Nucleic acid sequencing methods and devices

Abstract

A method for determining the nucleotide type on a nucleic acid sequence to be analyzed, and a method for nucleic acid sequencing. In this method, at least one modified nucleotide for nucleic acid synthesis is separated from the nucleic acid sequence to be analyzed and other components on opposite sides of a membrane. Under the influence of an electric field, the modified nucleotide is transported through a nanopore embedded in the membrane to the other side, where a synthesis reaction occurs. Simultaneously, the base type of the nucleotide is determined based on the changes in the electrical properties of the nanopore caused by the transport of the modified nucleotide, thereby achieving sequencing.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid sequencing. Specifically, this invention is a technique for sequencing nucleic acid during synthesis via nanopores (Sequencing by Synthesis). Specifically, it relates to a nucleic acid sequencing method in which at least one modified nucleotide for nucleic acid synthesis is separated from the nucleic acid sequence to be analyzed and other components on both sides of a bilayer membrane. Under the action of voltage, the modified nucleotide is transported through a nanopore embedded in the bilayer membrane to the other side of the membrane, where a synthesis reaction occurs. Simultaneously, the base type of the nucleotide is determined based on the changes in the electrical properties of the nanopore caused by the transport of the modified nucleotide, thereby achieving sequencing. Background Technology

[0002] The concept of nanopore-based nucleic acid sequencing was proposed in 1995. Researchers discovered that certain transmembrane proteins, such as the bacterial toxin α-hemolysin, can form stable channels with a diameter of approximately 1-2 nanometers on phospholipid membranes, called nanopores. Single-stranded DNA (or RNA) molecules, due to their inherent electrical charge, spontaneously pass through nanopores in an electric field. During this passage, they cause changes in the resistance of the nanopores, generating a so-called blocking current. The four different bases of DNA (RNA)—A, T (U), C, and G—have identifiable differences in their blocking effect on the current when passing through nanopores due to their differences in chemical structure, producing their respective characteristic blocking currents. Accurate detection of these characteristic blocking currents allows for the determination of the corresponding base types, thereby determining the nucleic acid sequence.

[0003] There are currently two main industrially viable nanopore sequencing methods. One method, represented by the Oxford Nanopore Technologies system, directly allows single-stranded DNA molecules to pass through a nanopore and sequentially reads the characteristic blocking current corresponding to each base. However, because the characteristic currents given by different bases are small in difference, multiple bases can simultaneously remain in the nanopore, making the characterization of blocking currents very complex. This places extremely high demands on the analysis of current data in the later stages of sequencing. More importantly, this system faces insurmountable difficulties in determining a DNA sequence (homogeneous polymer) with consecutive identical bases. The other method, represented by the system used by Genia Technologies (now part of Roche Sequencing Solutions), utilizes modified nucleotide analogs to perform sequencing simultaneously with nucleic acid synthesis. While tagging nucleotides used for replication can improve the recognition of characteristic blocking currents corresponding to different bases, and the time interval between individual nucleotide tags entering the nanopore also helps in determining the sequence of nucleic acids with consecutive identical bases (homogeneous sequences), this system struggles to guarantee that every nucleotide tag used for synthesis enters the nanopore to provide a blocking current, leading to deletion errors during sequencing. It also struggles to avoid background noise interference from tags of nucleotides that did not participate in the synthesis reaction, which can interfere with the reading of valid signals, or even generate redundant blocking current signals that are mistakenly read as valid sequence signals (insertion errors). Compared to these two existing methods, this invention proposes a new improved sequencing method that utilizes nanopores to transport nucleotides used for synthesis. This ensures that each nucleotide added to the nascent nucleic acid chain necessarily passes through the nanopore and records its characteristic blocking current, thus avoiding deletion errors. Furthermore, after potential contact with the polymerase, unreacted intact phosphate deoxynucleotides and polyphosphate groups generated during the reaction will again pass through the nanopore. These unreacted intact phosphate deoxynucleotides and polyphosphate groups will provide different blocking currents, helping to determine whether a particular deoxynucleotide has been incorporated into the nucleic acid product. This information will effectively prevent overread errors caused by the system mistakenly recording signals of nucleotides that did not participate in the synthesis reaction. Summary of the Invention

[0004] One aspect of the present invention provides a method for determining the nucleotide type on a nucleic acid sequence to be analyzed, comprising the following steps:

[0005] S01 consists of a first compartment and a second compartment separated by a membrane having at least one nanopore, wherein at least one nucleotide molecule is provided in the first compartment and the nucleic acid sequence to be analyzed is provided in the second compartment;

[0006] S02 applies an electric field to cause nucleotide molecules and / or portions thereof to pass through or insert into the nanopore in a first direction;

[0007] The nucleotide molecule or a portion thereof is identified by measuring the first current characteristic value through the membrane in the SO2 state in the SO3 state.

