WO2018041274A2 - Dna测序方法 - Google Patents

Dna测序方法 Download PDF

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WO2018041274A2
WO2018041274A2 PCT/CN2017/108325 CN2017108325W WO2018041274A2 WO 2018041274 A2 WO2018041274 A2 WO 2018041274A2 CN 2017108325 W CN2017108325 W CN 2017108325W WO 2018041274 A2 WO2018041274 A2 WO 2018041274A2
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dna
single molecule
dna polymerase
molecule device
sequencing
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WO2018041274A3 (zh
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郭雪峰
王晓龙
慈海娜
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北京大学
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Priority to US16/327,535 priority Critical patent/US20190330695A1/en
Priority to JP2019506126A priority patent/JP2020500002A/ja
Priority to EP17845576.2A priority patent/EP3508587A4/en
Publication of WO2018041274A2 publication Critical patent/WO2018041274A2/zh
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • H—ELECTRICITY
    • H10—SEMICONDUCTOR DEVICES; ELECTRIC SOLID-STATE DEVICES NOT OTHERWISE PROVIDED FOR
    • H10K—ORGANIC ELECTRIC SOLID-STATE DEVICES
    • H10K85/00—Organic materials used in the body or electrodes of devices covered by this subclass
    • H10K85/40—Organosilicon compounds, e.g. TIPS pentacene
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the invention relates to the field of biotechnology, and in particular to a DNA sequencing method.
  • DNA is the carrier of genetic information, and the specific order of the four base pairs is the essence of genetic information. Determining the specific sequence of base pairs in DNA is called DNA sequencing, which has a fundamental role in revealing the mysteries of life, controlling genetic processes, promoting disease diagnosis, and improving medical technology. So far, two generations of sequencing technologies have been successfully developed and widely used: the first generation of sequencing technology based on dideoxy chain termination method opened the door for humans to reveal genetic ceremonies and laid the most important foundation for genetic engineering; The generation of large-scale parallel synthetic fluorescence detection method is based on the first generation sequencing technology, using PCR amplification and fluorescence detection technology, which greatly improves the efficiency of sequencing, sequencing the first human genome. The completion of the work has made a great contribution.
  • the relatively mature DNA sequencing technology is based on the nanopore-based electrical test platform. Even commercial prototypes have appeared, but there are still many defects, such as difficulty in controlling DNA movement speed, low resolution of sequencing signals, inaccurate sequencing, etc. To make the development of sequencing technology based on this method into a bottleneck. With the emergence of various new nano-materials and the continuous upgrading of micro-nano processing technology, the device platform with single-molecule detection capability has developed rapidly, providing a source for the continuous advancement of DNA sequencing. Constant motivation and opportunity.
  • a first aspect of the present invention provides a DNA polymerase-modified single molecule device, wherein the single molecule device is a two-dimensional nano material single molecule device, preferably a graphene-based single molecule device; Or a low-dimensional nanomaterial single molecule device, preferably a one-dimensional nanomaterial single molecule device, more preferably a silicon nanowire single molecule device.
  • single molecule device may be any device in the prior art that is responsive to a single molecule change, i.e., one skilled in the art can know whether a corresponding molecule has changed by detecting or observing a change in the single molecule device.
  • the present invention preferably uses a single molecule device using graphene or silicon nanowires as electrodes, and connects it to the DNA polymerase, and by detecting the current change of the single molecule device, the DNA polymerase is known. Whether the conformation has changed.
  • the preparation process of the graphene-based single molecule device and the silicon nanowire single molecule device can be referred to the literature (Cao Y, Dong S, Liu S, He L, Gan L, Yu X, Steigerwald ML, Wu X, Liu Z, Guo X. Building high-throughput molecular junctions using indented graphene point contacts. Angew Chem Int Ed Engl. 2012 Dec 3; 51(49): 12228-32.).
  • the DNA polymerase has a binding site to the single molecule device, and the amino acid residue providing the linkage site is located in a conformational change region of the DNA polymerase.
  • providing that the amino acid residue of the ligation site is located in the conformational change region of the DNA polymerase means that the DNA polymerase generates a conformation of a specific region of the DNA polymerase every time a DNA is synthesized when the DNA polymer is synthesized. change.
  • a conformational change in the DNA polymerase causes a change in the current of the single molecule device.
  • amino acid residues located in the conformational change region of the DNA polymerase are preferably linked to a single molecule device.
  • attachment of the amino acid residue providing the attachment site to the single molecule device does not affect the conformational change of the DNA polymerase.
  • the DNA polymerase and the single The molecular device is only joined by a ligation site, preferably the amino acid residue providing the ligation site occurs only once in the DNA polymerase.
  • the amino acid residue providing the attachment site appears only once in the DNA polymerase means that in order to ensure that there is only one single attachment site, it is necessary to make the amino acid residue providing the linkage site It occurs only once in the DNA polymerase. If the DNA polymerase has an amino acid or amino acid sequence at the other position in addition to the junction site, the amino acid or amino acid sequence at other positions needs to be modified or mutated to prevent other positions. The amino acid or amino acid sequence is linked to a single molecule device. If the DNA polymerase has no amino acid or amino acid sequence identical to the unique ligation site except for the unique ligation site, it is not necessary to modify or mutate the amino acid or amino acid sequence at other positions.
  • modifications or mutations refer to modifications or mutations made in accordance with conventional knowledge of those skilled in the art without altering the DNA polymerase activity and conformational changes.
  • the amino acid sequence represented by SEQ ID NO. 1 is replaced with a cysteine residue at position 907 from the N terminus, and any amino acid residue other than a cysteine residue is substituted.
  • a serine residue is preferred.
  • DNA polymerases include, but are not limited to, E. coli DNA polymerase I, II, III, IV or V, or DNA polymerases ⁇ , ⁇ , ⁇ , ⁇ or ⁇ ; preferably E. coli DNA polymerase I.
  • E. coli DNA polymerase I the amino acid sequence of E. coli DNA polymerase I is set forth in SEQ ID NO.
  • nucleotide sequence of E. coli DNA polymerase I is as shown in SEQ ID NO.
  • the DNA polymerase is selected from the group consisting of
  • B1 a protein having the amino acid sequence set forth in SEQ ID NO. 3, or a functional mutant thereof;
  • B2 A protein having the amino acid sequence set forth in SEQ ID NO. 1, or a functional mutant thereof.
  • the term "functional mutant” refers to an amino acid sequence obtained by substitution and/or deletion and/or addition of one or several amino acid residues, and still retains its original biological activity, ie, directs DNA. Synthetic protein.
  • the functional mutant of the protein having the amino acid sequence set forth in SEQ ID NO. 3 is one or more amino acid residues of the amino acid sequence set forth in SEQ ID NO. Substituted and/or deleted and/or added proteins having the same function.
  • the functional mutant of the protein having the amino acid sequence of SEQ ID NO. 1 is the amino acid sequence of SEQ ID NO. 1 from position N of the N-terminus.
  • the cysteine residue is replaced with a non-cysteine residue, and the leucine residue at position 790 becomes a cysteine residue; preferably, SEQ ID NO.
  • the amino acid sequence shown in the amino acid sequence was replaced with a serine residue at position 907 from the N-terminus, and a leucine residue at position 790 was changed to a cysteine residue.
  • the DNA polymerase when the DNA polymerase is linked to the single molecule device, the DNA polymerase is ligated to the amino acid sequence of SEQ ID NO. 1 from the N-terminus at position 790. Cysteine.
  • the DNA polymerase and the single molecule device can be ligated using any linkage known to those skilled in the art, as long as the activity and conformational changes of the DNA polymerase are maintained.
  • the DNA polymerase and the graphene-based monomolecular device can be joined as follows:
  • the DNA polymerase and the silicon nanowire single molecule device can be joined as follows:
  • step (3) mixing the electrical circuit of step (3) with sodium salt of N-hydroxysuccinimide sulfonate to obtain an electrical circuit activated by Sulfo-NHS;
  • the DNA polymerase region of the DNA polymerase-modified single molecule device is coated by a microcavity, and preferably, the material of the microcavity is polydimethylene. Silicone.
  • each DNA polymerase is coated with a microcavity.
  • the DNA polymerase modified single molecule device is disposable.
  • the DNA polymerase modified single molecule device is recyclable.
  • a second aspect of the invention provides a DNA sequencing method comprising the steps of:
  • the DNA to be tested is single-stranded DNA or double-stranded DNA
  • the double-stranded DNA is first denatured and then mixed with the sequencing primer and dNTP.
  • the 3' end of the DNA to be tested is added with a tag sequence, the nucleotide sequence of the tag sequence and the sequencing primer
  • the nucleotide sequence is reverse-complementary; preferably, the nucleotide sequence of the sequencing primer is the sequence shown in SEQ ID NO. 4, and the nucleotide sequence of the tag sequence is SEQ ID NO. 4 from 5' The sequence shown at positions 51 to 68 ends.
  • step (2) in the step (2),
  • the DNA polymerase region of the DNA polymerase modified single molecule device is coated by a microcavity, preferably the material of the microcavity is polydimethylsiloxane;
  • reaction system of the step (1) is added to the microcavity.
  • step (4) comprises:
  • the sequencing method according to the invention separately sequences the following DNA to be tested:
  • the sequencing process specifically includes the following steps:
  • the nucleotide sequence of the tag sequence is inversely complementary to the nucleotide sequence of the sequencing primer;
  • step (2) the product is mixed with dATP, dCTP, dTTP and dGTP, respectively, to obtain four systems, each system is added to the DNA polymerase modified single molecule device, and the electrical signal is read, and other
  • the dNTPs in the system where the electrical signals of the three systems are significantly different are a1) or a2):
  • the nucleotide of the first segment of the 3' end of the DNA to be tested consists of N nucleotides, N being a natural number and greater than or equal to 2;
  • the nucleotide of the second segment of the 3' end of the DNA to be tested consists of M nucleotides, M being a natural number and greater than or equal to 2;
  • step (4) Repeat step (4) until the sequencing result of the DNA to be tested is obtained.
  • N is preferably 50.
  • M is preferably 50.
  • the DNA to be tested is single-stranded DNA.
  • the DNA to be tested is double-stranded DNA, it is first denatured and then mixed with the sequencing primer.
  • the nucleotide sequence of the sequencing primer is preferably the sequence shown in SEQ ID NO.
  • the nucleotide sequence of the tag sequence can be as shown in SEQ ID NO. 4 from positions 51 to 68 from the 5' end.
  • a third aspect of the invention provides a kit for DNA sequencing comprising a DNA polymerase and a single molecule device, wherein:
  • the single molecule device is a two-dimensional nanomaterial single molecule device, preferably a graphene single molecule device; or a low dimensional nano material single molecule device, preferably a one-dimensional nano material single molecule device, more preferably a silicon nanowire single molecule device.
