WO2024093421A1 - 表面处理方法、测序方法和试剂盒 - Google Patents

表面处理方法、测序方法和试剂盒 Download PDF

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WO2024093421A1
WO2024093421A1 PCT/CN2023/112186 CN2023112186W WO2024093421A1 WO 2024093421 A1 WO2024093421 A1 WO 2024093421A1 CN 2023112186 W CN2023112186 W CN 2023112186W WO 2024093421 A1 WO2024093421 A1 WO 2024093421A1
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formula
μmol
buffer
sequencing
concentration
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PCT/CN2023/112186
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English (en)
French (fr)
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卢柳燕
王文
赵楠楠
陈美容
尚欢
许定
何学森
刘磊
陈方
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深圳市真迈生物科技有限公司
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Priority claimed from CN202211351166.9A external-priority patent/CN117987520A/zh
Priority claimed from CN202211344450.3A external-priority patent/CN117987519A/zh
Application filed by 深圳市真迈生物科技有限公司 filed Critical 深圳市真迈生物科技有限公司
Publication of WO2024093421A1 publication Critical patent/WO2024093421A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention belongs to the technical field of nucleic acid sequencing, and relates to a surface treatment method and a sequencing method, a kit for extending nucleic acid molecules, an extension reagent buffer, an application of an extension reagent buffer, an extension kit, and a sequencing method.
  • the signal-to-noise ratio of the surface affects the recognition of the fluorescence signal.
  • a high signal-to-noise ratio may make it difficult to distinguish the fluorescence signal of the nucleotides involved in the sequencing reaction from the background signal on the surface, so that the fluorescence signal generated by the sample to be tested during the sequencing process cannot be effectively identified, which ultimately affects the accuracy of the sequencing results, or even makes it impossible to obtain the sequencing results.
  • the surface is used as a carrier, on which a large number of adapter sequences are randomly fixed.
  • the solution containing nucleotides and polymerase is introduced into the surface, the free nucleotides are easily randomly adsorbed on the chip surface, resulting in an increase in the surface signal-to-noise ratio.
  • the random adsorption generated on the surface is easily identified as a fluorescent signal point in the subsequent fluorescence imaging process, and when the fluorescent signal point is close to the fluorescent signal point generated by the real nucleic acid template site, it is easy to cause data analysis errors, which are manifested as a high error rate (insertion error ratio) and a reduction in the amount of effective data.
  • the present invention provides a surface treatment method, a sequencing method and a kit for extending nucleic acid molecules.
  • the present invention unexpectedly finds that the contact between a phosphate compound of a specific structure and the surface of a sequencing solid phase carrier can effectively reduce the random adsorption of nucleotide substrates on the surface of a sequencing chip during sequencing, thereby effectively reducing the error rate caused by the random adsorption of nucleotide substrates, which is of great significance to the field of gene sequencing.
  • the present invention adopts the following technical solutions:
  • the present invention provides a surface treatment method, the surface treatment method comprising:
  • the phosphate compound includes a compound having a structural formula as shown in formula (1) and/or formula (2);
  • R 1 and R 2 are each independently selected from any one of metal ions, hydrogen atoms, optionally substituted or unsubstituted hydrocarbon groups, optionally substituted or unsubstituted cycloalkyl groups, optionally substituted or unsubstituted heteroalkyl groups, optionally substituted or unsubstituted heterocycloalkyl groups, optionally substituted or unsubstituted aryl groups, and optionally substituted or unsubstituted heteroaryl groups;
  • R3 is selected from any substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, or any heteroaryl group;
  • n is an integer selected from 1-8.
  • the phosphate compound of the specific structure shown in formula (1) and (2) can be used to treat the surface bound to the oligonucleotide. After the phosphate compound contacts the surface bound to the oligonucleotide, the phosphate compound and the oligonucleotide bind to form a stable complex to block the reactivity of the oligonucleotide.
  • this method can reduce the signal interference caused by the reaction of the oligonucleotide with the substrate raw material, that is, reduce the nonspecific adsorption generated on the surface (this nonspecific adsorption is not what we expect to obtain).
  • this method When this method is applied to nucleic acid sequencing on a surface with a high signal-to-noise ratio, it can effectively reduce the random adsorption of the nucleotide substrate on the surface of the sequencing chip during the sequencing process (the adsorption reaction of the nucleotide substrate and the surface oligonucleotide), thereby effectively reducing the error rate caused by the random adsorption of the nucleotide substrate, and ultimately improving the sequencing quality and the sequencing read length.
  • R 1 and R 2 are a hydrogen atom or an aromatic group
  • R 3 is selected from an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group, and an aromatic group having 6 to 20 carbon atoms.
  • R 3 includes a cycloalkyl or a phenyl group.
  • the phosphate group in the phosphate compound is connected to the cycloalkyl or phenyl group, and the similarity of the structural type that is relatively similar to the nucleotide structure increases.
  • the nucleotide substrate such as a reversible terminator
  • oligonucleotide is a single-stranded structure, and the volume of the cycloalkyl or phenyl group is large, steric hindrance can be formed to a certain extent, which is also beneficial to reduce the probability of the reversible terminator and the oligonucleotide being combined.
  • formula (1) is selected from at least one of the compounds represented by formula (1-1) and formula (1-2):
  • the phosphate group in the phosphate compound is connected to a cycloalkyl or phenyl group having less than 10 carbon atoms, and the similarity with the nucleotide structure is further improved, so that when the above-mentioned phosphate compound and the nucleotide substrate (reversible terminator) are added at the same time, the phosphate compound can competitively bind to the oligonucleotide, thereby reducing the probability of the nucleotide substrate (reversible terminator) binding to the oligonucleotide.
  • R 2 contains at least one metal ion.
  • the metal ion in formula (1) helps the phosphate compound to form phosphate anions in the solution system, thereby more effectively binding to the oligonucleotide.
  • R2 contains n metal ions.
  • each phosphate group can form a phosphate anion, which increases the binding sites for the phosphate compound to react with the oligonucleotide.
  • the phosphate compound and the nucleotide substrate reversible terminator
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • n is selected from an integer of 2 to 6, including but not limited to 2, 3, 4, 5 or 6.
  • R 1 is selected from a hydrogen atom
  • R 2 is selected from a metal ion.
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • formula (2) is selected from at least one of the compounds represented by formula (2-1) and formula (2-2):
  • M is selected from Na or K.
  • the inventors have found that the phosphate compounds satisfying the above formula (2-1) and formula (2-2) can effectively bind to the oligonucleotides on the surface, so that when the oligonucleotides appear on the surface in an unexpected manner, the activity of the oligonucleotides can be inhibited by adding the phosphate compounds shown in the structures of formula (2-1) and formula (2-2), thereby inhibiting the interference caused by them.
  • the activity of the surface oligonucleotides can be inhibited by the phosphate compound, thereby reducing the specific adsorption of the nucleic acid molecules generated during the extension process, reducing the sequencing background signal, and thus improving the recognizability of the sequencing signal.
  • the phosphate compound is selected from any one or at least two of sodium phytate, disodium dihydrogen pyrophosphate and bisphenol A bis (diphenyl phosphate). These phosphate compounds are used for sequencing on a surface that does not require a high signal-to-noise ratio, and when amplified clustered nucleic acid molecules are sequenced, due to the small number of nucleic acids at each site, mostly one, the signal intensity generated by sequencing is weaker. At this point, the interference signal (background noise) has a significant impact on the recognition of sequencing signals.
  • the above-mentioned phosphate compound can be combined with the oligonucleotides on the surface to reduce the reactive activity of the oligonucleotides.
  • the binding activity of the former residence nucleotide sequence and the nucleotide substrate (reversible terminator) on the surface of the solid phase carrier is closed by the method, and the non-specific adsorption on the surface of the solid phase carrier can be significantly reduced, thereby reducing background noise, improving the recognizability of sequencing signals, reducing sequencing error rate, especially insertion type error rate, and ultimately promoting nucleic acid sequencing data volume.
  • sodium phytate has a chemical formula of C 6 H 6 Na 12 O 24 P 6 and a structural formula as shown in formula (3). It is in the form of white powder or crystal and is an important pure natural green additive. The most notable feature is that it has a strong chelating effect with metal ions, strong antioxidant and color protection properties, and is widely used as an antioxidant and color protection agent for fruit and vegetable juice drinks, meat products, and seafood.
  • Disodium dihydrogen pyrophosphate has a chemical formula of Na 2 H 2 P 2 O 7 and a structural formula as shown in formula (4). It is a white crystalline powder and is often used as a food additive.
  • Bisphenol A bis(diphenyl phosphate) (BDP for short) has a molecular formula of C 39 H 34 O 8 P 2 and a structural formula as shown in formula (5). It is often used as a flame retardant.
  • the contacting of the phosphate compound with the surface bound with the oligonucleotide comprises:
  • a first solution containing the phosphoric acid compound is flowed over the surface.
  • the concentration of the phosphate compound is 1 to 150 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 10 ⁇ mol/L, 20 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 91 ⁇ mol/L, 92 ⁇ mol/L, 95 ⁇ mol/L, 96 ⁇ mol/L, 98 ⁇ mol/L, 99 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L, 120 ⁇ mol/L, 130 ⁇ mol/L, 140 ⁇ mol/L or 150 ⁇ mol/L.
  • the excess phosphate compound will bind to other nucleotide molecules on the surface, such as occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain during nucleic acid sequencing. Since there is no polymerase in the reaction process and the phosphate compound cannot form hydrogen bonds with the complementary chain like nucleotides or nucleotide analogs, the reaction when the phosphate compound binds to other nucleotide molecules on the surface is not strong, but this will still have a certain impact on the sequencing reaction. In particular, when the concentration of the phosphate compound is too high, the excess phosphate compound may occupy the reaction position of the sequencing chain, and therefore, the throughput of nucleic acid sequencing may still be reduced.
  • the phosphate compound comprises sodium phytate, and in the first solution, the concentration of the sodium phytate is less than or equal to 15 ⁇ mol/L.
  • the phosphate compound is sodium phytate
  • the concentration of sodium phytate is 1 to 10 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 9 ⁇ mol/L or 10 ⁇ mol/L.
  • the adverse reactions that may be introduced by sodium phytate will also be reduced. For example, in the process of nucleic acid sequencing, the probability of sodium phytate occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain is reduced, and the sequencing flux is basically not affected or the impact produced can be ignored.
  • the phosphate compound comprises disodium dihydrogen pyrophosphate, and in the first solution, the concentration of the disodium dihydrogen pyrophosphate is less than or equal to 120 ⁇ mol/L.
  • the phosphate compound is disodium dihydrogen pyrophosphate
  • the concentration of disodium dihydrogen pyrophosphate is 10 to 120 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 20 ⁇ mol/L, 30 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 43 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L or 120 ⁇ mol/L.
  • the phosphoric acid compound comprises bisphenol A bis(diphenyl phosphate), and in the first solution, the concentration of bisphenol A bis(diphenyl phosphate) is less than or equal to 70 ⁇ mol/L.
  • the phosphate compound comprises bisphenol A bis(diphenyl phosphate), and in the first solution, the concentration of the bisphenol A bis(diphenyl phosphate) is 10 to 60 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 20 ⁇ mol/L, 30 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 43 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 52 ⁇ mol/L, 53 ⁇ mol/L, 54 ⁇ mol/L, 56 ⁇ mol/L, 58 ⁇ mol/L, 59 ⁇ mol/L or 60 ⁇ mol/L.
  • the present invention provides a sequencing method, comprising:
  • the surface is bound to a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule is a double-stranded nucleic acid molecule, and the first nucleic acid molecule contains at least one single-stranded end, and the second nucleic acid molecule is a single-stranded nucleic acid molecule;
  • the phosphate compound includes a compound having a structural formula as shown in formula (1) and/or formula (2):
  • R1 and R2 are each independently selected from any one of metal ion, hydrogen atom, any substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, any substituted or unsubstituted heteroaryl group;
  • R3 is selected from any one of optionally substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, and heteroaryl group; and n is selected from an integer of 1 to 8.
  • the compounds of the structures shown in formula (1) and formula (2) can competitively bind to the oligonucleotides remaining on the surface of the solid phase carrier (such as a biochip) (such as oligonucleotides bound to the surface of the solid phase carrier but not bound to the nucleic acid template), thereby reducing or inhibiting the binding of these oligonucleotides to the virtual terminator, that is, reducing the non-specific adsorption of the surface of the solid phase carrier, thereby reducing the signal interference caused by these oligonucleotides to nucleic acid sequencing (after the virtual terminator binds to these oligonucleotides, it will also generate a recognizable signal, thereby affecting the interference with the signal introduced into the nucleic acid template molecule), reducing the sequencing error rate caused by them, and further improving the sequencing accuracy.
  • the virtual terminator binds to these oligonucleotides remaining on the surface of the solid phase carrier but not bound to the nucleic acid template
  • At least one of R 1 and R 2 is a hydrogen atom or an aromatic group
  • R 3 is selected from an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group, and an aromatic group having 6 to 20 carbon atoms.
  • R 3 includes a cycloalkyl or a phenyl group.
  • the phosphate group in the phosphate compound is connected to the cycloalkyl or phenyl group, and the similarity of the structural type that is relatively similar to the nucleotide structure increases.
  • the nucleotide substrate such as a reversible terminator
  • oligonucleotide is a single-stranded structure, and the volume of the cycloalkyl or phenyl group is large, steric hindrance can be formed to a certain extent, which is also beneficial to reduce the probability of the reversible terminator and the oligonucleotide being combined.
  • formula (1) is selected from at least one of the compounds represented by formula (1-1) and formula (1-2):
  • the phosphate group in the phosphate compound is connected to a cycloalkyl or phenyl group having less than 10 carbon atoms, and the similarity with the nucleotide structure is further improved, so that when the above-mentioned phosphate compound and the nucleotide substrate (reversible terminator) are added at the same time, the phosphate compound can competitively bind to the oligonucleotide, thereby reducing the probability of the nucleotide substrate (reversible terminator) binding to the oligonucleotide.
  • R 2 contains at least one metal ion.
  • the metal ion in formula (1) helps the phosphate compound to form phosphate anions in the solution system, thereby more effectively binding to the oligonucleotide.
  • R2 contains n metal ions.
  • each phosphate group can form a phosphate anion, which increases the binding sites for the phosphate compound to react with the oligonucleotide.
  • the phosphate compound and the nucleotide substrate reversible terminator
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • n is selected from an integer of 2 to 6, including but not limited to 2, 3, 4, 5 or 6.
  • R 1 is selected from a hydrogen atom
  • R 2 is selected from a metal ion.
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • formula (2) is selected from at least one of the compounds having structural formulas such as formula (2-1) and formula (2-2):
  • M is selected from Na or K.
  • the inventors have found that the phosphate compounds satisfying the above formula (2-1) and formula (2-2) can effectively bind to the oligonucleotides on the surface, so that when the oligonucleotides appear on the surface in an unexpected manner, the activity of the oligonucleotides can be inhibited by adding the phosphate compounds shown in the structures of formula (2-1) and formula (2-2), thereby inhibiting the interference caused by them.
  • the activity of the surface oligonucleotides can be inhibited by the phosphate compound, thereby reducing the specific adsorption of the nucleic acid molecules generated during the extension process, reducing the sequencing background signal, and thus improving the recognizability of the sequencing signal.
  • the phosphate compound can be selected from any one or a combination of at least two of sodium phytate, disodium dihydrogen pyrophosphate and bisphenol A bis(diphenyl phosphate).
  • these phosphate compounds are used to sequence surface nucleic acid molecules with a high signal-to-noise ratio, especially nucleic acid molecules that do not need to be amplified into clusters, the signal intensity generated by sequencing is relatively weak due to the small number of nucleic acids at each site, or even only one. At this time, the interference signal (background noise) has a significant impact on the recognition of the sequencing signal.
  • the above-mentioned phosphate compound can bind to the oligonucleotide on the surface and reduce the reactive activity of the oligonucleotide.
  • the non-specific adsorption on the surface of the solid phase carrier can be significantly reduced, thereby reducing background noise, improving the recognizability of sequencing signals, reducing sequencing error rates, especially insertion type errors, and ultimately increasing the amount of nucleic acid sequencing data.
  • the phosphate compounds can be used alone or in combination, that is, two or even three compounds can be used together. Since these compounds compete with sequencing bases during sequencing, their effect in combination is expected to be similar to that of using one alone. The reason is that the surface area of the sequencing chip and the added sequencing bases are fixed. Therefore, when used in combination, in a relatively balanced competitive system, their effect of reducing adsorption on the surface of the sequencing chip is limited, and their maximum effect is similar to that of a single addition.
  • the step of contacting the second solution containing the phosphate compound with the surface of the solid phase carrier includes:
  • the second solution containing the phosphoric acid compound is allowed to flow over the surface of the solid support.
  • the concentration of the phosphate compound is 1 to 150 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 10 ⁇ mol/L, 20 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 91 ⁇ mol/L, 92 ⁇ mol/L, 95 ⁇ mol/L, 96 ⁇ mol/L, 98 ⁇ mol/L, 99 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L, 120 ⁇ mol/L, 130 ⁇ mol/L, 140 ⁇ mol/L or 150 ⁇ mol/L.
  • the excess phosphate compound will bind to other nucleotide molecules on the surface, such as occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain during nucleic acid sequencing. Since there is no polymerase in the reaction process and the phosphate compound cannot form hydrogen bonds with the complementary chain like nucleotides or nucleotide analogs, the reaction when the phosphate compound binds to other nucleotide molecules on the surface is not strong, but this will still have a certain impact on the sequencing reaction. In particular, when the concentration of the phosphate compound is too high, the excess phosphate compound may occupy the reaction position of the sequencing chain, and therefore, the throughput of nucleic acid sequencing may still be reduced.
  • the phosphate compound comprises sodium phytate, and in the second solution, the concentration of the sodium phytate is less than or equal to 15 ⁇ mol/L.
  • the phosphate compound is sodium phytate
  • the concentration of sodium phytate is 1 to 10 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 8 ⁇ mol/L, 9 ⁇ mol/L or 10 ⁇ mol/L.
  • Studies have found that when the content of sodium phytate is within this range, the activity of oligonucleotides on the surface can be effectively inhibited.
  • the adverse reactions that may be introduced by sodium phytate will also be reduced. For example, in the process of nucleic acid sequencing, the probability of sodium phytate occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain is reduced, and the sequencing flux is basically not affected or the impact produced can be ignored.
  • the phosphate compound comprises disodium dihydrogen pyrophosphate
  • the concentration of the disodium dihydrogen pyrophosphate is less than or equal to 120 ⁇ mol/L.
  • the phosphate compound is disodium dihydrogen pyrophosphate
  • the concentration of disodium dihydrogen pyrophosphate is 10-120 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L or 120
  • the phosphoric acid compound comprises bisphenol A bis(diphenyl phosphate), and in the second solution, the concentration of bisphenol A bis(diphenyl phosphate) is less than or equal to 70 ⁇ mol/L.
  • the phosphate compound comprises bisphenol A bis(diphenyl phosphate), and in the second solution, the concentration of bisphenol A bis(diphenyl phosphate) is 10 to 60 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 52 ⁇ mol/L, 53 ⁇ mol/L, 54 ⁇ mol/L, 56 ⁇ mol/L, 58 ⁇ mol/L, 59 ⁇ mol/L or 60 ⁇ mol/L.
  • controlling the amount of the phosphate compound used during the sequencing process can further effectively reduce the sequencing error rate while effectively controlling the cost.
  • the second solution includes at least one of a buffer, a surfactant and other additives.
  • the buffer is selected from at least one of tris buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, Hepes buffer, and N,N-dihydroxyethylglycine.
  • the buffer is used to adjust the pH value of the second solution, thereby providing a stable pH environment for the extension reaction of the nucleic acid molecule.
  • the other additives include at least one of ammonium ions, magnesium ions, potassium ions, sodium ions, dimethyl sulfoxide, and 1,3-dimethylthiourea.
  • ammonium ions, magnesium ions, potassium ions, and sodium ions can improve the activity of polymerase in promoting the extension of nucleic acid molecules in the solution; dimethyl sulfoxide can promote the binding ability of base hydrogen bonds during the extension of nucleic acid molecules; 1,3-dimethylthiourea helps to remove oxidative free radicals in the extension reagent buffer and provide a more stable buffer system.
  • the surfactant includes at least one of Triton X-100 and Tween-20.
  • the surfactant is used to adjust the surface tension of the second solution, especially when reacting on the surface of the solid phase carrier, which is conducive to the spreading of the second solution on the surface of the solid phase carrier.
  • the concentration of K + is 50-100 mmol/L
  • the concentration of NH4 + is 150-350 mmol/L
  • the pH of the second solution is 8.9-9.4.
  • the second solution may improve the polymerase reaction activity in the following ways: when the pH and the concentrations of K + and NH4 + in the extension reagent buffer are within the above range, the second solution changes the charge distribution on the surface of the polymerase protein, adjusts the isoelectric point of the polymerase, thereby adjusting the interaction between the polymerase and the nucleic acid molecule, and between the polymerase and the second solution, so that the polymerase can act on the nucleic acid molecule more easily; in addition, the second solution changes the spatial conformation of the nucleic acid molecule in the second solution, so that the polymerase can bind more easily to the reaction position of the nucleic acid molecule.
  • the concentration of K + can be 50mmol/L, 55mmol/L, 60mmol/L, 65mmol/L, 70mmol/L, 75mmol/L, 80mmol/L, 85mmol/L, 90mmol/L, 95mmol/L, 100mmol/L and the like in different embodiments.
  • the concentration of NH 4 + can be 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, 320mmol/L, 350mmol/L and the like in different embodiments; the concentration of NH 4 + can be 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, 320mmol/L, 350mmol/L and the like in different embodiments.
  • the second solution further comprises dNTP or NTP.
  • the dNTP is selected from at least one of dATP or its analogs, dTTP or its analogs, dGTP or its analogs, and dCTP or its analogs; the NTP is selected from at least one of ATP or its analogs, UTP or its analogs, GTP or its analogs, and CTP or its analogs.
  • a third solution containing dNTP or NTP is added to the mixed system.
  • the third solution includes at least one of a buffer, a surfactant, and other additives.
  • the buffer is selected from at least one of tris buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, Hepes buffer, and N,N-dihydroxyethylglycine.
  • the other additives include at least one of ammonium ions, magnesium ions, potassium ions, sodium ions, dimethyl sulfoxide, and 1,3-dimethylthiourea.
  • the surfactant includes at least one of Triton X-100 and Tween-20.
  • the concentration of K + is 50 to 100 mmol/L
  • the concentration of NH 4 + is 150 to 350 mmol/L
  • the pH of the third solution is 8.9 to 9.4.
  • the third solution further includes dNTP or NTP.
  • the dNTP is selected from at least one of dATP or its analogs (the examples are directly represented by dATP), dTTP or its analogs (the examples are directly represented by dTTP), dGTP or its analogs (the examples are directly represented by dGTP), dCTP or its analogs (the examples are directly represented by dCTP); the NTP is selected from at least one of ATP or its analogs, UTP or its analogs, GTP or its analogs, CTP or its analogs.
  • the functions of the components in the third solution can refer to the second solution.
  • the first nucleic acid molecule includes an oligonucleotide molecule and a nucleic acid template, and at least a portion of the sequence of the oligonucleotide molecule is complementary to the nucleic acid template, and the nucleic acid template is a nucleic acid molecule that has not been amplified.
  • the oligonucleotide is bound to the surface of the solid phase template, used to fix the nucleic acid template on the surface of the solid phase template, and can play the role of a primer.
  • the present invention provides a kit for extending a nucleic acid molecule, the kit comprising a first reagent, the first reagent comprising a phosphate compound, and the phosphate compound comprises a compound having a structural formula as shown in formula (1) and/or formula (2);
  • R 1 and R 2 are each independently selected from any one of metal ions, hydrogen atoms, optionally substituted or unsubstituted hydrocarbon groups, optionally substituted or unsubstituted cycloalkyl groups, optionally substituted or unsubstituted heteroalkyl groups, optionally substituted or unsubstituted heterocycloalkyl groups, optionally substituted or unsubstituted aryl groups, and optionally substituted or unsubstituted heteroaryl groups;
  • R3 is selected from any substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, or any heteroaryl group;
  • n is an integer selected from 1-8.
  • a kit for nucleic acid molecule extension is prepared using a phosphate compound of a specific structure.
  • the compounds of the structures shown in formula (1) and formula (2) can competitively bind to the oligonucleotides remaining on the surface of a solid phase carrier (such as a biochip) (such as oligonucleotides bound to the surface of the solid phase carrier but not bound to the nucleic acid template), thereby reducing or inhibiting the binding of these oligonucleotides to the virtual terminator, that is, reducing the nonspecific adsorption of the surface of the solid phase carrier, thereby reducing the signal interference caused by these oligonucleotides to nucleic acid sequencing (after the virtual terminator binds to these oligonucleotides, it will also generate a recognizable signal, thereby affecting the interference with the signal introduced into the nucleic acid template molecule), reducing the sequencing error rate caused by them, and further improving the sequencing accuracy
  • R 1 and R 2 are a hydrogen atom or an aromatic group
  • R 3 is selected from an alkyl group or a cycloalkyl group having 1 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms.
  • R 3 includes a cycloalkyl or a phenyl group.
  • the phosphate group in the phosphate compound is connected to the cycloalkyl or phenyl group, and the similarity of the structural type that is relatively similar to the nucleotide structure increases.
  • the nucleotide substrate such as a reversible terminator
  • oligonucleotide is a single-stranded structure, and the volume of the cycloalkyl or phenyl group is large, steric hindrance can be formed to a certain extent, which is also beneficial to reduce the probability of the reversible terminator and the oligonucleotide being combined.
  • formula (1) is selected from at least one of the compounds represented by formula (1-1) and formula (1-2):
  • the phosphate group in the phosphate compound is connected to a cycloalkyl or phenyl group having less than 10 carbon atoms, and the similarity with the nucleotide structure is further improved, so that when the above-mentioned phosphate compound and the nucleotide substrate (reversible terminator) are added at the same time, the phosphate compound can competitively bind to the oligonucleotide, thereby reducing the probability of the nucleotide substrate (reversible terminator) binding to the oligonucleotide.
  • R 2 contains at least one metal ion.
  • the metal ion in formula (1) helps the phosphate compound to form phosphate anions in the solution system, thereby more effectively binding to the oligonucleotide.
  • R2 contains n metal ions.
  • each phosphate group can form a phosphate anion, which increases the binding sites for the phosphate compound to react with the oligonucleotide.
  • the phosphate compound and the nucleotide substrate reversible terminator
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • n is selected from an integer of 2 to 6, including but not limited to 2, 3, 4, 5 or 6.
  • R 1 is selected from a hydrogen atom
  • R 2 is selected from a metal ion.
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • formula (2) is selected from at least one of the compounds represented by the structural formulas of formula (2-1) and formula (2-2):
  • M is selected from Na or K.
  • the inventors have found that the phosphate compounds satisfying the above formula (2-1) and formula (2-2) can effectively bind to the oligonucleotides on the surface, so that when the oligonucleotides appear on the surface in an unexpected manner, the activity of the oligonucleotides can be inhibited by adding the phosphate compounds shown in the structures of formula (2-1) and formula (2-2), thereby inhibiting the interference caused by them.
  • the activity of the surface oligonucleotides can be inhibited by the phosphate compound, thereby reducing the specific adsorption of the nucleic acid molecules generated during the extension process, reducing the sequencing background signal, and thus improving the recognizability of the sequencing signal.
  • the phosphate compound is selected from any one or at least two of sodium phytate, disodium dihydrogen pyrophosphate and bisphenol A bis (diphenyl phosphate). These phosphate compounds are used for the surface nucleic acid molecules with high signal-to-noise ratio, especially when the nucleic acid molecules that do not need to be amplified into clusters are sequenced, because the number of nucleic acids at each site is small, or even one, the signal intensity generated by sequencing is weak. At this time, the interference signal (background noise) has a significant impact on the recognition of sequencing signals.
  • the above-mentioned phosphate compound can be combined with the oligonucleotides on the surface to reduce the reactive activity of the oligonucleotides.
  • the binding activity of the former residence nucleotide sequence and the nucleotide substrate (reversible terminator) on the surface of the solid phase carrier is closed by the method, and the non-specific adsorption on the surface of the solid phase carrier can be significantly reduced, thereby reducing background noise, improving the recognizability of sequencing signals, reducing sequencing error rate, especially insertion type error rate, and finally improving the amount of nucleic acid sequencing data.
  • the kit includes a second reagent, and the second reagent includes at least one of a buffer, a surfactant and other additives.
  • the first reagent and the second reagent are placed in the same reagent tube in the reagent kit in the form of a mixture; or the first reagent and the second reagent are packaged in different reagent tubes in the reagent kit.
  • the buffer is selected from at least one of tris buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, Hepes buffer, and N,N-dihydroxyethylglycine.
  • the buffer is used to adjust the pH value of the second solution, thereby providing a stable pH environment for the extension reaction of the nucleic acid molecule.
  • the other additives include at least one of ammonium ions, magnesium ions, potassium ions, sodium ions, dimethyl sulfoxide, and 1,3-dimethylthiourea.
  • ammonium ions, magnesium ions, potassium ions, and sodium ions can improve the activity of polymerase in promoting the extension of nucleic acid molecules in the solution; dimethyl sulfoxide can promote the binding ability of base hydrogen bonds during the extension of nucleic acid molecules; 1,3-dimethylthiourea helps to remove oxidative free radicals in the extension reagent buffer and provide a more stable buffer system.
  • the surfactant includes at least one of Triton X-100 and Tween-20.
  • the surfactant is used to adjust the surface tension of the second solution, especially when reacting on the surface of the solid phase carrier, which is conducive to the spreading of the second solution on the surface of the solid phase carrier.
  • the concentration of K + is 50-100 mmol/L
  • the concentration of NH4 + is 150-350 mmol/L
  • the pH of the second reagent is 8.9-9.4.
  • the second reagent may improve the polymerase reaction activity in the following ways: when the pH and the concentrations of K + and NH4 + in the extension reagent buffer are within the above range, the second reagent changes the charge distribution on the surface of the polymerase protein, adjusts the isoelectric point of the polymerase, thereby adjusting the interaction between the polymerase and the nucleic acid molecule, and between the polymerase and the second reagent, so that the polymerase is more likely to act on the nucleic acid molecule; in addition, the second reagent changes the spatial conformation of the nucleic acid molecule in the second reagent, so that the polymerase is more likely to bind to the reaction position of the nucleic acid molecule.
  • the concentration of K + can be 50mmol/L, 55mmol/L, 60mmol/L, 65mmol/L, 70mmol/L, 75mmol/L, 80mmol/L, 85mmol/L, 90mmol/L, 95mmol/L, 100mmol/L and the like in different embodiments.
  • the concentration of NH 4 + can be 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, 320mmol/L, 350mmol/L and the like in different embodiments; the concentration of NH 4 + can be 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, 320mmol/L, 350mmol/L and the like in different embodiments.
  • the kit includes a third reagent and a fourth reagent, the third reagent includes dNTP or NTP, and the fourth reagent includes polymerase.
  • the dNTP is selected from at least one of dATP or its analogs, dTTP or its analogs, dGTP or its analogs, and dCTP or its analogs; the NTP is selected from at least one of ATP or its analogs, UTP or its analogs, GTP or its analogs, and CTP or its analogs.
  • the third reagent, the fourth reagent and the first reagent are each independently packaged in different reagent tubes in the kit.