[0008] S04 applies an electric field that is opposite in direction to or in the same direction as the electric field applied in step S02 but with a lower driving voltage, causing the nucleotide molecule and / or a portion thereof to pass through and / or exit the nanopore in a second direction opposite to the first direction;

[0009] S05. The second current characteristic value passing through the membrane is measured again to identify the nucleotide molecule or a portion thereof. This second current characteristic value is compared with the standard current characteristic value of a nucleotide molecule or polyphosphate molecule pre-determined in the S04 state to determine whether the nucleotide molecule or a portion thereof is attached to the nucleic acid sequence to be analyzed, thereby determining the type of nucleotide on the nucleic acid sequence to be analyzed in the second compartment. The polyphosphate molecule is a product of the nucleic acid synthesis reaction of the corresponding nucleotide molecule.

[0010] In some implementations, in step S04, the term "lower driving voltage" refers to a voltage difference of 0mV or more between an electric field with a lower voltage and an electric field with a higher voltage. For example, the voltage difference could be 0mV or more, 5mV or more, 10mV or more, 20mV or more, 30mV or more, 40mV or more, 50mV or more, 60mV or more, 70mV or more, 80mV or more, 90mV or more, 100mV or more, 110mV or more, 120mV or more, 130mV or more, 140mV or more, 150mV or more, 160mV or more, 170mV or more, 180mV or more, 190mV or more, 200mV or more, etc., or any value within any of the above voltage difference ranges.

[0011] In some embodiments, the second compartment also provides, but is not limited to, nucleic acid polymerases, template primers, etc. In some preferred embodiments, the nucleic acid polymerase is bound to the nanopore.

[0012] In some embodiments, the nucleotide molecule is a modified nucleotide molecule as shown in Formula I:

[0013] NXP-LB Type I

[0014] NXP (Nucleoside phosphate) represents deoxyphosphate nucleotides and / or phosphate nucleotides, wherein the deoxyphosphate nucleotides include dAXP (deoxyadenine phosphate), dTXP (deoxythymidine phosphate), dCXP (deoxycytosine phosphate), and dGXP (deoxyguanine phosphate); and the phosphate nucleotides include AXP (guanine phosphate), TXP / UXP (thymidine phosphate / uracil phosphate), CXP (cytosine phosphate), and GXP (guanine phosphate).

[0015] L represents a linker portion, which has a long chain segment for separating the NTP portion and the B portion in Formula I by a certain distance. In a preferred embodiment, the long chain segment is a biocompatible linear polymer, such as, but not limited to, PEG chains, polymers formed by phosphodiester bonds (similar to nucleic acid backbone structures), peptides, and suitable nanowires. The linker portion also has a connecting segment for specifically connecting large-volume steric hindrance portions, such as, but not limited to, biotin and maleimide.

[0016] B represents the large volume steric hindrance portion, which is selected from protein molecules, preferably globular protein molecules, which have the property of structural stability, and is connected to the linker portion through the above-mentioned linker segment, such as, but not limited to, avidin, which is connected to the linker through biotin; C is a xylanase with a cysteine ​​residue introduced at the end, which is connected to the linker through maleamide.

[0017] In some preferred embodiments, the biocompatible linear polymer segment is preferably polyethylene glycol (PEG) or a polypeptide composed of glycine and serine (e.g., GGSGSGGSSSSSSSSSSSSSSS...). In some preferred embodiments, the linker segment is biotin or maleamide. In some preferred embodiments, the bulky sterically hindered portion is preferably avidin, which is linked to the linker via biotin; or a xylanase with a cysteine ​​residue introduced at the C-terminus, which is linked to the linker via maleamide.

[0018] In some embodiments, the membrane is selected from natural lipid bilayer membranes and bilayer membranes formed from artificial amphipathic molecules. In some embodiments, the membrane has at least one nanopore. In some preferred embodiments, the membrane has one nanopore.

[0019] In some embodiments, the nanopores are selected from natural protein nanopores and nanopores prepared from artificial materials. In some embodiments, the nanopores are bacterial toxins α-hemolysin. In some embodiments, the nanopores are other types of protein pores, such as, but not limited to, MspA, CsgG, OmpF, etc. In some embodiments, the nanopores are modified variants of wild-type protein nanopores. In some embodiments, the nanopores can also be artificial nanopores prepared from artificial materials such as, but not limited to, silicon or graphene. An artificial material such as, but not limited to, silicon or graphene is formed into a membrane, and then nanopores are formed on the membrane.

[0020] Another aspect of the present invention provides a sequencing method for a nucleic acid sequence to be analyzed, comprising repeating steps S01 to S05 of the method as described in claim 1 to sequentially determine each nucleotide type of the nucleic acid sequence to be analyzed until the sequencing of the nucleic acid sequence to be analyzed is completed.

[0021] Another aspect of the present invention provides a nucleic acid sequencing apparatus for the above-described methods for determining the nucleotide types on a nucleic acid sequence to be analyzed and / or for nucleic acid sequencing methods, the apparatus comprising:

[0022] (a) A first compartment and a second compartment separated by a membrane having at least one nanopore;

[0023] (b) A component for applying an electric field through the membrane; and

[0024] (c) A component used to measure the current passing through the membrane.