  • the DNA polymerase is selected from the group consisting of
  • B1 a protein having the amino acid sequence set forth in SEQ ID NO. 3, or a functional mutant thereof;
  • B2 A protein having the amino acid sequence set forth in SEQ ID NO. 1, or a functional mutant thereof.
  • the DNA polymerase is present in the separate compartment with the single molecule device, which is ligated in situ when used; or the DNA polymerase is linked to The single molecule device; preferably the DNA polymerase has a binding site to the single molecule device, and the amino acid residue providing the linkage site is located in a conformational change region of the DNA polymerase; more preferably, the DNA The polymerase region is coated via a microcavity.
  • the kit further comprises sequencing primers, dNTPs, and/or instructions for use.
  • the method provided by the present invention can perform DNA sequencing, and the sequencing result shows that the synthesis speed of DNA is related to the existing literature (Tivoli J. Olsen, Yongki Choi, Patrick C. Sims, O. Tolga Gul, Brad L. Corso, Chengjun Dong, William A. Brown, Philip G. Collins and Gregory A. Weiss, Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment), Journal of the American Chemical Society 2013, 135, 7855-7860) Consistent.
  • the DNA sequencing method provided by the invention has important application value.
  • DNA polymerase-modified single molecule device, DNA sequencing method and kit according to the present invention can be widely applied to DNA sequencing, and have at least the following advantages:
  • the rate of sequencing depends on the rate of reaction of the DNA polymerase itself, and is therefore much faster than chemical sequencing;
  • DNA polymerases have inherent continuities, one can measure very long sequences, such as thousands of bases, while second-generation sequencing can only detect hundreds of bases.
  • Figure 1 is a flow diagram of the preparation of a DNA polymerase modified graphene single molecule device.
  • Figure 2 shows the electrical properties of the low-resolution probe station; where Vsd is the source-drain bias and Isd is the source-drain current.
  • FIG. 3 is a schematic illustration of a DNA polymerase modified graphene single molecule device and a DNA polymerase modified silicon nanowire single molecule device.
  • Figure 4 is a reaction scheme in which a cysteine and a treated graphene-based single molecule device are chemically reacted.
  • Figure 5 is a kinetic data and peak statistical results of DNA sequencing of template strand C using a DNA polymerase modified graphene single molecule device.
  • Figure 6 is a statistical analysis of the high-state and low-state time of DNA sequencing of template strand C using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 7 is a kinetic data and peak statistical results of DNA sequencing of template strand A using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 8 is a statistical analysis of the high-state and low-state time of DNA sequencing of template strand A using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 9 is a kinetic data and peak statistical results of DNA sequencing of template strand G using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 10 is a statistical analysis of the high-state and low-state time of DNA sequencing of the template strand G using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 11 is a kinetic data and peak statistical results of DNA sequencing of template strand T using a graphene-based single molecule device modified with DNA polymerase.
  • Figure 12 is a statistical analysis of the high-state and low-state time of DNA sequencing of the template strand T using a DNA polymerase-modified graphene single molecule device.
  • Figure 13 is a statistical analysis of the high-state and low-state time of DNA sequencing of a random strand using a graphene-based single molecule device modified with a DNA polymerase.
  • Figure 14 shows the preparation of a DNA polymerase modified silicon nanowire single molecule device.
  • the DTT buffer was pH 7.8, 10 mM Tris-HCl buffer containing 50 mM NaCl, 10 mM MgCl 2 and 1 mM DTT.
  • HF solution It is a mixture of 7 parts by volume of 40% (mass%) aqueous solution of NH4F and 1 part by volume of HF aqueous solution; wherein the concentration of HF aqueous solution is 40% (mass%), which is a product of Beijing Chemical Plant.
  • Single-stranded DNA molecule A 5'-(C)50ACTGGCCGTCGTTTTACA-3' (SEQ ID NO. 4).
  • Single-stranded DNA molecule B 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO. 5).
  • Single-stranded DNA molecule C 5'-(A)50ACTGGCCGTCGTTTTACA-3' (SEQ ID NO. 6).
  • Single-stranded DNA molecule D 5'-(G)50ACTGGCCGTCGTTTTACA-3' (SEQ ID NO. 7).
  • the single-stranded DNA molecule pentane 5'-(T)50ACTGGCCGTCGTTTTACA-3' (SEQ ID NO. 8).
  • Single-stranded DNA molecules have:
  • the template chain C is prepared by artificially synthesizing single-stranded DNA molecule A and single-stranded DNA molecule B, and then 10 ⁇ L of single-stranded DNA molecule A (single-stranded DNA molecule A concentration is 10 ⁇ M), 10 ⁇ L of single-stranded DNA molecule B (single The concentration of the strand DNA molecule B was 10 ⁇ M), mixed with 980 ⁇ L of DTT buffer, heated at 70 ° C for 1 h, and naturally cooled to room temperature to obtain a template chain C (5'-end double-stranded single-stranded form). Template chain C was prepared 3 h before the kinetic test.
  • Template strand A is prepared by artificially synthesizing single-stranded DNA molecule C and single-stranded DNA molecule B, and then 10 ⁇ L of single-stranded DNA molecule C (single-stranded DNA molecule C concentration is 10 ⁇ M), 10 ⁇ L of single-stranded DNA molecule B (single The concentration of the strand DNA molecule B was 10 ⁇ M), mixed with 980 ⁇ L of DTT buffer, heated at 70 ° C for 1 h, and naturally cooled to room temperature to obtain template chain A (5'-end double-stranded single-stranded form). Template chain A was prepared 3 h before the kinetic test.
  • the template strand G is prepared by artificially synthesizing a single-stranded DNA molecule and a single-stranded DNA molecule B, and then 10 ⁇ L of a single-stranded DNA molecule (single-stranded DNA molecule with a concentration of 10 ⁇ M) and 10 ⁇ L of a single-stranded DNA molecule B (single The concentration of the strand DNA molecule B was 10 ⁇ M), mixed with 980 ⁇ L of DTT buffer, heated at 70 ° C for 1 h, and naturally cooled to room temperature to obtain a template chain G (5'-end double-stranded single-stranded form).
  • the template chain G needs to be prepared 3 hours before the kinetic test.
  • the template strand T is prepared by artificially synthesizing a single-stranded DNA molecule penta and a single-stranded DNA molecule B, and then 10 ⁇ L of a single-stranded DNA molecule penta (a single-stranded DNA molecule having a concentration of 10 ⁇ M) and 10 ⁇ L of a single-stranded DNA molecule B (single The concentration of the strand DNA molecule B was 10 ⁇ M), mixed with 980 ⁇ L of DTT buffer, heated at 70 ° C for 1 h, and naturally cooled to room temperature to obtain a template chain T (5'-end double-stranded single-stranded form).
  • the template chain T needs to be prepared 3 h before the kinetic test.
  • the random strand is prepared by artificially synthesizing a single-stranded DNA molecule and a single-stranded DNA molecule B, and then 10 ⁇ L of a single-stranded DNA molecule (a single-stranded DNA molecule having a concentration of 10 ⁇ M) and 10 ⁇ L of a single-stranded DNA molecule B (single-stranded)
  • concentration of DNA molecule B was 10 ⁇ M), mixed with 980 ⁇ L of DTT buffer, heated at 70 ° C for 1 h, and naturally cooled to room temperature to obtain a random chain (5'-end double-stranded single-stranded form).
  • the random strands were prepared 3 h before the kinetic test.
  • the low-resolution probe station consists of a semiconductor parameter meter (Agilent's product, model 4155C) and a probe station (Karl Suess's product, model PM5).
  • the high-resolution probe station consists of two core components: a preamplifier (Digital Instruments, model DL1211) and a lock-in amplifier (Zurich Instruments, model HF2LI).
  • 1,3,5-tris(4-carbonylphenyloxy)benzene is a product of Jinan Henghua Technology Co., Ltd.
  • the dATP solution was obtained by dissolving dATP in DTT buffer.
  • the dCTP solution was obtained by dissolving dCTP in DTT buffer.
  • the dTTP solution was obtained by dissolving dTTP in DTT buffer.
  • the dGTP solution was obtained by dissolving dGTP in DTT buffer.
  • dATP, dCTP, dTTP and dGTP are products of Beijing Saibaisheng Gene Technology Co., Ltd.
  • Example 1 DNA sequencing using a graphene-based single molecule device modified with DNA polymerase
  • the preparation process of graphene-based single-molecule devices is as follows: Cao Y, Dong S, Liu S, He L, Gan L, Yu X, Steigerwald ML, Wu X, Liu Z, Guo X. Building high-throughput molecular junctions using Indented graphene point contacts.
  • a graphene electrode having a nanogap on a graphene-based monomolecular device hereinafter referred to as a graphene electrode, the structural formula of which is shown in Fig. 1 (1)).
  • step 1 the graphene-based monomolecular device and p-phenylenediamine (see (2) in Fig. 1) are mixed.
  • the ends of the graphene electrode are modified with an amino group.
  • the structural formula of the graphene electrode whose terminal is modified with an amino group is shown in (3) of Fig. 1.
  • step 2 1,3,5-tris(4-carbonylphenyloxy)benzene (see (4) in Fig. 1) and a graphene electrode whose terminal is modified with an amino group were mixed to obtain an electrical circuit.
  • the electrical properties of the electrical loop were examined using a low resolution probe station. The results are shown in the upper left panel of Figure 2.
  • the partial structural formula of the electrical circuit is shown in (5) of Fig. 1.
  • step 3 the electrical circuit was mixed with sodium salt of N-hydroxysuccinimide (NHS) sulfonate (Sulfo-NHS) (see (6) in Fig. 1) to obtain an electrical circuit activated by Sulfo-NHS.
  • NHS N-hydroxysuccinimide
  • Sulfo-NHS sodium salt of N-hydroxysuccinimide
  • the electrical properties of the electrical circuit activated by Sulfo-NHS were detected using a low resolution probe station. The results are shown in the upper right panel of Figure 2.
  • the partial structural formula of the electrical circuit activated by Sulfo-NHS is shown in (7) of Fig. 1.
  • step 4 the Sulfo-NHS activated electrical circuit and N-(2-aminoethyl)maleimide hydrochloride (see (8) in Fig. 1) are mixed to obtain a treated graphene single.
  • Molecular device The partial structural formula of the treated graphene-based single molecule device is shown in (9) of FIG.
  • the electrical properties of the treated graphene-based single molecule device were examined using a low resolution probe station. The results are shown in the lower left panel of Figure 2.
  • Figure 2 (1) is the conductivity of device 5 of Figure 1, indicating the successful preparation of molecular devices with carboxyl groups.
  • Figure 2 (1) is the conductivity of device 7 of Figure 1, indicating the successful preparation of molecular devices with active esters.