  • the kit includes a fifth reagent
  • the fifth reagent includes oligonucleotide A (hereinafter referred to as A chain), oligonucleotide T (hereinafter referred to as T chain), oligonucleotide G (hereinafter referred to as G chain), oligonucleotide C (hereinafter referred to as C chain), or the fifth reagent includes oligonucleotide A, oligonucleotide U, oligonucleotide G, and oligonucleotide C.
  • the fifth reagent and the first reagent are packaged in different reagent tubes in the reagent kit.
  • the concentration of the phosphate compound is calculated based on the concentration of the mixed solution formed by mixing the components in the kit, and is 1 to 150 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 10 ⁇ mol/L, 20 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 91 ⁇ mol/L, 92 ⁇ mol/L, 95 ⁇ mol/L, 96 ⁇ mol/L, 98 ⁇ mol/L, 99 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L, 120 ⁇ mol/L, 130 ⁇ mol/L, 140 ⁇ mol/L or 150 ⁇ mol/L.
  • the excess phosphate compound will bind to other nucleotide molecules on the surface, such as occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain during nucleic acid sequencing. Since there is no polymerase in the reaction process and the phosphate compound cannot form hydrogen bonds with the complementary chain like nucleotides or nucleotide analogs, the reaction when the phosphate compound binds to other nucleotide molecules on the surface is not strong, but this will still have a certain impact on the sequencing reaction. In particular, when the concentration of the phosphate compound is too high, the excess phosphate compound may occupy the reaction position of the sequencing chain, and therefore, the throughput of nucleic acid sequencing may still be reduced.
  • the phosphate compound comprises sodium phytate, and the concentration of the sodium phytate is less than or equal to 15 ⁇ mol/L.
  • the phosphate compound is sodium phytate
  • the concentration of the sodium phytate is 1 to 10 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 8 ⁇ mol/L, 9 ⁇ mol/L or 10 ⁇ mol/L.
  • Studies have found that when the content of sodium phytate is within this range, the activity of oligonucleotides on the surface can be effectively inhibited, the error rate can be reduced, and the sequencing flux can be increased.
  • the adverse reactions that may be introduced by sodium phytate will also be reduced. For example, in the process of nucleic acid sequencing, the probability of sodium phytate occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain is reduced, and the sequencing flux is basically not affected or the impact produced can be ignored.
  • the phosphate compound comprises disodium dihydrogen pyrophosphate, and the concentration of the disodium dihydrogen pyrophosphate is less than or equal to 120 ⁇ mol/L.
  • the phosphate compound is disodium dihydrogen pyrophosphate
  • the concentration of the disodium dihydrogen pyrophosphate is 10 to 120 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L or 120 ⁇ mol/L.
  • the phosphoric acid compound comprises bisphenol A bis(diphenyl phosphate), and the concentration of the bisphenol A bis(diphenyl phosphate) is less than or equal to 70 ⁇ mol/L.
  • the phosphoric acid compound comprises bisphenol A bis(diphenyl phosphate), and the concentration of the bisphenol A bis(diphenyl phosphate) is 10 to 60 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 52 ⁇ mol/L, 53 ⁇ mol/L, 54 ⁇ mol/L, 56 ⁇ mol/L, 58 ⁇ mol/L, 59 ⁇ mol/L
  • the concentration of the dNTP or NTP is 20 to 1000 ⁇ mol/L, including but not limited to 20 ⁇ mol/L, 21 ⁇ mol/L, 22 ⁇ mol/L, 23 ⁇ mol/L, 24 ⁇ mol/L, 25 ⁇ mol/L, 50 ⁇ mol/L, 100 ⁇ mol/L, 150 ⁇ mol/L, 200 ⁇ mol/L, 300 ⁇ mol/L, 400 ⁇ mol/L, 500 ⁇ mol/L, 600 ⁇ mol/L, 700 ⁇ mol/L, 800 ⁇ mol/L, 900 ⁇ mol/L, 910 ⁇ mol/L, 920 ⁇ mol/L, 950 ⁇ mol/L, 960 ⁇ mol/L, 970 ⁇ mol/L, 980 ⁇ mol/L or 1000 ⁇ mol/L.
  • the concentration of the polymerase is 5 to 10 U/mL, including but not limited to 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL or 10 U/mL.
  • Next generation sequencing also known as high-throughput sequencing or massively parallel sequencing, can determine the nucleic acid sequence of multiple samples in a single sequencing run.
  • the more common next generation sequencing methods are sequencing by ligation (SBL) and sequencing by synthesis (SBS).
  • SBL sequencing by ligation
  • SBS sequencing by synthesis
  • the platform based on SBS for nucleic acid sequencing is based on the principle of base pairing and uses DNA polymerase to connect nucleotides (including nucleotide analogs) to the 3' end of the primer bound to the template to controllably achieve single base extension.
  • the base arrangement order of the template is determined based on the changes in these collected signals.
  • a typical SBS method may include the following steps: (1) hybridizing the nucleic acid molecule to be tested with the probe on the surface of the solid phase carrier to connect the nucleic acid template to be tested to the surface of the solid phase carrier; (2) under the action of DNA polymerase and under conditions suitable for polymerase chain reaction, using the probe as a primer to incorporate a nucleotide analog with a fluorescent group into the nucleic acid template (forming a complementary chain), thereby achieving single base extension; (3) stimulating the fluorescent group on the nucleotide analog to emit light, and then imaging the surface to collect the luminescent signal on the surface; (4) removing the fluorescent group in the nucleotide analog bound to the nucleic acid chain to be tested; (5) repeating the above steps (2) and (4) to allow the nucleic acid chain to be tested to continue to be extended.
  • the SBS method also includes step (6): analyzing the optical signal obtained in step (3) to determine the type of nucleotide analog incorporated into the nucleic acid template in each round of extension reaction; reading the type of introduced nucleotide analog in sequence, and finally obtaining all nucleotide sequences of the chain to be tested.
  • phase errors are usually manifested as phase lag (phasing or phase, which means that the nucleotides that should react in cycle N and be incorporated into the nucleic acid template lag to cycle N+1 to participate in the reaction) and phase advance (prephasing or prephase, which means that the nucleotides that should react in cycle N and be incorporated into the nucleic acid template participate in the reaction in cycle N-1 in advance), that is, crosstalk will occur between adjacent cycles in the same channel.
  • phase lag phasing or phase
  • phase advance prephasing or prephase
  • This phase error accumulates and becomes stronger as the number of sequencing cycles increases.
  • the final result is that four nucleotides are assembled simultaneously and the brightness is uniform in an amplification cluster.
  • the algorithm will not be able to identify the correct sequencing signal in this round, that is, it will not be able to accurately obtain the sequence information of the nucleotide molecule to be tested.
  • Another object of the present application is to provide an extension reagent buffer and its application, an extension kit and a sequencing method, aiming to solve the problem that phase errors are easily generated in the current process of incorporating nucleotide analogs into nucleic acid templates, affecting the recognition of sequencing signals and thereby reducing the sequencing accuracy of the nucleotide molecules to be tested.
  • the present application provides an extension reagent buffer, comprising at least a basic buffer, K + and NH4 + , wherein the concentration of K + is 50-100 mmol/L, the concentration of NH4 + is 150-350 mmol/L, and the pH of the extension reagent buffer is 8.9-9.4.
  • the extension reagent buffer provided in the present application comprehensively balances the pH and the concentrations of K + and NH4 + in the buffer, so that the activity of the polymerase in promoting the extension of nucleic acid molecules in the extension reagent buffer can be improved, thereby improving the extension efficiency of nucleic acid molecules, reducing the phase lag phenomenon, and ultimately improving the sequencing accuracy of the nucleotide molecules to be tested.
  • the extension reagent buffer may improve the polymerase reaction activity in the following ways: when the pH and the concentrations of K + and NH4 + in the extension reagent buffer are within the above range, the extension reagent buffer changes the charge distribution on the surface of the polymerase protein, adjusts the isoelectric point of the polymerase, thereby adjusting the interaction between the polymerase and the nucleic acid molecule, and between the polymerase and the extension reagent buffer, so that the polymerase can act on the nucleic acid molecule more easily; in addition, the extension reagent buffer changes the spatial conformation of the nucleic acid molecule in the extension reagent buffer, so that the polymerase can bind more easily to the reaction position of the nucleic acid molecule.
  • the pH of the extension reagent buffer is 8.9-9.1
  • the concentration of K + is 50-70 mmol/L
  • the concentration of NH 4 + is 150-230 mmol/L.
  • the pH of the extension reagent buffer is 9.05-9.35
  • the concentration of K + is 60-90 mmol/L
  • the concentration of NH 4 + is 200-310 mmol/L.
  • the pH of the extension reagent buffer is 9.3-9.5
  • the concentration of K + is 80-100 mmol/L
  • the concentration of NH 4 + is 300-350 mmol/L.
  • extension reagent buffer has an improving effect on the phase lag phenomenon.
  • the K+ is provided by at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate. That is to say, in the embodiment of the present application, the extension reagent buffer contains at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate, or in the process of configuring the extension reagent buffer, at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate is added to provide K + .
  • potassium hydroxide as a source of K + , can also adjust the pH of the extension reagent buffer through its anion to enrich the selection of basic buffer and other reagents used to adjust pH or affect pH.
  • the NH 4 + is NH 4 + provided by at least one of ammonium sulfate, organic ammonium and ammonia water. That is to say, in the implementation of the present application, the extension reagent buffer contains at least one of ammonium sulfate, organic ammonium and ammonia water, or in the process of configuring the extension reagent buffer, at least one of ammonium sulfate, organic ammonium and ammonia water is added to at least provide NH 4 + .
  • ammonia water as a source of NH 4 + , can also adjust the pH of the extension reagent buffer to enrich the selection of basic buffer and other reagents used to adjust pH or affect pH.
  • the extension reagent buffer contains potassium hydroxide and ammonium sulfate.
  • potassium hydroxide provides K +
  • the OH - it produces can also adjust the pH of the extension reagent buffer.
  • ammonium sulfate at least provides part of the NH 4 + required for the extension reagent buffer, or the NH 4 + is completely derived from ammonium sulfate.
  • the content of other NH 4 + sources, especially ammonia water, in the extension reagent buffer can be reduced, and the ammonia water content can even be reduced to 0, that is, no ammonia water is added when configuring the extension reagent buffer.
  • the double-layer effect of potassium hydroxide and ammonia water on the pH of the extension reagent buffer can be reduced, which is beneficial to regulating the pH of the extension reagent buffer to obtain the expected pH value.
  • the extension reagent buffer comprises at least one of organic potassium carboxylate and potassium sulfate and ammonia water.
  • ammonia water can affect the pH of the extension reagent buffer, when K + is derived from organic potassium carboxylate and potassium sulfate, it can reduce or avoid The content of the reagent affecting pH by potassium hydroxide is reduced, so that the reagent affecting pH by potassium hydroxide and ammonia water can strengthen the influence on the pH of the extension reagent buffer, which is beneficial to regulate the extension reagent buffer to obtain the expected pH.
  • the basic buffer is selected from at least one of tris(hydroxymethyl)aminomethane buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, Hepes buffer, and N,N-dihydroxyethylglycine.
  • the above basic buffer is used to adjust the pH value of the extension reagent buffer, thereby providing a stable pH environment for the extension reaction of the nucleic acid molecule.
  • the extension reagent buffer also includes: at least one of a chelating agent, a polymerase catalyst, a surfactant and other auxiliary agents.
  • the chelating agent is used to chelate the metal impurity ions (such as metal ions formed by lead, iron or copper, etc.) that may be present in the extension reagent buffer, thereby eliminating the inhibitory effect of the impurity metal ions on the enzyme during nucleic acid sequencing.
  • the polymerase catalyst is used to increase the activity of the polymerase, thereby improving the efficiency of nucleic acid molecule extension, that is, to increase the efficiency of the introduced nucleotides or nucleotide analogs incorporated into the nucleic acid molecules through the polymerization reaction.
  • the surfactant adjusts the surface tension of the extension reagent buffer, especially when reacting on the surface of the solid phase carrier, which is conducive to the spreading of the extension reagent buffer on the surface of the solid phase carrier.
  • the complexing agent is selected from at least one of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid tetrasodium salt, and EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid).
  • the polymerase catalyst includes a magnesium salt.
  • the surfactant is selected from at least one of Tween-20 and Triton X-100.
  • the other auxiliary agents include at least one of dimethyl sulfoxide and 1,3-dimethylthiourea.
  • dimethyl sulfoxide can promote the binding ability of base hydrogen bonds during the extension of nucleic acid molecules; 1,3-dimethylthiourea helps to remove oxidative free radicals in the extension reagent buffer and provide a more stable buffer system.
  • the chelating agent is selected from at least one of ethylenediaminetetraacetic acid, disodium salt of ethylenediaminetetraacetic acid, tetrasodium salt of ethylenediaminetetraacetic acid, and EGTA
  • the polymerase catalyst includes a magnesium salt
  • the surfactant is selected from at least one of Tween-20 and Triton X-100
  • the other auxiliary agents include at least one of dimethyl sulfoxide and 1,3-dimethylthiourea.
  • the other auxiliary agent includes a phosphate compound
  • the phosphate compound includes a compound having a structural formula as shown in formula (1) and/or formula (2):
  • R 1 and R 2 are each independently selected from any one of metal ions, hydrogen atoms, optionally substituted or unsubstituted hydrocarbon groups, optionally substituted or unsubstituted cycloalkyl groups, optionally substituted or unsubstituted heteroalkyl groups, optionally substituted or unsubstituted heterocycloalkyl groups, optionally substituted or unsubstituted aryl groups, and optionally substituted or unsubstituted heteroaryl groups;
  • R3 is selected from any substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, or any heteroaryl group;
  • n is an integer selected from 1-8.
  • the compounds of the structures shown in formula (1) and formula (2) can competitively bind to the oligonucleotides remaining on the surface of the solid phase carrier (such as a biochip) (such as oligonucleotides bound to the surface of the solid phase carrier but not bound to the nucleic acid template), thereby reducing or inhibiting the binding of these oligonucleotides to the virtual terminator, that is, reducing the non-specific adsorption of the surface of the solid phase carrier, thereby reducing the signal interference caused by these oligonucleotides to nucleic acid sequencing (after the virtual terminator binds to these oligonucleotides, it will also generate a recognizable signal, thereby affecting the interference with the signal introduced into the nucleic acid template molecule), reducing the sequencing error rate caused by them, and further improving the sequencing accuracy.
  • the virtual terminator binds to these oligonucleotides remaining on the surface of the solid phase carrier but not bound to the nucleic acid template
  • At least one of R 1 and R 2 is a hydrogen atom, and R 3 is selected from one of an alkyl group with 1 to 10 carbon atoms, a cycloalkyl group, and an aryl group with 6 to 20 carbon atoms; or at least one of R 1 and R 2 is an aromatic group, and R 3 is selected from one of an alkyl group with 1 to 10 carbon atoms, a cycloalkyl group, and an aryl group with 6 to 20 carbon atoms.
  • the competitiveness of the structure of formula (1) can be improved, which is beneficial for reducing nonspecific adsorption on the surface of the solid phase carrier.
  • R 3 includes cycloalkyl or phenyl.
  • the phosphate group in the phosphate compound is connected to the cycloalkyl or phenyl, and the similarity of the structural type that is relatively close to the nucleotide structure increases.
  • the nucleotide substrate such as a reversible terminator
  • oligonucleotide is a single-stranded structure, and the volume of the cycloalkyl or phenyl is large, steric hindrance can be formed to a certain extent, which is also beneficial to reduce the probability of reversible terminator binding to the oligonucleotide.
  • formula (1) is selected from at least one of the compounds represented by the structural formulas (1-1) and (1-2):
  • the phosphate group in the phosphate compound is connected to a cycloalkyl or phenyl group having less than 10 carbon atoms, and the similarity with the nucleotide structure is further improved, so that when the above-mentioned phosphate compound and the nucleotide substrate (reversible terminator) are added at the same time, the phosphate compound can competitively bind to the oligonucleotide, thereby reducing the probability of the nucleotide substrate (reversible terminator) binding to the oligonucleotide.
  • R 2 contains at least one metal ion.
  • the metal ion in formula (1) helps the phosphate compound to form phosphate anions in the solution system, thereby more effectively binding to the oligonucleotide.
  • R 2 contains n metal ions.
  • each phosphate group can form a phosphate anion, which increases the binding position of the phosphate compound and the oligonucleotide reaction.
  • the phosphate compound and the nucleotide substrate reversible terminator
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • the n is selected from integers of 2 to 6, including but not limited to 3, 4 or 5.
  • R1 is selected from a hydrogen atom
  • R2 is selected from a metal ion.
  • the structure of formula (2) can compete with the reversible terminator and bind to the oligonucleotide on the surface of the solid phase carrier, thereby reducing the nonspecific adsorption on the surface of the solid phase carrier.
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion.
  • the monovalent metal ions include sodium ions and/or potassium ions.
  • formula (2) is selected from at least one of the compounds represented by the structural formulas (2-1) and (2-2):
  • M is selected from Na or K.
  • the inventors have found that the phosphate compounds satisfying the above formula (2-1) and formula (2-2) can effectively bind to the oligonucleotides on the surface, so that when the oligonucleotides appear on the surface in an unexpected manner, the activity of the oligonucleotides can be inhibited by adding the phosphate compounds shown in the structures of formula (2-1) and formula (2-2), thereby inhibiting the interference caused by them.
  • the activity of the surface oligonucleotides can be inhibited by the phosphate compound, thereby reducing the specific adsorption of the nucleic acid molecules generated during the extension process, reducing the sequencing background signal, and thus improving the recognizability of the sequencing signal.
  • the phosphate compound is selected from any one or a combination of at least two of sodium phytate, disodium dihydrogen pyrophosphate and bisphenol A bis (diphenyl phosphate).
  • the above-mentioned phosphate compound can bind to the oligonucleotides on the surface to reduce the reactive activity of the oligonucleotides.
  • the extension reagent buffer is used for the surface nucleic acid molecules with high signal-to-noise ratio, especially when the nucleic acid molecules that do not need to be amplified into clusters are sequenced, because the number of nucleic acids at each site is small, or even one, the signal intensity generated by sequencing is weak.
  • the interference signal (background noise) has a significant impact on the recognition of the sequencing signal.
  • the binding activity of the non-sequence to be measured on the surface of the solid phase carrier and the reversible terminator is closed by this method, which can significantly reduce the nonspecific adsorption on the surface of the solid phase carrier, thereby reducing background noise, improving the recognizability of the sequencing signal, reducing the sequencing error rate, especially the insertion type error rate, and improving the amount of nucleic acid sequencing data.
  • the present application provides a use of the extension reagent buffer provided in the fourth aspect of the present application in the field of nucleic acid sequencing.
  • the present application provides an extension kit, comprising the extension reagent buffer provided in the fourth aspect of the present application.
  • extension kit of the present application also includes a polymerase, a reversible terminator with a fluorescent group and/or a reversible terminator without a fluorescent group.
  • the present application provides a sequencing method, comprising:
  • the nucleic acid molecule comprises a nucleic acid template and an oligonucleotide bound to the nucleic acid template, and the reversible terminator is bound to the 3' end of the chain where the oligonucleotide is located.
  • the extension reagent buffer, the reversible terminator and the polymerase are introduced into the surface of the solid phase carrier bound with the nucleic acid molecule, including:
  • a mixed solution containing the extension reagent buffer, the reversible terminator and the polymerase is flowed over the surface.
  • At least one means one or more, and “plurality” means two or more. "At least one” or similar expressions refer to any combination of these items, including any combination of single items or plural items.
  • at least one of a, b, or c or “at least one of a, b and c” can all mean: a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be a single item or multiple items, respectively.
  • first and second are used for descriptive purposes only to distinguish purposes such as substances, directions, interfaces, messages, requests, and terminals from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated.
  • the first imaging process may also be referred to as the second imaging process, and similarly, the second imaging process may also be referred to as the first imaging process.
  • features defined as “first” and “second” may explicitly or implicitly include one or more of the features.
  • the concentration of the relevant components mentioned may not only refer to the specific content of each component, but also indicate the proportional relationship between the contents of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application.
  • the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
  • hydrocarbyl by itself or as part of a substituent refers to a straight (i.e., non-branched) carbon chain or a branched carbon chain (or carbon) or a combination thereof, and it should be understood that “hydrocarbyl” is a non-cyclized chain.
  • Hydrocarbyl can be a fully saturated group, or a monounsaturated group or a polyunsaturated group.
  • alkyl group i.e., a
  • alkyl by itself or as part of a substituent refers to a straight (i.e., non-branched) carbon chain or a branched carbon chain (or carbon) or a combination thereof that does not contain an unsaturated bond.
  • alkyl is a non-cyclized chain;
  • alkenyl by itself or as part of a substituent refers to a straight (i.e., non-branched) carbon chain or branched carbon chain (or carbon) or a combination thereof containing a carbon-carbon double bond;
  • alkynyl by itself or as part of a substituent refers to a straight (i.e., non-branched) carbon chain or branched carbon chain (or carbon) or a combination thereof containing a carbon-carbon triple bond.
  • unsubstituted hydrocarbon group refers to the "hydrocarbon group” itself.
  • "Unsubstituted hydrocarbon groups” include, but are not limited to, alkyl groups such as -CH3 , -C2H5 , -C3H7 , -C4H9 , -C5H11, -C6H13, -C7H15, -C8H17, -C9H19 , -C10H21 , -C15H31 , -C20H41 , -C25H51 , -C30H61 , and the like , and their homologs and isomers; and alkyl groups such as -C2H3 , -C3H5 , -C4H7 , -C5H9 , -C6H11 , -C7H13 , -C8H15 , -C9H17 , -C10H21
  • Substituted hydrocarbon group refers to a group formed by replacing one or more hydrogen atoms in the “hydrocarbon group” itself with other groups other than the “hydrocarbon group” itself.
  • “Substituted hydrocarbon group” includes, but is not limited to, groups obtained by introducing one or more atoms or substituents such as halogen, alkyl, alkenyl, alkynyl, hydroxyl, nitro, amino, carbonyl, carboxyl, sulfhydryl, acyl, alkoxy, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, monophosphate, diphosphate, triphosphate, etc.
  • alkoxy is an alkyl group connected to the rest of the molecule through an oxygen linker (-O-), and the alkyl part may contain substitution or unsubstitution.
  • acyl refers to -C(O)R, where R is one of substituted or unsubstituted hydrocarbon group, substituted or unsubstituted cyclic hydrocarbon group, substituted or unsubstituted hydrocarbon group containing heteroatoms, substituted or unsubstituted cyclic hydrocarbon group containing heteroatoms, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • R, R', R", R'' and R" each independently represent hydrogen, substituted or unsubstituted heteroatom-containing hydrocarbon group, substituted or unsubstituted cyclic hydrocarbon group, substituted or unsubstituted heteroatom-containing cyclic hydrocarbon group, substituted or unsubstituted aryl group (for example, aryl group substituted by 1-3 halogens), substituted or unsubstituted heteroaryl group, substituted or unsubstituted hydrocarbon group, alkoxy or thioalkoxy group, or arylalkyl group.
  • heteroalkyl refers to a heteroatom-containing alkyl.
  • the term “heteroalkyl” refers to a stable straight or branched chain or combination thereof comprising at least one carbon atom and at least one heteroatom (e.g., B, O, N, P, Si, and S), and wherein nitrogen atoms and sulfur atoms may be optionally oxidized, and nitrogen heteroatoms may be optionally quaternized.
  • the heteroatom e.g., B, O, N, S, Si, or P
  • heteroalkyl is a non-cyclized chain.
  • heteroatom-containing hydrocarbyl may include one heteroatom (e.g., B, O, N, S, Si or P), two optional different heteroatoms (e.g., B, O, N, S, Si or P), three optional different heteroatoms (e.g., B, O, N, S, Si or P), or even four, five or more optional different heteroatoms (e.g., B, O, N, S, Si or P).
  • the “hydrocarbon group containing heteroatoms” when the other groups except heteroatoms in the "hydrocarbon group containing heteroatoms" are alkyl groups, the “hydrocarbon group containing heteroatoms” is a heteroalkyl group.
  • the “hydrocarbon group containing heteroatoms” when the other groups except heteroatoms in the "hydrocarbon group containing heteroatoms" contain carbon-carbon double bonds, the “hydrocarbon group containing heteroatoms” may be referred to as a heteroalkenyl group.
  • the heteroalkenyl group may optionally contain more than one double bond and/or contain one or more triple bonds in addition to one or more double bonds.
  • heteroalkynyl group When the other groups except heteroatoms in the "hydrocarbon group containing heteroatoms" contain carbon-carbon triple bonds, the "hydrocarbon group containing heteroatoms" may be referred to as a heteroalkynyl group.
  • the heteroalkynyl group may optionally contain more than one triple bond and/or contain one or more double bonds in addition to one or more triple bonds.
  • cyclic hydrocarbon group and “heterocyclic hydrocarbon group” by themselves or in combination with other terms mean cyclic forms of "hydrocarbon group” and “hydrocarbon group containing heteroatoms”, respectively. It should be understood that “cyclic hydrocarbon group” and “cyclic hydrocarbon group containing heteroatoms” are not aromatic. In addition, for “cyclic hydrocarbon group containing heteroatoms", heteroatoms can occupy the position where the heterocycle is connected to the rest of the molecule.
  • cyclic hydrocarbon group examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, etc. .
  • substituted cyclic hydrocarbon group and “substituted heterocyclic hydrocarbon group” refer to groups formed by replacing one or more hydrogen atoms in the "cyclic hydrocarbon group” and “cyclic hydrocarbon group containing heteroatoms” themselves with substituents.
  • the substituents may replace any non-hydrogen atoms on the cyclic hydrocarbon group.
  • substituted cyclic hydrocarbon group and “substituted heterocyclic hydrocarbon group” include, but are not limited to, groups obtained by introducing one or more atoms or substituents such as the following on the basis of unsubstituted "cyclic hydrocarbon group” and "cyclic hydrocarbon group containing heteroatoms”: halogen, alkyl, alkenyl, alkynyl, hydroxyl, nitro, amino, carbonyl, carboxyl, thiol, acyl, alkoxy, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, monophosphate, diphosphate, triphosphate, etc.
  • alkoxy is an alkyl group connected to the rest of the molecule through an oxygen linker (-O-), and the alkyl part may contain substitutions or unsubstituted.
  • acyl refers to -C(O)R, wherein R is a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cyclic hydrocarbon group, a substituted or unsubstituted hydrocarbon group containing heteroatoms, a substituted or unsubstituted cyclic hydrocarbon group containing heteroatoms, a substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group.
  • the substituents of the cyclic hydrocarbon group and the cyclic hydrocarbon group containing heteroatoms are each independently selected from, but not limited to, one or more of the following groups:
  • aryl refers to a polyunsaturated aromatic hydrocarbon substituent, which may be a single ring or multiple rings (preferably 1-3 rings) fused together or covalently linked, i.e., a fused ring aryl.
  • a fused ring aryl refers to multiple rings fused together, wherein at least one fused ring is an aryl ring.
  • heteroaryl refers to an aryl group (or ring) containing at least one heteroatom (such as N, O or S), wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized.
  • heteroaryl includes fused ring heteroaryls (i.e., multiple rings fused together, wherein at least one of the fused rings is a heteroaromatic ring).
  • a heteroaryl group can be attached to the rest of the molecule via a carbon atom or a heteroatom.
  • substituted aryl and substituted heteroaryl refer to groups formed by replacing one or more hydrogen atoms in “aryl” and “heteroaryl” by substituents.
  • substituted aryl and substituted heteroaryl include, but are not limited to, groups obtained by introducing one or more atoms or substituents such as the following on the basis of unsubstituted “aryl” and “heteroaryl”: halogen, alkyl, alkenyl, alkynyl, hydroxyl, nitro, amino, carbonyl, carboxyl, thiol, acyl, alkoxy, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, monophosphate, diphosphate, triphosphate, etc.
  • alkoxy is an alkyl group connected to the rest of the molecule through an oxygen linker (-O-), and the alkyl part may contain substitutions or unsubstituted.
  • acyl refers to -C(O)R, wherein R is one of a substituted or unsubstituted hydrocarbon group, a substituted or unsubstituted cyclic hydrocarbon group, a substituted or unsubstituted heteroatom-containing hydrocarbon group, a substituted or unsubstituted heteroatom-containing cyclic hydrocarbon group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group.
  • R, R', R", R'" and R" each independently represent hydrogen, substituted or unsubstituted heteroatom-containing hydrocarbon group, substituted or unsubstituted cyclic hydrocarbon group, substituted or unsubstituted heteroatom-containing cyclic hydrocarbon group, substituted or unsubstituted aryl group (e.g., aryl group substituted by 1-3 halogens), substituted or unsubstituted heteroaryl group, substituted or unsubstituted hydrocarbon group, alkoxy or thioalkoxy group, or arylalkyl group.
  • aryl group e.g., aryl group substituted by 1-3 halogens
  • substituted or unsubstituted heteroaryl group substituted or unsubstituted hydrocarbon group, alkoxy or thioalkoxy group, or arylalkyl group.
  • the term “sequencing” may also be referred to as "nucleic acid sequencing” or “gene sequencing”, that is, the three are interchangeable in expression, and refer to the determination of base types and arrangement order in nucleic acid sequences; including synthesis sequencing (sequencing by synthesis, SBS) and/or ligation sequencing (sequencing by ligation, SBL), including DNA sequencing and/or RNA sequencing, including long fragment sequencing and/or short fragment sequencing, and the so-called long fragments and short fragment sequencing are relative, such as nucleic acid molecules longer than 1Kb, 2Kb, 5Kb or 10Kb can be called long fragments, and those shorter than 1Kb or 800bp can be called short fragments; including double-end sequencing, single-end sequencing and/or paired-end sequencing, etc., and the so-called double-end sequencing or paired-end sequencing can refer to the reading of any two segments or two parts of the same nucleic acid molecule that do not completely overlap; the so-called double-end sequencing or
  • Sequencing generally includes multiple rounds of processes to achieve the determination of the order of multiple nucleotides/bases on the template.
  • each round of “the process to achieve the determination of the order of multiple nucleotides/bases on the template” is referred to as “one round of sequencing”.
  • “One round of sequencing” (cycle) is also called “sequencing round”, which can be defined as one base extension of four nucleotides/bases. In other words, "one round of sequencing” can be defined as completing the determination of the base type at any specified position on the template.
  • one round of sequencing includes the process of implementing the binding of four nucleotides (including nucleotide analogs) to the so-called template and collecting the corresponding signals emitted;
  • the reaction system includes reaction substrate nucleotides, polymerase and template, and the template is bound to a sequence (sequencing primer).
  • one round of sequencing may include one or more base extensions (repeat), for example, four nucleotides are added to the reaction system in sequence, and base extensions and corresponding reaction signals are collected respectively, and one round of sequencing includes four base extensions; for another example, four nucleotides are added to the reaction system in any combination, such as two-by-two combinations or one-to-three combinations, and base extensions and corresponding reaction signals are collected respectively in two combinations, and one round of sequencing includes two base extensions; for another example, four nucleotides are added to the reaction system at the same time for base extension and reaction signal collection, and one round of sequencing includes one base extension.
  • base extensions replicates
  • nucleic acid molecule means: a polymer form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term may refer to single-stranded or double-stranded polynucleotides.
  • Nucleotides in nucleic acid molecules may include naturally occurring nucleotides and functionally alternative analogs thereof. Examples of analogs are capable of hybridizing with nucleic acids in a sequence-specific manner, or can be used as templates for replication of a specific nucleotide sequence.
  • Naturally occurring nucleotides typically have a backbone comprising a phosphodiester bond. Analog structures may have alternative backbone connections including any kind known in the art.