[0025] In some embodiments, the device of the present invention is a single-channel device based on the sequencing method of the present invention, intended to further illustrate the principles and implementation methods of the present invention. In practical applications, the present invention can also be extended to a multi-channel array device to simultaneously sequence several DNA molecules.

[0026] Brief description of the attached figures

[0027] Figure 1 A specific example of a modified nucleotide molecule according to the present invention is shown, specifically, deoxyribonucleotide triphosphates (dATP, dTTP, dCTP and dGTP) are respectively linked to a polypeptide sequence and form a tight nucleotide triphosphate complex through a biotin group and an avidin. Figure 1 The bottom left corner shows a simplified diagram of four nucleotide molecular complexes.

[0028] Figure 2The invention illustrates a first compartment and a second compartment separated by a membrane having at least one nanopore, wherein a modified nucleotide molecule is provided in the first compartment and a nucleic acid sequence to be analyzed, a nucleic acid polymerase, and a template primer are provided in the second compartment, wherein the nucleic acid polymerase is bound to the nanopore.

[0029] Figure 3 This demonstrates dNTP-L under higher voltage (180mV) driving. N After the nucleotides of the -B complex move from the first compartment to the second compartment, different complex molecules dATP-L A -B、dTTP-L T -B、dCTP-L C -B and dGTP-L G -B, representative current curve and standard blocking current characteristic value I when interacting with nanopores. N1 .

[0030] Figure 4 This demonstrates how, after a nucleotide transitions from the first compartment to the second compartment at 180 mV, the voltage is switched to a lower value (80 mV), resulting in dNTP-L N -B complex and PP-L N -Characteristic current curves I corresponding to different molecules during the process of B returning from the second compartment to the first compartment. N2 and I N3 For complexes with the same linker, I N2 and I N3 They can be clearly distinguished.

[0031] Figure 5 This invention illustrates how, by applying an electric field, modified nucleotide molecules are inserted into a nanopore in a first orientation. The nucleotide portion passes through the nanopore into a second compartment. The nucleotide molecule and its corresponding linker portion interact with the nanopore, and a corresponding current characteristic value can be obtained by measurement. The measured current characteristic value is then compared with the standard blocking current characteristic value I for different nucleotides. N1 By comparing the samples, the type of nucleotide that enters the second compartment from the first compartment can be determined. Figure 5 Example measured current characteristic value and I T1 If the match is accurate, the nucleotide type can be determined to be T.

[0032] Figures 6-7 This invention illustrates that, after a certain period of time, the system switches to a low-voltage electric field (e.g., 80 mV). The electric field driving force is insufficient to overcome the diffusion tendency of nucleotide molecules. Linkers with intact nucleotides or pyrophosphate groups resulting from enzymatic reactions pass through or exit the nanopore in a second direction, returning to the first compartment. During this process, the corresponding blocking current characteristic values ​​are recorded. When the current characteristic is consistent with I... N2When there is a characteristic match, it can be determined that the linker contains a complete nucleotide, and no synthesis reaction has been completed in the second compartment, for example. Figure 6 The situation is shown. When the current characteristics are similar to I... N3 When the characteristics match, it can be determined that the linker only has a pyrophosphate group (PP), and that a polymerase-catalyzed synthesis reaction occurred in the second compartment, previously via I... N1 The bases identified are complementary bases on the nucleic acid template in the second compartment, for example... Figure 7 As shown, it can be determined that the nucleotide entering the second compartment from the first compartment is A, and the DNA synthesis reaction is complete, with the corresponding position on the template being T.

[0033] Figure 8 This is a schematic diagram of the nucleic acid sequencing device of the present invention.

[0034] Figure 9 This is a current signal diagram obtained during sequencing. In the lower left magnified region 1, based on the current characteristic value at 180mV, the nucleotide entering the second compartment is identified as G. However, after switching to a lower voltage of 80mV, the current characteristic is different from that in region 1. G2 The corresponding information indicates that the linker contains a complete nucleotide, and no synthesis reaction occurred in the second compartment. Therefore, G is not a valid sequence signal at this location. In the magnified area 2 at the lower right, based on the current characteristic value at 180mV, the nucleotide entering the second compartment is identified as T. After switching to a lower voltage of 80mV, its current characteristic is similar to that of I. T3 Correspondingly, the synthesis reaction is complete, and C is determined to be a valid sequence signal. Detailed Implementation

[0035] It should be understood that different applications of the methods disclosed herein can be varied according to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention and is not intended to be limiting.