  • Figure 2 (1) is the conductivity of device 9 of Figure 1, indicating the successful preparation of a maleimide device.
  • Figure 2 (1) is the conductivity of device 11 of Figure 1, indicating the successful preparation of a DNA polymerase modified molecular device.
  • the amino acid sequence of Escherichia coli DNA polymerase I (hereinafter referred to as DNA polymerase I) is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO.
  • the modified DNA polymerase I needs to have the following characteristics: (1) having the biological activity of DNA polymerase I, that is, guiding the synthesis of DNA; (2) the modified DNA polymerase I can be treated with the graphene single after treatment.
  • the amino acid residue providing the ligation site (designated as a donor amino acid residue) should be located in the conformational region of the engineered DNA polymerase I (in order to increase the sensitivity of subsequent DNA sequencing); (3)
  • the amino acid corresponding to the donor amino acid residue and the amino acid residue that does not provide the attachment site (designated as a non-donor amino acid residue) differ in the amino acid species.
  • the modified DNA polymerase I is a cysteine residue at position 907 from the N-terminus of DNA polymerase I (amino acid sequence as shown in SEQ ID NO. 1), which is changed to a serine residue. The amino acid residue becomes a cysteine residue. The amino acid residue at position 790 from the N-terminus of SEQ ID NO. 3 is located in the conformational change region.
  • the treated graphene-based single molecule device and an excess of the modified DNA polymerase I are mixed to obtain a DNA polymerase-modified graphene-based single molecule device (Fig. 3 in the left panel), the partial structural formula of the DNA polymerase modified graphene single molecule device is shown in Figure 1 (11).
  • the electrical properties of the DNA polymerase modified graphene single molecule device were examined using a low resolution probe station. The results are shown in the lower right panel of Figure 2.
  • the reaction formula of the chemical reaction of cysteine in the modified DNA polymerase I and the treated graphene single molecule device is shown in Fig. 4.
  • the prepared DNA polymerase-modified graphene-based single molecule device was placed on a low-resolution probe station to observe conductivity.
  • the graphene-based single molecule device having conductivity was selected for the following experiment. Specific steps refer to the literature Tivoli J. Olsen, Yongki Choi, Patrick C. Sims, O. Tolga Gul, Brad L. Corso, Chengjun Dong, William A. Brown, Philip G. Collins and Gregory A. Weiss, Electronic Measurements of Single- Molecule Processing by DNA Polymerase I (Klenow Fragment), Journal of the American Chemical Society 2013, 135, The method reported in 7855-7860.
  • the microcavity is made of polydimethylsiloxane. , PDMS), to obtain a graphene-based single molecule device containing microcavities.
  • step 2 the graphene-based single-molecule device containing the microcavity is placed on the high-resolution probe station, and the sample is sampled for 5 minutes for kinetic testing (ie, the test source leakage current changes with time).
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of A in Figure 5, indicating that no oscillating signal of DNA synthesis was observed without sample conditions.
  • step 3 After completing step 1, add 10 ⁇ L of DTT buffer to the microcavity of the graphene-based single-molecule device containing the microcavity, and sample for 5 minutes for kinetic testing (ie, test the source leakage current as a function of time).
  • the experimental results are shown in the left panel of B in Fig. 5.
  • the preliminary peak statistic was then performed using MatLab software.
  • the experimental results are shown in the right panel of B in Figure 5, indicating that no oscillating signal of DNA synthesis was observed in the buffer without the sample.
  • step 4 After completing step 1, add 30 ⁇ L of DTT buffer containing 100 nM template chain C to the microcavity of the graphene-based single-molecule device containing microcavity, and sample for 5 minutes for kinetic testing (ie, test source leakage current with time). Change situation).
  • the experimental results are shown in the left panel of C in Fig. 5.
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of C in Figure 5, indicating that no oscillation signal of DNA synthesis was observed in the template chain and buffer without sample.
  • step 1 add 30 ⁇ L of DTT buffer containing 100 nM template chain C, 5 ⁇ L of dATP solution (concentration: 5 ⁇ M), and 5 ⁇ L of dTTP solution to the microcavity of the graphene-based monomolecular device containing microcavity.
  • dATP solution concentration: 5 ⁇ M
  • dTTP solution 5 ⁇ L
  • the sample was sampled for 10 minutes for kinetic testing (ie, test source leakage current as a function of time).
  • the experimental results are shown in the left diagram of D in Fig. 5.
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of D in Figure 5, indicating that there are template chains, nucleic acid molecules and buffers, but no oscillation signals of DNA synthesis were observed under base mismatch conditions.
  • step 1 After completing step 1, add 30 ⁇ L to the microcavity of the graphene-based single-molecule device containing the microcavity.
  • DTT buffer containing 100 nM template strand C and 5 ⁇ L of dGTP solution (concentration: 5 ⁇ M) were sampled for 10 minutes to test the change of source and drain current with time during DNA synthesis by polymerase, that is, when DNA polymerase was used for DNA polymerization.
  • Kinetic data The experimental results are shown in the left diagram of E in Fig. 5. The preliminary peak statistics were then performed using MatLab software. The experimental results are shown in the right panel of E in Fig. 5, indicating that there are template chains, nucleic acid molecules and buffers, and the oscillating signals of DNA synthesis were observed under base matching conditions.
  • the results show that the kinetic data in step 6 is apparently different from the kinetic data in other control experiments, that is, when there is deoxynucleotide in the system with template chain C and matching
  • the learning data can be different from the other four groups of control experiments.
  • step 6 Pull the kinetic data obtained in step 6 and use the Qub software to select the corresponding high current (ie, high current) and low current (ie, small current) (A in Figure 6).
  • the high-state time ( ⁇ hi) corresponding to the high-state current and the low-state time ( ⁇ dow) corresponding to the low-state current are statistically analyzed.
  • the experimental results are shown in B and C in Fig. 6, respectively. According to the average low-state time and the average high-state time, the synthesis rate of DNA in this system is calculated to be 62.62 dGTP nucleotides per second (see Table 1), compared with the existing literature (Tivoli J).
  • Template chain C Template chain G Template chain T Template chain A ⁇ low (ms) 0.156 0.122 0.710 2.825 ⁇ hi (ms) 15.813 6.823 3.585 3.575 Synthesis speed (pieces per second) 62.62 143.99 232.83 156.25
  • step 2 Same as step 2 in 2.
  • the experimental results of the kinetic test are shown in the left panel of A in Figure 7.
  • the experimental results of the peak statistics are shown in the right panel of A in Figure 7, indicating that no oscillating signal of DNA synthesis was observed in the absence of sample conditions.
  • step 3 Same as step 3 in step 3.
  • the experimental results of the kinetic test are shown in the left panel of B in Figure 7.
  • the experimental results of the peak statistics are shown in the right panel of B in Fig. 7, indicating that no oscillation signal of DNA synthesis was observed in the buffer without the sample.
  • step 4 of step 2 only the template chain C is replaced with the template chain A.
  • the experimental results of the kinetic test are shown in the left panel of C in Figure 7.
  • the experimental results of the peak statistics are shown in the right panel of C in Figure 7, indicating that only the template chain and the buffer have no observed oscillating signals for DNA synthesis.
  • step 1 add 30 ⁇ L of DTT buffer containing 100 nM template chain A, 5 ⁇ L of dATP solution (concentration of 5 ⁇ M), and 5 ⁇ L of dGTP solution to the microcavity of the microenventuring graphene-based monomolecular device.
  • dATP solution concentration of 5 ⁇ M
  • dGTP solution 5 ⁇ L
  • the sample was sampled for 10 minutes for kinetic testing (ie, test source leakage current as a function of time).
  • the experimental results are shown in the left diagram of D in Fig. 7.
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of D in Figure 7, which indicates that there are template chains, nucleic acid molecules and buffers, but no oscillation signals of DNA synthesis were observed under base mismatch conditions.
  • step 6 After completing step 1, add 30 ⁇ L of DTT buffer containing 100 nM template chain A and 5 ⁇ L of dTTP solution (concentration: 5 ⁇ M) to the microcavity of the graphene-based monomolecular device containing microcavity, and sample for 10 minutes to test the polymerization.
  • the change of source leakage current with time during DNA synthesis that is, the kinetic data of DNA polymerase during DNA polymerization.
  • the experimental results are shown in the left diagram of E in Fig. 7. The preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of E in Figure 7, which indicates that there are template chains, nucleic acid molecules and buffers, and the oscillating signal of DNA synthesis was observed under base matching conditions.
  • the results show that the kinetic data in step 6 is apparently different from the kinetic data in other control experiments, that is, when there is deoxynucleotide in the system with template chain A and matching
  • the learning data can be different from the other four groups of control experiments.
  • step 7 of step 7 the high-state time corresponding to the high-state current ( ⁇ hi) and the low-state time corresponding to the low-state current ( ⁇ low) are statistically analyzed.
  • the experimental results are shown in Fig. 8 (the left graph is the low-state time). Right For high state time).
  • the synthesis rate of DNA in this system was calculated to be 156.25 dTTP nucleotides per second (see Table 1), and the existing literature (Tivoli J. Olsen, Yongki Choi, Patrick) C. Sims, O. Tolga Gul, Brad L. Corso, Chengjun Dong, William A. Brown, Philip G. Collins and Gregory A. Weiss, Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment), Journal of The synthetic speeds described in the American Chemical Society 2013, 135, 7855-7860 are substantially the same.
  • step 2 Same as step 2 in 2.
  • the experimental results of the kinetic test are shown in the left panel of A in Figure 9.
  • the experimental results of the peak statistics are shown in the right panel of A in Fig. 9, indicating that no oscillating signal of DNA synthesis was observed under the condition of the sample.
  • step 3 Same as step 3 in step 3.
  • the experimental results of the kinetic test are shown in the left panel of B in Fig. 9.
  • the experimental results of the peak statistics are shown in the right panel of B in Fig. 9, indicating that no oscillation signal of DNA synthesis was observed in the buffer without the sample.
  • step 4 of step 2 only the template chain C is replaced with the template chain G.
  • the experimental results of the kinetic test are shown in the left panel of C in Figure 9.
  • the experimental results of the peak statistics are shown in the right panel of C in Figure 9, indicating that only the template chain and the buffer were not observed to have an oscillating signal for DNA synthesis.
  • step 1 add 30 ⁇ L of DTT buffer containing 100 nM template strand G, 5 ⁇ L of dATP solution (concentration: 5 ⁇ M), and 5 ⁇ L of dGTP solution to the microcavity of the graphene-based monomolecular device containing microcavity.
  • 5 ⁇ M 5 ⁇ L
  • 5 ⁇ L of dTTP solution concentration of 5 ⁇ M
  • the sample was sampled for 10 minutes for kinetic testing (ie, test source leakage current as a function of time).
  • the experimental results are shown in the left diagram of D in Fig. 9.