  • Naturally occurring nucleotides typically have deoxyribose (e.g., found in DNA) or ribose (e.g., found in RNA). Analog structures may have alternative sugar moieties including any kind known in the art. Nucleotides may include natural bases or non-natural bases. The bases in natural DNA may include one or more of adenine, thymine, cytosine, and/or guanine, and the bases of natural RNA may include one or more of adenine, uracil, cytosine, and/or guanine. Nucleotides may also use any unnatural bases or base analogs, such as locked nucleic acids (LNA) and bridged nucleic acids (BNA).
  • LNA locked nucleic acids
  • BNA bridged nucleic acids
  • a primer refers to an oligonucleotide or nucleic acid molecule that can hybridize with a target sequence of interest.
  • a primer acts as a substrate to which nucleotides can be polymerized by a polymerase.
  • a primer can be used as a starting point for DNA or RNA synthesis.
  • a sequencing primer can hybridize with a synthetic nucleic acid template strand to initiate the synthesis of a new strand complementary to the synthetic nucleic acid template strand.
  • a primer can include any combination of nucleotides or their analogs.
  • a primer is a single-stranded oligonucleotide or polynucleotide.
  • analog or “functional analog” refers to a chemical compound that is similar in structure to another compound (the so-called “reference” compound) but differs in composition, such as the replacement of one atom by an atom of a different element, or the presence of a specific functional group, or the replacement of one functional group by another.
  • XX or its analogues include “XX” and “XX analogues”
  • nucleotides or their analogues include nucleotides and nucleotide analogs
  • bases or their analogues include bases and base analogs
  • ribose groups or their analogues include ribose and ribose analogs
  • deoxyribose groups or their analogues include deoxyribose and deoxyribose analogs.
  • reversible terminator refers to a nucleotide analog with a reversible terminator group, which can block the reactive position of the pentose in the nucleotide analog (such as the 3'-OH position) to prevent the nucleotide analog from undergoing polymerization at that position; and the reversible terminator group can be removed by certain chemical methods or other methods to restore the reactivity of the nucleotide analog at the corresponding position of the pentose.
  • cluster refers to a location on a surface such as a chip. Generally speaking, this location can contain multiple nucleic acid molecules, but usually the multiple nucleic acid molecules at this location are derived from the same nucleic acid molecule, for example, an amplified cluster of multiple nucleic acid molecules formed by performing bridge PCR on the same nucleic acid molecule.
  • each nanowell on the surface of the nanowell array chip is a site.
  • the phosphate compound of the specific structure shown in formula (1) and (2) can be used to treat the surface bound to the oligonucleotide. After the phosphate compound contacts the surface bound to the oligonucleotide, the phosphate compound and the oligonucleotide bind to form a stable complex to block the reactivity of the oligonucleotide.
  • the present invention applies the above discovery to sequencing, and addresses the problems existing in the existing sequencing technology: a large number of specific linker sequences (oligonucleotides) are randomly fixed on the surface of the sequencing solid phase carrier. After the addition of nucleotide substrates (reversible terminators) and a polymerase mixture, the nucleotide substrates (reversible terminators) are easily randomly adsorbed on the carrier surface, resulting in the easy identification of fluorescent spots located by non-hybridized templates in the subsequent fluorescence imaging process, and when the fluorescent spots are close to the real hybridization template sites, data analysis errors are easily caused, which are manifested as a high error rate (insertion error ratio) and a reduction in the amount of effective data.
  • a high error rate insertion error ratio
  • the present invention utilizes a phosphate compound with a specific structure to treat the surface of the sequencing solid phase carrier, which can reduce the signal interference caused by the reaction of oligonucleotides with substrate raw materials, that is, reduce the nonspecific adsorption generated on the surface (this nonspecific adsorption is not what we expect to obtain).
  • this method When this method is applied to sequencing surface nucleic acid molecules with a high signal-to-noise ratio, especially nucleic acid molecules that do not need to be amplified into clusters, it can effectively reduce the random adsorption of nucleotide substrates on the surface of the sequencing chip during the sequencing process (the adsorption reaction between the nucleotide substrate and the surface oligonucleotides), thereby effectively reducing the error rate caused by the random adsorption of the nucleotide substrate, and ultimately improving the sequencing quality and the sequencing read length.
  • the present invention also develops a kit for nucleic acid molecule extension based on the phosphate compound.
  • the nucleic acid molecule extension reagent formula contains a phosphate compound in addition to sequencing bases, DNA polymerase, and buffer.
  • the phosphate compound can be used alone to treat the sequencing chip before the extension reaction, or it can be mixed with other extension reagent components to jointly perform the sequencing extension reaction.
  • the structure of the phosphate compound is similar to the structure of the sequencing base, and there is a "competitive" relationship with the sequencing base, which is equivalent to indirectly reducing the number of sequencing bases on the surface of the sequencing chip, thereby reducing its adsorption on the surface, and then reducing the error rate of the sequencing reaction.
  • the nucleotide phase will change during the process of incorporating nucleotide analogs into the nucleic acid template (nucleic acid molecule extension), resulting in phase errors.
  • cycle N sequencing before the end of the reaction, some nucleic acid molecules to be tested and the incorporated nucleotide analogs have not yet undergone polymerization reaction, resulting in the nucleotide analogs that should have been introduced in cycle N sequencing participating in the reaction only in cycle N+1, thus causing the sequencing number to lag (i.e., phasing or phase), and crosstalk between adjacent cycles in the same channel, which in turn affects the accurate identification of sequencing signals.
  • the inventors of the present application have repeatedly studied the reaction reagents in the extension reaction process and finally obtained a method that can effectively improve the nucleic acid sequencing process. Problems with phasing or phase of the extension reagent buffer.
  • the extension reagent buffer includes a basic buffer, which is the main component of the extension reagent buffer and is used to stabilize the reaction environment during the extension reaction, especially to stabilize the pH environment during the extension reaction, so that the efficiency, uniformity and stability of nucleotide analogs incorporated into nucleic acid molecules during the extension reaction are improved.
  • the basic buffer for nucleic acid extension can be used in the embodiments of the present application.
  • the basic buffer is selected from at least one of tris(hydroxymethyl)aminomethane buffer, glycine, ethanolamine, tetraethylethylenediamine, tetramethylethylenediamine, N-butyldiethanolamine, diethylaminoethanol, Hepes buffer, and N,N-dihydroxyethylglycine.
  • tris(hydroxymethyl)aminomethane buffer is also known as tris(hydroxymethyl)aminomethane hydrochloride or Tris buffer, abbreviated as Tris-HCl in English; Hepes buffer is a non-ionic amphoteric buffer, and its main component is 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid.
  • Tris buffer is a non-ionic amphoteric buffer, and its main component is 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid.
  • at least one of tris(hydroxymethyl)aminomethane buffer and glycine is used as the basic buffer in the extension reagent buffer.
  • the basic buffer is 10 to 160 mmol/L, which can be adjusted according to the type of basic buffer and the content of other components in the extension reagent buffer.
  • the basic buffer is tris hydroxymethylaminomethane buffer, and the concentration of the basic buffer is 60 to 150 mmol/L.
  • the concentration of tris hydroxymethylaminomethane buffer can be 60 mmol/L, 65 mmol/L, 70 mmol/L, 75 mmol/L, 80 mmol/L, 85 mmol/L, 90 mmol/L, 95 mmol/L, 100 mmol/L, 105 mmol/L, 110 mmol/L, 115 mmol/L, 120 mmol/L, 125 mmol/L, 130 mmol/L, 135 mmol/L, 140 mmol/L, 145 mmol/L, 150 mmol/L, 155 mmol/L, 160 mmol/L and other specific situations.
  • the basic buffer is glycine
  • the concentration of the basic buffer can be 10 to 60 mmol/L.
  • the concentration of glycine can be 10mmol/L, 15mmol/L, 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, 55mmol/L, 60mmol/L and the like.
  • the extension reagent buffer includes K + and NH 4 + , which can adjust the activity of the polymerase by changing the charge distribution on the protein surface of the polymerase, increase its binding activity with the reaction site of the nucleic acid molecule, and thus improve the catalytic activity of the polymerase.
  • K + is provided by at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate. That is to say, in the embodiments of the present application, the extension reagent buffer contains at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate, or in the process of preparing the extension reagent buffer, at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate is added, and the use of at least one of potassium hydroxide, potassium halide, organic potassium carboxylate and potassium sulfate includes at least providing K + .
  • the potassium halide is at least one of potassium chloride, potassium bromide, and potassium iodide;
  • the organic potassium carboxylate is at least one of potassium acetate (or potassium acetate) and potassium oxalate.
  • the extension reagent buffer provides K + through potassium hydroxide.
  • the extension reagent buffer contains K + and OH - .
  • Potassium hydroxide as a K + source, can also adjust the pH of the extension reagent buffer through its anion (OH - ). Due to the adjustment of the pH of the extension reagent buffer by potassium hydroxide, there is better flexibility in the selection of the basic buffer and other reagents for adjusting or affecting the pH. For example, when the extension reagent buffer contains potassium hydroxide, other reagents other than ammonia water can be selected as the NH 4 + source.
  • NH 4 + is NH 4 + provided by at least one of ammonium sulfate, organic ammonium and ammonia water. That is to say, in the embodiments of the present application, the extension reagent buffer contains at least one of ammonium sulfate, organic ammonium and ammonia water, or in the process of preparing the extension reagent buffer, at least one of ammonium sulfate, organic ammonium and ammonia water is added, and the use of at least one of ammonium sulfate, organic ammonium and ammonia water at least includes providing NH 4 + .
  • the organic ammonium is at least one of ammonium acetate (or ammonium acetate) and ammonium oxalate.
  • the extension reagent buffer provides NH 4 + through ammonia water.
  • the ammonia water in the extension reagent buffer does not provide OH - , and the pH of the extension reagent buffer is adjusted by OH - . Due to the adjustment of the pH of the extension reagent buffer by ammonia water, there is better flexibility in the selection of the basic buffer and other reagents for adjusting or affecting the pH. For example, when the extension reagent buffer contains ammonia water, other salts other than potassium hydroxide can be selected as the K + source.
  • the extension reagent buffer contains potassium hydroxide and ammonium sulfate.
  • potassium hydroxide provides K +
  • the OH - produced by it can also adjust the pH of the extension reagent buffer.
  • ammonium sulfate at least provides part of the NH 4 + required by the extension reagent buffer, or the NH 4 + is completely derived from ammonium sulfate. Therefore, the content of other NH 4 + sources in the extension reagent buffer, especially ammonia water, can be reduced, and the ammonia water content can even be reduced to 0, that is, no ammonia water is added when the extension reagent buffer is prepared. In this way, the double-layer effect of potassium hydroxide and ammonia water on the pH of the extension reagent buffer can be reduced, which is conducive to regulating the pH of the extension reagent buffer to obtain the expected pH value.
  • the K + source of the extension reagent buffer is potassium hydroxide
  • the NH 4 + source is ammonium sulfate
  • the extension reagent buffer provides K + through potassium hydroxide, and provides NH 4 + through ammonium sulfate.
  • the K + source of the extension reagent buffer is potassium hydroxide
  • the NH 4 + source is ammonium sulfate and ammonium acetate, that is, the extension reagent buffer provides K + through potassium hydroxide, and provides NH 4 + through ammonium sulfate and ammonium acetate.
  • the extension reagent buffer may also be added with other potassium salts such as but not limited to potassium chloride, potassium acetate, etc. as a K + source for providing K + .
  • the common point of the above-mentioned several embodiments is that potassium hydroxide and ammonia water are not added simultaneously when preparing the extension reagent buffer to improve the adjustability of the pH of the extension reagent buffer.
  • the researchers of the present application found that when ammonium acetate is used alone as the NH 4 + source (i.e., the extension reagent buffer provides NH 4 + through ammonium acetate), when the extension reagent buffer is used in nucleic acid sequencing, the occurrence of base mismatches will increase, that is, the base pairs originally paired according to the base complementary pairing principle (AT(U) pairing, GC pairing) will become other mismatches, such as GT pairing, CT pairing, etc., which will directly affect the accuracy of the nucleic acid sequencing results. Therefore, the present application embodiment will add NH 4 + source material while using ammonium acetate.
  • the extension reagent buffer includes at least one of organic potassium carboxylate and potassium sulfate and ammonia water.
  • the ammonia water provides NH 4 +
  • the OH - generated by it can also adjust the pH of the extension reagent buffer.
  • at least one of the organic potassium carboxylate and potassium sulfate provides part of the NH 4 + required for the extension reagent buffer, or the NH 4 + is completely derived from at least one of the organic potassium carboxylate and potassium sulfate.
  • the content of other K + sources in the extension reagent buffer especially potassium hydroxide
  • the potassium hydroxide content can even be reduced to 0, that is, potassium hydroxide is not added when the extension reagent buffer is prepared.
  • the double-layer effect of potassium hydroxide and ammonia water on the pH of the extension reagent buffer can be reduced, which is conducive to regulating the pH of the extension reagent buffer to obtain the expected pH value.
  • the K + source of the extension reagent buffer is organic potassium carboxylate and potassium sulfate
  • the NH4 + source is aqueous ammonia
  • the extension reagent buffer provides K + through organic potassium carboxylate and potassium sulfate, and provides NH4 + through aqueous ammonia.
  • the organic potassium carboxylate can be selected from one or more of the organic potassium carboxylates listed above.
  • the K + source of the extension reagent buffer is organic potassium carboxylate and potassium sulfate
  • the NH 4 + source is aqueous ammonia and ammonium sulfate
  • the extension reagent buffer provides K + through organic potassium carboxylate and potassium sulfate, and provides NH 4 + through aqueous ammonia and ammonium sulfate.
  • the K + source of the extension reagent buffer is organic potassium carboxylate and potassium sulfate
  • the NH 4 + source is aqueous ammonia and ammonium acetate
  • the extension reagent buffer provides K + through organic potassium carboxylate and potassium sulfate, and provides NH 4 + through aqueous ammonia and ammonium acetate.
  • the K + source of the extension reagent buffer is organic potassium carboxylate and potassium sulfate
  • the NH 4 + source is ammonia water, ammonium acetate and ammonium phosphate
  • the extension reagent buffer provides K + through organic potassium carboxylate and potassium sulfate, and provides NH 4 + through ammonia water, ammonium acetate and ammonium phosphate.
  • the concentration of K + is 50-100 mmol/L
  • the concentration of NH 4 + is 150-350 mmol/L.
  • the extension reagent buffer may improve the polymerase reaction activity in the following manner: when the pH and the concentrations of K + and NH4 + in the extension reagent buffer are within the above ranges, the extension reagent buffer changes the charge distribution on the surface of the polymerase protein, adjusts the isoelectric point of the polymerase, thereby adjusting the interaction between the polymerase and the nucleic acid molecule, and between the polymerase and the extension reagent buffer, making it easier for the polymerase to act on the nucleic acid molecule; in addition, the extension reagent buffer changes the spatial conformation of the nucleic acid molecule in the extension reagent buffer, making it easier for the polymerase to bind to the reaction position of the nucleic acid molecule.
  • the concentrations of K + and NH 4 + can be adjusted according to the changes of other components in the extension reagent buffer, but still within the above range. Therefore, the concentration of K + can be 50mmol/L, 55mmol/L, 60mmol/L, 65mmol/L, 70mmol/L, 75mmol/L, 80mmol/L, 85mmol/L, 90mmol/L, 95mmol/L, 100mmol/L, etc. in different embodiments.
  • the concentration of NH 4 + can be 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 200mmol/L, 220mmol/L, 250mmol/L, 280mmol/L, 300mmol/L, 320mmol/L, 350mmol/L, etc. in different embodiments.
  • the extension reagent buffer provided in the embodiment of the present application is basically determined by selecting a suitable basic buffer and adjusting the concentration of the basic buffer, and on this basis, the pH of the extension reagent buffer is finally determined under the action of other reagents affecting pH, such as K + and NH 4 + .
  • the pH of the extension reagent buffer is 8.9 to 9.4.
  • the pH of the extension reagent buffer is 8.9-9.0
  • the concentration of K + is 50-60 mmol/L
  • the concentration of NH 4 + is 150-190 mmol/L.
  • the pH of the extension reagent buffer is 9.0-9.1
  • the concentration of K + is 60-70 mmol/L
  • the concentration of NH 4 + is 190-230 mmol/L.
  • the pH of the extension reagent buffer is 9.1-9.3, the concentration of K + is 70-90 mmol/L, and the concentration of NH 4 + is 230-310 mmol/L.
  • the pH of the extension reagent buffer is 9.3-9.4, the concentration of K + is 90-100 mmol/L, and the concentration of NH 4 + is 310-350 mmol/L.
  • the pH of the extension reagent buffer is 8.9-9.1
  • the concentration of K + is 50-70 mmol/L
  • the concentration of NH 4 + is 150-230 mmol/L.
  • the pH of the extension reagent buffer is 9.05-9.35
  • the concentration of K + is 60-90 mmol/L
  • the concentration of NH 4 + is 200-310 mmol/L.
  • the pH of the extension reagent buffer is 9.3-9.5
  • the concentration of K + is 80-100 mmol/L
  • the concentration of NH 4 + is 300-350 mmol/L.
  • the extension reagent buffer prepared in the above manner can more obviously show the improvement effect of the extension reagent buffer on the phase lag phenomenon.
  • the extension reagent buffer may further include a polymerase catalyst.
  • the polymerase catalyst is used to increase the activity of the polymerase, thereby increasing the efficiency of nucleic acid molecule extension, that is, increasing the efficiency of the introduced nucleotides or nucleotide analogs being incorporated into the nucleic acid molecule through polymerization.
  • the polymerase catalyst is selected from magnesium salts, and specifically, the magnesium ions in the magnesium salts play a role in increasing the activity of the polymerase.
  • the magnesium salt may be at least one of magnesium sulfate, magnesium chloride, etc., but is not limited thereto.
  • the concentration of the polymerase catalyst in the extension reagent buffer is 2.0 to 5.0 mmol/L, and within this concentration range, it can be adjusted according to the concentration of the polymerase used in the nucleic acid molecule extension process.
  • the polymerase catalyst is magnesium sulfate
  • its concentration can be 2.0 mmol/L, 2.5 mmol/L, 3.0 mmol/L, 3.5 mmol/L, 4.0 mmol/L, 4.5 mmol/L, 5.0 mmol/L, and the like.
  • the extension reagent buffer may further include a complexing agent.
  • the complexing agent is used to complex the metal impurity ions (such as metal ions formed by lead, iron or copper) that may be present in the extension reagent buffer, thereby eliminating the inhibitory effect of the impurity metal ions on the enzyme during nucleic acid sequencing.
  • the complexing agent can be ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid tetrasodium salt, EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) and the like.
  • the concentration of the complexing agent in the extension reagent buffer is 0.5-2.0 mmol/L.
  • the complexing agent is ethylenediaminetetraacetic acid
  • its concentration can be 0.5 mmol/L, 1.0 mmol/L, 1.5 mmol/L, 2.0 mmol/L, etc.
  • the extension reagent buffer may further include a surfactant.
  • the surfactant is used to adjust the surface tension of the extension reagent buffer, especially when reacting on the surface of a solid phase carrier, which is conducive to the spreading of the extension reagent buffer on the surface of the solid phase carrier.
  • the surfactant is selected from at least one of Tween-20 and Triton X-100.
  • the volume percentage of the surfactant in the extension reagent buffer is 0.010-0.100%.
  • the surfactant is Tween-20
  • its volume percentage can be 0.010%, 0.015%, 0.020%, 0.025%, 0.050%, 0.075%, 0.100%, etc.
  • the extension reagent buffer may also include other auxiliary agents that perform one or more specific functions.
  • the other auxiliary agents include at least one of dimethyl sulfoxide and 1,3-dimethylthiourea.
  • dimethyl sulfoxide can promote the binding ability of base hydrogen bonds during the extension of nucleic acid molecules; 1,3-dimethylthiourea helps to remove oxidative free radicals in the extension reagent buffer and provide a more stable buffer system.
  • the chelating agent is selected from at least one of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, tetrasodium ethylenediaminetetraacetic acid, and EGTA
  • the polymerase catalyst includes a magnesium salt
  • the surfactant is selected from at least one of Tween-20 and Triton X-100
  • other auxiliary agents include at least one of dimethyl sulfoxide and 1,3-dimethylthiourea.
  • the other auxiliary agent in the extension reagent buffer includes a phosphate compound
  • the phosphate compound includes a compound having a structural formula as shown in Formula (1) and/or Formula (2):
  • R 1 and R 2 are each independently selected from any one of metal ions, hydrogen atoms, optionally substituted or unsubstituted hydrocarbon groups, optionally substituted or unsubstituted cycloalkyl groups, optionally substituted or unsubstituted heteroalkyl groups, optionally substituted or unsubstituted heterocycloalkyl groups, optionally substituted or unsubstituted aryl groups, and optionally substituted or unsubstituted heteroaryl groups;
  • R3 is selected from any substituted or unsubstituted hydrocarbon group, any substituted or unsubstituted cycloalkyl group, any substituted or unsubstituted heteroalkyl group, any substituted or unsubstituted heterocycloalkyl group, any substituted or unsubstituted aryl group, or any heteroaryl group;
  • n is an integer selected from 1-8.
  • the phosphate compound in the extension reagent buffer forms an ionic state, including metal cations and phosphate anions. Therefore, in essence, in the present application, the anions corresponding to the compounds shown in formula (1) and formula (2) also belong to the category of phosphorus compounds in the embodiments of the present application.
  • the phosphate anions include at least the following situations:
  • the compounds of the structures shown in formula (1) and formula (2) can competitively bind to the oligonucleotides remaining on the surface of the solid phase carrier (such as a biochip) (such as oligonucleotides bound to the surface of the solid phase carrier but not bound to the nucleic acid template), thereby reducing or inhibiting the binding of these oligonucleotides to the virtual terminator, that is, reducing the non-specific adsorption of the surface of the solid phase carrier, thereby reducing the signal interference caused by these oligonucleotides to nucleic acid sequencing (after the virtual terminator binds to these oligonucleotides, it will also generate a recognizable signal, thereby affecting the interference with the signal introduced into the nucleic acid template molecule), reducing the sequencing error rate caused by them, and further improving the sequencing accuracy.
  • the virtual terminator binds to these oligonucleotides remaining on the surface of the solid phase carrier but not bound to the nucleic acid template
  • R 1 and R 2 are a hydrogen atom
  • R 3 is selected from an alkyl group or a cycloalkyl group having 1 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms.
  • the competitiveness of the structure of formula (1) can be improved, which is beneficial for reducing nonspecific adsorption on the surface of the solid phase carrier.
  • the hydrogen (H) in the above structure can be free in the extension reagent buffer in the form of H + , while the phosphate compound exists in the form of phosphate anion.
  • R 1 and R 2 are aromatic groups
  • R 3 is selected from an alkyl group or a cycloalkyl group having 1 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms.
  • R3 includes a cycloalkyl group or a phenyl group.
  • the phosphate group in the phosphate compound is connected to the cycloalkyl group or the phenyl group to form a structure type that is relatively similar to the nucleotide structure, which is more conducive to improving its competitiveness with the oligonucleotide.
  • the oligonucleotide is a single-stranded structure, the steric hindrance caused by the above-mentioned bulky group is also conducive to reducing the probability of the reversible terminator binding to the oligonucleotide.
  • formula (1) is selected from at least one of the compounds represented by formula (1-1) and formula (1-2):
  • the phosphate group is connected to the carbon atom of the alkyl ring of the cycloalkyl group.
  • the hydrogen (H) in the above structure can be free in the extension reagent buffer in the form of H + , and the phosphate compound exists in the form of phosphate anion.
  • the phosphate group is connected to the aromatic carbon atom.
  • at least one of R 1 and R 2 is an aromatic group.
  • the phosphate group in the phosphate compound is connected to a cycloalkyl or phenyl group having less than 10 carbon atoms, and the similarity with the nucleotide structure is further enhanced, so that when the above phosphate compound and the nucleotide substrate (reversible terminator) are added at the same time, the phosphate compound can competitively bind to the oligonucleotide, thereby reducing the probability of the nucleotide substrate (reversible terminator) binding to the oligonucleotide.
  • the phosphate compounds shown in formula (1-1) and formula (1-2) contain metal ions, such as: R 2 contains at least one metal ion. It should be understood that at least one metal ion in R 2 means that the phosphate compounds shown in the above formula (1-1) and formula (1-2) contain n R 2.
  • the metal ions in formula (1) help the phosphate compound to form phosphate anions in the solution system, thereby more effectively binding to the oligonucleotide.
  • n 1
  • R 2 in the corresponding phosphate compound is a metal ion
  • n>1 at least one R 2 among the multiple R 2 in the corresponding phosphate compound is a metal ion, and of course, multiple or even all R 2 can be metal ions.
  • R 2 contains n metal ions.
  • each phosphate group can form a phosphate anion, which increases the binding position of the phosphate compound and the oligonucleotide reaction.
  • the phosphate compound and the nucleotide substrate reversible terminator
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion. It should be understood that when the metal ion is a divalent metal ion, every two phosphoric acid units in the structure of formula (1) (including formula (1-1) and formula (1-2)) share one R 2 , as shown in the following formula:
  • the corresponding n is an even number.
  • the metal ion when the metal ion is a monovalent metal ion, the monovalent metal ion includes a sodium ion and/or a potassium ion.
  • the phosphoric acid compound represented by formula (1) contains a plurality of R 2 , each of the plurality of R 2 can be a sodium ion or a potassium ion.
  • n is selected from an integer of 2 to 6, including but not limited to 2, 3, 4, 5 or 6.
  • n is selected from an integer of 2 to 6, including but not limited to 2, 3, 4, 5 or 6.
  • multiple phosphate groups participate in the competition, which is beneficial to improve the competitiveness of the phosphate compound to bind to the oligonucleotide on the surface of the solid phase carrier.
  • R3 is a cycloalkyl or phenyl group, and each carbon atom on the alkane ring or aromatic ring is bound to a phosphate structure, that is, n is the number of carbon atoms on the alkane ring or aromatic ring.
  • the number of phosphate groups distributed on the alkane ring or aromatic ring is large, providing more reaction sites for reacting with oligonucleotides, thereby enhancing their reaction competitiveness; on the other hand, the phosphate group is connected to the alkane ring or aromatic ring, which has a certain similarity to the structure of the phosphate group connected to the pentose and base in the nucleotide, and can also enhance the competitive advantage of the phosphate compound.
  • the binding ability of the phosphate compound shown in formula (1) to the oligonucleotide on the solid phase carrier can be effectively improved, thereby reducing nonspecific adsorption during the extension of the nucleic acid molecule, improving the recognizability of the sequencing signal, and improving the sequencing accuracy.
  • R1 is selected from a hydrogen atom
  • R2 is selected from a metal ion.
  • the structure of formula (2) can compete with the reversible terminator and bind to the oligonucleotide on the surface of the solid phase carrier, thereby reducing the nonspecific adsorption on the surface of the solid phase carrier.
  • the hydrogen (H) of formula (2) can be free in the extension reagent buffer in the form of H +
  • the metal ion can also form a metal ion
  • the phosphate compound exists in the form of a phosphate anion.
  • the metal ion is selected from a monovalent metal ion or a divalent metal ion. It should be understood that when the metal ion is a divalent metal ion, the two phosphate units in the structure of formula (2) share one R 2 , as shown in the following formula:
  • the metal ion when the metal ion is a monovalent metal ion, the monovalent metal ion may be a sodium ion and/or a potassium ion.
  • the two R 2 in the phosphate compound represented by formula (2) may be a sodium ion or a potassium ion, or a sodium ion and a potassium ion, respectively.
  • formula (2) is selected from at least one of the compounds represented by formula (2-1) and formula (2-2):
  • M is selected from Na or K.
  • the inventors have found that the phosphate compounds satisfying the above formula (2-1) and formula (2-2) can effectively bind to the oligonucleotides on the surface, so that when the oligonucleotides appear on the surface in an unexpected manner, the activity of the oligonucleotides can be inhibited by adding the phosphate compounds shown in the structures of formula (2-1) and formula (2-2), thereby inhibiting the interference caused by them.
  • the activity of the surface oligonucleotides can be inhibited by the phosphate compound, thereby reducing the specific adsorption of the nucleic acid molecules generated during the extension process, reducing the sequencing background signal, and thus improving the recognizability of the sequencing signal.
  • the phosphate compound is selected from any one or a combination of at least two of the following formula (3), formula (4) and formula (5).
  • the above-mentioned phosphate compound can reduce the error rate in the nucleic acid sequencing process.
  • the extension reagent buffer is used for sequencing of surface nucleic acid molecules with high signal-to-noise ratio, especially nucleic acid molecules that do not need to be amplified into clusters, the signal intensity generated by sequencing is weak due to the small number of nucleic acids at each site, or even one.
  • the interference signal background noise
  • the non-specific adsorption on the surface of the solid phase carrier can be significantly reduced, thereby reducing the sequencing error rate, especially the insertion type error rate, and increasing the amount of nucleic acid sequencing data.
  • the phosphate compounds can be used alone or in combination, that is, two or even three compounds can be used together. Since these compounds compete with sequencing bases during sequencing, their effect in combination is expected to be similar to that of using one alone. The reason is that the surface area of the sequencing chip and the added sequencing bases are fixed. Therefore, when used in combination, in a relatively balanced competitive system, their effect of reducing adsorption on the surface of the sequencing chip is limited, and their maximum effect is similar to that of a single addition.
  • the concentration of the phosphate compound in the extension reagent buffer is 1 to 150 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 10 ⁇ mol/L, 20 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 91 ⁇ mol/L, 92 ⁇ mol/L, 95 ⁇ mol/L, 96 ⁇ mol/L, 98 ⁇ mol/L, 99 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L, 120 ⁇ mol/L, 130 ⁇ mol/L, 140 ⁇ mol/L or 150 ⁇ mol/L.
  • the concentration of the phosphate compound in the extension reagent buffer is too high, such as exceeding 150 ⁇ mol/L, the excess phosphate compound will bind to other nucleotide molecules on the surface, such as occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain during nucleic acid sequencing. Since there is no polymerase in the reaction process and the phosphate compound cannot form hydrogen bonds with the complementary chain like nucleotides or nucleotide analogs, the reaction when the phosphate compound binds to other nucleotide molecules on the surface is not strong, but this will still have a certain impact on the sequencing reaction. In particular, when the concentration of the phosphate compound is too high, the excess phosphate compound may occupy the reaction position of the sequencing chain, and therefore, the throughput of nucleic acid sequencing may still be reduced.
  • the phosphate compound comprises sodium phytate, and in the extension reagent buffer, the concentration of the sodium phytate is less than or equal to 15 ⁇ mol/L.
  • the phosphate compound is sodium phytate
  • the concentration of the sodium phytate is 1 to 10 ⁇ mol/L, including but not limited to 1 ⁇ mol/L, 2 ⁇ mol/L, 3 ⁇ mol/L, 4 ⁇ mol/L, 5 ⁇ mol/L, 6 ⁇ mol/L, 7 ⁇ mol/L, 8 ⁇ mol/L, 9 ⁇ mol/L or 10 ⁇ mol/L.
  • Studies have found that when the content of sodium phytate is within this range, the activity of oligonucleotides on the surface can be effectively inhibited.
  • the adverse reactions that may be introduced by sodium phytate will also be reduced. For example, in the process of nucleic acid sequencing, the probability of sodium phytate occupying the reaction position of nucleotides or nucleotide analogs in the sequencing chain is reduced, and the sequencing flux is basically not affected or the impact produced can be ignored.
  • the phosphate compound comprises disodium dihydrogen pyrophosphate
  • the concentration of the disodium dihydrogen pyrophosphate is less than or equal to 120 ⁇ mol/L.