[0036] This invention provides a method for determining the nucleotide type in a nucleic acid sequence using nanopores on a bilayer membrane and modified nucleotides. This invention also provides a method for sequencing nucleic acid sequences using nanopores on a bilayer membrane and modified nucleotides. The modified nucleotides are complexes as shown in Formula I:

[0037] NXP-LB Type I

[0038] NXP (Nucleoside phosphate) represents deoxyphosphate and / or phosphate nucleotides, including dAXP, dTXP, dCXP and dGXP; and phosphate nucleotides including AXP, TXP / UXP, CXP and GXP.

[0039] In this invention, the term "phosphate deoxynucleotide" includes, but is not limited to, monophosphate deoxynucleotides and polyphosphate deoxynucleotides, wherein polyphosphate deoxynucleotides include, but are not limited to, triphosphate deoxynucleotides, tetraphosphate deoxynucleotides, pentaphosphate deoxynucleotides, and hexaphosphate deoxynucleotides, preferably triphosphate deoxynucleotides. In this invention, the term "phosphate nucleotide" includes, but is not limited to, monophosphate nucleotides and polyphosphate nucleotides, wherein polyphosphate nucleotides include, but are not limited to, triphosphate nucleotides, tetraphosphate nucleotides, pentaphosphate nucleotides, and hexaphosphate nucleotides, preferably triphosphate nucleotides.

[0040] The phosphate nucleotides and / or deoxyphosphate nucleotides used in this invention can be modified at multiple sites, such as, but not limited to, the second position on ribose, the fifth position on pyrimidine, and the seventh position on purine. Any modification, as long as the modified phosphate nucleotides and / or deoxyphosphate nucleotides can still support the efficient conduct of nucleic acid synthesis reactions, can be considered as candidate modification schemes in this invention to help obtain better sequencing results. Based on their corresponding blocking currents, appropriate modification methods can be screened to make the characteristic blocking currents of different nucleotides more distinguishable, and the signals clearer and easier to identify. In addition, modification can also change the size or charge properties of the phosphate nucleotides and / or deoxyphosphate nucleotide molecules, thereby slowing down their entry, passage, and / or exit time from the nanopore, helping to obtain clearer and more accurate current measurements.

[0041] In the method of this invention, the nucleic acid sequence to be analyzed can be a DNA nucleic acid sequence or an RNA nucleic acid sequence. When determining the nucleotide type in a DNA nucleic acid sequence or sequencing a DNA nucleic acid sequence, either a DNA polymerase can be used with modified deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP), or an RNA polymerase can be used with a DNA template and modified nucleotides (ATP, TTP / UTP, CTP, and GTP) to synthesize an RNA chain. When determining the nucleotide type in an RNA nucleic acid sequence or sequencing an RNA nucleic acid sequence, a reverse transcriptase can be used with the aforementioned deoxyribonucleotides as a template to synthesize a cDNA chain complementary to it.

[0042] L represents the connector sub-section, which has a long chain segment and a connector segment.

[0043] The long-chain segment is preferably a biocompatible linear polymer. Biocompatible linear polymers that can be used in this invention include, but are not limited to, linear peptides, preferably linear peptides. Besides linear peptides, other chemically chained polymers such as polymers formed by phosphodiester bonds (similar to nucleic acid backbone structures), polyethylene glycol (PEG), and suitable nanowires can also be used in this invention. The length range of the long-chain segment can be adjusted according to the scale of the selected nanoporous protein and the different coupling methods between the nanopore and the nucleic acid polymerase. In this invention, the length range of the long-chain segment can be in the range of 5-25 nanometers, preferably in the range of 8-15 nanometers.

[0044] In this invention, different connectors can be distinguished by subscript letters, such as L. N L A L T L U L C L G The symbols A, T, U, C, and G represent the types of nucleotides linked to the linker part L, while N is a general representation of nucleotides, which can be any of A, T, U, C, and G.

[0045] Linkages that can be used in this invention include, but are not limited to, biotin, maleamide, etc. These linkers have the ability to specifically bind to specific proteins, such as biotin specifically binding to avidin.

[0046] B represents the steric hindrance portion. As used herein, the term "steric hindrance portion" refers to a biomolecular portion with a diameter greater than 1-2 nm, preferably greater than 2 nm, and more preferably greater than 3 nm. The steric hindrance portion is selected from protein molecules, preferably globular protein molecules, which possess structural stability and are connected to the linker portion via the aforementioned linker segment. Examples include, but are not limited to, avidin, which is linked to the linker via biotin. Besides using biotin / avidin to couple proteins to the ends of the linker chain, other proteins or other blockers with diameters larger than nanopores, as well as other coupling methods, are also applicable to this invention. For example, maleimide active groups can be added to the ends of the linker via chemical synthesis as a linker segment. Then, any stable globular protein with a size larger than the nanopore diameter can be selected. For example, in some preferred embodiments, xylanase can be used as the steric hindrance portion. A cysteine ​​residue is introduced at the C-terminus of the recombinant protein, and its thiol group -SH is covalently coupled to the maleimide linker segment.