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of D in Figure 9, indicating that there are template strands, nucleic acid molecules and buffers, but no oscillation signals of DNA synthesis were observed under base mismatch conditions.
  • step 6 After completing step 1, add 30 ⁇ L of DTT buffer containing 100 nM template strand G and 5 ⁇ L of dCTP solution (concentration: 5 ⁇ M) to the microcavity of the graphene-based monomolecular device containing microcavity, and sample. 10 minutes, test the change of source leakage current with time during DNA synthesis, that is, the kinetic data of DNA polymerase during DNA polymerization.
  • the experimental results are shown in the left diagram of E in Fig. 9. The preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of E in Figure 9, indicating that there are template strands, nucleic acid molecules and buffers, and the oscillation signal of DNA synthesis was observed under base matching conditions.
  • step 6 The results show that the kinetic data in step 6 is essentially different from the kinetic data in other control experiments, that is, when there is deoxynucleotide in the system with the template chain G and its matching, the power of the system
  • the learning data can be different from the other four groups of control experiments.
  • step 7 in step 2 the high-state time corresponding to the high-state current ( ⁇ hi) and the low-state time corresponding to the low-state current ( ⁇ low) are statistically analyzed.
  • the experimental results are shown in Fig. 10 (the left graph is the low-state time). The picture on the right is high time).
  • the synthesis rate of DNA in this system was calculated to be 143.99 dCTP nucleotides per second (see Table 1), and the existing literature (Tivoli J. Olsen, Yongki Choi, Patrick) C. Sims, O. Tolga Gul, Brad L. Corso, Chengjun Dong, William A. Brown, Philip G. Collins and Gregory A. Weiss, Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment), Journal of The synthetic speeds described in the American Chemical Society 2013, 135, 7855-7860 are substantially the same.
  • step 2 Same as step 2 in 2.
  • the experimental results of the kinetic test are shown in the left panel of A in Figure 11.
  • the experimental results of the peak statistics are shown in the right panel of A in Figure 11, indicating that no oscillating signal of DNA synthesis was observed without sample conditions.
  • step 3 Same as step 3 in step 3.
  • the experimental results of the kinetic test are shown in the left panel of B in Figure 11.
  • the experimental results of the peak statistics are shown in the right panel of B in Figure 11, indicating that no oscillation signal of DNA synthesis was observed in the buffer without the sample.
  • step 4 of step 2 only the template chain C is replaced with the template chain T.
  • the experimental results of the kinetic test are shown in the left panel of C in Figure 11.
  • the experimental results of the peak statistics are shown in the right graph of C in Figure 11, the table It is clear that only the template chain and the buffer have no observed oscillating signal for DNA synthesis under the condition of the sample.
  • step 1 add 30 ⁇ L of DTT buffer containing 100 nM template strand T, 5 ⁇ L of dCTP solution (concentration: 5 ⁇ M), and 5 ⁇ L of dGTP solution (concentration) to the microcavity of the graphene-based monomolecular device containing microcavity.
  • 5 ⁇ M 5 ⁇ L
  • 5 ⁇ L of dTTP solution concentration of 5 ⁇ M
  • the sample was sampled for 10 minutes for kinetic testing (ie, test source leakage current as a function of time).
  • the experimental results are shown in the left panel of D in Figure 11.
  • the preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of D in Figure 11, which indicates that there are template chains, nucleic acid molecules and buffers, but no oscillation signals of DNA synthesis were observed under base mismatch conditions.
  • step 6 After completing step 1, add 30 ⁇ L of DTT buffer containing 100 nM template strand T and 5 ⁇ L of dATP solution (concentration: 5 ⁇ M) to the microcavity of the graphene-based monomolecular device containing microcavity, and sample for 10 minutes to test the polymerization.
  • the change of source leakage current with time during DNA synthesis that is, the kinetic data of DNA polymerase during DNA polymerization.
  • the experimental results are shown in the left diagram of E in Fig. 11. The preliminary peak statistics were then performed using MatLab software.
  • the experimental results are shown in the right panel of E in Figure 11, which indicates that there are template strands, nucleic acid molecules and buffers. At the same time, the oscillating signal of DNA synthesis was observed under base matching conditions.
  • step 6 The results show that the kinetic data in step 6 is apparently different from the kinetic data in other control experiments, that is, when there is deoxynucleotide in the system with the template chain T and its matching, the power of the system
  • the learning data can be different from the other four groups of control experiments.
  • step 7 in step 2 the high-state time corresponding to the high-state current ( ⁇ hi) and the low-state time corresponding to the low-state current ( ⁇ low) are statistically analyzed.
  • the experimental results are shown in Fig. 12 (the left graph is the low-state time). The picture on the right is high time).
  • the synthesis rate of DNA in this system was calculated to be 232.83 dATP nucleotides per second (see Table 1), and the existing literature (Tivoli J. Olsen, Yongki Choi, Patrick) C. Sims, O. Tolga Gul, Brad L. Corso, Chengjun Dong, William A. Brown, Philip G. Collins and Gregory A. Weiss, Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment), Journal of The synthetic speeds described in the American Chemical Society 2013, 135, 7855-7860 are substantially the same.
  • step 1 After completion of step 1, 30 ⁇ L of DTT buffer containing 100 nM random strand (SEQ ID NO. 9) and 5 ⁇ L of solution (containing dATP, dGCTP, dATP and dTTP) were added to the microcavity of the microcavity graphene-based single molecule device. The concentration was 5 ⁇ M), and the sample was sampled for 10 minutes to test the change of source leakage current with time during DNA synthesis, that is, the kinetic data of DNA polymerase during DNA polymerization. The experimental results are shown in Figure 13, and four different oscillating signals are seen. The preliminary peak statistics were then performed using MatLab software. The experimental results are shown in the table in Figure 13. The data in this table and the data in Table 1 can determine the bases corresponding to different currents, thereby determining the sequence of the DNA according to the change in current. Therefore, DNA sequencing can be performed using a DNA polymerase modified graphene single molecule device.
  • silicon nanowire single-molecule devices The preparation process of silicon nanowire single-molecule devices is as follows: Cao Y, Dong S, Liu S, He L, Gan L, Yu X, Steigerwald ML, Wu X, Liu Z, Guo X. Building high-throughput molecular junctions using Indented graphene point contacts. Angew Chem Int Ed Engl. 2012 Dec 3;51(49):12228-32.
  • a silicon nanowire electrode (hereinafter referred to as a silicon nanowire electrode) having a nanogap on a silicon nanowire single molecule device.
  • step 1 the silicon nanowire single molecule device and the HF solution are mixed to obtain an etched silicon nanowire single molecule device.
  • a schematic structural view of a partially etched silicon nanowire single molecule device is shown in (1) of FIG.
  • step 2 After completion of step 2, the etched silicon nanowire single molecule device and 10-undecynoic acid (see (2) in Fig. 13) are mixed to obtain an electrical circuit.
  • a partial structural schematic diagram of the electrical circuit is shown in (3) of FIG.
  • step 3 the electrical circuit was mixed with sodium salt of N-hydroxysuccinimide sulfonate (Sulfo-NHS) (see (4) in Fig. 13) to obtain an electrical circuit activated by Sulfo-NHS.
  • Sulfo-NHS N-hydroxysuccinimide sulfonate
  • the partial structural formula of the Sulfo-NHS activated electrical circuit is shown in (5) of FIG.
  • step 4 the Sulfo-NHS activated electrical circuit and N-(2-aminoethyl)maleimide hydrochloride (see (6) in Fig. 13) are mixed to obtain a treated silicon nanowire single.
  • Molecular device A partial structural formula of the treated silicon nanowire single molecule device is shown in (7) of FIG.
  • step 6 the treated silicon nanowire single molecule device and the excess modified DNA polymerase I (see (8) in Fig. 7) are mixed to obtain a DNA polymerase-modified silicon nanowire single molecule device (see The right picture in Figure 3).
  • a schematic diagram of the structure of a partially DNA polymerase-modified silicon nanowire single molecule device is shown in (7) of FIG.
  • the prepared DNA polymerase-modified silicon nanowire single molecule device was placed on a low resolution probe station to observe conductivity, and a conductive DNA polymerase-modified silicon nanowire single molecule device was used in the following experiment.
  • the DNA polymerase modified graphene single molecule device is replaced by the DNA polymerase modified silicon nanowire single molecule device, and the other steps are unchanged, and the synthesis speed of the DNA in the system is obtained.
  • the DNA polymerase-modified graphene-based single-molecule device is replaced by the DNA polymerase-modified silicon nanowire single-molecule device, and the other steps are unchanged, and the ontology is obtained.
  • the rate of DNA synthesis in the system was 117.27 TTP nucleotides per second, and there was no significant difference in DNA sequencing of template strand A with a graphene-based single molecule device modified with DNA polymerase.
  • the DNA polymerase modified graphene single molecule device is replaced by the DNA polymerase modified silicon nanowire single molecule device, and the other steps are unchanged, and the synthesis speed of the DNA in the system is obtained.
  • the DNA polymerase modified graphene single molecule device is replaced by the DNA polymerase modified silicon nanowire single molecule device, and the other steps are unchanged, and the synthesis speed of the DNA in the system is obtained.
  • the DNA polymerase-modified graphene-based single-molecule device was replaced with a DNA polymerase-modified silicon nanowire single-molecule device according to the method of the fifth step of the first embodiment.
  • the other steps were unchanged, and the measured oscillating signal of the DNA synthesis was
  • the oscillating signal measured by the DNA polymerase modified graphene single molecule device is consistent; the synthesis rate of DNA in this system is 234.60 dATP nucleotides per second, and the graphene-based single molecule device modified by DNA polymerase There was no significant difference in the results of DNA sequencing of the template strand T.
  • DNA nanoparticle single molecule devices modified with DNA polymerase can be used for DNA sequencing.