  • the phosphate compound is disodium dihydrogen pyrophosphate
  • the concentration of disodium dihydrogen pyrophosphate is 10-120 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 60 ⁇ mol/L, 70 ⁇ mol/L, 80 ⁇ mol/L, 90 ⁇ mol/L, 100 ⁇ mol/L, 110 ⁇ mol/L
  • the phosphate compound comprises bisphenol A bis(diphenyl phosphate), and in the extension reagent buffer, the concentration of bisphenol A bis(diphenyl phosphate) is less than or equal to 70 ⁇ mol/L.
  • the phosphate compound comprises bisphenol A bis(diphenyl phosphate), and in the extension reagent buffer, the concentration of bisphenol A bis(diphenyl phosphate) is 10 to 60 ⁇ mol/L, including but not limited to 10 ⁇ mol/L, 11 ⁇ mol/L, 12 ⁇ mol/L, 13 ⁇ mol/L, 14 ⁇ mol/L, 15 ⁇ mol/L, 16 ⁇ mol/L, 18 ⁇ mol/L, 22 ⁇ mol/L, 25 ⁇ mol/L, 28 ⁇ mol/L, 30 ⁇ mol/L, 34 ⁇ mol/L, 36 ⁇ mol/L, 38 ⁇ mol/L, 40 ⁇ mol/L, 42 ⁇ mol/L, 44 ⁇ mol/L, 46 ⁇ mol/L, 48 ⁇ mol/L, 49 ⁇ mol/L, 50 ⁇ mol/L, 52 ⁇ mol/L, 53 ⁇ mol/L, 54 ⁇ mol/L, 56 ⁇ mol/L, 58 ⁇ mol/L, 59 ⁇ mol/L or 60 ⁇ mol/L.
  • controlling the amount of the phosphate compound used during the sequencing process can further effectively reduce the sequencing error rate while effectively controlling the cost.
  • the extension reagent buffer provided in the embodiment of the present application can be used in the field of nucleic acid sequencing.
  • the application of the extension reagent buffer in the field of nucleic acid sequencing includes that during the extension process of nucleic acid molecules, the extension reagent buffer carries a reversible terminator and a polymerase, reacts with a nucleic acid template complex bound to the surface of a solid phase substrate, and the reversible terminator is bound to the nucleic acid template complex.
  • the nucleic acid template complex includes a probe and a nucleic acid template molecule, one end of the probe is bound to the surface of a solid phase carrier, and the other end of the probe is bound to the nucleic acid template molecule.
  • the extension of the nucleic acid molecule is carried out on the chain where the probe is located, and nucleic acid sequencing is achieved by introducing reversible terminators one by one in the chain where the probe is located and identifying the introduced reversible terminators.
  • the extension reagent buffer provided in the embodiments of the present application can also be used to carry the reversible terminator and the polymerase to participate in the nucleic acid molecule extension reaction.
  • the extension reagent buffer is used as a reagent kit for nucleic acid extension reaction, and is added to the reaction system together with the raw materials and other reagents of the nucleic acid extension reaction.
  • the extension reagent buffer is used as a reagent kit for nucleic acid extension reaction, and is flowed into the surface of the solid phase carrier to be reacted together with the raw materials and other reagents of the nucleic acid extension reaction.
  • the present embodiment provides an extension kit, including the extension reagent buffer provided in the embodiment of the present application.
  • the composition, selection of each component, concentration, pH and other conditions of the extension reagent buffer provided in the first aspect can all be referred to above. In order to save space, it will not be repeated here.
  • the extension kit also includes a reversible terminator.
  • the reversible terminator is a reversible terminator with a fluorescent group (such as HN-A, HN-T, HN-G, HN-C mentioned below).
  • a fluorescent group such as HN-A, HN-T, HN-G, HN-C mentioned below.
  • the reversible terminator is incorporated through a polymerization reaction, and the type of the incorporated reversible terminator is determined by identifying the fluorescent group of the reversible terminator, or the base type of the incorporated reversible terminator is determined. Therefore, in the embodiments of the present application, the identification or confirmation of the type of nucleotide or nucleotide analog can be understood as the identification or confirmation of the base type.
  • the type of nucleotide or its analog can be interchanged with the expression of the base type.
  • dATP is expressed as a deoxyribonucleotide containing base A
  • ATP is expressed as a ribonucleotide containing base A; others are similar.
  • the reversible terminator is a reversible terminator without a fluorescent group (such as CN-A, CN-T, CN-G, CN-C mentioned below).
  • the reversible terminator is used to fill the position of the nucleic acid molecule that does not participate in the reaction after the reversible terminator with a fluorescent group undergoes polymerization reaction, so as to reduce the phase lag phenomenon.
  • the reversible terminator includes a reversible terminator with a fluorescent group and a reversible terminator without a fluorescent group.
  • the reversible terminator without a fluorescent group can be combined with the probe chain to form a base pair or a nucleotide pair with the nucleotide molecule in the non-sequencing region of the chain where the template molecule is located.
  • the reversible terminator with a fluorescent group is complementary to the nucleotide in the sequencing region of the chain where the template molecule is located.
  • the type of the corresponding reversible terminator is determined by the fluorescent group on the reversible terminator.
  • the extension kit includes oligonucleotide A, oligonucleotide T, oligonucleotide G, and oligonucleotide C, or the fifth reagent includes oligonucleotide A, oligonucleotide U, oligonucleotide G, and oligonucleotide C.
  • the extension kit also includes a polymerase, which is used to promote the binding of the reversible terminator to the nucleic acid template complex, especially to the chain where the probe is located.
  • the extension reagent buffer, the reversible terminator and the polymerase are each independently packaged in a specific reagent tube, and when entering the nucleic acid molecule extension reaction step, they are respectively extracted from different reagent tubes and mixed into the area where the nucleic acid template complex is located.
  • the extension reagent buffer, the reversible terminator and the polymerase are respectively extracted from different reagent tubes by a pump, and the branches converge to mix the reagents, and finally flow through the surface of the solid phase carrier with the nucleic acid template complex bound to the surface, so that the reversible terminator is bound to the nucleic acid template complex.
  • the content of magnesium ions in the extension reagent buffer can be adjusted according to the concentration and flux of the polymerase.
  • concentration of the polymerase in the extension reagent mixed by the extension kit is 0.02-0.05 mg/mL
  • the concentration of magnesium ions is 2.0-5.0 mmol/L.
  • the present application provides a sequencing method, comprising:
  • the extension reagent buffer, the reversible terminator and the polymerase provided in the embodiment of the present application are introduced to allow the reversible terminator to bind to the nucleic acid molecule;
  • the nucleic acid molecule includes a nucleic acid template and an oligonucleotide bound to the nucleic acid template, and a reversible terminator is bound to the 3' end of the chain where the oligonucleotide is located.
  • the present embodiment provides a gene sequencing kit, which contains tris hydroxymethylaminomethane (Tris base), ammonium sulfate, magnesium sulfate, potassium acetate, Triton X-100, sodium phytate, disodium dihydrogen pyrophosphate, BDP, A chain solution, T chain solution, C chain solution, G chain solution, DNA polymerase, dATP, dTTP, dCTP and dTGP.
  • Tris base tris hydroxymethylaminomethane
  • Ammonium sulfate ammonium sulfate
  • magnesium sulfate magnesium sulfate
  • potassium acetate Triton X-100
  • sodium phytate sodium phytate
  • disodium dihydrogen pyrophosphate BDP
  • a chain solution T chain solution, C chain solution, G chain solution
  • DNA polymerase dATP, dTTP, dCTP and dTGP.
  • Example 1 the kit in Example 1 was used for sequencing.
  • the sequencing Tris buffer add final concentrations of 10mmol/L ammonium sulfate, 3mmol/L magnesium sulfate, 50mmol/L potassium acetate, 0.1% Triton X-100, 10nmol/L A chain solution, 10nmol/L T chain solution, 10nmol/L C chain solution, and 10nmol/L G chain solution to the 20mmol/LTris buffer. Then divide the above solutions into four groups, one of which is used as the control group, and the other three groups are added with the following additives to prepare four extension reagents respectively: 1 Sodium phytate: Add final concentration ...
  • 2BDP add 10 ⁇ mol/L BDP to the above sequencing Tris buffer, mix well, filter through 0.22 ⁇ m filter membrane, cool the reagent in 4°C environment, and then add 250nmol/LdATP, 250nmol/LdTTP, 250nmol/LdCTP, 250nmol/LdTGP, and 5U/mL DNA polymerase, and mix well;
  • 3Disodium dihydrogen pyrophosphate add 100 ⁇ mol/L disodium dihydrogen pyrophosphate to the above sequencing Tris buffer, mix well, filter through 0.22 ⁇ m filter membrane, cool the reagent in 4°C environment, and then add 250nmol/LdATPT, 250nmol/LdCTPG, and 5U/mL DNA polymerase, and mix well; DNA polymerase, mix well.
  • Example 1 the kit in Example 1 was used for sequencing.
  • extension reagents 1 Sodium phytate: Add sodium phytate at a final concentration of 10 ⁇ mol/L ( ⁇ mol/L) to the above sequencing Tris buffer, mix well, filter through a 0.22 ⁇ m filter membrane, cool the reagent at 4°C, and then add 500nmol/L dATP, 250nmol/L dTTP, 500nmol/L dCTP, 250nmol/L dTGP, and 5U/mL DNA polymerase, respectively, and mix well; 2 BDP: Add 50 ⁇ mol/L BDP to the above sequencing Tris buffer, mix well, filter through a 0.22 ⁇ m filter membrane, cool the reagent at 4°C, and then add 500nmol/L dATP, 250nmol/L dTT P, 500nmol/LdCTP, 250nmol/LdTGP, 5U/mL DNA poly
  • extension reagents 1 Sodium phytate: Add final concentrations of 0 ⁇ mol/L, 0.5 ⁇ mol/L, 1 ⁇ mol/L, and 12 ⁇ mol/L sodium phytate to the above sequencing Tris buffer, mix well, filter with a 0.22 ⁇ m filter membrane, cool the reagent in a 4°C environment, and then add 500nmol/LdATP, 250nmol/LdTTP, 500nmol/LdCTP, 250nmol/LdTGP, and 5U/mL DNA polymerase respectively, and mix well; 2 BDP: Add 0 ⁇ mol/L, 5 ⁇ mol/L, and 55 ⁇ mol/L BDP to the above sequencing Tris buffer respectively, mix well, Filter through a 0.22 ⁇ m filter membrane, cool the reagents at 4°C, add 500nmol/LdATP, 250nmol/LdTTP, 500nmol/LdC
  • Example 2 The operation steps were referred to Example 2, and the sequencing was performed using extension reagents with different additive formulations. The comparison results of error rate and sequencing flux are shown in Table 5.
  • the final concentration of sodium phytate is controlled to be 1-10 ⁇ mol/L.
  • the sequencing flux is improved and the error rate (insertion ratio) is not significantly reduced; when the concentration is 55 ⁇ mol/L, the sequencing flux is improved and the error rate (insertion ratio) is reduced, but the improvement effect is similar to 50 ⁇ mol/L.
  • the final concentration of BDP is controlled to be 10-50 ⁇ mol/L.
  • disodium dihydrogen pyrophosphate additive to the extension reagent, when the concentration was 40 ⁇ mol/L, the sequencing throughput was improved and the error rate (insertion ratio) was not significantly reduced; when the concentration was 110 ⁇ mol/L, the sequencing throughput was improved and the error rate (insertion ratio) was reduced, but the improvement effect was similar to that of 100 ⁇ mol/L. Therefore, considering the cost, the final concentration of disodium dihydrogen pyrophosphate was controlled to 50-100 ⁇ mol/L.
  • the embodiments of the present invention aim to solve the problem that the nucleotide substrate (reversible terminator) is easily randomly adsorbed on the surface of the sequencing chip during the sequencing process, which leads to the problem that the fluorescent spots located by the non-hybridized template are easily identified in the subsequent fluorescence imaging process. It is unexpectedly found that the compounds having the structures shown in formula (1) and formula (2) compete with the virtual terminator nucleotide substrate (reversible terminator) in the sequencing process and compete for adsorption on the surface of the sequencing chip, thereby reducing the random adsorption of the nucleotide substrate (reversible terminator) on the surface of the sequencing chip, reducing the sequencing error rate caused by it, and improving the sequencing throughput.
  • the present invention discovered that by adjusting the pH and the concentrations of K + and NH4 + in the extension reagent formula to meet the following conditions: the concentration of K + is 50-100 mmol/L, the concentration of NH4 + is 150-350 mmol/L, and the pH of the extension reagent buffer is 8.9-9.4, the activity of nucleic acid molecule extension can be promoted and the phase lag (phase or phasing, which means that the nucleotides that should react in cycle N and be incorporated into the nucleic acid template lag behind to cycle N+1 to participate in the reaction) generated during nucleic acid sequencing can be reduced.
  • phase lag phase or phasing, which means that the nucleotides that should react in cycle N and be incorporated into the nucleic acid template lag behind to cycle N+1 to participate in the reaction
  • Extension reagent buffer was prepared as follows:
  • the prepared buffer system was divided into 4 parts, one of which remained unchanged and was named Extension Reagent Buffer 1-1. Potassium acetate was added to the other 3 parts so that the final concentrations of K + in the buffer system were 50mmol/L, 70mmol/L and 100mmol/L, respectively.
  • the corresponding buffer systems were named Extension Reagent Buffer 1-2, Extension Reagent Buffer 1-3 and Extension Reagent Buffer 1-4, and the pH of each buffer was 9.25.
  • the extension reagent buffer was mixed and filtered through a 0.22 ⁇ m microporous membrane filter.
  • extension reagent buffer 1-1, extension reagent buffer 1-2, extension reagent buffer 1-3 and extension reagent buffer 1-4 provided in Example 5 are used to prepare extension reagent 1-1A and extension reagent 1-1B, extension reagent 1-2A and extension reagent 1-2B, extension reagent 1-3A and extension reagent 1-3B, extension reagent 1-4A and extension reagent 1-4B according to the final concentrations of the components provided in Tables 2 and 3 below, respectively, wherein the extension reagents named with A correspond to the formula in Table 6, and the extension reagents named with B correspond to the formula in Table 7.
  • the final concentration of the extension reagent buffer in Table 6 and Table 7 is represented by "-", which means that the extension reagent buffer in the extension reagent is equivalent to the solvent, and other components can be added to the extension reagent buffer and their final concentrations can be controlled.
  • concentration of the extension reagent buffer does not need to be characterized.
  • HN-N represents a nucleotide containing a fluorescent group in its structure
  • CN-N represents a nucleotide not containing a fluorescent group in its structure, wherein N is selected from A, T, G, and C.
  • Extension reagent 1-1A, Extension reagent 1-2A, Extension reagent 1-3A and Extension reagent 1-4A were used to replace synthesis reagent 3 in the Genolab M sequencing reaction universal kit
  • Extension reagent 1-1B, Extension reagent 1-2B, Extension reagent 1-3B and Extension reagent 1-4B were used to replace synthesis reagent 4 in the Genolab M sequencing reaction universal kit for SE100 sequencing.
  • the phasing that occurs during the sequencing process is statistically analyzed.
  • the statistical method of phasing is as follows: the data of phasing changing with the cycle is statistically analyzed, and the slope is output through linear regression fitting. The slope is the corresponding phasing value.
  • Extension reagent buffer was prepared as follows:
  • Tris buffer 100.248mmol/L, K + concentration 72.139mmol/L, ethylenediaminetetraacetic acid 1.040mmol/L, NH4 + concentration (the raw material contains ammonium sulfate and ammonia water) 124.0mmol/L, magnesium sulfate 3.124mmol/L, dimethyl sulfoxide 5.208% (volume percentage), 1,3-dimethylthiourea 10.392mmol/L, lipoic acid 2mmol/L, and Tween-20 0.052% (volume percentage).
  • the prepared buffer system was divided into 4 parts, one of which remained unchanged (NH 4 + concentration was 124 mmol/L) and was named extension reagent buffer 2-1. Ammonium acetate was added to the other 3 parts so that the final concentrations of NH 4 + in the buffer system were 184 mmol/L, 244 mmol/L and 304 mmol/L, respectively.
  • the corresponding buffer systems were named extension reagent buffer 2-2, extension reagent buffer 2-3 and extension reagent buffer 2-4, respectively.
  • the pH of each buffer was 9.25.
  • the extension reagent buffer was mixed and filtered through a 0.22 ⁇ m microporous membrane filter.