[0047] After the modified nucleotide molecule passes through or inserts into the nanopore, the linker confines the phosphate nucleotide moiety near the nanopore opening. Furthermore, the linker also helps control the time it takes for the phosphate nucleotide or phosphate group to pass through the nanopore in the reverse direction. The linker can be chemically modified to introduce charged groups, such as positively charged -NH2 groups or negatively charged -PO4 groups within the system's operating pH range (6-9). Variations in the charge density and charge sites introduced onto the chain allow the entire modified nucleotide molecule—the phosphate nucleotide / phosphate deoxynucleotide-linker-bulk steric hindrance molecule complex—to experience different driving forces in an electric field, thus affecting the timing and manner of the interaction between the nucleotide moiety and the nanopore, as well as its direction of movement at a given voltage. For example, introducing a positive charge near the linker-phosphate nucleotide connection site can reduce the net negative charge density of the complex, decreasing the electric field driving force on the nucleotide within the nanopore. This allows it to exit the nanopore in a second direction even at higher voltages, facilitating the recording of the current characteristics during exit at higher voltages and improving the signal-to-noise ratio.

[0048] As used herein, the term "lipid bilayer membrane" refers to a membrane prepared based on the tendency of lipid molecules, such as, but not limited to, phospholipid molecules, to form a stable lipid bilayer membrane in an aqueous phase. Lipid bilayer membranes that can be used in this invention include, but are not limited to, phospholipid bilayer membranes.

[0049] The term "nanopore" as used in this paper refers to a pore of nanoscale size that can be generated from porous proteins or pores in synthetic materials such as silicon or graphene. Protein pores in electrically insulating membranes or artificial solid pores fabricated using insulating materials can be used as single-molecule detectors. It can be a biological protein channel in a high-resistivity lipid bilayer, a protein channel in a synthetic membrane, or directly an artificial solid pore. Currently, there are two general methods for preparing nanopores for nucleic acid analysis: (1) organic nanopores prepared from naturally occurring molecules, such as α-hemolysin pores; and (2) synthetic solid nanopores produced by a variety of conventional and unconventional manufacturing techniques.

[0050] The terms “nucleotide” and “NXP” as used herein encompass both nucleotides and deoxynucleotides, unless otherwise specified in the context.

[0051] In some embodiments, the sequencing system of the present invention involves the formation of a phospholipid bilayer to separate and insulate the cis and trans sides of the bilayer, and to provide a suitable chemical environment for the embedding of nanopores, forming pores with a diameter of 1.4-2 nanometers to connect the cis and trans sides, thereby allowing ions to pass through under the influence of an electric field to form a microcurrent. Various methods are currently available, such as coating, folding, and microfluidics, for forming phospholipid bilayers ranging in size from several micrometers to 200 micrometers across hydrophobic surface materials, such as polyimide and polyethylene. Numerous publications have described the materials and steps involved in implementing these methods, which will not be elaborated upon here.

[0052] This invention is highly adaptable, and theoretically, various transmembrane nanopores can be used to achieve the transport and recognition of phosphate nucleotides, such as, but not limited to, α-hemolysin, γ-hemolysin, MspA, OmpF, etc. As long as the nanopore structure is stable after embedding in the phospholipid membrane, the corresponding current is relatively stable and measurable, and the pore size is suitable—allowing phosphate nucleotides and linkers to pass through while blocking large proteins—then it can be used for sequencing in the manner described in this invention. In addition to wild-type nanoporous proteins, this invention can also use modified variant nanoporous proteins. Finally, besides protein nanopores, this invention is also applicable to artificial solid-state nanopores, provided they meet the prerequisites of stable current and appropriate pore size.

[0053] Applying a voltage across the insulating lipid bilayer, such as 50-300mV, 75mV-275mV, 100mV-250mV, 125mV-225mV, 150mV-200mV, or 80mV, 90mV, 100mV, 125mV, 150mV, 175mV, 180mV, 190mV, 200mV, or any value between these ranges, will create a transmembrane potential gradient along the inner diameter of the nanopore. Driven by this potential gradient, the electrolyte moves directionally within the nanopore, generating a current—the open nanopore current. When negatively charged nucleotides approach the nanopore opening through a combination of free diffusion and electrophoretic motion, they are captured by the potential gradient along the nanopore and transported across the narrow nanopore under the influence of an electric field. During this process, nucleotides interfere with the movement of electrolytes within the nanopore to a certain extent, thereby altering the current magnitude and forming a so-called blocking current. The blocking currents formed by the interaction of the four different bases (A, T(U), C, and G) of different nucleotides with the nanopore can be distinguished due to differences in structure, physicochemical properties, and linker structures. Recording and identifying this characteristic blocking current can be used to determine the type of nucleotide base passing through the nanopore.