Abstract

本发明公开了一种DNA测序方法。本发明所提供的DNA测序方法依次包括如下步骤：（1）在待测DNA的3'末端增加标签序列，形成含有标签序列的待测DNA；所述标签序列的核苷酸序列与测序引物的核苷酸序列反向互补；（2）将所述含有标签序列的待测DNA和所述测序引物混合，形成5'末端双链主体单链形式的产物；（3）完成步骤（2）后，将产物分别与dATP、dCTP、dTTP和dGTP混合，得到四个体系，分别将每个体系加入DNA聚合酶修饰的单分子器件，读取电信号。实验证明，采用本发明提供的方法可进行DNA测序，具有重要的应用价值。

Description

DNA测序方法
本申请要求于2016年8月31日提交中国专利局、申请号为201610798113.X发明名称为“DNA测序方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，具体涉及一种DNA测序方法。
背景技术
DNA是遗传信息的载体，其内四种碱基对的特定排列顺序是遗传信息的本质所在。确定DNA中碱基对的特定排列顺序被称为DNA测序，其对于揭示生命奥秘、控制遗传过程、推动疾病诊断、提升医疗技术有着根本的促进作用。迄今为止，先后已经有两代测序技术被成功研发并得到广泛应用：第一代基于双脱氧链终止法的测序技术打开了人类揭示遗传奥秘的大门，奠定了基因工程最为重要的基础；第二代大规模平行合成荧光检测法，则是在第一代测序技术的基础上，利用PCR扩增及荧光检测技术，该方法在很大程度上提高了测序的效率，为第一个人类基因组测序的完成做出了巨大贡献。尽管前两代测序技术发展的很成熟，但是仍旧有许多弊端亟待解决，如PCR扩增耗时长、成本高，荧光标记及检测过程复杂不稳定等等。同时，随着人类对生命本质的理解加深和医学领域技术手段的不断进步，个性化治疗方案及分子诊断逐渐成为热点，这对DNA测序提出了更高的要求。因此，如何快速、准确、低成本进行DNA测序已经成为炙手可热的研究领域。
目前相对成熟的DNA测序技术是基于纳米孔的电学测试平台，甚至已经有商业化测试仪原型出现，但还存在诸多缺陷，如DNA移动速度难以控制、测序信号分辨率低、测序不准确等等，使基于此方法的测序技术的发展陷入瓶颈。随着各种新型纳米材料的不断涌现，以及微纳加工技术的不断升级进步，具有单分子检测能力的器件平台迅猛发展，为DNA测序的持续推进提供源源 不断的动力和机会。
发明内容
为解决现有技术的问题，本发明的第一方面提供了一种DNA聚合酶修饰的单分子器件，其中所述单分子器件是二维纳米材料单分子器件，优选石墨烯基单分子器件；或低维纳米材料单分子器件，优选一维纳米材料单分子器件，更优选硅纳米线单分子器件。
本文所用的术语“单分子器件”可以是现有技术中任何能响应单分子变化的器件，即本领域技术人员通过检测或观察该单分子器件的变化，便能知晓相应的分子是否发生变化。本发明优选地使用了以石墨烯或硅纳米线作为电极的单分子器件，并使其与所述DNA聚合酶连接，通过检测该单分子器件的电流变化，就可知晓所述DNA聚合酶的构象是否发生了变化。
石墨烯基单分子器件和所述硅纳米线单分子器件的制备过程均可参考文献(Cao Y，Dong S，Liu S，He L，Gan L，Yu X，Steigerwald ML，Wu X，Liu Z，Guo X.Building high-throughput molecular junctions using indented graphene point contacts.Angew Chem Int Ed Engl.2012 Dec 3；51(49)：12228-32.)中记载的方法。
在本发明第一方面的一个优选实施方案中，所述DNA聚合酶具有与所述单分子器件的连接位点，且提供所述连接位点的氨基酸残基位于DNA聚合酶的构象变化区。
在本文中，“提供该连接位点的氨基酸残基位于所述DNA聚合酶的构象变化区”是指：DNA聚合酶在合成DNA时，每接纳一个dNTP，DNA聚合酶特定区域的构象会发生改变。将DNA聚合酶与单分子器件连接后，DNA聚合酶的构象变化会引起单分子器件的电流变化。为了提高后续DNA测序的灵敏度，优选地将位于DNA聚合酶的构象变化区中的氨基酸残基与单分子器件连接。另外，提供连接位点的氨基酸残基与单分子器件连接后不影响所述DNA聚合酶的构象变化。
在本发明第一方面的另一个优选实施方案中，所述DNA聚合酶与所述单 分子器件仅通过一个连接位点连接，优选地，提供所述连接位点的氨基酸残基在所述DNA聚合酶中仅出现一次。
在本文中，“DNA聚合酶与所述单分子器件仅通过一个连接位点连接”指的是只有一个氨基酸残基与单分子器件连接。
在本文中，“提供所述连接位点的氨基酸残基在所述DNA聚合酶中仅出现一次”指的是：为了保证只有唯一一个连接位点，需要使得提供该连接位点的氨基酸残基在所述DNA聚合酶中仅出现一次。若所述DNA聚合酶中除了该连接位点外，在其它位置还有与该连接位点一致的氨基酸或氨基酸序列，则需将其它位置的氨基酸或氨基酸序列进行修饰或突变，以防止其它位置的氨基酸或氨基酸序列与单分子器件连接。如果所述DNA聚合酶中，除了该唯一连接位点外，其余位置没有与该唯一连接位点一致的氨基酸或氨基酸序列，则无需将其它位置的氨基酸或氨基酸序列进行修饰或突变。所述修饰或突变指的是在不改变所述DNA聚合酶活性和构象变化的情况下，结合本领域技术人员的常规知识，进行的修饰或突变。例如本发明的一个优选实施方案中，将SEQ ID NO.1所示的氨基酸序列自N末端起第907位的半胱氨酸残基替换为半胱氨酸残基以外的任意氨基酸残基，优选丝氨酸残基。
在本发明的情况下，DNA聚合酶包括但不限于：大肠杆菌DNA聚合酶I、II、III、IV或V，或者DNA聚合酶α、β、γ、δ或ε；优选大肠杆菌DNA聚合酶I。优选地大肠杆菌DNA聚合酶I的氨基酸序列如SEQ ID NO.1所示。进一步优选地，大肠杆菌DNA聚合酶I的的核苷酸序列如SEQ ID NO.2所示。
在本发明第一方面的又一个优选实施方案中，所述DNA聚合酶选自：
b1)具有如SEQ ID NO.3所示的氨基酸序列的蛋白质，或其功能性突变体；或者
b2)具有如SEQ ID NO.1所示的氨基酸序列的蛋白质，或其功能性突变体。
在本文中，术语“功能性突变体”是指氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的，且仍然保持其原有生物学活性、即指导DNA的合成的蛋白质。
在本发明第一方面的优选实施方案中，具有如SEQ ID NO.3所示的氨基酸序列的蛋白质的功能性突变体是将SEQ ID NO.3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质。
在本发明第一方面的优选实施方案中，具有如SEQ ID NO.1所示的氨基酸序列的蛋白质的功能性突变体是将SEQ ID NO.1所示的氨基酸序列自N末端起第907位的半胱氨酸残基替换为非半胱氨酸残基，第790位的亮氨酸残基变为半胱氨酸残基所得到的蛋白质；优选地，是将SEQ ID NO.1所示的氨基酸序列自N末端起第907位的半胱氨酸残基替换为丝氨酸残基，第790位的亮氨酸残基变为半胱氨酸残基所得到的蛋白质。
在本发明第一方面的优选实施方案中，DNA聚合酶与单分子器件的连接时，所述DNA聚合酶的连接位点为SEQ ID NO.1所示的氨基酸序列自N末端起第790位的半胱氨酸。
DNA聚合酶和单分子器件可以采用本领域技术人员公知公用的任何连接方式连接，只要保持所述DNA聚合酶的活性和构象变化即可。
优选地，DNA聚合酶和石墨烯基单分子器件可以按照下述步骤进行连接：
(1)制备石墨烯基单分子器件；
(2)将石墨烯基单分子器件和对苯二胺混合，得到末端被氨基修饰的石墨烯电极；
(3)将1，3，5-tris(4-carbonylphenyloxy)benzene和所述末端被氨基修饰的石墨烯电极混合，得到电学回路；
(4)将电学回路和N-羟基琥珀酰亚胺磺酸钠盐混合，得到Sulfo-NHS活化的电学回路；
(5)将Sulfo-NHS活化的电学回路和N-(2-氨乙基)马来酰亚胺盐酸盐混合，得到处理后的石墨烯基单分子器件；
(6)将处理后的石墨烯基单分子器件和过量的DNA聚合酶混合，得到DNA聚合酶修饰的石墨烯基单分子器件。
优选地，DNA聚合酶和硅纳米线单分子器件可以按照下述步骤进行连接：
(1)制备硅纳米线单分子器件；
(2)将硅纳米线单分子器件和HF溶液混合，得到刻蚀的硅纳米线单分子器件；所述HF溶液由7体积份的40％(质量百分比)的NH4F水溶液和1体积份的HF水溶液混合而成，其中HF水溶液的浓度为40％(质量百分比)，具体可为北京化工厂的产品。
(3)将所述刻蚀的硅纳米线单分子器件和10-十一碳炔酸混合，得到电学回路；
(4)将步骤(3)所述电学回路和N-羟基琥珀酰亚胺磺酸钠盐混合，得到Sulfo-NHS活化的电学回路；
(5)将步骤(4)所述Sulfo-NHS活化的电学回路和N-(2-氨乙基)马来酰亚胺盐酸盐混合，得到处理后的硅纳米线单分子器件；
(6)将所述处理后的硅纳米线单分子器件和过量的所述DNA聚合酶混合，得到DNA聚合酶修饰的硅纳米线单分子器件。
在本发明第一方面的特别优选的实施方案中，所述DNA聚合酶修饰的单分子器件的DNA聚合酶区域是经微腔包被的，优选地，所述微腔的材质是聚二甲基硅氧烷。
优选地，每一个DNA聚合酶用一个微腔包被。
在本发明第一方面的一个具体实施方案中，所述DNA聚合酶修饰的单分子器件是一次性的。
在本发明第一方面的另一个具体实施方案中，所述DNA聚合酶修饰的单分子器件是可重复利用的。
本发明的第二方面提供一种DNA测序方法，其包括以下步骤：
(1)将待测DNA与测序引物和dNTP混合，得到反应体系；优选地，在所述待测DNA的3’末端增加与测序引物配对的序列；
(2)将步骤(1)的反应体系与权利要求1-5中任一项所述的DNA聚合酶修饰的单分子器件混合；
(3)检测DNA合成时所述DNA聚合酶修饰的单分子器件源漏电流随时间变化的电信号；
(4)分析所述电信号，获得测序结果。
在本发明第二方面的一个优选的实施方案中，在步骤(1)中，所述待测DNA为单链DNA或双链DNA，
其中当待测DNA为双链DNA时，需先将双链DNA变性，再与所述测序引物和dNTP混合。
在本发明第二方面的另一个优选的实施方案中，在步骤(1)中，所述待测DNA的3’末端增加有标签序列，所述标签序列的核苷酸序列与所述测序引物的核苷酸序列反向互补；优选地，所述测序引物的核苷酸序列为SEQ ID NO.4所示的序列，所述标签序列的核苷酸序列为SEQ ID NO.4自5’末端起第51至68位所示的序列。
在本发明第二方面的再一个优选的实施方案中，在步骤(2)中，
所述DNA聚合酶修饰的单分子器件的DNA聚合酶区域是经微腔包被的，优选地所述微腔的材质是聚二甲基硅氧烷；
并且将步骤(1)的反应体系加入到所述微腔中。
在本发明第二方面的又一个优选的实施方案中，其中步骤(4)包括：
统计分析源漏电流的峰值，根据各峰值对应的碱基，获得测序结果；或者
统计分析高态电流对应的高态时间和低态电流对应的低态时间，计算平均低态时间和平均高态时间，根据各平均低态时间和平均高态时间对应的碱基，获得测序结果。
在本发明的一个具体实施方案中，根据本发明的测序方法分别对以下待测DNA进行测序：
5’-CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC-3’；
5’-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’；
5’-GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG-3’；
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’；和