  • extension reagent buffer 2-1, extension reagent buffer 2-2, extension reagent buffer 2-3 and extension reagent buffer 2-4 provided in Example 8 were respectively prepared according to the final concentrations of the components provided in Tables 2 and 3 above to prepare extension reagents 2-1A and 2-1B, extension reagents 2-2A and 2-2B, extension reagents 2-3A and 2-3B, Extension reagent 2-4A and extension reagent 2-4B, wherein the extension reagent named with A corresponds to the formula in Table 2, and the extension reagent named with B corresponds to the formula in Table 7.
  • Extension reagent 2-1A, Extension reagent 2-2A, Extension reagent 2-3A and Extension reagent 2-4A were used to replace synthesis reagent 3 in the Genolab M sequencing reaction universal kit
  • Extension reagent 2-1B, Extension reagent 2-2B, Extension reagent 2-3B and Extension reagent 2-4B were used to replace synthesis reagent 4 in the Genolab M sequencing reaction universal kit for SE100 sequencing.
  • Extension reagent buffer was prepared as follows:
  • Tris buffer 100.248mmol/L, ethylenediaminetetraacetic acid 1.040mmol/L, magnesium sulfate 3.124mmol/L, dimethyl sulfoxide 5.208% (volume percentage), 1,3-dimethylthiourea 10.392mmol/L, lipoic acid 2mmol/L, Tween-20 0.052% (volume percentage).
  • the prepared buffer system was divided into three parts, and the pH was adjusted with potassium hydroxide, potassium salt, ammonium salt and ammonia water. While maintaining the K + concentration of 72.139 mmol/L and the NH4 + concentration of 244.868 mmol/L in each buffer, the pH in the buffer system was adjusted to 8.90, 9.25 and 9.50, respectively.
  • the corresponding buffer systems were named Extension Reagent Buffer 3-1, Extension Reagent Buffer 3-2 and Extension Reagent Buffer 3-3, respectively.
  • the extension reagent buffer was mixed and filtered through a 0.22 ⁇ m microporous membrane filter.
  • extension reagent buffer 3-1, extension reagent buffer 3-2 and extension reagent buffer 3-3 provided in Example 8 are used to prepare extension reagent 3-1A and extension reagent 3-1B, extension reagent 3-2A and extension reagent 3-2B, extension reagent 3-3A and extension reagent 3-3B according to the final concentrations of the components provided in Table 2 and Table 3 above, respectively, wherein the extension reagents named with A in the name correspond to the formula in Table 6, and the extension reagents named with B correspond to the formula in Table 7.
  • Extension reagent 3-1A, extension reagent 3-2A and extension reagent 3-3A were used to replace synthesis reagent 3 in the Genolab M sequencing reaction universal kit, and extension reagent 3-1B, extension reagent 3-2B and extension reagent 3-3B were used to replace synthesis reagent 4 in the Genolab M sequencing reaction universal kit for SE100 sequencing.
  • phase lag phase or phasing
  • the present invention is inspired by another inventive idea and finds that: in the single-molecule sequencing process, by adding a phosphate compound having a structure represented by formula (1) and/or formula (2) at a concentration of 1 to 150 ⁇ mol/L to the extension reagent formula, the sequencing error rate can be reduced and the sequencing throughput can be improved.
  • the sequencing Tris buffer add 3mmol/L magnesium sulfate, 50mmol/L potassium acetate, 0.1% Triton X-100, 10nmol/L A chain solution, 10nmol/L T chain solution, 10nmol/L C chain solution, and 10nmol/L G chain solution to a final concentration of 20mmol/L Tris buffer, and add ammonium salt and ammonia water to adjust the NH 4 + concentration in the buffer to 180mmol/L and the pH to 9.0.
  • An X-bot 16 automatic sample injector was used for single-molecule sequencing chip pretreatment and library sample loading; the prepared extension reagent was placed in the sequencing reaction universal kit (single-molecule fluorescence sequencing method), 72 cycles of sequencing were performed, and 400 FOV of photography was taken.
  • the sequencing error rate and sequencing flux were calculated using four different extension reagents.
  • the comparison results of the error rate (Insertion rate) and sequencing flux using extension reagents containing different additives are shown in Table 11.
  • the sequencing throughput can be improved and the error rate (insertion ratio) can be reduced, especially for sequencing of nucleic acid molecules that do not need to be amplified into clusters.
  • the additives reduce the random adsorption of nucleotide substrates (reversible terminators) on the surface of the sequencing chip during the sequencing process, resulting in the problem that the fluorescent spots located by the non-hybridized template are easily identified in the subsequent fluorescence imaging process.
  • the compounds with structures shown in formulas (1) and (2) compete with the virtual terminator nucleotide substrates (reversible terminators) in the sequencing process and compete for adsorption on the surface of the sequencing chip, thereby reducing the random adsorption of nucleotide substrates (reversible terminators) on the surface of the sequencing chip, reducing the sequencing error rate caused by them, and improving the sequencing throughput.
  • the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented.
  • Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

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Abstract

提供了一种表面处理方法及测序方法和用于核酸分子延伸的试剂盒。所述表面处理方法包括：将磷酸化合物与结合有寡聚核苷酸的表面接触；所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物。针对测序过程中碱基容易在测序芯片表面随机吸附的问题，意外发现具有特定结构的化合物能够与测序过程中的虚拟终止子碱基竞争吸附在测序芯片表面，从而减少碱基在测序芯片表面的随机吸附，降低由其引起的测序错误率，并提高测序通量。

Description

表面处理方法、测序方法和试剂盒
优先权信息
本申请请求2022年10月31日向中国国家知识产权局提交的、专利申请号为202211351166.9和202211344450.3的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明属于核酸测序技术领域，涉及一种表面处理方法及测序方法和用于核酸分子延伸的试剂盒，以及一种延伸试剂缓冲液，一种延伸试剂缓冲液的应用，一种延伸试剂盒，以及一种测序方法。
背景技术
基于荧光信号对表面的核酸分子进行测序的过程中，表面的信噪比高低影响荧光信号的识别。具体的，高的信噪比可能使得参与测序反应的核苷酸的荧光信号难以与表面具有的背景信号进行区分，从而不能有效识别测序过程中待测样本产生的荧光信号，最终导致测序结果的准确性受到影响，甚至无法获得测序结果。相较于扩增成簇的荧光信号位点，核酸模板数量较少甚至只有一条核酸分子的位点受表面信噪比的影响更为严重。其中一个原因在于：表面作为载体，其上面随机固定了大量接头序列。在表面引入含有核苷酸及聚合酶的溶液后，游离的核苷酸容易在芯片表面随机吸附，导致表面信噪比增加。表面产生的随机吸附，在后续的荧光成像过程易被识别成荧光信号点，并且当该荧光信号点靠近真正的核酸模板位点产生的荧光信号点时，易造成数据分析错误，表现为错误率(insertion error ratio)偏高，有效数据量降低。
综上所述，如何解决由于碱基随机吸附而引起的错误率偏高的问题，是基因测序领域亟需解决问题之一。
申请内容
针对现有技术的不足和实际需求，本发明提供一种表面处理方法及测序方法和用于核酸分子延伸的试剂盒，本发明意外发现利用特定结构的磷酸化合物与测序固相载体的表面接触，能够有效降低测序过程中核苷酸底物在测序芯片表面的随机吸附，从而有效降低由于核苷酸底物随机吸附而引起的错误率，对于基因测序领域具有重要意义。
为达上述目的，本发明采用以下技术方案：
第一方面，本发明提供一种表面处理方法，所述表面处理方法包括：
将磷酸化合物与结合有寡聚核苷酸的表面接触；
所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物；
其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
n选自1～8的整数。
本发明中，发现如式(1)、(2)所示的特定结构的磷酸化合物能够用于处理结合有寡聚核苷酸的表面。所述磷酸化合物与结合有寡聚核苷酸的表面接触后，磷酸化合物和寡聚核苷酸结合并形成稳定的复合体，以封闭寡聚核苷酸的反应活性。当在将结合有寡聚核苷酸的表面应用于特定场景(如用于核酸测序)，而表面的寡聚核苷酸又不是预期保留的物质时，通过这种方法，可以减少寡聚核苷酸与底物原料反应带来的信号干扰，即降低表面产生的非特异性吸附(这种非特异性吸附并非我们期望获得的)。当将该方法应用于在信噪比高的表面进行核酸测序时，能够有效降低测序过程中核苷酸底物在测序芯片表面的随机吸附(核苷酸底物和表面寡聚核苷酸的吸附反应)，从而有效降低由于核苷酸底物随机吸附而引起的错误率，最终提高测序质量，提高测序读长。
作为本申请表面处理方法的一种可能的实施情形，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基、碳原子数为6～20的芳基中的一种。
在一些实施方式中，R3包括环烷基或苯基。此时，磷酸化合物中的磷酸基团连接环烷基或苯基，与核苷酸结构较为近似的结构类型的相似度增加，在将磷酸化合物与核苷酸底物(如可逆终止子)同时添加时，有利于提高磷酸化合物与寡聚核苷酸的竞争性。同时，由于寡聚核苷酸为单链结构，而环烷基或苯基的体积较大，可以一定程度形成空间位阻，也有利于降低可逆终止子与寡聚核苷酸的结合概率。
在一些实施方式中，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。此时，式(1)中，磷酸化合物中的磷酸基团连接碳原子数小于10的环烷基或苯基，与核苷酸结构的相似性进一步提升，使得同时添加上述磷酸化合物和核苷酸底物(可逆终止子)时，磷酸化合物能够竞争性地结合寡聚核苷酸，从而降低核苷酸底物(可逆终止子)与寡聚核苷酸结合的概率。
在一些实施方式中，R2中至少含有一个金属离子。式(1)中的金属离子，有助于磷酸化合物在溶液体系中形成磷酸负离子，从而更有效地结合寡聚核苷酸。
在一些实施方式中，R2中含有n个金属离子。此时，每个磷酸基团都能形成一个磷酸负离子，增加了磷酸化合物与寡聚核苷酸反应的结合位置。特别是将磷酸化合物和核苷酸底物(可逆终止子)同时添加至表面时，有利于提高磷酸化合物与表面反应的竞争性。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
在一些实施方式中，所述n选自2～6的整数，包括但不限于2、3、4、5或6。
在一些实施方式中，式(2)中，R1选自氢原子，R2选自金属离子。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
作为本申请表面处理方法的一种可能的实施情形，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
式(2-1)中，M选自Na或K。
发明人研究发现，满足上述式(2-1)、式(2-2)结构所示的磷酸化合物，能够有效结合表面的寡聚核苷酸，从而在寡聚核苷酸以非预期的方式出现在表面时，可以通过添加式(2-1)、式(2-2)结构所示的磷酸化合物来抑制寡聚核苷酸的活性，从而抑制其带来的干扰。如在在信噪比高的表面进行核酸测序时，即可通过该磷酸化合物抑制表面寡聚核苷酸的活性，从而核酸分子延伸过程中产生的费特异性吸附，降低测序背景信号，从而提高测序信号的可识别性。
作为本申请表面处理方法的一种可能的实施情形，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。将这些磷酸化合物用于不需要经过信噪比高的表面进行测序，特别是扩增成簇的核酸分子进行测序时，由于每一位点的核酸数量少，多为一条，测序产生的信号强度较弱。此时，干扰信号(背景噪声)对测序信号的识别影响显著。上述磷酸化合物可以结合表面的寡聚核苷酸，降低寡聚核苷酸的可反应活性。通过该方法封闭固相载体表面的故居核苷酸序列与核苷酸底物(可逆终止子)的结合活性，可以显著减少固相载体表面的非特异性吸附，从而降低背景噪音，提高测序信号的可识别性，降低测序错误率，尤其是insertion类型错误率，最终提升核酸测序数据量。
本发明中，植酸钠化学式为C6H6Na12O24P6，结构式如式(3)，白色粉末状或结晶状，是一种重要的纯天然绿色添加剂，最显著的特征是与金属离子有极强的鳌合作用，较强的抗氧化性和护色性，广泛被用作果蔬汁饮料、肉制品、海产品的抗氧化和护色剂；焦磷酸二氢二钠化学式为Na2H2P2O7，结构式如式(4)，白色结晶性粉末，常被用作食品添加剂；双酚A双(二苯基磷酸酯)(简称BDP)分子式为C39H34O8P2，结构式如式(5)，常被用作阻燃剂。
作为本申请表面处理方法的一种可能的实施情形，所述将磷酸化合物与结合有寡聚核苷酸的表面接触，包括：
在所述表面加含有所述磷酸化合物的第一溶液；或
将所述表面置于含有所述磷酸化合物的第一溶液中；或
使含有所述磷酸化合物的第一溶液流经所述表面。
作为本申请表面处理方法的一种可能的实施情形，所述第一溶液中，所述磷酸化合物的浓度为1～150μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、10μmol/L、20μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、91μmol/L、92μmol/L、95μmol/L、96μmol/L、98μmol/L、99μmol/L、100μmol/L、110μmol/L、120μmol/L、130μmol/L、140μmol/L或150μmol/L。若第一溶液中磷酸化合物的浓度过高，如超过150μmol/L，则过量的磷酸化合物会结合到表面其他核苷酸分子，如在核酸测序过程中占据测序链中核苷酸或核苷酸类似物的反应位置。由于反应过程中不含有聚合酶，且磷酸化合物无法像核苷酸或核苷酸类似物一样与互补链形成氢键，磷酸化合物结合到表面其他核苷酸分子时的反应不牢固，但这仍然会对测序反应造成一定的影响。特别是磷酸化合物的浓度过高时，由于过量的磷酸化合物可能占据测序链反应位置，因此，还是可能会降低核酸测序的通量。
在一些实施方式中，所述磷酸化合物包含植酸钠，且所述第一溶液中，所述植酸钠的浓度小于或等于15μmol/L。
在一些实施方式中，所述磷酸化合物为植酸钠，且所述第一溶液中，所述植酸钠的浓度为1～10μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、9μmol/L或10μmol/L。研究发现，当植酸钠含量在此范围时，可以有效抑制表面的寡聚核苷酸活性，降低错误率，提高测序通量。此外，由于浓度较低，植酸钠可能引入的不良反应也会降低。如在核酸测序过程中，植酸钠占据测序链中核苷酸或核苷酸类似物的反应位置的概率降低，对测序通量基本不产生影响或产生的影响可以忽略。
在一些实施方式中，所述磷酸化合物包含焦磷酸二氢二钠，且所述第一溶液中，所述焦磷酸二氢二钠的浓度小于或等于120μmol/L。
在一些实施方式中，所述磷酸化合物为焦磷酸二氢二钠，且所述第一溶液中，所述焦磷酸二氢二钠的浓度为10～120μmol/L，包括但不限于10μmol/L、11μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、20μmol/L、30μmol/L、40μmol/L、42μmol/L、43μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、100μmol/L、110μmol/L或120μmol/L。研究发现，当单独采用焦磷酸二氢二钠作为抑制寡聚核苷酸活性的添加剂时，焦磷酸二氢二钠的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第一溶液中，所述双酚A双(二苯基磷酸酯)的浓度小于或等于70μmol/L。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第一溶液中，所述双酚A双(二苯基磷酸酯)的浓度为10～60μmol/L，包括但不限于10μmol/L、11μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、20μmol/L、30μmol/L、40μmol/L、42μmol/L、43μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、52μmol/L、53μmol/L、54μmol/L、56μmol/L、58μmol/L、59μmol/L或60μmol/L。研究发现，当双酚A双(二苯基磷酸酯)作为抑制寡聚核苷酸活性的添加剂时，双酚A双(二苯基磷酸酯)的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
第二方面，本发明提供一种测序方法，所述测序方法包括：
将含有磷酸化合物的第二溶液与固相载体的表面接触，形成混合体系；
其中，所述表面结合有第一核酸分子和第二核酸分子，所述第一核酸分子为双链核酸分子，且所述第一核酸分子至少含有一个单链末端，所述第二核酸分子为单链核酸分子；
所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物：
其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；n选自1～8的整数。
在核酸分子延伸过程中，式(1)和式(2)所示结构的化合物可竞争性地与固相载体(如生物芯片)表面残留的寡聚核苷酸(如结合在固相载体表面、但没有结合核酸模板结合的寡聚核苷酸)结合，从而降低或抑制这些寡聚核苷酸与虚拟终止子的结合，即：降低固相载体表面的非特异性吸附，由此，可减少这些寡聚核苷酸对核酸测序带来的信号干扰(虚拟终止子与这些寡聚核苷酸结合后，同样会产生可识别的信号，从而影响对核酸模板分子中引入的信号的干扰)，降低由其引起的测序错误率，进一步提高测序准确性。
作为本申请测序方法的一种可能的实施情形，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基、碳原子数为6～20的芳基中的一种。
在一些实施方式中，R3包括环烷基或苯基。此时，磷酸化合物中的磷酸基团连接环烷基或苯基，与核苷酸结构较为近似的结构类型的相似度增加，在将磷酸化合物与核苷酸底物(如可逆终止子)同时添加时，有利于提高磷酸化合物与寡聚核苷酸的竞争性。同时，由于寡聚核苷酸为单链结构，而环烷基或苯基的体积较大，可以一定程度形成空间位阻，也有利于降低可逆终止子与寡聚核苷酸的结合概率。
在一些实施方式中，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。此时，式(1)中，磷酸化合物中的磷酸基团连接碳原子数小于10的环烷基或苯基，与核苷酸结构的相似性进一步提升，使得同时添加上述磷酸化合物和核苷酸底物(可逆终止子)时，磷酸化合物能够竞争性地结合寡聚核苷酸，从而降低核苷酸底物(可逆终止子)与寡聚核苷酸结合的概率。
在一些实施方式中，R2中至少含有一个金属离子。式(1)中的金属离子，有助于磷酸化合物在溶液体系中形成磷酸负离子，从而更有效地结合寡聚核苷酸。
在一些实施方式中，R2中含有n个金属离子。此时，每个磷酸基团都能形成一个磷酸负离子，增加了磷酸化合物与寡聚核苷酸反应的结合位置。特别是将磷酸化合物和核苷酸底物(可逆终止子)同时添加至表面时，有利于提高磷酸化合物与表面反应的竞争性。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
在一些实施方式中，所述n选自2～6的整数，包括但不限于2、3、4、5或6。
在一些实施方式中，式(2)中，R1选自氢原子，R2选自金属离子。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
作为本申请测序方法的一种可能的实施情形，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
式(2-1)中，M选自Na或K。
发明人研究发现，满足上述式(2-1)、式(2-2)结构所示的磷酸化合物，能够有效结合表面的寡聚核苷酸，从而在寡聚核苷酸以非预期的方式出现在表面时，可以通过添加式(2-1)、式(2-2)结构所示的磷酸化合物来抑制寡聚核苷酸的活性，从而抑制其带来的干扰。如在对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，即可通过该磷酸化合物抑制表面寡聚核苷酸的活性，从而核酸分子延伸过程中产生的费特异性吸附，降低测序背景信号，从而提高测序信号的可识别性。
作为本申请测序方法的一种可能的实施情形，所述磷酸化合物可选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。将这些磷酸化合物用于对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，由于每一位点的核酸数量少，甚至为一条，测序产生的信号强度较弱。此时，干扰信号(背景噪声)对测序信号的识别影响显 著。上述磷酸化合物可以结合表面的寡聚核苷酸，降低寡聚核苷酸的可反应活性。通过该方法封闭固相载体表面的故居核苷酸序列与核苷酸底物(可逆终止子)的结合活性，可以显著减少固相载体表面的非特异性吸附，从而降低背景噪音，提高测序信号的可识别性，降低测序错误率，尤其是insertion类型错误率，最终提升核酸测序数据量。
在测序过程中，所述磷酸化合物可单独使用一种，也可以组合使用，即组合2种甚至3种共同作用，因这类化合物在测序中与测序碱基发生竞争，因此在组合使用中预期其作用效果与单独使用一种作用类似，原因为测序芯片表面积及添加的测序碱基是固定量的，因此在组合使用时，在相对平衡的竞争体系中，其降低测序芯片表面吸附的效果是有极限的，其最大作用效果与单一添加效果类似。
作为本申请测序方法的一种可能的实施情形，所述将含有磷酸化合物的第二溶液与固相载体的表面接触，包括：
在所述固相载体的表面加含有所述磷酸化合物的第二溶液；或
将所述固相载体的表面置于含有所述磷酸化合物的第二溶液中；或
使含有所述磷酸化合物的第二溶液流经所述固相载体的表面。
作为本申请测序方法的一种可能的实施情形，所述第二溶液中，所述磷酸化合物的浓度为1～150μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、10μmol/L、20μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、91μmol/L、92μmol/L、95μmol/L、96μmol/L、98μmol/L、99μmol/L、100μmol/L、110μmol/L、120μmol/L、130μmol/L、140μmol/L或150μmol/L。若第二溶液中磷酸化合物的浓度过高，如超过150μmol/L，则过量的磷酸化合物会结合到表面其他核苷酸分子，如在核酸测序过程中占据测序链中核苷酸或核苷酸类似物的反应位置。由于反应过程中不含有聚合酶，且磷酸化合物无法像核苷酸或核苷酸类似物一样与互补链形成氢键，磷酸化合物结合到表面其他核苷酸分子时的反应不牢固，但这仍然会对测序反应造成一定的影响。特别是磷酸化合物的浓度过高时，由于过量的磷酸化合物可能占据测序链反应位置，因此，还是可能会降低核酸测序的通量。
在一些实施方式中，所述磷酸化合物包含植酸钠，且所述第二溶液中，所述植酸钠的浓度小于或等于15μmol/L。
在一些实施方式中，所述磷酸化合物为植酸钠，且所述第二溶液中，所述植酸钠的浓度为1～10μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、8μmol/L、9μmol/L或10μmol/L。研究发现，当植酸钠含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。此外，由于浓度较低，植酸钠可能引入的不良反应也会降低。如在核酸测序过程中，植酸钠占据测序链中核苷酸或核苷酸类似物的反应位置的概率降低，对测序通量基本不产生影响或产生的影响可以忽略。
在一些实施方式中，所述磷酸化合物包含焦磷酸二氢二钠，且所述第二溶液中，所述焦磷酸二氢二钠的浓度小于或等于120μmol/L。
在一些实施方式中，所述磷酸化合物为焦磷酸二氢二钠，且所述第二溶液中，所述焦磷酸二氢二钠的浓度为10～120μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、100μmol/L、110μmol/L或120μmol/L。研究发现，当单独采用焦磷酸二氢二钠作为抑制寡聚核苷酸活性的添加剂时，焦磷酸二氢二钠的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第二溶液中，所述双酚A双(二苯基磷酸酯)的浓度小于或等于70μmol/L。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第二溶液中，所述双酚A双(二苯基磷酸酯)的浓度为10～60μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、52μmol/L、53μmol/L、54μmol/L、56μmol/L、58μmol/L、59μmol/L或60μmol/L。研究发现，当双酚A双(二苯基磷酸酯)作为抑制寡聚核苷酸活性的添加剂时，双酚A双(二苯基磷酸酯)的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
本发明中，在测序过程中控制磷酸化合物的用量，能够进一步有效降低测序错误率，同时有效控制成本。
作为本申请测序方法的一种可能的实施情形，所述第二溶液包括缓冲液、表面活性剂和其他添加剂中的至少一种。
在一些实施方式中，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。上述缓冲液用于调节第二溶液的pH值，从而为核酸分子的延伸反应提供稳定的pH环境。
在一些实施方式中，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。其中，铵根离子、镁离子、钾离子、钠离子，可提高聚合酶在溶液中促进核酸分子延伸的活性；二甲基亚砜可以在核酸分子延伸过程中，促进碱基氢键的结合能力；1,3-二甲基硫脲有助于清除延伸试剂缓冲液中的氧化自由基，提供更稳定的缓冲体系。
在一些实施方式中，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。上述表面活性剂用于调节第二溶液的表面张力，特别是在固相载体表面反应时，有利于第二溶液在固相载体表面的铺展。
在一些实施方式中，所述第二溶液中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第二溶液的pH为8.9～9.4。综合第二溶液中pH以及K+和NH4 +的浓度，可使得聚合酶在延伸试剂缓冲液中促进核酸分子延伸的活性提高，进而提高核酸分子的延伸效率，降低相位滞后现象，最终提高待测核苷酸分子的测序准确率。该第二溶液提高聚合酶反应活性的方式可能为：延伸试剂缓冲液中pH以及K+和NH4 +的浓度在上述范围时，第二溶液改变了聚合酶蛋白表面的电荷分布，调整聚合酶的等电点，从而调整聚合酶与核酸分子之间、聚合酶与第二溶液之间的相互作用，使聚合酶更容易作用于核酸分子；另外，第二溶液改变核酸分子在第二溶液中的空间构象，使聚合酶更容易结合在核酸分子的反应位置。
其中，K+的浓度在不同实施例中可以为50mmol/L、55mmol/L、60mmol/L、65mmol/L、70mmol/L、75mmol/L、80mmol/L、85mmol/L、90mmol/L、95mmol/L、100mmol/L等具体情形。NH4 +的浓度在不同实施例中可以为150mmol/L、160mmol/L、170mmol/L、180mmol/L、200mmol/L、220mmol/L、250mmol/L、280mmol/L、300mmol/L、320mmol/L、350mmol/L等具体情形；NH4 +的浓度在不同实施例中可以为150mmol/L、160mmol/L、170mmol/L、180mmol/L、200mmol/L、220mmol/L、250mmol/L、280mmol/L、300mmol/L、320mmol/L、350mmol/L等具体情形。
在一些实施方式中，所述第二溶液中还包括dNTP或NTP。
在一些实施方式中，所述dNTP选自dATP或其类似物、dTTP或其类似物、dGTP或其类似物、dCTP或其类似物中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
作为本申请测序方法的一种可能的实施情形，在所述将含有磷酸化合物的第二溶液与固相载体的表面接触的步骤之后，在所述混合体系中加入含有dNTP或NTP的第三溶液。
在一些实施方式中，所述第三溶液包括缓冲液、表面活性剂和其他添加剂中的至少一种。
在一些实施方式中，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。
在一些实施方式中，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。
在一些实施方式中，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。
在一些实施方式中，所述第三溶液中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第三溶液的pH为8.9～9.4。
在一些实施方式中，所述第三溶液中还包括dNTP或NTP。
在一些实施方式中，所述dNTP选自dATP或其类似物(实施例直接用dATP表示)、dTTP或其类似物(实施例直接用dTTP表示)、dGTP或其类似物(实施例直接用dGTP表示)、dCTP或其类似物(实施例直接用dCTP表示)中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
第三溶液中各成分的作用，可以参考第二溶液。
作为本申请测序方法的一种可能的实施情形，所述第一核酸分子包括寡聚核酸分子和核酸模板，且所述寡聚核酸分子的至少部分序列与所述核酸模板互补，且所述核酸模板为未经扩增反应的核酸分子。其中，寡聚核苷酸结合在固相模板表面，用于将核酸模板固定在固相模板表面，并可以发挥引物的作用。
第三方面，本发明提供一种用于核酸分子延伸的试剂盒，所述试剂盒包括第一试剂，所述第一试剂包括磷酸化合物，且所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物；
其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
n选自1～8的整数。
本发明中，利用特定结构的磷酸化合物制备用于核酸分子延伸的试剂盒，在核酸分子延伸过程中，式(1)和式(2)所示结构的化合物可竞争性地与固相载体(如生物芯片)表面残留的寡聚核苷酸(如结合在固相载体表面、但没有结合核酸模板结合的寡聚核苷酸)结合，从而降低或抑制这些寡聚核苷酸与虚拟终止子的结合，即：降低固相载体表面的非特异性吸附，由此，可减少这些寡聚核苷酸对核酸测序带来的信号干扰(虚拟终止子与这些寡聚核苷酸结合后，同样会产生可识别的信号，从而影响对核酸模板分子中引入的信号的干扰)，降低由其引起的测序错误率，进一步提高测序准确性。
作为本申请试剂盒的一种可能的实施情形，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。
在一些实施方式中，R3包括环烷基或苯基。此时，磷酸化合物中的磷酸基团连接环烷基或苯基，与核苷酸结构较为近似的结构类型的相似度增加，在将磷酸化合物与核苷酸底物(如可逆终止子)同时添加时，有利于提高磷酸化合物与寡聚核苷酸的竞争性。同时，由于寡聚核苷酸为单链结构，而环烷基或苯基的体积较大，可以一定程度形成空间位阻，也有利于降低可逆终止子与寡聚核苷酸的结合概率。
在一些实施方式中，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。此时，式(1)中，磷酸化合物中的磷酸基团连接碳原子数小于10的环烷基或苯基，与核苷酸结构的相似性进一步提升，使得同时添加上述磷酸化合物和核苷酸底物(可逆终止子)时，磷酸化合物能够竞争性地结合寡聚核苷酸，从而降低核苷酸底物(可逆终止子)与寡聚核苷酸结合的概率。
在一些实施方式中，R2中至少含有一个金属离子。式(1)中的金属离子，有助于磷酸化合物在溶液体系中形成磷酸负离子，从而更有效地结合寡聚核苷酸。
在一些实施方式中，R2中含有n个金属离子。此时，每个磷酸基团都能形成一个磷酸负离子，增加了磷酸化合物与寡聚核苷酸反应的结合位置。特别是将磷酸化合物和核苷酸底物(可逆终止子)同时添加至表面时，有利于提高磷酸化合物与表面反应的竞争性。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
在一些实施方式中，所述n选自2～6的整数，包括但不限于2、3、4、5或6。
在一些实施方式中，式(2)中，R1选自氢原子，R2选自金属离子。
在一些实施方式中，所述金属离子选自一价金属离子或二价金属离子。
在一些实施方式中，所述一价金属离子包括钠离子和/或钾离子。
作为本申请试剂盒的一种可能的实施情形，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
式(2-1)中，M选自Na或K。