[0054] In the method of this invention, under the influence of a strong electric field, the modified nucleotide portion connected to the linker passes through the nanopore. However, the large steric hindrance portion connected by the linker cannot pass through the nanopore due to its size and thus remains on one side of the nanopore, while the linker portion remains within the nanopore. When the applied voltage decreases and the electric field weakens, the nucleotide or its enzymatically catalyzed pyrophosphate portion will exit the nanopore in the second direction described above. During the exit process, the intact nucleotide and pyrophosphate will provide distinct current characteristics, helping to determine whether the exiting group on the linker is an intact nucleotide or the product of the enzymatic reaction, pyrophosphate. By recording different characteristic currents flowing through the nanopore, the method of this invention can accurately identify the type of nucleotide passing through the nanopore and determine whether the nucleotide is captured by a nucleic acid polymerase and participates in nucleic acid synthesis. The method records the current values ​​of the nanopore in the fully open state. At higher voltages, such as not less than 160mV, 170mV, 180mV, 190mV, and 200mV, the current characteristics of the interaction between nucleotides (including nucleotides and deoxynucleotides) and linkers and nanopores are recorded. At lower voltages, such as not more than 100mV, 90mV, 80mV, 70mV, 60mV, and 50mV, the current characteristics of the linker and nucleotides / / or pyrophosphate exiting the nanopore in a second direction are recorded.

[0055] Furthermore, by analyzing the logical relationship between the aforementioned current values ​​and the changes in system state over time, the type of nucleotides passing through the nanopore and whether they successfully participated in nucleic acid synthesis can be inferred. This method has significant potential and value in reducing deletion errors and insertion errors in nucleic acid sequencing using nanopores, thereby improving sequencing accuracy.

[0056] The electrolyte solution used in this invention is suitable for nucleic acid synthesis, such as a KCl solution, specifically a 0.1-1M KCl solution, for example, a 0.3M KCl solution or a 0.5M KCl solution. Under the pH conditions of the electrolyte solution of this invention, the nucleotide moiety in the complex of this invention is negatively charged and can move down the potential gradient from the cis side (first compartment) of the phospholipid membrane through the nanopore to the trans side (second compartment). Once the nucleotide moiety reaches the trans side, it can diffuse freely and be captured and utilized by a nucleic acid polymerase coupled to the nanopore. Furthermore, the nucleotide moiety interacts with the nanopore during entry / passage, generating a characteristic blocking current that is recorded by a microcurrent measurement system connected to the nanopore, thereby determining the base type of the nucleotide transported by the nanopore.

[0057] This invention provides a method for allowing phosphate nucleotides (or pyrophosphates, if the synthesis reaction has been completed) that have passed through a nanopore to the trans side of the phospholipid membrane to return to the cis side, thus enabling a second detection by the nanopore. In some embodiments of this invention, the phosphate nucleotides are linked by PEG or similar long chains and a biotin / avidin complex. Due to their size being much larger than the nanopore diameter, the biotin / avidin complex cannot pass through the nanopore and remains on the cis side, with the nucleotide portion confined near the trans side opening of the nanopore by the PEG linker across the nanopore. When the electric field weakens, the nucleotides near the trans side opening of the nanopore will pass through the nanopore again to return to the cis side under thermal motion or diffusion, generating a second characteristic blocking current. Whether the second characteristic blocking current corresponds to the intact nucleotide or the pyrophosphate after the synthesis reaction is complete can be used to determine whether the nucleotide participated in the nucleic acid synthesis reaction.

[0058] This invention relates to a method of covalently coupling a nucleic acid polymerase and a nanopore on the trans side. This helps limit the distance between the polymerase active site and the nanopore trans-side opening to within the range of 1-3 nanometers. When phosphate nucleotides enter the trans side through the nanopore, they are also confined nearby by the linker chain, which effectively increases the local effective concentration of polymerase and nucleotides. This significantly shortens the time for phosphate nucleotides to be captured by the polymerase, thus contributing to the sequencing efficiency of this invention.

[0059] Besides using characteristic blocking currents to determine the base type of the nucleotide transported by the nanopore, this invention can also infer whether the transported nucleotide participated in DNA synthesis. As mentioned earlier, phosphate nucleotides transported to the trans side of the nanopore are confined near the nanopore opening by the linker chain. After a certain period of time, regardless of whether it participated in the synthesis reaction, this nucleotide (if the nucleotide participated in the nucleic acid synthesis reaction and was completed, it is pyrophosphate) will eventually return to the cis side through the nanopore by diffusion. If this nucleotide did not participate in the synthesis reaction, the system will again record the characteristic blocking current corresponding to the complete nucleotide exiting the nanopore; conversely, the system will detect the blocking current caused by pyrophosphate exiting the nanopore.

[0060] In summary, this invention ensures that every nucleotide involved in nucleic acid synthesis is transported through a nanopore and its characteristic blocking current is recorded, thereby obtaining information about its base type. After a certain period, molecules transported or entering the trans side pass through the nanopore back to the cis side, and during this process, it is determined whether the original nucleotide participated in the synthesis reaction. By monitoring each nucleotide that may come into contact with the polymerase and inferring its participation in the reaction, this invention can obtain the sequence of the template DNA (or RNA) to be tested, effectively avoiding missed and multiple read errors during sequencing and improving accuracy.