5’-TGCTAGCTGCTAATTGTCTCCATGTCATGTAGCTAGCTGTCACAGT-3’。
测序过程具体包括如下步骤：
(1)在待测DNA的3’末端增加标签序列，形成含有标签序列的待测DNA；所述标签序列的核苷酸序列与测序引物的核苷酸序列反向互补；
(2)将所述含有标签序列的待测DNA和所述测序引物混合，形成5’末端双链主体单链形式的产物；
(3)完成步骤(2)后，将产物分别与dATP、dCTP、dTTP和dGTP混合，得到四个体系，分别将每个体系加入DNA聚合酶修饰的单分子器件，读取电信号，与其它三个体系的电信号显著不同的体系中的dNTP即为a1)或a2)：
a1)所述待测DNA的3’末端的第一个核苷酸；
a2)所述待测DNA的3’末端的第一区段的核苷酸；所述第一区段由N个核苷酸组成，N为自然数且大于等于2；
(4)将前一步骤中与其它三个体系的电信号显著不同的体系的产物分别与dATP、dCTP、dTTP和dGTP混合，得到四个体系，分别将每个体系加入所述DNA聚合酶修饰的单分子器件，读取电信号，与其它三个体系的电信号显著不同的体系中的dNTP即为b1)或b2)：
b1)所述待测DNA的3’末端的第二个核苷酸；
b2)所述待测DNA的3’末端的第二区段的核苷酸；所述第二区段由M个核苷酸组成，M为自然数且大于等于2；
(5)重复步骤(4)，直至获得所述待测DNA的测序结果。
上述测序过程中，所述a2)中，N优选为50。
上述测序过程中，所述b2)中，M优选为50。
上述测序过程中，所述待测DNA为单链DNA。当待测DNA为双链DNA时，需先变性，再和所述测序引物混合。
所述测序引物的核苷酸序列优选如SEQ ID NO.4所示的序列。
所述标签序列的核苷酸序列可如SEQ ID NO.4自5’末端起第51至68位所示的序列。
本发明的第三方面提供一种用于DNA测序的试剂盒，其包括DNA聚合酶和单分子器件，其中：
所述单分子器件是二维纳米材料单分子器件，优选石墨烯基单分子器件；或低维纳米材料单分子器件，优选一维纳米材料单分子器件，更优选硅纳米线单分子器件。
在本发明第三方面的一个优选的实施方案中，所述DNA聚合酶选自：
b1)具有如SEQ ID NO.3所示的氨基酸序列的蛋白质，或其功能性突变体；或者
b2)具有如SEQ ID NO.1所示的氨基酸序列的蛋白质，或其功能性突变体。
在本发明第三方面的另一个优选的实施方案中，其中所述DNA聚合酶与所述单分子器件存在于分开的隔室中，其在使用时现场连接；或者所述DNA聚合酶连接到所述单分子器件；优选地所述DNA聚合酶具有与所述单分子器件的连接位点，且提供所述连接位点的氨基酸残基位于DNA聚合酶的构象变化区；更优选地，DNA聚合酶区域是经微腔包被的。
在本发明第三方面的再一个优选的实施方案中，试剂盒还包括测序引物、dNTP和/或使用说明书。
本发明所提供的方法可进行DNA测序，测序结果显示，DNA的合成速度与已有文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)记载的合成速度基本一致。本发明所提供的DNA测序方法具有重要的应用价值。
根据本发明的DNA聚合酶修饰的单分子器件、DNA测序方法和试剂盒可以广泛地应用于DNA测序，并且至少具有以下优势：
测序速度依赖于DNA聚合酶内在自身的反应速度，因此远远快于化学法测序；
DNA聚合酶具有内在自身的延续性，一个反应就可以测非常长的序列，例如几千个碱基，而二代测序仅可以测到几百个碱基。
由于没有PCR扩增步骤，不存在扩增引入的碱基错误，因此本测序精确度和分辨率非常高。
附图说明
图1为DNA聚合酶修饰的石墨烯基单分子器件的制备流程图。
图2为低分辨探针台检测电学性质；其中Vsd为源漏偏压，Isd为源漏电流。
图3为DNA聚合酶修饰的石墨烯基单分子器件和DNA聚合酶修饰的硅纳米线单分子器件的示意图。
图4为半胱氨酸和处理后的石墨烯基单分子器件进行化学反应的反应式。
图5为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链C进行DNA测序的动力学数据和峰值统计结果。
图6为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链C进行DNA测序的高态时间和低态时间的统计分析结果。
图7为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链A进行DNA测序的动力学数据和峰值统计结果。
图8为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链A进行DNA测序的高态时间和低态时间的统计分析结果。
图9为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链G进行DNA测序的动力学数据和峰值统计结果。
图10为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链G进行DNA测序的高态时间和低态时间的统计分析结果。
图11为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链T进行DNA测序的动力学数据和峰值统计结果。
图12为采用DNA聚合酶修饰的石墨烯基单分子器件对模板链T进行DNA测序的高态时间和低态时间的统计分析结果。
图13为采用DNA聚合酶修饰的石墨烯基单分子器件对随机链进行DNA测序的高态时间和低态时间的统计分析结果。
图14为DNA聚合酶修饰的硅纳米线单分子器件的制备。
具体实施方式
为使本发明的目的、技术方案、及优点更加清楚明白，以下参照附图并举实施例，对本发明进一步详细说明。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为本领域的常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
DTT缓冲液为含50mM NaCl、10mM MgCl2和1mM DTT的pH7.8、10mM Tris-HCl缓冲液。
HF溶液：由7体积份的40％(质量百分比)的NH4F水溶液和1体积份的HF水溶液混合而成；其中HF水溶液的浓度为40％(质量百分比)，为北京化工厂的产品。
单链DNA分子甲：5’-(C)50ACTGGCCGTCGTTTTACA-3’(SEQ ID NO.4所示)。
单链DNA分子乙：5’-TGTAAAACGACGGCCAGT-3’(SEQ ID NO.5所示)。
单链DNA分子丙：5’-(A)50ACTGGCCGTCGTTTTACA-3’(SEQ ID NO.6所示)。
单链DNA分子丁：5’-(G)50ACTGGCCGTCGTTTTACA-3’(SEQ ID NO.7所示)。
单链DNA分子戊：5’-(T)50ACTGGCCGTCGTTTTACA-3’(SEQ ID NO.8所示)。
单链DNA分子己：
5’-TGCTAGCTGCTAATTGTCTCCATGTCATGTAGCTAGCTGTCACAG TACTGGCCGTCGTTTTACA-3’(SEQ ID NO.9所示)。
模板链C的制备方法为：人工合成单链DNA分子甲和单链DNA分子乙，然后将10μL单链DNA分子甲(单链DNA分子甲的浓度为10μM)、10μL单链DNA分子乙(单链DNA分子乙的浓度为10μM)和980μL DTT缓冲液混合，70℃加热1h，自然冷却至室温，即获得模板链C(5’末端双链主体单链形式)。模板链C需在动力学测试前3h制备。
模板链A的制备方法为：人工合成单链DNA分子丙和单链DNA分子乙，然后将10μL单链DNA分子丙(单链DNA分子丙的浓度为10μM)、10μL单链DNA分子乙(单链DNA分子乙的浓度为10μM)和980μL DTT缓冲液混合，70℃加热1h，自然冷却至室温，即获得模板链A(5’末端双链主体单链形式)。模板链A需在动力学测试前3h制备。
模板链G的制备方法为：人工合成单链DNA分子丁和单链DNA分子乙，然后将10μL单链DNA分子丁(单链DNA分子丁的浓度为10μM)、10μL单链DNA分子乙(单链DNA分子乙的浓度为10μM)和980μL DTT缓冲液混合，70℃加热1h，自然冷却至室温，即获得模板链G(5’末端双链主体单链形式)。模板链G需在动力学测试前3h制备。
模板链T的制备方法为：人工合成单链DNA分子戊和单链DNA分子乙，然后将10μL单链DNA分子戊(单链DNA分子戊的浓度为10μM)、10μL单链DNA分子乙(单链DNA分子乙的浓度为10μM)和980μL DTT缓冲液混合，70℃加热1h，自然冷却至室温，即获得模板链T(5’末端双链主体单链形式)。模板链T需在动力学测试前3h制备。
随机链的制备方法为：人工合成单链DNA分子己和单链DNA分子乙，然后将10μL单链DNA分子己(单链DNA分子己的浓度为10μM)、10μL单链DNA分子乙(单链DNA分子乙的浓度为10μM)和980μL DTT缓冲液混合，70℃加热1h，自然冷却至室温，即获得随机链(5’末端双链主体单链形式)。随机链需在动力学测试前3h制备。
低分辨探针台由半导体参数仪(安捷伦公司的产品，型号为4155C)和探针台(Karl Suess公司的产品，型号为PM5)两个部分组成。
高分辨探针台由前置放大器(Digital Instruments公司的产品，型号为DL1211)和锁相放大器(Zurich Instruments公司的产品，型号为HF2LI)两个核心部件组成。
1，3，5-tris(4-carbonylphenyloxy)benzene为济南恒化科技有限公司的产品。
dATP溶液由DTT缓冲液溶解dATP获得。dCTP溶液由DTT缓冲液溶解dCTP获得。dTTP溶液由DTT缓冲液溶解dTTP获得。dGTP溶液由DTT缓冲液溶解dGTP获得。dATP、dCTP、dTTP和dGTP均为北京赛百盛基因技术有限公司的产品。
实施例1、采用DNA聚合酶修饰的石墨烯基单分子器件进行DNA测序
一、DNA聚合酶修饰的石墨烯基单分子器件的制备
1、制备石墨烯基单分子器件
石墨烯基单分子器件的制备过程参考如下文献：Cao Y，Dong S，Liu S，He L，Gan L，Yu X，Steigerwald ML，Wu X，Liu Z，Guo X.Building high-throughput molecular junctions using indented graphene point contacts.Angew Chem Int Ed Engl.2012 Dec 3；51(49)：12228-32。石墨烯基单分子器件上具有纳米间隙的石墨烯电极(以下简称石墨烯电极，其结构式如图1中(1))所示。
按照以下文献中报道的方法进行步骤2-7，Yang Cao，Shaohua Dong，Song Liu，Li He，Lin Gan，Xiaoming Yu，Michael L.Steigerwald，Xiaosong Wu，Zhongfan Liu and Xuefeng Guo*，Building High-Throughput Molecular Junctions Using Indented Graphene Point Contacts，Angew.Chem.Int.Ed.2012，51，12228和Xuefeng Guo，Alon Gorodetsky，Jacqueline K.Barton，James Hone，Colin Nuckolls*，Conductivity of a single DNA duplex bridging a carbon nanotube gap，Nat.Nanotechnol.2008，3，163。
2、末端氨基修饰
完成步骤1后，将所述石墨烯基单分子器件和对苯二胺(见图1中(2))混合， 石墨烯电极末端被氨基修饰。末端被氨基修饰的石墨烯电极的结构式见图1中(3)所示。