发明人研究发现，满足上述式(2-1)、式(2-2)结构所示的磷酸化合物，能够有效结合表面的寡聚核苷酸，从而在寡聚核苷酸以非预期的方式出现在表面时，可以通过添加式(2-1)、式(2-2)结构所示的磷酸化合物来抑制寡聚核苷酸的活性，从而抑制其带来的干扰。如在对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，即可通过该磷酸化合物抑制表面寡聚核苷酸的活性，从而核酸分子延伸过程中产生的费特异性吸附，降低测序背景信号，从而提高测序信号的可识别性。
作为本申请试剂盒的一种可能的实施情形，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。将这些磷酸化合物用于对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，由于每一位点的核酸数量少，甚至为一条，测序产生的信号强度较弱。此时，干扰信号(背景噪声)对测序信号的识别影响显著。上述磷酸化合物可以结合表面的寡聚核苷酸，降低寡聚核苷酸的可反应活性。通过该方法封闭固相载体表面的故居核苷酸序列与核苷酸底物(可逆终止子)的结合活性，可以显著减少固相载体表面的非特异性吸附，从而降低背景噪音，提高测序信号的可识别性，降低测序错误率，尤其是insertion类型错误率，最终提升核酸测序数据量。
作为本申请试剂盒的一种可能的实施情形，所述试剂盒包括第二试剂，所述第二试剂包括缓冲液、表面活性剂和其他添加剂中的至少一种。
在一些实施方式中，所述第一试剂与所述第二试剂以混合物的方式置于所述试剂盒中的同一个试剂管中；或所述第一试剂和所述第二试剂分装于所述试剂盒中不同的试剂管中。
在一些实施方式中，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。上述缓冲液用于调节第二溶液的pH值，从而为核酸分子的延伸反应提供稳定的pH环境。
在一些实施方式中，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。其中，铵根离子、镁离子、钾离子、钠离子，可提高聚合酶在溶液中促进核酸分子延伸的活性；二甲基亚砜可以在核酸分子延伸过程中，促进碱基氢键的结合能力；1,3-二甲基硫脲有助于清除延伸试剂缓冲液中的氧化自由基，提供更稳定的缓冲体系。
在一些实施方式中，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。上述表面活性剂用于调节第二溶液的表面张力，特别是在固相载体表面反应时，有利于第二溶液在固相载体表面的铺展。
在一些实施方式中，所述第二试剂中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第二试剂的pH为8.9～9.4。综合第二试剂中pH以及K+和NH4 +的浓度，可使得聚合酶在第二试剂中促进核酸分子延伸的活性提高，进而提高核酸分子的延伸效率，降低相位滞后现象，最终提高待测核苷酸分子的测序准确率。该第二试剂提高聚合酶反应活性的方式可能为：延伸试剂缓冲液中pH以及K+和NH4 +的浓度在上述范围时，第二试剂改变了聚合酶蛋白表面的电荷分布，调整聚合酶的等电点，从而调整聚合酶与核酸分子之间、聚合酶与第二试剂之间的相互作用，使聚合酶更容易作用于核酸分子；另外，第二试剂改变核酸分子在第二试剂中的空间构象，使聚合酶更容易结合在核酸分子的反应位置。
其中，K+的浓度在不同实施例中可以为50mmol/L、55mmol/L、60mmol/L、65mmol/L、70mmol/L、75mmol/L、80mmol/L、85mmol/L、90mmol/L、95mmol/L、100mmol/L等具体情形。NH4 +的浓度在不同实施例中可以为150mmol/L、160mmol/L、170mmol/L、180mmol/L、200mmol/L、220mmol/L、250mmol/L、280mmol/L、300mmol/L、320mmol/L、350mmol/L等具体情形；NH4 +的浓度在不同实施例中可以为150mmol/L、160mmol/L、170mmol/L、180mmol/L、200mmol/L、220mmol/L、250mmol/L、280mmol/L、300mmol/L、320mmol/L、350mmol/L等具体情形。
作为本申请试剂盒的一种可能的实施情形，所述试剂盒包括第三试剂和第四试剂，所述第三试剂包括dNTP或NTP，所述第四试剂包括聚合酶。
在一些实施方式中，所述dNTP选自dATP或其类似物、dTTP或其类似物、dGTP或其类似物、dCTP或其类似物中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
在一些实施方式中，所述第三试剂、所述第四试剂和所述第一试剂各自独立地分装于所述试剂盒中不同的试剂管中。
在一些实施方式中，所述试剂盒包括第五试剂，所述第五试剂包括寡聚核苷酸A(下文简称A链)、寡聚核苷酸T(下文简称T链)、寡聚核苷酸G(下文简称G链)、寡聚核苷酸C(下文简称C链)，或所述第五试剂包括寡聚核苷酸A、寡聚核苷酸U、寡聚核苷酸G、寡聚核苷酸C。
在一些实施方式中，所述第五试剂和所述第一试剂分装于所述试剂盒中不同的试剂管中。
作为本申请试剂盒的一种可能的实施情形，以所述试剂盒中各成分混合形成的混合溶液的浓度计算，所述磷酸化合物的浓度为1～150μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、10μmol/L、20μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、91μmol/L、92μmol/L、95μmol/L、96μmol/L、98μmol/L、99μmol/L、100μmol/L、110μmol/L、120μmol/L、130μmol/L、140μmol/L或150μmol/L。若混合溶液中磷酸化合物的浓度过高，如超过150μmol/L，则过量的磷酸化合物会结合到表面其他核苷酸分子，如在核酸测序过程中占据测序链中核苷酸或核苷酸类似物的反应位置。由于反应过程中不含有聚合酶，且磷酸化合物无法像核苷酸或核苷酸类似物一样与互补链形成氢键，磷酸化合物结合到表面其他核苷酸分子时的反应不牢固，但这仍然会对测序反应造成一定的影响。特别是磷酸化合物的浓度过高时，由于过量的磷酸化合物可能占据测序链反应位置，因此，还是可能会降低核酸测序的通量。
在一些实施方式中，所述磷酸化合物包含植酸钠，且所述植酸钠的浓度小于或等于15μmol/L。
在一些实施方式中，所述磷酸化合物为植酸钠，且所述植酸钠的浓度为1～10μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、8μmol/L、9μmol/L或10μmol/L。研究发现，当植酸钠含量在此范围时，可以有效抑制表面的寡聚核苷酸活性，降低错误率，提高测序通量。此外，由于浓度较低，植酸钠可能引入的不良反应也会降低。如在核酸测序过程中，植酸钠占据测序链中核苷酸或核苷酸类似物的反应位置的概率降低，对测序通量基本不产生影响或产生的影响可以忽略。
在一些实施方式中，所述磷酸化合物包含焦磷酸二氢二钠，且所述焦磷酸二氢二钠的浓度小于或等于120μmol/L。
在一些实施方式中，所述磷酸化合物为焦磷酸二氢二钠，且所述焦磷酸二氢二钠的浓度为10～120μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、 30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、100μmol/L、110μmol/L或120μmol/L。研究发现，当单独采用焦磷酸二氢二钠作为抑制寡聚核苷酸活性的添加剂时，焦磷酸二氢二钠的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述双酚A双(二苯基磷酸酯)的浓度小于或等于70μmol/L。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述双酚A双(二苯基磷酸酯)的浓度为10～60μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、52μmol/L、53μmol/L、54μmol/L、56μmol/L、58μmol/L、59μmol/L或60μmol/L。研究发现，当双酚A双(二苯基磷酸酯)作为抑制寡聚核苷酸活性的添加剂时，双酚A双(二苯基磷酸酯)的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
在一些实施方式中，所述dNTP或NTP的浓度为20～1000μmol/L，包括但不限于20μmol/L、21μmol/L、22μmol/L、23μmol/L、24μmol/L、25μmol/L、50μmol/L、100μmol/L、150μmol/L、200μmol/L、300μmol/L、400μmol/L、500μmol/L、600μmol/L、700μmol/L、800μmol/L、900μmol/L、910μmol/L、920μmol/L、950μmol/L、960μmol/L、970μmol/L、980μmol/L或1000μmol/L。
在一些实施方式中，所述聚合酶的浓度为5～10U/mL，包括但不限于5U/mL、6U/mL、7U/mL、8U/mL、9U/mL或10U/mL。
下一代测序，也称为高通量测序或者大规模平行测序，能够在一次测序运行中测定多个样本的核酸序列。比较常见的下一代测序方法有边连接边测序(sequencing by ligation,SBL)和边合成边测序(Sequencing by Synthesis,SBS)。基于SBS实现核酸测序的平台，是基于碱基配对原则、利用DNA聚合酶使核苷酸(包括核苷酸类似物)连接到与模板结合的引物的3'末端以可控地实现单碱基延伸，通过采集每次核苷酸类似物结合后所引起的信号变化，进而基于该些采集的信号的变化确定模板的碱基排列次序。一种典型的SBS方法可以包括如下步骤：(1)使待测核酸分子与固相载体表面上的探针杂交以将待测核酸模板连接至该固相载体表面；(2)在DNA聚合酶的作用下、在适于聚合酶链式反应的条件下，以探针为引物使带有荧光基团的核苷酸类似物掺入到核酸模板上(形成互补链)，从而实现单碱基延伸；(3)激发核苷酸类似物上的荧光基团发光，进而对表面进行成像以采集表面上的发光信号；(4)去除结合到待测核酸链上的核苷酸类似物中的荧光基团；(5)重复上述步骤(2)和步骤(4)使待测核酸链得以继续延伸。进一步的，SBS方法还包括步骤(6)：通过对步骤(3)中得到的光学信号进行分析，以确定每轮延伸反应中掺入到核酸模板上的核苷酸类似物的类型；按顺序读取引入的核苷酸类似物类型，最终实现获得待测链的所有核苷酸序列。
将核苷酸类似物掺入到核酸模板的过程中，由于引入的核苷酸类似物掺入的效率影响等，核苷酸相位会发生变化，导致相位误差。相位误差通常表现为相位滞后(phasing或phase，是指：本应在cycle N反应并掺入到核酸模板的核苷酸，却滞后到cycle N+1参与反应)与相位超前(prephasing或prephase，是指：本应在Cycle N反应并掺入到核酸模板的核苷酸，却提前在Cycle N-1参与反应)，即同一个通道内相邻cycle之间会出现串扰。这种相位误差，随着测序循环数的增加，不断积累变强。最终的结果是，在一个扩增簇(Cluster)中四种核苷酸同时组装且亮度均匀。在这种情况下，算法将无法识别该轮下正确的测序信号，即无法准确获得待测核苷酸分子的序列信息。
本申请的另一目的在于提供一种延伸试剂缓冲液及其应用、延伸试剂盒和测序方法，旨在解决目前将核苷酸类似物掺入到核酸模板的过程中容易产生相位误差，影响测序信号的识别，进而降低待测核苷酸分子的测序准确率的问题。
为实现上述申请目的，本申请实施例采用的技术方案如下：
第四方面，本申请提供一种延伸试剂缓冲液，至少包括基础缓冲液、K+和NH4 +，其中，所述K+的浓度为50～100mmol/L，所述NH4 +的浓度为150～350mmol/L，所述延伸试剂缓冲液的pH为8.9～9.4。
本申请提供的延伸试剂缓冲液，综合平衡缓冲液中pH以及K+和NH4 +的浓度，可使得聚合酶在延伸试剂缓冲液中促进核酸分子延伸的活性提高，进而提高核酸分子的延伸效率，降低相位滞后现象，最终提高待测核苷酸分子的测序准确率。该延伸试剂缓冲液提高聚合酶反应活性的方式可能为：延伸试剂缓冲液中pH以及K+和NH4 +的浓度在上述范围时，延伸试剂缓冲液改变了聚合酶蛋白表面的电荷分布，调整聚合酶的等电点，从而调整聚合酶与核酸分子之间、聚合酶与延伸试剂缓冲液之间的相互作用，使聚合酶更容易作用于核酸分子；另外，延伸试剂缓冲液改变核酸分子在延伸试剂缓冲液中的空间构象，使聚合酶更容易结合在核酸分子的反应位置。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液的pH为8.9～9.1，所述K+的浓度为50～70mmol/L，所述NH4 +的浓度为150～230mmol/L。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液的pH为9.05～9.35，所述K+的浓度为60～90mmol/L，所述NH4 +的浓度为200～310mmol/L。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液的pH为9.3～9.5，所述K+的浓度为80～100mmol/L，所述NH4 +的浓度为300～350mmol/L。
结合上述三种实施方式配制的延伸试剂缓冲液，可以更明显地看到延伸试剂缓冲液对相位滞后现象的改善作用。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述K+为氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种提供的K+。也就意味着，在本申请的实施方式中，延伸试剂缓冲液中含有氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种，或者在配置延伸试剂缓冲液的过程中，添加氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种，至少用于提供K+。其中，氢氧化钾作为K+源，还可以通过其阴离子调节延伸试剂缓冲液的pH，以丰富基础缓冲液以及其他用于调节pH或对pH有影响的试剂的选择。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述NH4 +为硫酸铵、有机铵和氨水中的至少一种提供的NH4 +。也就意味着，在本申请的实施方式中，延伸试剂缓冲液中含有硫酸铵、有机铵和氨水中的至少一种，或者在配置延伸试剂缓冲液的过程中，添加硫酸铵、有机铵和氨水中的至少一种，至少用于提供NH4 +。其中，氨水作为NH4 +源，同样可以调节延伸试剂缓冲液的pH，以丰富基础缓冲液以及其他用于调节pH或对pH有影响的试剂的选择。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液含有氢氧化钾和硫酸铵。此时，配置延伸试剂缓冲液时，氢氧化钾提供K+，且其产生的OH-还能调节延伸试剂缓冲液的pH。同时，硫酸铵至少提供延伸试剂缓冲液所需的部分NH4 +，或者NH4 +完全来源于硫酸铵，因此，可以降低延伸试剂缓冲液中其他NH4 +源特别是氨水的含量，甚至可将氨水含量降至0，即配置延伸试剂缓冲液时不加氨水。由此，可以降低氢氧化钾和氨水对延伸试剂缓冲液pH的双层影响，有利于调控延伸试剂缓冲液的pH，以获得预期的pH值。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液包括有机羧酸钾和硫酸钾中的至少一种和氨水。此时，延伸试剂缓冲液的配置过程中，由于氨水可影响延伸试剂缓冲液pH，因此，当K+来源于有机羧酸钾和硫酸钾，可降低或避免诸如 氢氧化钾的影响pH的试剂含量，从而降低诸如氢氧化钾的影响pH的试剂与氨水联合作用加强对延伸试剂缓冲液pH的影响，从而有利于调控延伸试剂缓冲液，获得预期的pH。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述基础缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。上述基础缓冲液用于调节延伸试剂缓冲液的pH值，从而为核酸分子的延伸反应提供稳定的pH环境。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述延伸试剂缓冲液还包括：络合剂、聚合酶催化剂、表面活性剂和其他助剂中的至少一种。其中，络合剂用于络合延伸试剂缓冲液中可能存在的金属杂离子(如铅、铁或铜等形成的金属离子)，从而在核酸测序过程中，消除杂质金属离子对酶的抑制作用。在核酸分子延伸过程中，聚合酶催化剂用于提高聚合酶的活性，从而提高核酸分子延伸的效率，即提高引入的核苷酸或核苷酸类似物通过聚合反应掺入到核酸分子中的效率。表面活性剂调节延伸试剂缓冲液的表面张力，特别是在固相载体表面反应时，有利于延伸试剂缓冲液在固相载体表面的铺展。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述络合剂选自乙二胺四乙酸、乙二胺四乙酸二钠盐、乙二胺四乙酸四钠盐、EGTA(乙二醇双(2-氨基乙基醚)四乙酸)中的至少一种。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述聚合酶催化剂包括镁盐。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述表面活性剂选自吐温-20、Triton X-100中的至少一种。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述其他助剂包括二甲基亚砜、1,3-二甲基硫脲中的至少一种。其中，二甲基亚砜可以在核酸分子延伸过程中，促进碱基氢键的结合能力；1,3-二甲基硫脲有助于清除延伸试剂缓冲液中的氧化自由基，提供更稳定的缓冲体系。作为本申请延伸试剂缓冲液的一种可能的实施方式，所述络合剂选自乙二胺四乙酸、乙二胺四乙酸二钠盐、乙二胺四乙酸四钠盐、EGTA中的至少一种，所述聚合酶催化剂包括镁盐，所述表面活性剂选自吐温-20、Triton X-100中的至少一种，所述其他助剂包括二甲基亚砜、1,3-二甲基硫脲中的至少一种。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述其他助剂包括磷酸化合物，且所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物：
其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
n选自1～8的整数。
在核酸分子延伸过程中，式(1)和式(2)所示结构的化合物可竞争性地与固相载体(如生物芯片)表面残留的寡聚核苷酸(如结合在固相载体表面、但没有结合核酸模板结合的寡聚核苷酸)结合，从而降低或抑制这些寡聚核苷酸与虚拟终止子的结合，即：降低固相载体表面的非特异性吸附，由此，可减少这些寡聚核苷酸对核酸测序带来的信号干扰(虚拟终止子与这些寡聚核苷酸结合后，同样会产生可识别的信号，从而影响对核酸模板分子中引入的信号的干扰)，降低由其引起的测序错误率，进一步提高测序准确性。
作为本申请延伸试剂缓冲液的一种可能的实施方式，式(1)中，R1、R2中至少一个为氢原子，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种；或R1、R2中至少一个为芳香基，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。在这种情形下，可以提高式(1)结构的竞争力，有利于其降低固相载体表面的非特异性吸附。
作为本申请延伸试剂缓冲液的一种可能的实施方式，式(1)中，R3包括环烷基或苯基。此时，磷酸化合物中的磷酸基团连接环烷基或苯基，与核苷酸结构较为近似的结构类型的相似度增加，在将磷酸化合物与核苷酸底物(如可逆终止子)同时添加时，有利于提高磷酸化合物与寡聚核苷酸的竞争性。同时，由于寡聚核苷酸为单链结构，而环烷基或苯基的体积较大，可以一定程度形成空间位阻，也有利于降低可逆终止子与寡聚核苷酸的结合概率。
作为本申请延伸试剂缓冲液的一种可能的实施方式，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。此时，式(1-1)中，磷酸化合物中的磷酸基团连接碳原子数小于10的环烷基或苯基，与核苷酸结构的相似性进一步提升，使得同时添加上述磷酸化合物和核苷酸底物(可逆终止子)时，磷酸化合物能够竞争性地结合寡聚核苷酸，从而降低核苷酸底物(可逆终止子)与寡聚核苷酸结合的概率。
作为本申请延伸试剂缓冲液的一种可能的实施方式，R2中至少含有一个金属离子。式(1)中的金属离子，有助于磷酸化合物在溶液体系中形成磷酸负离子，从而更有效地结合寡聚核苷酸。
作为本申请延伸试剂缓冲液的一种可能的实施方式，R2中含有n个金属离子。此时，每个磷酸基团都能形成一个磷酸负离子，增加了磷酸化合物与寡聚核苷酸反应的结合位置。特别是将磷酸化合物和核苷酸底物(可逆终止子)同时添加至表面时，有利于提高磷酸化合物与表面反应的竞争性。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述金属离子选自一价金属离子或二价金属离子。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述一价金属离子包括钠离子和/或钾离子。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述n选自2～6的整数，包括但不限于3、4或5。
作为本申请延伸试剂缓冲液的一种可能的实施方式，式(2)中，R1选自氢原子，R2选自金属离子。此时，式(2)结构能与可逆终止子竞争，结合固相载体表面的寡聚核苷酸，从而降低固相载体表面的非特异性吸附。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述金属离子选自一价金属离子或二价金属离子。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述一价金属离子包括钠离子和/或钾离子。
作为本申请延伸试剂缓冲液的一种可能的实施方式，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
式(2-1)中，M选自Na或K。
发明人研究发现，满足上述式(2-1)、式(2-2)结构所示的磷酸化合物，能够有效结合表面的寡聚核苷酸，从而在寡聚核苷酸以非预期的方式出现在表面时，可以通过添加式(2-1)、式(2-2)结构所示的磷酸化合物来抑制寡聚核苷酸的活性，从而抑制其带来的干扰。如在对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，即可通过该磷酸化合物抑制表面寡聚核苷酸的活性，从而核酸分子延伸过程中产生的费特异性吸附，降低测序背景信号，从而提高测序信号的可识别性。
作为本申请延伸试剂缓冲液的一种可能的实施方式，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。上述磷酸化合物可以结合表面的寡聚核苷酸，降低寡聚核苷酸的可反应活性。特别是将延伸试剂缓冲液用于对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，由于每一位点的核酸数量少，甚至为一条，测序产生的信号强度较弱。此时，干扰信号(背景噪声)对测序信号的识别影响显著。而通过该方法封闭固相载体表面的非待测序列与可逆终止子的结合活性，可以显著减少固相载体表面的非特异性吸附，从而降低背景噪音，提高测序信号的可识别性，降低测序错误率，尤其是insertion类型错误率，并且提升核酸测序数据量。
第五方面，本申请提供一种如本申请第四方面提供的延伸试剂缓冲液在核酸测序领域中的应用。
第六方面，本申请提供一种延伸试剂盒，包括本申请第四方面提供的延伸试剂缓冲液。
作为本申请延伸试剂盒的一种可能的实施方式，还包括聚合酶，带荧光基团的可逆终止子和/或不带荧光基团的可逆终止子。
第七方面，本申请提供一种测序方法，包括：
于结合有核酸分子的固相载体的表面，通入本申请第四方面提供的延伸试剂缓冲液、可逆终止子和聚合酶，使所述可逆终止子结合在所述核酸分子上；
其中，所述核酸分子包括核酸模板和与所述核酸模板结合的寡聚核苷酸，且所述可逆终止子结合在所述寡聚核苷酸所在链的3’端。
作为本申请测序方法的一种可能的实施方式，所述于结合有核酸分子的固相载体的表面，通入所述延伸试剂缓冲液、可逆终止子和聚合酶，包括：
在所述固相载体的表面加含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液；或
将所述固相载体置于含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液中；或
使含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液流经所述表面。
本申请第五方面至第七方面的技术效果，由本申请第四方面提供的方法获得，此处不再赘述。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本申请中，在本申请实施例中使用的术语是仅仅出于描述特定实施例的目的，而非旨在限制本申请。术语“和/或”，描述关联对象的关联关系，表示可以存在三种关系，例如，A和/或B，可以表示：单独存在A，同时存在A和B，单独存在B的情况。其中A，B可以是单数或者复数。字符“/”一般表示前后关联对象是一种“和”的关系。
术语“至少一个”是指一个或者多个，“多个”是指两个或两个以上。“至少一种(个)”或其类似表达，是指的这些项中的任意组合，包括单项(个)或复数项(个)的任意组合。例如，“a，b，或c中的至少一种(个)”，或，“a，b和c中的至少一种(个)”，均可以表示：a，b，c，a-b(即a和b)，a-c，b-c，或a-b-c，其中a，b，c分别可以是单项(个)，也可以是多项(个)。
在本申请实施例和所附权利要求书中所使用的单数形式的“一种”、“所述”和“该”也旨在包括多数形式，除非上下文清楚地表示其他含义。
术语“第一”、“第二”仅用于描述目的，用来将目的如物质、方位、界面、消息、请求和终端彼此区分开，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。例如，在不脱离本申请实施例范围的情况下，第一成像处理也可以被称为第二成像处理，类似地，第二成像处理也可以被称为第一成像处理。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。
在本申请的描述中，所提到的相关成分的浓度不仅仅可以指代各组分的具体含量，也可以表示各组分间含量的比例关系，因此，只要是按照本申请实施例说明书相关组分的含量按比例放大或缩小均在本申请实施例说明书公开的范围之内。
应理解，在本申请的各种实施例中，上述各过程的序号的大小并不意味着执行顺序的先后，部分或全部步骤可以并行执行或先后执行，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。
本文使用的缩写具有其在化学和生物学领域中的常规含义。本文的化学结构和化学式是根据化学领域已知的化学价的标准规则构建的。
除非另有说明，否则术语“烃基”本身或作为取代基的一部分是指直链(即非支链)碳链或支链碳链(或碳)或其组合，应当理解的是，“烃基”是一个非环化链。“烃基”可以为完全饱和基团，也可以为单不饱和基团或多不饱和基团。当“烃基”为完全饱和基团(即烃基的不饱和度为0)时，“烃基”为烷基，即：烷烃分子中少掉一个氢原子而成的基团；当“烃基”为单不饱和基团(即烃基的不饱和度为1)时，“烃基”为含有一个碳碳双键的烯基，即：烷基中的一个碳碳单键但被一个碳碳双键取代；当“烃基”为多不饱和基团(即烃基的不饱和度大于或等于2)时，“烃基”为烯基或炔基，且烯基包括两个或两个以上的双键和/或除两个或两个以上之外还包括一个或多个三键；炔基可以包括至少一个三键和/或除至少一个三键之外还包括一个或多个双键。“烯基”可以包括具有指定数目的碳原子(如：C1～C100表示1～100个碳)的单价、二价和多价基团。
其中，术语“烷基”本身或作为取代基的一部分是指不含不饱和键的直链(即非支链)碳链或支链碳链(或碳)或其组合，应当理解 的是，“烷基”是一个非环化链；术语“烯基”本身或作为取代基的一部分是指含有碳碳双键的直链(即非支链)碳链或支链碳链(或碳)或其组合；术语“炔基”本身或作为取代基的一部分是指含有碳碳三键的直链(即非支链)碳链或支链碳链(或碳)或其组合。
本申请实施例中，“未取代的烃基”是指“烃基”本身。“未取代的烃基”包括但不限于诸如-CH3、-C2H5、-C3H7、-C4H9、-C5H11、-C6H13、-C7H15、-C8H17、-C9H19、-C10H21、-C15H31、-C20H41、-C25H51、-C30H61等的同系物和异构体的烷基；包括但不限于诸如-C2H3、-C3H5、-C4H7、-C5H9、-C6H11、-C7H13、-C8H15、-C9H17、-C10H19、-C15H29、-C20H39、-C25H49、-C30H59等的同系物和异构体的单不饱和烯基；包括但不限于诸如-C2H、-C3H3、-C4H5、-C4H3、-C5H7、-C5H5、-C5H3、-C6H11、-C6H9、-C6H9、-C6H7、-C7H13、-C7H11、-C7H9、-C7H7、-C7H5、-C7H3、-C8H15、-C8H13、-C8H11、-C8H9、-C8H7、-C8H5、-C9H17、-C9H15、-C9H13、-C9H11、-C9H9、-C9H7、-C10H17、-C10H15、-C10H13、-C10H11、-C10H9、-C15H27、-C15H25、-C15H23、-C15H21、-C15H19、-C15H17、-C15H15、-C20H37、-C20H35、-C20H33、-C20H31、-C20H29、-C20H27、-C20H25、-C20H23、-C20H21、-C20H19、-C25H47、-C25H45、-C25H43、-C25H41、-C25H39、-C25H37、-C25H35、-C25H33、-C25H31、-C30H57、-C30H55、-C30H53、-C30H51、-C30H49、-C30H47、-C30H45、-C30H43、-C30H41、-C30H39等的同系物和异构体的多不饱和烯基或炔基。其中，术语“异构体”是指具有相同的原子数量和原子种类的化合物，因此具有相同的分子量，但在原子的结构排列或构型方面不同。
“取代的烃基”是指“烃基”本身中的一个或多个氢原子被除“烃基”本身以外的其他基团取代形成的基团。“取代的烃基”包括但不限于在未取代的烃基的基础上引入一个或多个诸如以下原子或取代基获得的的基团：卤素、烷基、烯基、炔基、羟基、硝基、氨基、羰基、羧基、巯基、酰基、烷氧基、环烷基、杂烷基、杂环烷基、芳基、杂芳基、单磷酸基、双磷酸基、三磷酸基等。其中，术语“烷氧基”是通过氧接头(-O-)与分子其余部分连接的烷基，烷基部分可以含有取代或未取代。术语“酰基”是指-C(O)R，其中R是取代或未取代的烃基、取代或未取代的环状烃基、取代或未取代的含杂原子的烃基、取代或未取代的含杂原子的环状烃基、取代或未取代的芳基，以及取代或未取代的杂芳基中的一种。示例性的，烃基的取代基可以选自但不限于以下各种基团的一种或多种：-OR’、＝O、＝NR’、＝N-OR’、-NR’R”、-SR’、-卤素、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR’C(O)2R”、-NR-C(NR’R”R”’)＝NR””、-NR-C(NR’R”)＝NR”’、-S(O)R’、-S(O)2R’、-S(O)2NR’R”、-NRSO2R’、-NR’NR”R”’、-ONR’R”、-NR’C(O)NR”NR”’R””、-CN、-NO2、-NR’SO2R”、-NR’C(O)R”、-NR’C(O)-OR”、-NR’OR”，其数量范围从0到(2m’+1)，其中m’是这种基团中碳原子的总数。R、R’、R”、R”’和R””各自独立地表示氢、取代或未取代的含杂原子的烃基、取代或未取代的环状烃基、取代或未取代的含杂原子的环状烃基、取代或未取代的芳基(例如，由1-3个卤素取代的芳基)、取代或未取代的杂芳基、取代或未取代的烃基、烷氧基或硫代烷氧基、或芳基烷基。
除非另有说明，属于“杂烃基”是指含杂原子的烃基。术语“含杂原子的烃基”是指包括至少一个碳原子和至少一个杂原子(例如B、O、N、P、Si和S)形成的稳定的直链或支链或其组合，并且其中氮原子和硫原子可以任选地被氧化，氮杂原子可以任选地被季铵化。杂原子(例如，B、O、N、S、Si或P)可以位于杂烷基的任何内部位置处或在烷基与分子的其余部分连接的位置。应当理解的是，杂烷基是一个非环化链。杂烷基的实例包括但不限于：-CH2-CH2-O-CH3、-CH2-CH2-NH-CH3、-CH2-CH2-N(CH3)-CH3、-CH2-SCH2-CH3、-CH2-S(O)-CH3、-CH2-CH2-S(O)2-CH3、-CH＝CH-O-CH3、-CH2-Si(CH3)3、-CH2-O-Si(CH3)3、-CH2-CH＝NOCH3、-CH＝CH-N(CH3)-CH3、-CH2-O-CH3、-CH2-O-CH2-CH3、-CH2-CN、-CH2-O-CH2-N3等等。多达两个或三个的杂原子可以是连续的，例如-CH2-NH-OCH3和-CH2-O-Si(CH3)3。“含杂原子的烃基”部分可以包括一个杂原子(例如B、O、N、S、Si或P)，也可以包括两个任选的不同杂原子(例如B、O、N、S、Si或P)，还可以包括三个任选的不同杂原子(例如B、O、N、S、Si或P)，甚至四个、五个或更多个任选的不同杂原子(例如B、O、N、S、Si或P)。
本申请实施例中，当“含杂原子的烃基”中除杂原子以外的其他基团为烷基，“含杂原子的烃基”为杂烷基。当“含杂原子的烃基”中除杂原子以外的其他基团中含有碳碳双键，“含杂原子的烃基”可称为杂烯基。杂烯基可以任选地包含多于一个的双键和/或除一个或多个双键之外还包含一个或多个三键。当“含杂原子的烃基”中除杂原子以外的其他基团中含有碳碳三键，“含杂原子的烃基”可称为杂炔基。杂炔基可以任选地包含多于一个三键和/或除一个或多个三键之外还包含一个或多个双键。
除非另有说明，否则术语“环状烃基”和“杂环烃基”本身或与其它术语组合意指分别为“烃基”和“含杂原子的烃基”的环状形式。应当理解的是，“环状烃基”和“含杂原子的环状烃基”不是芳香族的。另外，对于“含杂原子的环状烃基”，杂原子可以占据杂环与分子其余部分连接的位置。“环状烃基”的实例包括但不限于环丙基、环丁基、环戊基、环己基、1-环己烯基、3-环己烯基、环庚基等。。
术语“取代的环状烃基”和“取代的杂环烃基”分别是指“环状烃基”和“含杂原子的环状烃基”本身中的一个或多个氢原子被取代基取代形成的基团。取代基可以取代环状烃基上的任意非氢原子。“取代的环状烃基”和“取代的杂环烃基”的实例包括但不限于在未取代的“环状烃基”和“含杂原子的环状烃基”的基础上引入一个或多个诸如以下原子或取代基获得的的基团：卤素、烷基、烯基、炔基、羟基、硝基、氨基、羰基、羧基、巯基、酰基、烷氧基、环烷基、杂烷基、杂环烷基、芳基、单磷酸基、双磷酸基、三磷酸基等。其中，术语“烷氧基”是通过氧接头(-O-)与分子其余部分连接的烷基，烷基部分可以含有取代或未取代。术语“酰基”是指-C(O)R，其中R是取代或未取代的烃基、取代或未取代的环状烃基、取代或未取代的含杂原子的烃基、取代或未取代的含杂原子的环状烃基、取代或未取代的芳基，以及取代或未取代的杂芳基中的一种。示例性的，环状烃基和含杂原子的环状烃基的取代基各自独立地选自但不限于以下各种基团的一种或多种：
除非另有说明，否则术语“芳基”是指多不饱和的芳族烃取代基，其可以是单环或稠合在一起的或共价连接的多个环(优选1-3个环)，即稠环芳基。稠环芳基是指稠合在一起的多个环，其中至少一个稠合的环是芳基环。
除非另有说明，否则术语“杂芳基”是指含有至少一个杂原子(如N、O或S)的芳基(或环)，其中氮和硫原子任选被氧化，氮原子任选被季铵化。因此，术语“杂芳基”包括稠环杂芳基(即稠合在一起的多个环，其中至少一个稠合的环是杂芳环)。杂芳基可以通过碳原子或杂原子连接到分子的其余部分。
术语“取代的芳基”和“取代的杂芳基”分别是指“芳基”和“杂芳基”本身中的一个或多个氢原子被取代基取代形成的基团。“取代的芳基”和“取代的杂芳基”的实例包括但不限于在未取代的“芳基”和“杂芳基”的基础上引入一个或多个诸如以下原子或取代基获得的的基团：卤素、烷基、烯基、炔基、羟基、硝基、氨基、羰基、羧基、巯基、酰基、烷氧基、环烷基、杂烷基、杂环烷基、芳基、单磷酸基、双磷酸基、三磷酸基等。其中，术语“烷氧基”是通过氧接头(-O-)与分子其余部分连接的烷基，烷基部分可以含有取代或未取代。术语“酰基”是指-C(O)R，其中R是取代或未取代的烃基、取代或未取代的环状烃基、取代或未取代的含杂原子的烃基、取代或未取代的含杂原子的环状烃基、取代或未取代的芳基，以及取代或未取代的杂芳基中的一种。
示例性的，芳基和杂芳基的取代基各自独立地选自但不限于以下各种基团的一种或多种：-OR’、-NR’R”、-SR’、-卤素、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)2R’、-NR-C(NR’R”R”’)＝NR””、-NR’-C(NR”R”’)＝NR””、-S(O)R’、-S(O)2R’、-S(O)2NR’R”、-NRSO2R’、-NR’NR”R”’、-ONR’R”、-NR’C(O)NR”NR”’R””、-CN、-NO2、-R’、-N3、-CH(Ph)2、卤代(C1-C4)烷氧基和卤代(C1-C4)烷基、-NR’SO2R”、-NR’C(O)R”、-NR’C(O)-OR”、-NR’OR”， 其数量范围从0至芳环体系上的开放化合价的总数。R、R’、R”、R”’和R””各自独立地表示氢、取代或未取代的含杂原子的烃基、取代或未取代的环状烃基、取代或未取代的含杂原子的环状烃基、取代或未取代的芳基(例如，由1-3个卤素取代的芳基)、取代或未取代的杂芳基、取代或未取代的烃基、烷氧基或硫代烷氧基、或芳基烷基。
在本申请的实施方式中，术语“测序”又可称为“核酸测序”或“基因测序”，即三者在表述上可以互换，指核酸序列中碱基类型和排列顺序的测定；包括合成测序(边合成边测序，SBS)和/或连接测序(边连接边测序，SBL)，包括DNA测序和/或RNA测序，包括长片段测序和/或短片段测序，所称的长片段和短片段是相对的，如长于1Kb、2Kb、5Kb或者10Kb的核酸分子可称为长片段，短于1Kb或者800bp的可称为短片段；包括双末端测序、单末端测序和/或配对末端测序等，所称的双末端测序或者配对末端测序可以指同一核酸分子的不完全重叠的任意两段或两个部分的读出；所称的测序包括使核苷酸(包括核苷酸类似物)结合到模板并采集核苷酸(包括类似物)上发出的相应的信号的过程。
测序一般包括多轮以实现模板上的多个核苷酸/碱基的次序的测定的过程，本申请实施例将每一轮“以实现模板上的多个核苷酸/碱基的次序的测定的过程”称为“一轮测序”。“一轮测序”(cycle)也称为“测序轮”，可定义为四种核苷酸/碱基的一次碱基延伸，换句话说，“一轮测序”可定义为完成模板上任意一个指定位置的碱基类型的测定。对于基于聚合或连接反应实现测序的测序平台，一轮测序包括实现一次四种核苷酸(包括核苷酸类似物)结合到所称的模板并采集发出的相应的信号的过程；对于基于聚合反应实现测序的平台，反应体系包括反应底物核苷酸、聚合酶和模板，模板上结合有一段序列(测序引物)，基于碱基配对原则和聚合反应原理，加入的反应底物核苷酸在聚合酶的催化下，连接到测序引物上实现该核苷酸与模板的特定位置的结合；通常地，一轮测序可包括一次或多次碱基延伸(repeat)，例如，四种核苷酸依次加入到反应体系中，分别进行碱基延伸和相应的反应信号的采集，一轮测序包括四次碱基延伸；又例如，四种核苷酸任意组合加入到反应体系中，例如两两组合或者一三组合，两个组合分别进行碱基延伸和相应的反应信号的采集，一轮测序包括两次碱基延伸；再例如，四种核苷酸同时加入到反应体系中进行碱基延伸和反应信号的采集，一轮测序包括一次碱基延伸。