[0061] The present invention is further illustrated by the following embodiments, which should not be construed as limiting.

[0062] Example

[0063] Example 1: Design and preparation of nucleotide-linker-bulk steric hindrance complex

[0064] In this embodiment, a long-chain polypeptide is selected as the linker portion. One end of the polypeptide is linked to the third phosphate group of a deoxyribonucleotide triphosphate (dNTP), and the other end is linked to maleamide. Different nucleotides (A, T, C, G) are linked to polypeptides with different sequences, which facilitates the determination of base types based on different characteristic blocking current values.

[0065] The bulk steric hindrance protein portion is a globular protein xylanase with a stable structure. A cysteine ​​residue was introduced at the C-terminus of the xylanase via protein recombination, and its thiol-SH group formed a stable covalent bond with the maleamide of the linker portion, thereby synthesizing a nucleotide-linker-bulk steric hindrance protein complex.

[0066] Example 2: Preparation of a phospholipid bilayer membrane with nanopores

[0067] The compartment system conforming to the requirements of this invention can be purchased from Warner Instruments. The opening diameter between the left and right compartments is 150 micrometers. A hydrophobic material suitable for phospholipid adhesion is placed near the opening. 0.3M KCl solution is added to each of the left and right compartments, with the liquid level below the opening. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DphPC) is dissolved in the organic solvent pentane to a final concentration of 10 mg / ml. This phospholipid solution is added to the existing KCl solution in the compartment, forming an organic phase covering the KCl surface. 0.3M KCl solution is added along the outer wall of the compartment; the organic phase containing the phospholipid layer will rise and submerge the pore between the two compartments. During this process, the phospholipid combines with the hydrophobic material near the pore and spontaneously forms a phospholipid bilayer across the pore.

[0068] Add nanoporous solution to the first compartment until the final concentration is 1 nM. Simultaneously test the current between the two compartments. Once a single-molecule nanopore spontaneously inserts into the phospholipid bilayer membrane, the system will detect the opening current of the nanopore. Remove excess nanoporous solution promptly to ensure that only single-molecule nanopore channels remain on the phospholipid membrane.

[0069] The trans end of the aforementioned nanopore is incorporating a cysteine ​​residue. After successful embedding of the phospholipid membrane within the nanopore, maleimide-PEG-maleimide (available from nanosoft polymers) can be used as a ligation medium to couple it with a DNA polymerase that also contains a cysteine ​​residue. The coupled polymerase, along with the corresponding DNA template and primer components, are all located in the second compartment.

[0070] The above steps can be used to prepare phospholipid membranes containing nanopores and DNA replication components (excluding nucleotides).

[0071] Example 3: Determination of Standard Current Characteristic Values ​​and Obtaining Standard Current Characteristic Curves

[0072] Following the steps described in Example 2, manufacture as follows: Figure 8 The nucleic acid sequencing device described herein, in which 0.3M KCl buffer is added to the first and second compartments, measures the characteristic current I passing through the phospholipid bilayer membrane when the nanopore is fully open.o .

[0073] Add 5 μM of dATP-L as prepared in Example 1 to the first compartment. A The -B complex was used, with a voltage of 180 mV, and the current changes passing through the nanopore were recorded at a sampling frequency of over 1 kHz. During this process, the system first recorded the open nanopore I... o Then, the dATP portion passes through the nanopore under the influence of the electric field, entering the second compartment from the first compartment, and remains relatively stable under the driving force of the electric field. At this time, the system records dATP-L. A The current I generated by the interaction of the -B complex with the nanopore A1 After 400 ms, the voltage was reduced to 80 mV and maintained for 200 ms. The characteristic current curve I caused by dATP exiting the nanopore from the second compartment to the first compartment was recorded. A2 Repeat the voltage stimulation protocol described above and continuously record current changes for 15 minutes to obtain a sufficiently large sample size and obtain dATP-L. A -B's characteristic blocking current value I at 180mV voltage A1 Range, and clearly defined current curve I A2 The reproducibility of the features presented.

[0074] The standard current characteristic value I corresponding to dTTP, dCTP, and dGTP is determined in the same manner. T1 I C1 、、I G1 The respective numerical ranges were determined, and the characteristic current curves of the corresponding nucleotides exiting the nanopore were recorded. T2 I C2 and I G2 .

[0075] Record PP-L in the above manner. A -B、PP-L T -B、PP-L C -B and PP-L G The characteristic current curves (I) of the four composites (-B) at 80 mV during the process of the PP moiety exiting the nanopore from the second compartment and returning to the first compartment are shown in Figure 1. A3 I B3 I C3 and I G3 .

[0076] Example 4: Determination of Nucleic Acid Sequence

[0077] The nucleic acid sequence to be tested was synthesized from Beijing Qingke Biotechnology Co., Ltd., as described in SEQ ID NO.1.