3、电学回路的构建
完成步骤2后，将1，3，5-tris(4-carbonylphenyloxy)benzene(见图1中(4))和末端被氨基修饰的石墨烯电极混合，得到电学回路。用低分辨探针台检测电学回路的电学性质，结果见图2中左上图。电学回路的部分结构式如图1中(5)所示。
4、Sulfo-NHS活化
完成步骤3后，将电学回路和N-羟基琥珀酰亚胺(NHS)磺酸钠盐(Sulfo-NHS)(见图1中(6))混合，得到Sulfo-NHS活化的电学回路。用低分辨探针台检测Sulfo-NHS活化的电学回路的电学性质，结果见图2中右上图。Sulfo-NHS活化的电学回路的部分结构式如图1中(7)所示。
5、处理后的石墨烯基单分子器件的获得
完成步骤4后，将Sulfo-NHS活化的电学回路和N-(2-氨乙基)马来酰亚胺盐酸盐(见图1中(8))混合，得到处理后的石墨烯基单分子器件。处理后的石墨烯基单分子器件的部分结构式如图1中(9)所示。用低分辨探针台检测处理后的石墨烯基单分子器件的电学性质，结果见图2中左下图。
图2(1)是图1中器件5的导电性，表明带羧基的分子器件的成功制备。图2(1)是图1中器件7的导电性，表明带活泼酯的分子器件的成功制备。图2(1)是图1中器件9的导电性，表明带马来酰亚胺器件的成功制备。图2(1)是图1中器件11的导电性，表明DNA聚合酶修饰的分子器件的成功制备。
6、大肠杆菌DNA聚合酶I的改造
(1)改造原则
大肠杆菌DNA聚合酶I(以下简称DNA聚合酶I)的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示。改造后的DNA聚合酶I需同时具有如下特点：(1)具有DNA聚合酶I的生物活性，即指导DNA的合成；(2)改造后的DNA聚合酶I可与处理后的石墨烯基单分子器件进行连接且仅有 一个连接位点，提供该连接位点的氨基酸残基(命名为供体氨基酸残基)应位于改造后的DNA聚合酶I的构象变化区(目的为提高后续DNA测序的灵敏度)；(3)供体氨基酸残基对应的氨基酸和非提供连接位点的氨基酸残基(命名为非供体氨基酸残基)对应的氨基酸种类不同。
(2)改造后的DNA聚合酶I的获得
采用文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)中报道的方法，经过大量实验，得到改造后的DNA聚合酶I。改造后的DNA聚合酶I(氨基酸序列如SEQ ID NO.3所示)，由生工生物工程(上海)股份有限公司制备。改造后的DNA聚合酶I为将DNA聚合酶I(氨基酸序列如SEQ ID NO.1所示)自N末端起第907位的半胱氨酸残基变为丝氨酸残基，第790位的亮氨酸残基变为半胱氨酸残基。SEQ ID NO.3自N末端起第790位氨基酸残基位于构象变化区。
7、DNA聚合酶修饰的石墨烯基单分子器件的制备
完成步骤6后，将处理后的石墨烯基单分子器件和过量的改造后的DNA聚合酶I(见图1中(10))混合，得到DNA聚合酶修饰的石墨烯基单分子器件(图3中左图)，DNA聚合酶修饰的石墨烯基单分子器件的部分结构式见图1中(11)。用低分辨探针台检测DNA聚合酶修饰的石墨烯基单分子器件的电学性质，结果见图2中右下图。改造后的DNA聚合酶I中半胱氨酸和处理后的石墨烯基单分子器件进行化学反应的反应式见图4。
将制备的DNA聚合酶修饰的石墨烯基单分子器件置于低分辨探针台，观察导电性。选择具有导电性的石墨烯基单分子器件进行下述实验。具体步骤参照文献Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135， 7855-7860中报道的方法。
二、采用DNA聚合酶修饰的石墨烯基单分子器件对模板链C进行DNA测序
1、取DNA聚合酶修饰的石墨烯基单分子器件，将改造后的DNA聚合酶I的区域用体积为40μL的微腔进行包被，微腔的材质为聚二甲基硅氧烷(polydimethylsiloxane，PDMS)，得到含有微腔的石墨烯基单分子器件。
2、完成步骤1后，将含有微腔的石墨烯基单分子器件置于高分辨探针台，采样5分钟，进行动力学测试(即测试源漏电流随时间的变化情况)，实验结果见图5中A的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图5中A的右图，表明没有样品条件下没有观察到DNA合成的震荡信号。
3、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入10μL DTT缓冲液，采样5分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图5中B的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图5中B的右图，表明只有缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
4、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链C的DTT缓冲液，采样5分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图5中C的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图5中C的右图，表明只有模板链和缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
5、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链C的DTT缓冲液、5μL的dATP溶液(浓度为5μM)、5μL的dTTP溶液(浓度为5μM)和5μL的dCTP溶液(浓度为5μM)，采样10分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图5中D的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图5中D的右图，表明有模板链、核酸分子和缓冲液，但是碱基不匹配条件下没有观察到DNA合成的震荡信号。
6、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL 含100nM模板链C的DTT缓冲液和5μL的dGTP溶液(浓度为5μM)，采样10分钟，测试聚合酶在进行DNA合成时源漏电流随时间的变化情况，亦即DNA聚合酶进行DNA聚合时的动力学数据。实验结果见图5中E的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图5中E的右图，表明有模板链、核酸分子和缓冲液，同时碱基匹配条件下观察到了DNA合成的震荡信号。
结果表明，步骤6中的动力学数据与其它对照实验中的动力学数据在表观上有本质区别，即当体系中存在与模板链C以及与之匹配的脱氧核苷酸时，体系的动力学数据才能与其余四组对照实验不同。
7、将步骤6获得的动力学数据拉展，采用Qub软件选定相应的高态电流(即大电流)及低态电流(即小电流)(图6中A)。将高态电流对应的高态时间(τhi)和低态电流对应的低态时间(τdow)进行统计分析，实验结果分别见图6中B和C。根据平均低态时间和平均高态时间，通过统计出现地台的次数计算得到本体系中DNA的合成速度是每秒62.62个dGTP核苷酸(见表1)，与已有的文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)记载的合成速度基本一致。值得说明的是，这里提及的平均，并不是简单意义上的线性平均，而是在指数分布基础上的泊松平均，具体的计算过程，由MatLab软件进行。
表1
  模板链C 模板链G 模板链T 模板链A
τlow(ms) 0.156 0.122 0.710 2.825
τhi(ms) 15.813 6.823 3.585 3.575
合成速度(个/秒) 62.62 143.99 232.83 156.25
三、采用DNA聚合酶修饰的石墨烯基单分子器件对模板链A进行DNA测序
1、同步骤二中1。
2、同步骤二中2。动力学测试的实验结果见图7中A的左图。峰值统计的实验结果见图7中A的右图，表明没有样品条件下没有观察到DNA合成的震荡信号。
3、同步骤二中3。动力学测试的实验结果见图7中B的左图。峰值统计的实验结果见图7中B的右图，表明只有缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
4、同步骤二中4的方法，仅将模板链C替换为模板链A。动力学测试的实验结果见图7中C的左图。峰值统计的实验结果见图7中C的右图，表明只有模板链和缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
5、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链A的DTT缓冲液、5μL的dATP溶液(浓度为5μM)、5μL的dGTP溶液(浓度为5μM)和5μL的dCTP溶液(浓度为5μM)，采样10分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图7中D的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图7中D的右图，表明有模板链、核酸分子和缓冲液，但是碱基不匹配条件下没有观察到DNA合成的震荡信号。
6、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链A的DTT缓冲液和5μL的dTTP溶液(浓度为5μM)，采样10分钟，测试聚合酶在进行DNA合成时源漏电流随时间的变化情况，亦即DNA聚合酶进行DNA聚合时的动力学数据。实验结果见图7中E的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图7中E的右图，表明有模板链、核酸分子和缓冲液，同时碱基匹配条件下观察到了DNA合成的震荡信号。
结果表明，步骤6中的动力学数据与其它对照实验中的动力学数据在表观上有本质区别，即当体系中存在与模板链A以及与之匹配的脱氧核苷酸时，体系的动力学数据才能与其余四组对照实验不同。
7、按照步骤二中7的方法，将高态电流对应的高态时间(τhi)和低态电流对应的低态时间(τlow)进行统计分析，实验结果见图8(左图为低态时间，右图 为高态时间)。根据平均低态时间和平均高态时间，计算得到本体系中DNA的合成速度是每秒156.25个dTTP核苷酸(见表1)，与已有的文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)记载的合成速度基本一致。
四、采用DNA聚合酶修饰的石墨烯基单分子器件对模板链G进行DNA测序
1、同步骤二中1。
2、同步骤二中2。动力学测试的实验结果见图9中A的左图。峰值统计的实验结果见图9中A的右图，表明没有样品条件下没有观察到DNA合成的震荡信号。
3、同步骤二中3。动力学测试的实验结果见图9中B的左图。峰值统计的实验结果见图9中B的右图，表明只有缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
4、同步骤二中4的方法，仅将模板链C替换为模板链G。动力学测试的实验结果见图9中C的左图。峰值统计的实验结果见图9中C的右图，表明只有模板链和缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