术语“核酸分子”表示：任何长度的核苷酸的聚合物形式，并且可以包括核糖核苷酸、脱氧核糖核苷酸、其类似物或其混合物。该术语可以指单链或双链多核苷酸。核酸分子中的核苷酸可以包括天然存在的核苷酸及其功能上可替代的类似物。类似物的实例能够以序列特异性方式与核酸杂交，或者能够用作特定核苷酸序列的复制的模板。天然存在的核苷酸通常具有包含磷酸二酯键的主链。类似物结构可以具有包括本领域中已知的任何种类的替代的主链连接。天然存在的核苷酸通常具有脱氧核糖(例如，在DNA中发现的)或核糖(例如，在RNA中发现的)。类似物结构可以具有替代的糖部分，包括本领域中已知的任何种类。核苷酸可以包括天然碱基或非天然碱基。天然DNA中的碱基可以包括腺嘌呤、胸腺嘧啶、胞嘧啶和/或鸟嘌呤中的一种或更多种，并且天然RNA的碱基可以包括腺嘌呤、尿嘧啶、胞嘧啶和/或鸟嘌呤中的一种或更多种。核苷酸也可以使用任何非天然碱基或碱基类似物，诸如锁定核酸(LNA)和桥接核酸(BNA)。
术语“探针”又称“引物”，是指：可以与感兴趣的靶序列杂交的寡聚核苷酸或核酸分子。在实施例中，引物起底物(substrate)的作用，核苷酸可以通过聚合酶聚合到该底物上。例如，引物可以用作用于DNA或RNA合成的起点。例如，测序引物可以与合成的核酸模板链杂交，以便引发与合成的核酸模板链互补的新链的合成。引物可以包括核苷酸或其类似物的任何组合。在一些实例中，引物是单链寡聚核苷酸或多核苷酸。
术语“类似物”或“功能类似物”是指与另一种化合物(即所谓的“参比”化合物)在结构上相似但组成不同的化学化合物，不同组成例如一个原子被不同元素的原子置换，或存在的特定官能团不同，或一个官能团被另一个官能团置换。
本申请实施例中，应当理解，“XX或其类似物”包括“XX”和“XX类似物”，示例性的：核苷酸或其类似物包括核苷酸和核苷酸类似物，碱基或其类似物包括碱基和碱基类似物，核糖基或其类似物包括核糖和核糖类似物，脱氧核糖基或其类似物包括脱氧核糖和脱氧核糖类似物。
术语“可逆终止子”是指带有可逆终止基团的核苷酸类似物，该可逆终止基团可以封闭该核苷酸类似物中五碳糖的反应位置(如3’-OH位置)，以阻止该核苷酸类似物在该位置发生聚合反应；且可逆终止基团可以通过一定的化学方法或其他方法去除，以恢复该核苷酸类似物在五碳糖对应位置的反应活性。
术语“簇”(cluster)是指在表面如芯片上的一个位置，通常而言，这个位置可以包含多个核酸分子，但通常该位置的多个核酸分子是衍生自同一个核酸分子，例如通过对同一个核酸分子进行桥式PCR所形成的多个核酸分子的扩增簇。
术语“位点”可以理解为表面用于固定核酸分子或待酸分子扩增簇的一个点，相邻的两个这样的点之间存在一定的距离，且该距离足够大于同一位点内核酸分子之间的距离，使得两个点产生的测序信号在图像上能够被区分。示例性的，纳米井点阵芯片表面的每一纳米井为一个位点。
本发明中，发现如式(1)、(2)所示的特定结构的磷酸化合物能够用于处理结合有寡聚核苷酸的表面。所述磷酸化合物与结合有寡聚核苷酸的表面接触后，磷酸化合物和寡聚核苷酸结合并形成稳定的复合体，以封闭寡聚核苷酸的反应活性。
进一步地，本发明将上述发现应用于测序中，针对现有测序技术中存在问题：测序固相载体表面随机固定了大量特定接头序列(寡聚核苷酸)，在加入核苷酸底物(可逆终止子)及聚合酶混合液后，核苷酸底物(可逆终止子)容易在载体表面随机吸附，导致在后续的荧光成像过程易识别到非杂交模板定位的荧光点，并且当该荧光点靠近真正的杂交模板位点，易造成数据分析错误，表现为错误率(insertion error ratio)偏高，有效数据量降低；本发明利用特定结构的磷酸化合物处理测序固相载体的表面，可以减少寡聚核苷酸与底物原料反应带来的信号干扰，即降低表面产生的非特异性吸附(这种非特异性吸附并非我们期望获得的)。当将该方法应用于对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，能够有效降低测序过程中核苷酸底物在测序芯片表面的随机吸附(核苷酸底物和表面寡聚核苷酸的吸附反应)，从而有效降低由于核苷酸底物随机吸附而引起的错误率，最终提高测序质量，提高测序读长。
本发明还开发基于所述磷酸化合物的用于核酸分子延伸的试剂盒，核酸分子延伸试剂配方除测序碱基、DNA聚合酶、缓冲液外，还含有磷酸化合物。磷酸化合物可在延伸反应前单独使用处理测序芯片，亦可与其他延伸试剂组分混合共同进行测序延伸反应，磷酸化合物的结构类似测序碱基结构，与测序碱基存在“竞争”关系，相当于间接减少测序碱基在测序芯片表面的数量，从而减少其在表面的吸附，进而减少测序反应错误率。
对待测核酸分子进行SBS测序时，将核苷酸类似物掺入到核酸模板(核酸分子延伸)的过程中，核苷酸相位会发生变化，导致相位误差。比如，在cycle N测序中，反应结束前，部分待测核酸分子与掺入的核苷酸类似物还没进行聚合反应，导致本应在cycle N测序引入的核苷酸类似物，在cycle N+1才参与反应，从而导致测序序号滞后(即phasing或phase)，同一个通道内相邻cycle之间会出现串扰，进而影响测序信号的准确识别。
有鉴于此，本申请发明人通过反复研究，对延伸反应过程中的反应试剂进行研究，最终获得一种能够有效改善核酸测序过程中 的phasing或phase问题的延伸试剂缓冲液。
本申请实施例中，延伸试剂缓冲液包括基础缓冲液，基础缓冲液为延伸试剂缓冲液的主要成分，用于稳定延伸反应过程中的反应环境，特别是稳定延伸反应过程中的pH环境，使得延伸反应过程中，核苷酸类似物掺入核酸分子中的效率均匀性和稳定性提高。
用于核酸延伸的基础缓冲液均可用于本申请实施例中。在一些实施例中，基础缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。其中，三羟甲基氨基甲烷缓冲液又名三羟甲基氨基甲烷盐酸盐或Tris缓冲液，英文缩写为Tris-HCl；Hepes缓冲液是一种非离子两性缓冲液，其主要成分是羟乙基哌嗪乙硫磺酸(2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonicacid)。在优选实施例中，采用三羟甲基氨基甲烷缓冲液和甘氨酸中的至少一种作为延伸试剂缓冲液中的基础缓冲液。
本申请实施例中，基础缓冲液为10～160mmol/L，具体可视延伸试剂缓冲液中基础缓冲液的类型以及其他成分的含量进行调整。示例性的，基础缓冲液为三羟甲基氨基甲烷缓冲液，基础缓冲液的浓度为60～150mmol/L。示例性的，三羟甲基氨基甲烷缓冲液的浓度可以为60mmol/L、65mmol/L、70mmol/L、75mmol/L、80mmol/L、85mmol/L、90mmol/L、95mmol/L、100mmol/L、105mmol/L、110mmol/L、115mmol/L、120mmol/L、125mmol/L、130mmol/L、135mmol/L、140mmol/L、145mmol/L、150mmol/L、155mmol/L、160mmol/L等具体情形。示例性的，基础缓冲液为甘氨酸，基础缓冲液的浓度可以为10～60mmol/L。示例性的，甘氨酸的浓度可以为10mmol/L、15mmol/L、20mmol/L、25mmol/L、30mmol/L、35mmol/L、40mmol/L、45mmol/L、50mmol/L、55mmol/L、60mmol/L等具体情形。
本申请实施例中，延伸试剂缓冲液包括K+和NH4 +，K+和NH4 +可以通过改变聚合酶的蛋白表面的电荷分布，调整聚合酶的活性，增加其与核酸分子反应位置的结合活性，从而提高聚合酶的催化活性。
在一些实施例中，K+为氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种提供的K+。也就意味着，在本申请的实施例中，延伸试剂缓冲液中含有氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种，或者在配置延伸试剂缓冲液的过程中，添加氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种，且氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种的用途，至少包括提供K+。
示例性的，卤化钾为氯化钾、溴化钾、碘化钾中的至少一种；有机羧酸钾为乙酸钾(或醋酸钾)、草酸钾中的至少一种。
在一个实施例中，延伸试剂缓冲液通过氢氧化钾提供K+，此时，延伸试剂缓冲液中含有K+，以及OH-。氢氧化钾作为K+源，还可以通过其阴离子(OH-)调节延伸试剂缓冲液的pH。由于氢氧化钾对延伸试剂缓冲液pH的调节，因此，在基础缓冲液以及其他用于调节pH或对pH有影响的试剂的选择方面，具有更好的灵活性。如当延伸试剂缓冲液中含有氢氧化钾时，可以选择氨水以外的其他试剂作为NH4 +源。
在一些实施例中，NH4 +为硫酸铵、有机铵和氨水中的至少一种提供的NH4 +。也就意味着，在本申请的实施方式中，延伸试剂缓冲液中含有硫酸铵、有机铵和氨水中的至少一种，或者在配置延伸试剂缓冲液的过程中，添加硫酸铵、有机铵和氨水中的至少一种，且硫酸铵、有机铵和氨水中的至少一种的用途至少包括提供NH4 +。其中，有机铵为乙酸铵(或醋酸铵)、草酸铵中的至少一种。
在一个实施例中，延伸试剂缓冲液通过氨水提供NH4 +，此时，延伸试剂缓冲液中的氨水还没提供OH-，进而通过OH-调节延伸试剂缓冲液的pH。由于氨水对延伸试剂缓冲液pH的调节，因此，在基础缓冲液以及其他用于调节pH或对pH有影响的试剂的选择方面，具有更好的灵活性。如当延伸试剂缓冲液中含有氨水时，可以选择氢氧化钾以外的其他加盐作为K+源。
考虑到提供K+的K+源和提供NH4 +的NH4 +源对延伸试剂缓冲液的pH的影响，在选择K+源和NH4 +源时，可以进行更合理的搭配，使得在满足K+和NH4 +浓度的前提下，有利于调节延伸试剂缓冲液的pH。
在一些实施例中，延伸试剂缓冲液含有氢氧化钾和硫酸铵。此时，配置延伸试剂缓冲液时，氢氧化钾提供K+，且其产生的OH-还能调节延伸试剂缓冲液的pH。同时，硫酸铵至少提供延伸试剂缓冲液所需的部分NH4 +，或者NH4 +完全来源于硫酸铵，因此，可以降低延伸试剂缓冲液中其他NH4 +源特别是氨水的含量，甚至可将氨水含量降至0，即配置延伸试剂缓冲液时不加氨水。由此，可以降低氢氧化钾和氨水对延伸试剂缓冲液pH的双层影响，有利于调控延伸试剂缓冲液的pH，以获得预期的pH值。
在一种实施例中，延伸试剂缓冲液的K+源为氢氧化钾，NH4 +源为硫酸铵，即：延伸试剂缓冲液通过氢氧化钾提供K+，通过硫酸铵提供NH4 +。
在一种实施例中，延伸试剂缓冲液的K+源为氢氧化钾，NH4 +源为硫酸铵和乙酸铵，即：延伸试剂缓冲液通过氢氧化钾提供K+，通过硫酸铵和乙酸铵提供NH4 +。
在上述两种实施例的基础上，延伸试剂缓冲液还可以增加诸如但不限于氯化钾、乙酸钾等其他钾盐作为K+源，用于提供K+。
应当理解的是，上述几种实施例的共同点是：在配制延伸试剂缓冲液时，不同时添加氢氧化钾和氨水，以提高延伸试剂缓冲液pH的可调节性。此外，本申请研究人员发现，单独采用乙酸铵作为NH4 +源(即：延伸试剂缓冲液通过乙酸铵提供NH4 +)，将延伸试剂缓冲液用于核酸测序中时，碱基发生错配的情况会增加，即原本按照碱基互补配对原则(A-T(U)配对，G-C配对)进行配对的碱基对变成其他错配情形，如G-T配对、C-T配对等，这将直接影响核酸测序结果的准确性，因此，本申请实施例使用乙酸铵的同时，会添加NH4 +源物质。
在一些实施例中，延伸试剂缓冲液包括有机羧酸钾和硫酸钾中的至少一种和氨水。此时，配置延伸试剂缓冲液时，氨水提供NH4 +，且其产生的OH-还能调节延伸试剂缓冲液的pH。同时，有机羧酸钾和硫酸钾中的至少一种提供延伸试剂缓冲液所需的部分NH4 +，或者NH4 +完全来源于有机羧酸钾和硫酸钾中的至少一种，因此，可以降低延伸试剂缓冲液中其他K+源特别是氢氧化钾的含量，甚至可将氢氧化钾含量降至0，即配置延伸试剂缓冲液时不加氢氧化钾。由此，可以降低氢氧化钾和氨水对延伸试剂缓冲液pH的双层影响，有利于调控延伸试剂缓冲液的pH，以获得预期的pH值。
在一种实施例中，延伸试剂缓冲液的K+源为有机羧酸钾和硫酸钾，NH4 +源为氨水，即：延伸试剂缓冲液通过有机羧酸钾和硫酸钾共同提供K+，通过氨水提供NH4 +。示例性的，有机羧酸钾可以选自上文列举的有机羧酸钾中的一种或多种。
在一种实施例中，延伸试剂缓冲液的K+源为有机羧酸钾和硫酸钾，NH4 +源为氨水和硫酸铵，即：延伸试剂缓冲液通过有机羧酸钾和硫酸钾共同提供K+，通过氨水和硫酸铵共同提供NH4 +。
在一种实施例中，延伸试剂缓冲液的K+源为有机羧酸钾和硫酸钾，NH4 +源为氨水和乙酸铵，即：延伸试剂缓冲液通过有机羧酸钾和硫酸钾共同提供K+，通过氨水和乙酸铵共同提供NH4 +。
在一种实施例中，延伸试剂缓冲液的K+源为有机羧酸钾和硫酸钾，NH4 +源为氨水、乙酸铵和磷酸铵，即：延伸试剂缓冲液通过有机羧酸钾和硫酸钾共同提供K+，通过氨水、乙酸铵和磷酸铵共同提供NH4 +。
应当理解的是，上述几种实施例的共同点是：在配制延伸试剂缓冲液时，不同时添加氢氧化钾和氨水，以提高延伸试剂缓冲液 pH的可调节性。
本申请实施例中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L。通过综合平衡缓冲液中pH以及K+和NH4 +的浓度对相位滞后的影响，发现：调控pH为8.9～9.4，且K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L时，可使得聚合酶在延伸试剂缓冲液中促进核酸分子延伸的活性提高，进而提高核酸分子的延伸效率，降低相位滞后现象，最终提高待测核苷酸分子的测序准确率。该延伸试剂缓冲液提高聚合酶反应活性的方式可能为：延伸试剂缓冲液中pH以及K+和NH4 +的浓度在上述范围时，延伸试剂缓冲液改变了聚合酶蛋白表面的电荷分布，调整聚合酶的等电点，从而调整聚合酶与核酸分子之间、聚合酶与延伸试剂缓冲液之间的相互作用，使聚合酶更容易作用于核酸分子；另外，延伸试剂缓冲液改变核酸分子在延伸试剂缓冲液中的空间构象，使聚合酶更容易结合在核酸分子的反应位置。
应当理解的是，K+和NH4 +的浓度可以根据延伸试剂缓冲液中其他成分的变化调整，但仍在上述范围内。因此，K+的浓度在不同实施例中可以为50mmol/L、55mmol/L、60mmol/L、65mmol/L、70mmol/L、75mmol/L、80mmol/L、85mmol/L、90mmol/L、95mmol/L、100mmol/L等具体情形。NH4 +的浓度在不同实施例中可以为150mmol/L、160mmol/L、170mmol/L、180mmol/L、200mmol/L、220mmol/L、250mmol/L、280mmol/L、300mmol/L、320mmol/L、350mmol/L等具体情形。
本申请实施例提供的延伸试剂缓冲液，通过选择合适的基础缓冲液并调控基础缓冲液浓度，基本确定延伸试剂缓冲液pH，在此基础上，在诸如K+和NH4 +等其他影响pH的试剂的作用下，最终确定延伸试剂缓冲液的pH。本申请实施例中，延伸试剂缓冲液的pH为8.9～9.4。
在一种实施情形中，延伸试剂缓冲液的pH为8.9～9.0，K+的浓度为50～60mmol/L，NH4 +的浓度为150～190mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为9.0～9.1，K+的浓度为60～70mmol/L，NH4 +的浓度为190～230mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为9.1～9.3，K+的浓度为70～90mmol/L，NH4 +的浓度为230～310mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为9.3～9.4，K+的浓度为90～100mmol/L，NH4 +的浓度为310～350mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为8.9～9.1，K+的浓度为50～70mmol/L，NH4 +的浓度为150～230mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为9.05～9.35，K+的浓度为60～90mmol/L，NH4 +的浓度为200～310mmol/L。
在一种实施情形中，延伸试剂缓冲液的pH为9.3～9.5，K+的浓度为80～100mmol/L，NH4 +的浓度为300～350mmol/L。
按照上述方式配制的延伸试剂缓冲液，可以更明显地看到延伸试剂缓冲液对相位滞后现象的改善作用。
在一些实施例中，延伸试剂缓冲液还可以包括聚合酶催化剂。在核酸分子延伸过程中，聚合酶催化剂用于提高聚合酶的活性，从而提高核酸分子延伸的效率，即提高引入的核苷酸或核苷酸类似物通过聚合反应掺入到核酸分子中的效率。在一些实施例中，聚合酶催化剂选用镁盐，具体的，镁盐中的镁离子发挥提高聚合酶活性的作用。示例性的，镁盐可以为硫酸镁、氯化镁等中的至少一种，但不限于此。
在一些实施例中，延伸试剂缓冲液中聚合酶催化剂的浓度为2.0～5.0mmol/L，在该浓度范围内，可以根据核酸分子延伸过程中使用的聚合酶的浓度进行调整。示例性的，当聚合酶催化剂为硫酸镁时，其浓度可以为2.0mmol/L、2.5mmol/L、3.0mmol/L、3.5mmol/L、4.0mmol/L、4.5mmol/L、5.0mmol/L等具体情形。
在一些实施例中，延伸试剂缓冲液还可以包括络合剂。络合剂用于络合延伸试剂缓冲液中可能存在的金属杂离子(如铅、铁或铜等形成的金属离子)，从而在核酸测序过程中，消除杂质金属离子对酶的抑制作用。示例性的，络合剂可以为乙二胺四乙酸、乙二胺四乙酸二钠盐、乙二胺四乙酸四钠盐、EGTA(乙二醇双(2-氨基乙基醚)四乙酸)等。
在一些实施例中，延伸试剂缓冲液中络合剂的浓度为0.5～2.0mmol/L。示例性的，当络合剂为乙二胺四乙酸时，其浓度可以为0.5mmol/L、1.0mmol/L、1.5mmol/L、2.0mmol/L等具体情形。
在一些实施例中，延伸试剂缓冲液还可以包括表面活性剂。表面活性剂用于调节延伸试剂缓冲液的表面张力，特别是在固相载体表面反应时，有利于延伸试剂缓冲液在固相载体表面的铺展。示例性的，表面活性剂选自吐温-20、Triton X-100中的至少一种。
在一些实施例中，延伸试剂缓冲液中表面活性剂的体积百分含量为0.010-0.100％。示例性的，当表面活性剂为吐温-20时，其体积百分含量可以为0.010％、0.015％、0.020％、0.025％、0.050％、0.075％、0.100％等具体情形。
在一些实施例中，延伸试剂缓冲液还可以包括发挥一种或多种特定功能的其他助剂。在一些实施例中，其他助剂包括二甲基亚砜、1,3-二甲基硫脲中的至少一种。其中，二甲基亚砜可以在核酸分子延伸过程中，促进碱基氢键的结合能力；1,3-二甲基硫脲有助于清除延伸试剂缓冲液中的氧化自由基，提供更稳定的缓冲体系。
作为本申请延伸试剂缓冲液的一种实施例中，络合剂选自乙二胺四乙酸、乙二胺四乙酸二钠盐、乙二胺四乙酸四钠盐、EGTA中的至少一种，聚合酶催化剂包括镁盐，表面活性剂选自吐温-20、Triton X-100中的至少一种，其他助剂包括二甲基亚砜、1,3-二甲基硫脲中的至少一种。
在一些实施例中，延伸试剂缓冲液中的其他助剂包括磷酸化合物，且磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物：
其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
n选自1～8的整数。
应当理解的是，本申请实施例中，式(1)、式(2)所示的化合物中，当R1、R2中的至少一种选自金属离子，延伸试剂缓冲液中磷酸化合物形成离子状态，包括金属阳离子和磷酸阴离子。因此，本质上，本申请中，式(1)、式(2)所示的化合物对应的阴离子也属于本申请实施例中磷化合物的范畴。示例性的，磷酸阴离子至少包括如下情形：
在核酸分子延伸过程中，式(1)和式(2)所示结构的化合物可竞争性地与固相载体(如生物芯片)表面残留的寡聚核苷酸(如结合在固相载体表面、但没有结合核酸模板结合的寡聚核苷酸)结合，从而降低或抑制这些寡聚核苷酸与虚拟终止子的结合，即：降低固相载体表面的非特异性吸附，由此，可减少这些寡聚核苷酸对核酸测序带来的信号干扰(虚拟终止子与这些寡聚核苷酸结合后，同样会产生可识别的信号，从而影响对核酸模板分子中引入的信号的干扰)，降低由其引起的测序错误率，进一步提高测序准确性。
在一些实施例中，式(1)中，R1、R2中至少一个为氢原子，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。在这种情形下，可以提高式(1)结构的竞争力，有利于其降低固相载体表面的非特异性吸附。
示例性的，式(1)结构可以为下述情形：
当然，应当理解的是，上述结构中的氢(H)可以以H+的形式游离于延伸试剂缓冲液中，而磷酸化合物以磷酸阴离子的形式存在。
在另一些实施例中，式(1)中，R1、R2中至少一个为芳香基，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。
在一些实施例中，式(1)中，R3包括环烷基或苯基。此时，磷酸化合物中的磷酸基团连接环烷基或苯基，形成与核苷酸结构较为近似的结构类型，更有助于提高其与寡聚核苷酸的竞争性。同时，由于寡聚核苷酸为单链结构，因此，此外，上述大体积基团造成的空间位阻，也有利于降低可逆终止子与寡聚核苷酸的结合概率。
在一些实施例中，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n。此时，式(1-1)中，磷酸基团连接在环烷基的烷环碳原子上。同样的，上述结构中的氢(H)可以以H+的形式游离于延伸试剂缓冲液中，而磷酸化合物以磷酸阴离子的形式存在。
式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。此时，式(1-1)中，磷酸基团连接在芳基碳原子上。优选的，式(1-2)中，R1、R2中至少一个为芳基。
上述结构对应的式(1)中，磷酸化合物中的磷酸基团连接碳原子数小于10的环烷基或苯基，与核苷酸结构的相似性进一步提升，使得同时添加上述磷酸化合物和核苷酸底物(可逆终止子)时，磷酸化合物能够竞争性地结合寡聚核苷酸，从而降低核苷酸底物(可逆终止子)与寡聚核苷酸结合的概率。
在一些实施例中，式(1-1)和式(1-2)所示的磷酸化合物中含有金属离子，如：R2中至少含有一个金属离子。应该理解的是，R2中至少含有一个金属离子是指，上述式(1-1)和式(1-2)所示的磷酸化合物中，含有n个R2。式(1)中的金属离子，有助于磷酸化合物在溶液体系中形成磷酸负离子，从而更有效地结合寡聚核苷酸。当n＝1时，对应的磷酸化合物中R2为金属离子；当n＞1时，对应的磷酸化合物中的多个R2中，至少一个R2为金属离子，当然，也可以为多个甚至所有R2均为金属离子。在一个实施例中，R2中含有n个金属离子。此时，每个磷酸基团都能形成一个磷酸负离子，增加了磷酸化合物与寡聚核苷酸反应的结合位置。特别是将磷酸化合物和核苷酸底物(可逆终止子)同时添加至表面时，有利于提高磷酸化合物与表面反应的竞争性。
上述式(1)(包括式(1-1)和式(1-2))结构中，金属离子选自一价金属离子或二价金属离子。应当理解的是，当金属离子为二价金属离子时，式(1)(包括式(1-1)和式(1-2))结构中的每两个磷酸单元共用一个R2，如下式所示：
此时，对应的n为偶数。
示例性的，当金属离子为一价金属离子时，一价金属离子包括钠离子和/或钾离子。当式(1)所示磷酸化合物中含有多个R2时，多个R2可以各自为钠离子或钾离子。
在本申请实施例中，考虑到磷酸化合物的空间结构对其竞争性的影响，n选自2～6的整数，包括但不限于2、3、4、5或6。此时，多个磷酸基团参与竞争，有利于提高磷酸化合物与固相载体表面的寡聚核苷酸结合的竞争力。
在一些实施例中，R3为环烷基或苯基，烷环或芳环上的碳原子各结合一个磷酸结构，即n为烷环或芳环上的碳原子的个数。一方面，分布在烷环或芳环上磷酸基团数量较多，提供了较多与寡聚核苷酸反应的反应位置，增强了其反应竞争性；另一方面，磷酸基团连接烷环或芳环，与核苷酸中的磷酸基团连接五碳糖及碱基的结构有一定的类似性，也能增强磷酸化合物的竞争优势。由此，通过双层作用，可以有效提高式(1)所示磷酸化合物与固相载体上寡聚核苷酸的结合能力，从而在核酸分子延伸过程中降低非特异性吸附，提高测序信号的可识别性，提高测序准确性。
在一些实施例中，式(2)中，R1选自氢原子，R2选自金属离子。此时，式(2)结构能与可逆终止子竞争，结合固相载体表面的寡聚核苷酸，从而降低固相载体表面的非特异性吸附。应当理解的是，式(2)的氢(H)可以以H+的形式游离于延伸试剂缓冲液中，金属离子也可以形成金属离子，而磷酸化合物以磷酸阴离子的形式存在。
作为本申请延伸试剂缓冲液的一种可能的实施方式，金属离子选自一价金属离子或二价金属离子。应当理解的是，当金属离子为二价金属离子时，式(2)结构中的两个磷酸单元共用一个R2，如下式所示：
当金属离子为一价金属离子时，一价金属离子可以为钠离子和/或钾离子。式(2)所示磷酸化合物中的2个R2，可以各自为钠离子或钾离子，或者分别为钠离子和钾离子。示例性的，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
式(2-1)中，M选自Na或K。
发明人研究发现，满足上述式(2-1)、式(2-2)结构所示的磷酸化合物，能够有效结合表面的寡聚核苷酸，从而在寡聚核苷酸以非预期的方式出现在表面时，可以通过添加式(2-1)、式(2-2)结构所示的磷酸化合物来抑制寡聚核苷酸的活性，从而抑制其带来的干扰。如在对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，即可通过该磷酸化合物抑制表面寡聚核苷酸的活性，从而核酸分子延伸过程中产生的费特异性吸附，降低测序背景信号，从而提高测序信号的可识别性。
在一些实施例中，磷酸化合物选自如下式(3)、式(4)和式(5)中的任意一种或至少两种的组合。
上述磷酸化合物可以降低核酸测序过程中的错误率。特别是将延伸试剂缓冲液用于对信噪比高的表面核酸分子，特别是不需要经过扩增成簇的核酸分子进行测序时，由于每一位点的核酸数量少，甚至为一条，测序产生的信号强度较弱。此时，干扰信号(背景噪声)对测序信号的识别影响显著。而通过该方法封闭固相载体表面的非待测序列与可逆终止子的结合活性，可以显著减少固相载体表面的非特异性吸附，从而降低测序错误率，尤其是insertion类型错误率，并且提升核酸测序数据量。
在测序过程中，所述磷酸化合物可单独使用一种，也可以组合使用，即组合2种甚至3种共同作用，因这类化合物在测序中与测序碱基发生竞争，因此在组合使用中预期其作用效果与单独使用一种作用类似，原因为测序芯片表面积及添加的测序碱基是固定量的，因此在组合使用时，在相对平衡的竞争体系中，其降低测序芯片表面吸附的效果是有极限的，其最大作用效果与单一添加效果类似。
在一些实施例中，延伸试剂缓冲液中，磷酸化合物的浓度为1～150μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、10μmol/L、20μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、91μmol/L、92μmol/L、95μmol/L、96μmol/L、98μmol/L、99μmol/L、100μmol/L、110μmol/L、120μmol/L、130μmol/L、140μmol/L或150μmol/L。若延伸试剂缓冲液中磷酸化合物的浓度过高，如超过150μmol/L，则过量的磷酸化合物会结合到表面其他核苷酸分子，如在核酸测序过程中占据测序链中核苷酸或核苷酸类似物的反应位置。由于反应过程中不含有聚合酶，且磷酸化合物无法像核苷酸或核苷酸类似物一样与互补链形成氢键，磷酸化合物结合到表面其他核苷酸分子时的反应不牢固，但这仍然会对测序反应造成一定的影响。特别是磷酸化合物的浓度过高时，由于过量的磷酸化合物可能占据测序链反应位置，因此，还是可能会降低核酸测序的通量。
在一些实施方式中，磷酸化合物包含植酸钠，且延伸试剂缓冲液中，所述植酸钠的浓度小于或等于15μmol/L。
在一些实施方式中，所述磷酸化合物为植酸钠，且延伸试剂缓冲液中，所述植酸钠的浓度为1～10μmol/L，包括但不限于1μmol/L、2μmol/L、3μmol/L、4μmol/L、5μmol/L、6μmol/L、7μmol/L、8μmol/L、9μmol/L或10μmol/L。研究发现，当植酸钠含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。此外，由于浓度较低，植酸钠可能引入的不良反应也会降低。如在核酸测序过程中，植酸钠占据测序链中核苷酸或核苷酸类似物的反应位置的概率降低，对测序通量基本不产生影响或产生的影响可以忽略。
在一些实施方式中，所述磷酸化合物包含焦磷酸二氢二钠，且延伸试剂缓冲液中，所述焦磷酸二氢二钠的浓度小于或等于120μmol/L。
在一些实施方式中，所述磷酸化合物为焦磷酸二氢二钠，且延伸试剂缓冲液中，所述焦磷酸二氢二钠的浓度为10～120μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、60μmol/L、70μmol/L、80μmol/L、90μmol/L、100μmol/L、110μmol/L或120μmol/L。研究发现，当单独采用焦磷酸二氢二钠作为抑制寡聚核苷酸活性的添加剂时，焦磷酸二氢二钠的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且延伸试剂缓冲液中，所述双酚A双(二苯基磷酸酯)的浓度小于或等于70μmol/L。
在一些实施方式中，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且延伸试剂缓冲液中，所述双酚A双(二苯基磷酸酯)的浓度为10～60μmol/L，包括但不限于10μmol/L、11μmol/L、12μmol/L、13μmol/L、14μmol/L、15μmol/L、16μmol/L、18μmol/L、22μmol/L、25μmol/L、28μmol/L、30μmol/L、34μmol/L、36μmol/L、38μmol/L、40μmol/L、42μmol/L、44μmol/L、46μmol/L、48μmol/L、49μmol/L、50μmol/L、52μmol/L、53μmol/L、54μmol/L、56μmol/L、58μmol/L、59μmol/L或60μmol/L。研究发现，当双酚A双(二苯基磷酸酯)作为抑制寡聚核苷酸活性的添加剂时，双酚A双(二苯基磷酸酯)的含量在此范围时，可以有效抑制表面的寡聚核苷酸活性。
本发明中，在测序过程中控制磷酸化合物的用量，能够进一步有效降低测序错误率，同时有效控制成本。
本申请实施例提供的延伸试剂缓冲液可以用于核酸测序领域中。延伸试剂缓冲液在核酸测序领域的应用，包括在核酸分子延伸过程中，延伸试剂缓冲液携带可逆终止子和聚合酶，与结合在固相基底表面的核酸模板复合体反应，使可逆终止子结合到核酸模板复合体上。本申请实施例中，核酸模板复合体包括探针和核酸模板分子，探针的一端结合在固相载体表面，探针的另一端与核酸模板分子结合。核酸分子延伸在探针所在的链进行，通过在探针所在链逐一引入可逆终止子并进行对引入的可逆终止子进行识别，实现核酸测序。
理论上，用于核酸分子延伸的其他情形，也可以采用本申请实施例提供的延伸试剂缓冲液来携带可逆终止子和聚合酶参与核酸分子延伸反应。
延伸试剂缓冲液用于核酸测序领域的一种实施情形是：延伸试剂缓冲液作为核酸延伸反应的一个试剂盒，与核酸延伸反应的原料和其他试剂共同加入待反应体系中，在一种实施例中，延伸试剂缓冲液作为核酸延伸反应的一个试剂盒，与核酸延伸反应的原料和其他试剂共同流入待反应的固相载体表面。
对应的，本申请实施例提供一种延伸试剂盒，包括本申请实施例前文提供的延伸试剂缓冲液。第一方面提供的延伸试剂缓冲液的组成、各成分的选择、浓度、pH等情形，均可参照上文。为了节约篇幅，此处不再赘述。
本申请实施例中，延伸试剂盒还包括可逆终止子。
在一种情形中，可逆终止子为带有荧光基团的可逆终止子(如下文涉及的HN-A、HN-T、HN-G、HN-C)，此时，在核酸分子延伸过程中，可逆终止子通过聚合反应掺入，通过对可逆终止子的荧光基团进行辨别，确定掺入的可逆终止子的类型，或者说确定掺入的可逆终止子的碱基类型。因此，本申请实施例中，核苷酸或核苷酸类似物类型的识别或确认，又可以理解为碱基类型的识别或确认。或者说，本申请实施例中，核苷酸或其类似物的类型可以与碱基类型的表述互换。如dATP表示为含有碱基A的脱氧核糖核苷酸，ATP表示为含有碱基A的核糖核苷酸；其他类似。
在另一种情形中，可逆终止子为不带荧光基团的可逆终止子(如下文涉及的CN-A、CN-T、CN-G、CN-C)。此时，可逆终止子用于在带有荧光基团的可逆终止子发生聚合反应之后，填补部分没有参与反应的核酸分子位置，以减少相位滞后现象。
在再一种情形中，可逆终止子包括带荧光基团的可逆终止子和不带荧光基团的可逆终止子。其中，不带荧光基团的可逆终止子可以结合在探针所在链，与模板分子所在链非测序区的核苷酸分子形成碱基对或核苷酸对。而带荧光基团的可逆终止子与模板分子所在链测序区的核苷酸互补配对。平通过可逆终止子上的荧光基团，确定对应的可逆终止子的类型。
在一些实施例中，延伸试剂盒包括寡聚核苷酸A、寡聚核苷酸T、寡聚核苷酸G、寡聚核苷酸C，或所述第五试剂包括寡聚核苷酸A、寡聚核苷酸U、寡聚核苷酸G、寡聚核苷酸C。
本申请实施例中，延伸试剂盒还包括聚合酶，聚合酶用于促进可逆终止子结合到核酸模板复合体中，特别是结合到探针所在链上。
本申请实施例提供的延伸试剂盒中，延伸试剂缓冲液、可逆终止子和聚合酶各自独立分装于特定的试剂管中，待进入核酸分子延伸反应步骤时，分别从不同的试剂管中抽出，经混合进入核酸模板复合体所在区域。示例性的，通过泵从不同试剂管中分别抽取延伸试剂缓冲液、可逆终止子和聚合酶，各支管汇流使各试剂混合，最终流经表面结合有核酸模板复合体的固相载体表面，使可逆终止子结合到核酸模板复合体上。
在一些实施例中，根据聚合酶的浓度以及通量，可以调节延伸试剂缓冲液中镁离子的含量。示例性的，当延伸试剂盒混合形成的延伸试剂中，聚合酶的浓度为0.02～0.05mg/mL时，镁离子的浓度为2.0～5.0mmol/L。
作为延伸试剂缓冲液的一种应用实施例，本申请提供一种测序方法，包括：
于结合有核酸分子的固相载体的表面，通入本申请实施例提供的延伸试剂缓冲液、可逆终止子和聚合酶，使可逆终止子结合在核酸分子上；
其中，核酸分子包括核酸模板和与核酸模板结合的寡聚核苷酸，且可逆终止子结合在寡聚核苷酸所在链的3’端。
本发明具体实施例中以三种具体的式(1)、式(2)所示结构的化合物(植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯))为例进行验证，所使用试剂如表1所示，仪器如表2所示。
表1
表2
实施例1
本实施例提供一种基因测序试剂盒，试剂盒中含有三羟甲基氨基甲烷(Tris base)、硫酸铵、硫酸镁、乙酸钾、Triton X-100、植酸钠、焦磷酸二氢二钠、BDP、A链溶液、T链溶液、C链溶液、G链溶液、DNA聚合酶、dATP、dTTP、dCTP和dTGP。
实施例2
本实施例利用实施例1中试剂盒进行测序。
1、试剂配制方法
首先配制测序Tris缓冲液：在20mmol/LTris缓冲液中，添加终浓度为10mmol/L硫酸铵、3mmol/L硫酸镁、50mmol/L乙酸钾、0.1％Triton X-100、10nmol/L A链溶液、10nmol/L T链溶液、10nmol/L C链溶液、10nmol/L G链溶液。然后将上述溶液分为四组，其中一组作为对照组，其他三组分别加入以下添加剂，分别配制4种延伸试剂：①植酸钠：上述测序Tris缓冲液中加入终浓 度5μmol/L植酸钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/LdATP、250nmol/LdTTP、250nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；②BDP：上述测序Tris缓冲液中加入10μmol/L BDP，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/LdATPT、250nmol/LdCTPG、5U/mL DNA聚合酶，混匀；③焦磷酸二氢二钠：上述测序Tris缓冲液中加入100μmol/L焦磷酸二氢二钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/LdATPT、250nmol/LdCTPG、5U/mL DNA聚合酶，混匀。④对照组测序Tris缓冲液中加入250nmol/LdATP、250nmol/LdTTP、250nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀，作为对照。
2、测序流程
2.1采用X-bot 16自动进样仪进行单分子测序芯片预处理和文库样本加载；
2.2将配制好的延伸试剂放置测序反应通用试剂盒内，进行72cycles测序，拍照400FOV。
3、结果分析
分别计算采用4种不同的延伸试剂的测序错误率及测序通量，含不同添加剂的延伸试剂测序，错误率(Insertion rate)、测序通量对比结果如表3所示。
表3
由表3可知，在延伸试剂中加入添加剂后，采用GenoCare1600基因测序仪测序，测序结果表现为错误率(insertion ratio)相比无添加剂组降低，测序通量有提升，表明本发明实施例使用式(1)所示结构的化合物能够有效降低测序的错误率并提高测序通量。
实施例3
本实施例利用实施例1中试剂盒进行测序。
1、试剂配制方法：
首先配制测序Tris缓冲液：在20mmol/L(mmol/L)Tris缓冲液中，添加终浓度为30mmol/L硫酸铵、3mmol/L硫酸镁、50mmol/L乙酸钾、0.1％Triton X-100、10nmol/L(nmol/L)A链溶液、10nmol/L T链溶液、10nmol/L C链溶液、10nmol/L G链溶液。然后将上述溶液分为四组，其中一组作为对照组，其他三组分别加入以下添加剂，分别配制4种延伸试剂，①植酸钠：上述测序Tris缓冲液中加入终浓度10μmol/L(μmol/L)植酸钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；②BDP：上述测序Tris缓冲液中加入50μmol/L BDP，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；③焦磷酸二氢二钠：上述测序Tris缓冲液中加入50μmol/L焦磷酸二氢二钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；④对照组测序Tris缓冲液中加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀，作为对照。
2、测序流程
操作步骤参照实施例2。
3、结果分析
操作步骤参照实施例2，不同添加剂配方的延伸试剂测序错误率、测序通量对比结果如表4所示。
表4
结果如表4所示，在延伸试剂中加入添加剂后，采用GenoCare1600基因测序仪测序，测序结果表现为错误率(insertion ratio)相比无添加剂组降低，测序通量有提升。
实施例4
本实施例进一步考察添加剂浓度对测序结果的影响。
1、试剂配制方法