[0078] SEQ ID NO.1:

[0079] 5'ATAGACGC GGCCAAATTACGGCCGAT 3'

[0080] The underlined portion represents the complementary sequence of the binding primer. The primer sequence is as described in SEQ ID NO.2.

[0081] SEQ ID NO.2:

[0082] 5'ATCGGCCGTAATTTGGCC 3'

[0083] In such Figure 8 In the sequencing device shown, the nucleic acid sequence to be tested and the primer sequence are dissolved in 0.3M KCl solution and added to the second compartment of the nucleic acid sequencing device. The nucleic acid polymerase complex is coupled to the nanopore and is located near the opening on the trans side of the nanopore in the second compartment.

[0084] The nucleotide complex prepared according to Example 1 was dissolved in 0.3M KCl solution and added to the first compartment of the nucleic acid sequencing device.

[0085] First loop:

[0086] A positive electric field V1 = 180mV is applied from the first compartment to the second compartment for a duration of t1 = 400ms, and the current change is recorded.

[0087] A positive electric field V2 = 80mV is applied from the first compartment to the second compartment for a duration of t2 = 200ms, and the current change is recorded.

[0088] An electric field of -180mV is applied from the second compartment to the first compartment for a duration of t3 = 5ms to ensure that all macromolecules completely exit the nanopore.

[0089] Second cycle:

[0090] ...

[0091] Until sequencing is complete.

[0092] Based on the current change graphs obtained for each cycle, the measured current values ​​are compared with the standard current characteristic values ​​measured in Example 3 to determine the type of nucleotide that passes through the nanopore in each cycle, and whether the nucleotide has been successfully synthesized onto the nucleic acid sequence to be analyzed.

[0093] Based on the above analysis and judgment, it can be determined that the nucleotides involved in the synthesis reaction are GCGTCTAT in sequence, therefore the original nucleic acid sequence to be tested from 5' to 3' is... ATAGACGC.

[0094] This invention is not limited to the specific embodiments described herein, which are intended as a single illustration of various aspects of the invention. Many modifications and variations can be made to the invention without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the invention, other than those listed herein, will become apparent to those skilled in the art from the foregoing description. These modifications and variations are intended to fall within the scope of the appended claims. The invention is limited only by the terminology of the appended claims and the full scope of their equivalents. It should be understood that the invention is not limited to the specific methods, reagents, compound compositions, or biological systems that may, of course, be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not restrictive.

Claims

1. A method for determining the nucleotide type on a nucleic acid sequence to be analyzed, comprising the following steps: S01 consists of a first compartment and a second compartment separated by a membrane having at least one nanopore, wherein at least one nucleotide molecule is provided in the first compartment and the nucleic acid sequence to be analyzed is provided in the second compartment; S02 applies an electric field to cause nucleotide molecules and / or portions thereof to pass through or insert into the nanopore in a first direction; S03 The first current characteristic value of the membrane in the SO2 state is used to identify the nucleotide molecule or a portion thereof; S04 Apply an electric field in the opposite direction to the electric field applied in step S02, or an electric field in the same direction but with a lower driving voltage, to cause the nucleotide molecule and / or a portion thereof to pass through and / or exit the nanopore in a second direction, the second direction being opposite to the first direction; S05 The second current characteristic value through the membrane is measured again to identify the nucleotide molecule or its part. The second current characteristic value is compared with the standard current characteristic value of the nucleotide molecule or polyphosphate molecule in the S04 state as determined in advance to determine whether the nucleotide molecule or its part is attached to the nucleic acid sequence to be analyzed, thereby determining the type of nucleotide on the nucleic acid sequence to be analyzed in the second compartment, wherein the polyphosphate molecule is the product after the corresponding nucleotide molecule undergoes a nucleic acid synthesis reaction. The nucleotide molecule mentioned therein is a modified nucleotide molecule as shown in Formula I: NXP-LB Type I Wherein NXP represents phosphate deoxynucleotides and / or phosphate nucleotides, the phosphate deoxynucleotides including dAXP, dTXP, dCXP and dGXP; the phosphate nucleotides including AXP, TXP / UXP, CXP and GXP; L represents a connecting sub-part, which has a long chain segment and a connecting segment; B represents the large volume steric hindrance portion, which is selected from protein molecules and is connected to the linker portion through the linker segment.

2. The method according to claim 1, wherein the membrane is selected from natural lipid bilayer membranes or bilayer membranes formed by artificial amphipathic molecules.

3. The method according to claim 1, wherein the nanopore is selected from natural protein nanopores or nanopores prepared from artificial materials.

4. The method of claim 1, wherein a nucleic acid polymerase and template primers are also provided in the second compartment.

5. A sequencing method for a nucleic acid sequence to be analyzed, comprising repeating steps S01 to S05 of the method as described in claim 1 to sequentially determine each nucleotide type of the nucleic acid sequence to be analyzed until the sequencing of the nucleic acid sequence to be analyzed is completed.

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