymerization reaction solution are a group of four nucleotide analogs in Embodiment 10.
  • the phasing value and pre-phasing value of the excision reagent in the sequencing of Examples 14 to 16 are similar to those in Example 13, both lower than the corresponding control ratios.
  • Q30 is also similar to that in Example 13, both higher than the control ratios, which will not be repeated here.
  • the nucleotide analogs provided in the examples of the present application can stably protect the 3′-OH under the sequencing reaction conditions, and are quickly cleaved off to expose the 3′-OH after contact with the cleavage reagent, thereby not only reducing the phasing value in the sequencing process, but also ultimately reducing the error rate, improving the accuracy, and greatly shortening the sequencing time.
  • This embodiment provides a method for chemical synthesis of DNA, which is based on the phosphite-amide solid-phase chemical synthesis method on a DNA chip using the nucleotides of Examples 7 to 10 as reaction raw materials or their precursor compounds to synthesize a DNA sequence of a certain length through reaction steps such as deblocking, activation coupling, capping, and oxidation.

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Abstract

La présente invention concerne un composé ayant une formule développée telle que représentée dans la formule (I) et son utilisation dans le séquençage. Le composé introduit un groupe de clivage orthogonal sur un nucléotide, de telle sorte que lorsque le composé est utilisé en tant qu'amorce d'incorporation alternative d'un terminateur inversible pour extension, le groupe de clivage orthogonal servant de groupe protecteur peut être rapidement tronqué au moyen d'une réaction de Diels-Alder à demande d'électrons inverse (IEDDA), de façon à exposer 3'-OH pour une réaction biochimique du cycle suivant, ce qui permet de raccourcir ainsi de manière considérable le temps de réaction dans un procédé de séquençage, de réduire la valeur de phasage, de réduire enfin le taux d'erreur dans le procédé de séquençage, et d'améliorer la précision de séquençage.
PCT/CN2024/073078 2023-02-07 2024-01-18 Analogue nucléotidique et son utilisation dans le séquençage Ceased WO2024164823A1 (fr)

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CN117924392A (zh) * 2024-01-16 2024-04-26 深圳太古语科技有限公司 一种核苷酸衍生物及其应用

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