首先配制测序Tris缓冲液：在20mmol/L(mmol/L)Tris缓冲液中，添加终浓度为30mmol/L硫酸铵、3mmol/L硫酸镁、50mmol/L乙酸钾、0.1％Triton X-100、10nmol/L(nmol/L)A链溶液、10nmol/L T链溶液、10nmol/L C链溶液、10nmol/L G链溶液。然后将上述溶液分为三组，分别加入以下添加剂，并设置梯度浓度的添加剂，分别配制延伸试剂，①植酸钠：上述测序Tris缓冲液中加入终浓度0μmol/L、0.5μmol/L、1μmol/L、12μmol/L植酸钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；②BDP：上述测序Tris缓冲液中分别加入0μmol/L、5μmol/L、55μmol/L BDP，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀；③焦磷酸二氢二钠：上述测序Tris缓冲液中加入0μmol/L、40μmol/L、110μmol/L焦磷酸二氢二钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入500nmol/LdATP、250nmol/LdTTP、500nmol/LdCTP、250nmol/LdTGP、5U/mL DNA聚合酶，混匀。
2、测序流程
操作步骤参照实施例2。
3、结果分析
操作步骤参照实施例2，不同添加剂配方的延伸试剂测序，错误率、测序通量对比结果如表5所示。
表5
由表5可知，在延伸试剂中加入植酸钠添加剂后，当浓度为1μmol/L，其测序通量有提升，错误率(insertion ratio)降低；当浓度为12μmol/L，其测序通量也提升，错误率(insertion ratio)降低，但提升效果与10μmol/L相似，因此从成本考虑，控制添加植酸钠终浓度1～10μmol/L。在延伸试剂中加入BDP添加剂后，当浓度为5μmol/L，其测序通量提升、错误率(insertion ratio)降低不显著；当浓度为55μmol/L，其测序通量有提升，错误率(insertion ratio)降低，但提升效果与50μmol/L相似，因此从成本考虑，控制添加BDP终浓度为10～50μmol/L。在延伸试剂中加入焦磷酸二氢二钠添加剂后，当浓度为40μmol/L，其测序通量提升、错误率(insertion ratio)降低不显著；当浓度为110μmol/L，其测序通量有提升，错误率(insertion ratio)降低，但提升效果与100μmol/L相似，因此从成本考虑，控制添加焦磷酸二氢二钠终浓度为50～100μmol/L。
综上所述，本发明实施例针对测序过程中核苷酸底物(可逆终止子)容易在测序芯片表面随机吸附，导致在后续的荧光成像过程易识别到非杂交模板定位的荧光点的问题，意外发现具有如式(1)和式(2)所示结构的化合物与测序过程中的虚拟终止子核苷酸底物(可逆终止子)存在竞争作用，竞争吸附在测序芯片表面，从而减少核苷酸底物(可逆终止子)在测序芯片表面的随机吸附，降低由其引起的测序错误率，并提高测序通量。
本发明受另一发明思路的启发，发现：通过调整延伸试剂配方中的pH以及K+和NH4 +的浓度，使其满足：K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，延伸试剂缓冲液的pH为8.9～9.4时，还可以促进核酸分子延伸的活性，降低核酸测序过程中产生的相位滞后(phase或phasing，是指：本应在cycle N反应并掺入到核酸模板的核苷酸，却滞后到cycle N+1参与反应)现象。
在实施例2的基础上，采用硫酸铵和氨水共同提供NH4 +，并调节延伸试剂中NH4 +的浓度为180mmol/L，统计调整前后测序过程中出现的phasing，发现：调整后，测序产生的相位滞后(phase或phasing)现象有不同程度的降低，降低0.02～0.08％之间。
实施例5
延伸试剂缓冲液，通过下述方法配制：
在Tris缓冲液中添加氢氧化钾、乙二胺四乙酸、硫酸铵、硫酸镁、二甲基亚砜、1,3-二甲基硫脲、硫辛酸和吐温-20，使缓冲液体系中各成分的终浓度或含量满足：Tris缓冲液100.248mmol/L，氢氧化钾25mmol/L，乙二胺四乙酸1.040mmol/L，NH4 +浓度(原料含有硫酸铵和氨水)224.0mmol/L，硫酸镁3.124mmol/L，二甲基亚砜5.208％(体积百分比)，1,3-二甲基硫脲10.392mmol/L，硫辛酸2mmol/L，吐温-200.052％(体积百分比)。
将配制好的缓冲液体系分成4份，其中一份保持不变，命名为延伸试剂缓冲液1-1，其他3份加入乙酸钾，使K+在缓冲液体系中的终浓度分别为50mmol/L、70mmol/L和100mmol/L，将对应的缓冲体系分别命名为延伸试剂缓冲液1-2、延伸试剂缓冲液1-3和延伸试剂缓冲液1-4，各缓冲液的pH 9.25。
延伸试剂缓冲液混匀后经0.22μm微孔滤膜过滤器过滤。
实施例6
延伸试剂：
将实施例5提供的延伸试剂缓冲液1-1、延伸试剂缓冲液1-2、延伸试剂缓冲液1-3和延伸试剂缓冲液1-4，按照下表2和下表3提供的各成分终浓度，分别配制延伸试剂1-1A和延伸试剂1-1B、延伸试剂1-2A和延伸试剂1-2B、延伸试剂1-3A和延伸试剂1-3B、延伸试剂1-4A和延伸试剂1-4B，其中，命名中含A的延伸试剂对应表6的配方，命名为B的延伸试剂对应表7的配方。
表6
表7
表6、表7中延伸试剂缓冲液的终浓度用“-”表示，是指：延伸试剂中延伸试剂缓冲液相当于溶剂，在延伸试剂缓冲液中添加其他成分并控制其终浓度即可，延伸试剂缓冲液的浓度无需表征。
表6和表7中，HN-N表示结构中含有荧光基团的核苷酸，CN-N表示结构中不含有荧光基团的核苷酸，其中，N选自A、T、G、C。
实施例7
测序方法：
按照Genolab M测序反应通用试剂盒(可逆末端终止测序法)说明书，准备好生物芯片(或测序芯片)、试剂盒及文库；
分别采用延伸试剂1-1A、延伸试剂1-2A、延伸试剂1-3A和延伸试剂1-4A替换Genolab M测序反应通用试剂盒中的合成试剂3，分别采用延伸试剂1-1B、延伸试剂1-2B、延伸试剂1-3B和延伸试剂1-4B替换Genolab M测序反应通用试剂盒中的合成试剂4，进行SE100测序。
统计测序过程中出现的phasing，其中，phasing的统计方法如下：统计phasing随cycle变化的数据，通过线性回归拟合输出斜率(slope)，该斜率即为对应的phasing值。
结果如下表8所示。
表8
由表8可见，在pH为9.25、NH4 +浓度为224mmol/L的条件下，相较于对照组，K+浓度升高至50mmol/L、70mmol/L和100mmol/L，核酸测序过程中出现的相位滞后(phase或phasing)降低。
实施例8
延伸试剂缓冲液，通过下述方法配制：
在Tris缓冲液中添加氢氧化钾、乙二胺四乙酸、硫酸铵、硫酸镁、二甲基亚砜、1,3-二甲基硫脲、硫辛酸和吐温-20，使缓冲液体系中各成分的终浓度或含量满足：Tris缓冲液100.248mmol/L，K+浓度72.139mmol/L，乙二胺四乙酸1.040mmol/L，NH4 +浓度(原料含有硫酸铵和氨水)124.0mmol/L，硫酸镁3.124mmol/L，二甲基亚砜5.208％(体积百分比)，1,3-二甲基硫脲10.392mmol/L，硫辛酸2mmol/L，吐温-200.052％(体积百分比)。
将配制好的缓冲液体系分成4份，其中一份保持不变(NH4 +浓度为124mmol/L)，命名为延伸试剂缓冲液2-1，其他3份加入乙酸铵，使NH4 +在缓冲液体系中的终浓度分别为184mmol/L、244mmol/L和304mmol/L，将对应的缓冲体系分别命名为延伸试剂缓冲液2-2、延伸试剂缓冲液2-3和延伸试剂缓冲液2-4，各缓冲液的pH 9.25。
延伸试剂缓冲液混匀后经0.22μm微孔滤膜过滤器过滤。
实施例9
延伸试剂：
将实施例8提供的延伸试剂缓冲液2-1、延伸试剂缓冲液2-2、延伸试剂缓冲液2-3和延伸试剂缓冲液2-4，按照上表2和上表3提供的各成分终浓度，分别配制延伸试剂2-1A和延伸试剂2-1B、延伸试剂2-2A和延伸试剂2-2B、延伸试剂2-3A和延伸试剂2-3B、 延伸试剂2-4A和延伸试剂2-4B，其中，命名中含A的延伸试剂对应表2的配方，命名为B的延伸试剂对应表7的配方。
实施例10
测序方法：
按照Genolab M测序反应通用试剂盒(可逆末端终止测序法)说明书，准备好生物芯片(或测序芯片)、试剂盒及文库；
分别采用延伸试剂2-1A、延伸试剂2-2A、延伸试剂2-3A和延伸试剂2-4A替换Genolab M测序反应通用试剂盒中的合成试剂3，分别采用延伸试剂2-1B、延伸试剂2-2B、延伸试剂2-3B和延伸试剂2-4B替换Genolab M测序反应通用试剂盒中的合成试剂4，进行SE100测序。
统计测序过程中出现的phasing，结果如下表9所示。
表9
由表9可见，在pH为9.25、K+浓度为72.139mmol/L的条件下，相较于对照组，NH4 +浓度升高至184mmol/L、244mmol/L和304mmol/L，核酸测序过程中出现的相位滞后(phase或phasing)降低。
实施例11
延伸试剂缓冲液，通过下述方法配制：
在Tris缓冲液中添加氢氧化钾、乙二胺四乙酸、硫酸铵、硫酸镁、二甲基亚砜、1,3-二甲基硫脲、硫辛酸和吐温-20，使缓冲液体系中各成分的终浓度或含量满足：Tris缓冲液100.248mmol/L，，乙二胺四乙酸1.040mmol/L，，硫酸镁3.124mmol/L，二甲基亚砜5.208％(体积百分比)，1,3-二甲基硫脲10.392mmol/L，硫辛酸2mmol/L，吐温-200.052％(体积百分比)。
将配制好的缓冲液体系分成3份，采用氢氧化钾、钾盐、铵盐和氨水调节pH，在保持各份缓冲液中K+浓度为72.139mmol/L和NH4 +浓度为244.868mmol/L的情况下，使缓冲液体系中的pH分别为8.90、9.25和9.50，将对应的缓冲体系分别命名为延伸试剂缓冲液3-1、延伸试剂缓冲液3-2和延伸试剂缓冲液3-3。
延伸试剂缓冲液混匀后经0.22μm微孔滤膜过滤器过滤。
实施例12
延伸试剂：
将实施例8提供的延伸试剂缓冲液3-1、延伸试剂缓冲液3-2和延伸试剂缓冲液3-3，按照上表2和上表3提供的各成分终浓度，分别配制延伸试剂3-1A和延伸试剂3-1B、延伸试剂3-2A和延伸试剂3-2B、延伸试剂3-3A和延伸试剂3-3B，其中，命名中含A的延伸试剂对应表6的配方，命名为B的延伸试剂对应表7的配方。
实施例13
测序方法：
按照Genolab M测序反应通用试剂盒(可逆末端终止测序法)说明书，准备好生物芯片(或测序芯片)、试剂盒及文库；
分别采用延伸试剂3-1A、延伸试剂3-2A和延伸试剂3-3A替换Genolab M测序反应通用试剂盒中的合成试剂3，分别采用延伸试剂3-1B、延伸试剂3-2B和延伸试剂3-3B替换Genolab M测序反应通用试剂盒中的合成试剂4，进行SE100测序。
统计测序过程中出现的phasing，结果如下表10所示。
表10
由表10可见，在NH4 +浓度为244.868mmol/L、K+浓度为72.139mmol/L的条件下，pH在8.90～9.50范围内，核酸测序过程中出现的相位滞后(phase或phasing)现象较低。
此外，本发明受另一发明思路的启发，发现：在单分子测序过程中，通过在延伸试剂配方中添加浓度为1～150μmol/L的结构为式(1)和/或式(2)所示的磷酸化合物，还可以降低测序错误率，提高测序通量。
实施例14
(1)、试剂配制方法
首先配制测序Tris缓冲液：在20mmol/L Tris缓冲液中，添加终浓度为3mmol/L硫酸镁、50mmol/L乙酸钾、0.1％Triton X-100、10nmol/L A链溶液、10nmol/L T链溶液、10nmol/L C链溶液、10nmol/L G链溶液，增加铵盐和氨水调节缓冲液中NH4 +的浓度为180mmol/L，pH为9.0。然后将上述溶液分为四组，其中一组作为对照组，其他三组分别加入以下添加剂，并设置梯度浓度的添加剂，分别配制延伸试剂，①植酸钠：上述测序Tris缓冲液中加入终浓度5μmol/L植酸钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/L碱基A、250nmol/L碱基T、250nmol/L碱基C、250nmol/L碱基G、5U/mL DNA聚合酶，混匀；②BDP：上述测序Tris缓冲液中加入10μmol/L BDP，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/L碱基AT、250nmol/L碱基CG、5U/mL DNA聚合酶，混匀；③焦磷酸二氢二钠：上述测序Tris缓冲液中加入100μmol/L焦磷酸二氢二钠，混匀，0.22μm滤膜过滤，将试剂放在4℃环境冷却，再分别加入250nmol/L碱基AT、250nmol/L碱基CG、5U/mL DNA聚合酶，混匀。④对照组测序Tris缓冲液中加入250nmol/L dATP、250nmol/L dTTP、250nmol/L dCTP、250nmol/L dGTP、5U/mL DNA聚合酶，混匀，作为对照。
(2)、测序流程
采用X-bot 16自动进样仪进行单分子测序芯片预处理和文库样本加载；将配制好的延伸试剂放置测序反应通用试剂盒(单分子荧光测序法)内，进行72cycles测序，拍照400FOV。
3)、结果分析
分别计算采用4种不同的延伸试剂的测序错误率及测序通量，含不同添加剂的延伸试剂测序，错误率(Insertion rate)、测序通量对比结果如表11所示。
表11
由表11可知，在延伸试剂中加入添加剂后，可以提高测序的通量，降低错误率(insertion ratio)，特别是对于不需要经过扩增成簇的核酸分子进行的测序更为明显。这可能归因于：添加剂降低测序过程中核苷酸底物(可逆终止子)容易在测序芯片表面随机吸附，导致在后续的荧光成像过程易识别到非杂交模板定位的荧光点的问题，具有如式(1)和式(2)所示结构的化合物与测序过程中的虚拟终止子核苷酸底物(可逆终止子)存在竞争作用，竞争吸附在测序芯片表面，从而减少核苷酸底物(可逆终止子)在测序芯片表面的随机吸附，降低由其引起的测序错误率，并提高测序通量。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (123)

  1. 一种表面处理方法，其特征在于，所述表面处理方法包括：
    将磷酸化合物与结合有寡聚核苷酸的表面接触；
    所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物；
    其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
    R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
    n选自1～8的整数。
  2. 根据权利要求1所述的表面处理方法，其特征在于，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基、碳原子数为6～20的芳基中的一种。
  3. 根据权利要求2所述的表面处理方法，其特征在于，R3包括环烷基或苯基。
  4. 根据权利要求3所述的表面处理方法，其特征在于，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
    式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。
  5. 根据权利要求4所述的表面处理方法，其特征在于，R2中至少含有一个金属离子。
  6. 根据权利要求5所述的表面处理方法，其特征在于，R2中含有n个金属离子。
  7. 根据权利要求5所述的表面处理方法，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  8. 根据权利要求7所述的表面处理方法，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  9. 根据权利要求4所述的表面处理方法，其特征在于，所述n选自2～6的整数。
  10. 根据权利要求1所述的表面处理方法，其特征在于，式(2)中，R1选自氢原子，R2选自金属离子。
  11. 根据权利要求10所述的表面处理方法，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  12. 根据权利要求11所述的表面处理方法，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  13. 根据权利要求1所述的表面处理方法，其特征在于，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
    式(2-1)中，M选自Na或K。
  14. 根据权利要求1所述的表面处理方法，其特征在于，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。
  15. 根据权利要求1-14任一项所述的表面处理方法，其特征在于，所述将磷酸化合物与结合有寡聚核苷酸的表面接触，包括：
    在所述表面加含有所述磷酸化合物的第一溶液；或
    将所述表面置于含有所述磷酸化合物的第一溶液中；或
    使含有所述磷酸化合物的第一溶液流经所述表面。
  16. 根据权利要求15所述的表面处理方法，其特征在于，所述第一溶液中，所述磷酸化合物的浓度为1～150μmol/L。
  17. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物包含植酸钠，且所述第一溶液中，所述植酸钠的浓度小于或等于15μmol/L。
  18. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物为植酸钠，且所述第一溶液中，所述植酸钠的浓度为1～10μmol/L。
  19. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物包含焦磷酸二氢二钠，且所述第一溶液中，所述焦磷酸二氢二钠的浓度小于或等于120μmol/L。
  20. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物为焦磷酸二氢二钠，且所述第一溶液中，所述焦磷酸二氢二钠的浓度为10～120μmol/L。
  21. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第一溶液中，所述双酚A双(二苯基磷酸酯)的浓度小于或等于70μmol/L。
  22. 根据权利要求15所述的表面处理方法，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第一溶液中，所述双酚A双(二苯基磷酸酯)的浓度为10～60μmol/L。
  23. 一种测序方法，其特征在于，所述测序方法包括：
    将含有磷酸化合物的第二溶液与固相载体的表面接触，形成混合体系；
    其中，所述表面结合有第一核酸分子和第二核酸分子，所述第一核酸分子为双链核酸分子，且所述第一核酸分子至少含有一个单链末端，所述第二核酸分子为单链核酸分子；
    所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物：
    其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；n选自1～8的整数。
  24. 根据权利要求23所述的测序方法，其特征在于，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基、碳原子数为6～20的芳基中的一种。
  25. 根据权利要求24所述的测序方法，其特征在于，R3包括环烷基或苯基。
  26. 根据权利要求25所述的测序方法，其特征在于，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
    式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。
  27. 根据权利要求26所述的测序方法，其特征在于，R2中至少含有一个金属离子。
  28. 根据权利要求27所述的测序方法，其特征在于，R2中含有n个金属离子。
  29. 根据权利要求27所述的测序方法，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  30. 根据权利要求29所述的测序方法，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  31. 根据权利要求26所述的测序方法，其特征在于，所述n选自2～6的整数。
  32. 根据权利要求23所述的测序方法，其特征在于，式(2)中，R1选自氢原子，R2选自金属离子。
  33. 根据权利要求32所述的测序方法，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  34. 根据权利要求33所述的测序方法，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  35. 根据权利要求23所述的测序方法，其特征在于，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
    式(2-1)中，M选自Na或K。
  36. 根据权利要求23所述的测序方法，其特征在于，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。
  37. 根据权利要求23-36任一项所述的测序方法，其特征在于，所述将含有磷酸化合物的第二溶液与固相载体的表面接触，包括：
    在所述固相载体的表面加含有所述磷酸化合物的第二溶液；或
    将所述固相载体的表面置于含有所述磷酸化合物的第二溶液中；或
    使含有所述磷酸化合物的第二溶液流经所述固相载体的表面。
  38. 根据权利要求37所述的测序方法，其特征在于，所述第二溶液中，所述磷酸化合物的浓度为1～150μmol/L。
  39. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物包含植酸钠，且所述第二溶液中，所述植酸钠的浓度小于或等于10μmol/L。
  40. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物为植酸钠，且所述第二溶液中，所述植酸钠的浓度为1～10μmol/L。
  41. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物包含焦磷酸二氢二钠，且所述第二溶液中，所述焦磷酸二氢二钠的浓度小于或等于50μmol/L。
  42. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物为焦磷酸二氢二钠，且所述第二溶液中，所述焦磷酸二氢二钠的浓度为10～50μmol/L。
  43. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第二溶液中，所述双酚A双(二苯基磷酸酯)的浓度小于或等于50μmol/L。
  44. 根据权利要求37所述的测序方法，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述第二溶液中，所述双酚A双(二苯基磷酸酯)的浓度为10～50μmol/L。
  45. 根据权利要求23-44任一项所述的测序方法，其特征在于，所述第二溶液包括缓冲液、表面活性剂和其他添加剂中的至少一种。
  46. 根据权利要求45所述的测序方法，其特征在于，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙 二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。
  47. 根据权利要求45所述的测序方法，其特征在于，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。
  48. 根据权利要求45所述的测序方法，其特征在于，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。
  49. 根据权利要求45所述的测序方法，其特征在于，所述第二溶液中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第二溶液的pH为8.9～9.4。
  50. 根据权利要求45所述的测序方法，其特征在于，所述第二溶液中还包括dNTP或NTP。
  51. 根据权利要求50所述的测序方法，其特征在于，所述dNTP选自dATP或其类似物、dTTP或其类似物、dGTP或其类似物、dCTP或其类似物中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
  52. 根据权利要求23-51任一项所述的测序方法，其特征在于，在所述将含有磷酸化合物的第二溶液与固相载体的表面接触的步骤之后，在所述混合体系中加入第三溶液；
    所述第三溶液包括缓冲液、表面活性剂和其他添加剂中的至少一种。
  53. 根据权利要求52所述的测序方法，其特征在于，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。
  54. 根据权利要求52所述的测序方法，其特征在于，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。
  55. 根据权利要求52所述的测序方法，其特征在于，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。
  56. 根据权利要求52所述的测序方法，其特征在于，所述第三溶液中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第三溶液的pH为8.9～9.4。
  57. 根据权利要求52所述的测序方法，其特征在于，所述第三溶液中还包括dNTP或NTP。
  58. 根据权利要求57所述的测序方法，其特征在于，所述dNTP选自dATP或其类似物、dTTP或其类似物、dGTP或其类似物、dCTP或其类似物中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
  59. 根据权利要求23-58任一项所述的测序方法，其特征在于，所述第一核酸分子包括寡聚核酸分子和核酸模板，且所述寡聚核酸分子的至少部分序列与所述核酸模板互补，且所述核酸模板为未经扩增反应的核酸分子。
  60. 一种用于核酸分子延伸的试剂盒，其特征在于，所述试剂盒包括第一试剂，所述第一试剂包括磷酸化合物，且所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物；
    其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
    R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
    n选自1～8的整数。
  61. 根据权利要求60所述的试剂盒，其特征在于，式(1)中，R1、R2中至少一个为氢原子或芳香基，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。
  62. 根据权利要求61所述的试剂盒，其特征在于，R3包括环烷基或苯基。
  63. 根据权利要求62所述的试剂盒，其特征在于，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
    式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。
  64. 根据权利要求63所述的试剂盒，其特征在于，R2中至少含有一个金属离子。
  65. 根据权利要求64所述的试剂盒，其特征在于，R2中含有n个金属离子。
  66. 根据权利要求64所述的试剂盒，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  67. 根据权利要求66所述的试剂盒，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  68. 根据权利要求63所述的试剂盒，其特征在于，所述n选自2～6的整数。
  69. 根据权利要求60所述的试剂盒，其特征在于，式(2)中，R1选自氢原子，R2选自金属离子。
  70. 根据权利要求69所述的试剂盒，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  71. 根据权利要求70所述的试剂盒，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  72. 根据权利要求69所述的试剂盒，其特征在于，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
    式(2-1)中，M选自Na或K。
  73. 根据权利要求69所述的试剂盒，其特征在于，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。
  74. 根据权利要求69-73任一项所述的试剂盒，其特征在于，所述试剂盒包括第二试剂，所述第二试剂包括缓冲液、表面活性剂和其他添加剂中的至少一种。
  75. 根据权利要求74所述的试剂盒，其特征在于，所述第一试剂与所述第二试剂以混合物的方式置于所述试剂盒中的同一个试剂管中；或所述第一试剂和所述第二试剂分装于所述试剂盒中不同的试剂管中。
  76. 根据权利要求74所述的试剂盒，其特征在于，所述缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。
  77. 根据权利要求74所述的试剂盒，其特征在于，所述其他添加剂包括铵根离子、镁离子、钾离子、钠离子、二甲基亚砜、1,3-二甲基硫脲中的至少一种。
  78. 根据权利要求74所述的试剂盒，其特征在于，所述表面活性剂包括Triton X-100、吐温-20中的至少一种。
  79. 根据权利要求74所述的试剂盒，其特征在于，所述第二试剂中，K+的浓度为50～100mmol/L，NH4 +的浓度为150～350mmol/L，所述第二试剂的pH为8.9～9.4。
  80. 根据权利要求69-79任一项所述的试剂盒，其特征在于，所述试剂盒包括第三试剂和第四试剂，所述第三试剂包括dNTP或NTP，所述第四试剂包括聚合酶。
  81. 根据权利要求80所述的试剂盒，其特征在于，所述dNTP选自dATP或其类似物、dTTP或其类似物、dGTP或其类似物、dCTP或其类似物中的至少一种；所述NTP选自ATP或其类似物、UTP或其类似物、GTP或其类似物、CTP或其类似物中的至少一种。
  82. 根据权利要求80所述的试剂盒，其特征在于，所述第三试剂、所述第四试剂和所述第一试剂各自独立地分装于所述试剂盒中不同的试剂管中。
  83. 根据权利要求69-82任一项所述的试剂盒，其特征在于，所述试剂盒包括第五试剂，所述第五试剂包括寡聚核苷酸A、寡聚核苷酸T、寡聚核苷酸G、寡聚核苷酸C，或所述第五试剂包括寡聚核苷酸A、寡聚核苷酸U、寡聚核苷酸G、寡聚核苷酸C。
  84. 根据权利要求83所述的试剂盒，其特征在于，所述第五试剂和所述第一试剂分装于所述试剂盒中不同的试剂管中。
  85. 根据权利要求69-84任一项所述的试剂盒，其特征在于，以所述试剂盒中各成分混合形成的混合溶液的浓度计算，所述磷酸化合物的浓度为1～150μmol/L。
  86. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物包含植酸钠，且所述植酸钠的浓度小于或等于10μmol/L。
  87. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物为植酸钠，且所述植酸钠的浓度为1～10μmol/L。
  88. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物包含焦磷酸二氢二钠，且所述焦磷酸二氢二钠的浓度小于或等于50μmol/L。
  89. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物为焦磷酸二氢二钠，且所述焦磷酸二氢二钠的浓度为10～50μmol/L。
  90. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述双酚A双(二苯基磷酸酯)的浓度小于或等于50μmol/L。
  91. 根据权利要求85所述的试剂盒，其特征在于，所述磷酸化合物包含双酚A双(二苯基磷酸酯)，且所述双酚A双(二苯基磷酸酯)的浓度为10～50μmol/L。
  92. 根据权利要求85所述的试剂盒，其特征在于，所述dNTP或NTP的浓度为20～1000μmol/L。
  93. 根据权利要求85所述的试剂盒，其特征在于，所述聚合酶的浓度为5～10U/mL。
  94. 一种延伸试剂缓冲液，其特征在于，至少包括基础缓冲液、K+和NH4 +，其中，所述K+的浓度为50～100mmol/L，所述NH4 +的浓度为150～350mmol/L，所述延伸试剂缓冲液的pH为8.9～9.4。
  95. 如权利要求94所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液的pH为8.9～9.1，所述K+的浓度为50～70mmol/L，所述NH4 +的浓度为150～230mmol/L。
  96. 如权利要求94所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液的pH为9.05～9.35，所述K+的浓度为60～90mmol/L，所述NH4 +的浓度为200～310mmol/L。
  97. 如权利要求94所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液的pH为9.3～9.5，所述K+的浓度为80～100mmol/L，所述NH4 +的浓度为300～350mmol/L。
  98. 如权利要求94至97任一项所述的延伸试剂缓冲液，其特征在于，所述K+为氢氧化钾、卤化钾、有机羧酸钾和硫酸钾中的至少一种提供的K+。
  99. 如权利要求94至97任一项所述的延伸试剂缓冲液，其特征在于，所述NH4 +为硫酸铵、有机铵和氨水中的至少一种提供的NH4 +。
  100. 如权利要求94至97任一项所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液含有氢氧化钾和硫酸铵。
  101. 如权利要求94至97任一项所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液包括有机羧酸钾和硫酸钾中的至少一种和氨水。
  102. 如权利要求94至101任一项所述的延伸试剂缓冲液，其特征在于，所述基础缓冲液选自三羟甲基氨基甲烷缓冲液、甘氨酸、乙醇胺、四乙基乙二胺、四甲基乙二胺、N-丁基二乙醇胺、二乙氨基乙醇、Hepes缓冲液、N,N-二羟乙基甘氨酸中的至少一种。
  103. 如权利要求94至101任一项所述的延伸试剂缓冲液，其特征在于，所述延伸试剂缓冲液还包括：络合剂、聚合酶催化剂、表面活性剂和其他助剂中的至少一种。
  104. 如权利要求103所述的延伸试剂缓冲液，其特征在于，所述络合剂选自乙二胺四乙酸、乙二胺四乙酸二钠盐、乙二胺四乙酸四钠盐、EGTA中的至少一种；和/或
    所述聚合酶催化剂包括镁盐；和/或
    所述表面活性剂选自吐温-20、Triton X-100中的至少一种；和/或
    所述其他助剂包括二甲基亚砜、1,3-二甲基硫脲中的至少一种。
  105. 如权利要求103所述的延伸试剂缓冲液，其特征在于，所述其他助剂包括磷酸化合物，且所述磷酸化合物包括结构式如式(1)和/或式(2)所示的化合物：
    其中，R1、R2各自独立地选自金属离子、氢原子、任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、任意取代或未取代的杂芳基中的任意一种；
    R3选自任意取代或未取代的烃基、任意取代或未取代的环烃基、任意取代或未取代的杂烃基、任意取代或未取代的杂环烃基、任意取代或未取代的芳基、杂芳基中的一种；
    n选自1～8的整数。
  106. 如权利要求105所述的延伸试剂缓冲液，其特征在于，式(1)中，R1、R2中至少一个为氢原子，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种；或
    R1、R2中至少一个为芳香基，R3选自碳原子数为1～10的烷基、环烷基，碳原子数为6～20的芳基中的一种。
  107. 如权利要求105所述的延伸试剂缓冲液，其特征在于，式(1)中，R3包括环烷基或苯基。
  108. 如权利要求105至107任一项所述的延伸试剂缓冲液，其特征在于，式(1)选自结构式如式(1-1)、式(1-2)所示的化合物中的至少一种：
    式(1-1)中，x选自大于或等于n的整数，且x小于10，y的取值满足：y＝2x-n；式(1-2)中，x选自大于或等于n的整数，且x小于10，z的取值满足：z＝2x-n+2。
  109. 如权利要求108所述的延伸试剂缓冲液，其特征在于，R2中至少含有一个金属离子。
  110. 如权利要求108所述的延伸试剂缓冲液，其特征在于，R2中含有n个金属离子。
  111. 如权利要求109或110所述的延伸试剂缓冲液，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  112. 如权利要求111所述的延伸试剂缓冲液，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  113. 如权利要求110所述的延伸试剂缓冲液，其特征在于，所述n选自2～6的整数。
  114. 如权利要求105所述的延伸试剂缓冲液，其特征在于，式(2)中，R1选自氢原子，R2选自金属离子。
  115. 如权利要求114所述的延伸试剂缓冲液，其特征在于，所述金属离子选自一价金属离子或二价金属离子。
  116. 如权利要求115所述的延伸试剂缓冲液，其特征在于，所述一价金属离子包括钠离子和/或钾离子。
  117. 如权利要求114至116任一项所述的延伸试剂缓冲液，其特征在于，式(2)选自结构式如式(2-1)、式(2-2)所示的化合物中的至少一种：
    式(2-1)中，M选自Na或K。
  118. 如权利要求105至117任一项所述的延伸试剂缓冲液，其特征在于，所述磷酸化合物选自植酸钠、焦磷酸二氢二钠和双酚A双(二苯基磷酸酯)中的任意一种或至少两种的组合。
  119. 如权利要求94至118任一项所述的延伸试剂缓冲液在核酸测序领域中的应用。
  120. 一种延伸试剂盒，其特征在于，包括如权利要求94至119任一项所述的延伸试剂缓冲液。
  121. 如权利要求120所述的延伸试剂盒，其特征在于，还包括聚合酶，带荧光基团的可逆终止子和/或不带荧光基团的可逆终止子。
  122. 一种测序方法，其特征在于，包括：
    于结合有核酸分子的固相载体的表面，通入如权利要求94至119任一项所述的延伸试剂缓冲液、可逆终止子和聚合酶，使所述可逆终止子结合在所述核酸分子上；
    其中，所述核酸分子包括核酸模板和与所述核酸模板结合的寡聚核苷酸，且所述可逆终止子结合在所述寡聚核苷酸所在链的3’端。
  123. 如权利要求122所述的测序方法，其特征在于，所述于结合有核酸分子的固相载体的表面，通入所述延伸试剂缓冲液、可逆终止子和聚合酶，包括：
    在所述固相载体的表面加含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液；或
    将所述固相载体置于含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液中；或
    使含有所述延伸试剂缓冲液、所述可逆终止子和所述聚合酶的混合溶液流经所述表面。
PCT/CN2023/112186 2022-10-31 2023-08-10 表面处理方法、测序方法和试剂盒 WO2024093421A1 (zh)

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