5、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链G的DTT缓冲液、5μL的dATP溶液(浓度为5μM)、5μL的dGTP溶液(浓度为5μM)和5μL的dTTP溶液(浓度为5μM)，采样10分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图9中D的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图9中D的右图，表明有模板链、核酸分子和缓冲液，但是碱基不匹配条件下没有观察到DNA合成的震荡信号
6、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链G的DTT缓冲液和5μL的dCTP溶液(浓度为5μM)，采样 10分钟，测试聚合酶在进行DNA合成时源漏电流随时间的变化情况，亦即DNA聚合酶进行DNA聚合时的动力学数据。实验结果见图9中E的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图9中E的右图，表明有模板链、核酸分子和缓冲液，同时碱基匹配条件下观察到了DNA合成的震荡信号
结果表明，步骤6中的动力学数据与其它对照实验中的动力学数据在表观上有本质区别，即当体系中存在与模板链G以及与之匹配的脱氧核苷酸时，体系的动力学数据才能与其余四组对照实验不同。
7、按照步骤二中7的方法，将高态电流对应的高态时间(τhi)和低态电流对应的低态时间(τlow)进行统计分析，实验结果见图10(左图为低态时间，右图为高态时间)。根据平均低态时间和平均高态时间，计算得到本体系中DNA的合成速度是每秒143.99个dCTP核苷酸(见表1)，与已有的文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)记载的合成速度基本一致。
五、采用DNA聚合酶修饰的石墨烯基单分子器件对模板链T进行DNA测序
1、同步骤二中1。
2、同步骤二中2。动力学测试的实验结果见图11中A的左图。峰值统计的实验结果见图11中A的右图，表明没有样品条件下没有观察到DNA合成的震荡信号。
3、同步骤二中3。动力学测试的实验结果见图11中B的左图。峰值统计的实验结果见图11中B的右图，表明只有缓冲液没有样品条件下没有观察到DNA合成的震荡信号
4、同步骤二中4的方法，仅将模板链C替换为模板链T。动力学测试的实验结果见图11中C的左图。峰值统计的实验结果见图11中C的右图，表 明只有模板链和缓冲液没有样品条件下没有观察到DNA合成的震荡信号。
5、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链T的DTT缓冲液、5μL的dCTP溶液(浓度为5μM)、5μL的dGTP溶液(浓度为5μM)和5μL的dTTP溶液(浓度为5μM)，采样10分钟，进行动力学测试(即测试源漏电流随时间的变化情况)。实验结果见图11中D的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图11中D的右图，表明有模板链、核酸分子和缓冲液，但是碱基不匹配条件下没有观察到DNA合成的震荡信号
6、完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM模板链T的DTT缓冲液和5μL的dATP溶液(浓度为5μM)，采样10分钟，测试聚合酶在进行DNA合成时源漏电流随时间的变化情况，亦即DNA聚合酶进行DNA聚合时的动力学数据。实验结果见图11中E的左图。然后采用MatLab软件进行初步的峰值统计，实验结果见图11中E的右图，表明有模板链、核酸分子和缓冲液，同时碱基匹配条件下观察到了DNA合成的震荡信号
结果表明，步骤6中的动力学数据与其它对照实验中的动力学数据在表观上有本质区别，即当体系中存在与模板链T以及与之匹配的脱氧核苷酸时，体系的动力学数据才能与其余四组对照实验不同。
7、按照步骤二中7的方法，将高态电流对应的高态时间(τhi)和低态电流对应的低态时间(τlow)进行统计分析，实验结果见图12(左图为低态时间，右图为高态时间)。根据平均低态时间和平均高态时间，计算得到本体系中DNA的合成速度是每秒232.83个dATP核苷酸(见表1)，与已有的文献(Tivoli J.Olsen，Yongki Choi，Patrick C.Sims，O.Tolga Gul，Brad L.Corso，Chengjun Dong，William A.Brown，Philip G.Collins and Gregory A.Weiss，Electronic Measurements of Single-Molecule Processing by DNA Polymerase I(Klenow Fragment)，Journal of the American Chemical Society 2013，135，7855-7860)记载的合成速度基本一致。
六、采用DNA聚合酶修饰的石墨烯基单分子器件对随机序列进行DNA 测序
完成步骤1后，向含有微腔的石墨烯基单分子器件的微腔中加入30μL含100nM随机链(SEQ ID NO.9)的DTT缓冲液和5μL的溶液(含有dATP、dGCTP、dATP和dTTP，浓度均为5μM)，采样10分钟，测试聚合酶在进行DNA合成时源漏电流随时间的变化情况，亦即DNA聚合酶进行DNA聚合时的动力学数据。实验结果见图13，看到了四种不同的震荡信号。然后采用MatLab软件进行初步的峰值统计，实验结果见图13中的表格。此表数据与表1数据，可以确定不同电流对应的碱基，从而根据电流的变化确定DNA的序列。因此，采用DNA聚合酶修饰的石墨烯基单分子器件可进行DNA测序。
实施例2、采用DNA聚合酶修饰的硅纳米线单分子器件进行DNA测序
一、DNA聚合酶修饰的硅纳米线单分子器件的制备
1、制备硅纳米线单分子器件
硅纳米线单分子器件的制备过程参考如下文献：Cao Y，Dong S，Liu S，He L，Gan L，Yu X，Steigerwald ML，Wu X，Liu Z，Guo X.Building high-throughput molecular junctions using indented graphene point contacts.Angew Chem Int Ed Engl.2012 Dec 3；51(49)：12228-32。硅纳米线单分子器件上具有纳米间隙的硅纳米线电极(以下简称硅纳米线电极)。
按照文献(Gen He，Jie Li，Haina Ci，Chuanmin Qi*，and Xuefeng Guo*，Direct measurement of single-molecule DNA hybridization dynamic s with single-base resolution，Angew.Chem.Int.Ed.2016，55，9036)中报道的方法进行以下步骤2-7。
2、刻蚀
完成步骤1后，将硅纳米线单分子器件和HF溶液混合，得到刻蚀的硅纳米线单分子器件。部分刻蚀的硅纳米线单分子器件的结构示意图如图13中(1)所示。
3、电学回路的构建
完成步骤2后，将刻蚀的硅纳米线单分子器件和10-十一碳炔酸(见图13中(2))混合，得到电学回路。电学回路的部分结构示意图如图13中(3)所示。
4、Sulfo-NHS活化
完成步骤3后，将电学回路和N-羟基琥珀酰亚胺磺酸钠盐(Sulfo-NHS)(见图13中(4))混合，得到Sulfo-NHS活化的电学回路。Sulfo-NHS活化的电学回路的部分结构式如图13中(5)所示。
5、处理后的硅纳米线单分子器件的获得
完成步骤4后，将Sulfo-NHS活化的电学回路和N-(2-氨乙基)马来酰亚胺盐酸盐(见图13中(6))混合，得到处理后的硅纳米线单分子器件。处理后的硅纳米线单分子器件的部分结构式如图13中(7)所示。
6、大肠杆菌DNA聚合酶I的改造
同实施例1步骤一中的6。
7、DNA聚合酶修饰的硅纳米线单分子器件的制备
完成步骤6后，将处理后的硅纳米线单分子器件和过量的改造后的DNA聚合酶I(见图7中(8))混合，得到DNA聚合酶修饰的硅纳米线单分子器件(见图3中右图)。部分DNA聚合酶修饰的硅纳米线单分子器件的结构示意图如图7中(7)所示。
将制备的DNA聚合酶修饰的硅纳米线单分子器件置于低分辨探针台，观察导电性，将具有导电性的DNA聚合酶修饰的硅纳米线单分子器件用于下述实验。
二、采用DNA聚合酶修饰的硅纳米线单分子器件对模板链C进行DNA测序
按照实施例1步骤二的方法，将DNA聚合酶修饰的石墨烯基单分子器件替换为DNA聚合酶修饰的硅纳米线单分子器件，其它步骤均不变，得到本体系中DNA的合成速度是每秒65.30个dGTP核苷酸，与采用DNA聚合酶修饰的石墨烯基单分子器件对模板链C进行DNA测序的结果无显著差异。
三、采用DNA聚合酶修饰的硅纳米线单分子器件对模板链A进行DNA测序
按照实施例1步骤三的方法，将DNA聚合酶修饰的石墨烯基单分子器件替换为DNA聚合酶修饰的硅纳米线单分子器件，其它步骤均不变，得到本体 系中DNA的合成速度是每秒117.27个TTP核苷酸，与采用DNA聚合酶修饰的石墨烯基单分子器件对模板链A进行DNA测序的结果无显著差异。
四、采用DNA聚合酶修饰的硅纳米线单分子器件对模板链G进行DNA测序
按照实施例1步骤四的方法，将DNA聚合酶修饰的石墨烯基单分子器件替换为DNA聚合酶修饰的硅纳米线单分子器件，其它步骤均不变，得到本体系中DNA的合成速度是每秒154.51个dCTP核苷酸，与采用DNA聚合酶修饰的石墨烯基单分子器件对模板链G进行DNA测序的结果无显著差异。
五、采用DNA聚合酶修饰的硅纳米线单分子器件对模板链T进行DNA测序
按照实施例1步骤五的方法，将DNA聚合酶修饰的石墨烯基单分子器件替换为DNA聚合酶修饰的硅纳米线单分子器件，其它步骤均不变，测得的DNA合成的震荡信号与用DNA聚合酶修饰的石墨烯基单分子器件测得的震荡信号一致；得到本体系中DNA的合成速度是每秒234.60个dATP核苷酸，与采用DNA聚合酶修饰的石墨烯基单分子器件对模板链T进行DNA测序的结果无显著差异。
因此，采用DNA聚合酶修饰的硅纳米线单分子器件可进行DNA测序。

Claims (15)

  1. 一种DNA聚合酶修饰的单分子器件，其中所述单分子器件是二维纳米材料单分子器件，优选石墨烯基单分子器件；或低维纳米材料单分子器件，优选一维纳米材料单分子器件，更优选硅纳米线单分子器件。
  2. 根据权利要求1所述的DNA聚合酶修饰的单分子器件，其中所述DNA聚合酶具有与所述单分子器件的连接位点，且提供所述连接位点的氨基酸残基位于DNA聚合酶的构象变化区。
  3. 根据权利要求1或2所述的DNA聚合酶修饰的单分子器件，其中，所述DNA聚合酶与所述单分子器件仅通过一个连接位点连接，优选地，提供所述连接位点的氨基酸残基在所述DNA聚合酶中仅出现一次。
  4. 根据前述权利要求中任一项所述的DNA聚合酶修饰的单分子器件，其中所述DNA聚合酶选自：
    b1)具有如SEQ ID NO.3所示的氨基酸序列的蛋白质，或其功能性突变体；或者
    b2)具有如SEQ ID NO.1所示的氨基酸序列的蛋白质，或其功能性突变体。
  5. 根据权利要求4所述的DNA聚合酶修饰的单分子器件，其中提供所述连接位点的氨基酸残基为SEQ ID NO.1所示的氨基酸序列自N末端起第790位的半胱氨酸残基。
  6. 根据前述权利要求中任一项所述的DNA聚合酶修饰的单分子器件，其中所述DNA聚合酶修饰的单分子器件的DNA聚合酶区域是经微腔包被的，优选地，所述微腔的材质是聚二甲基硅氧烷。
  7. 一种DNA测序方法，其包括以下步骤：
    (1)将待测DNA与测序引物和dNTP混合，得到反应体系；
    (2)使步骤(1)的反应体系与权利要求1-6中任一项所述的DNA聚合酶修饰的单分子器件接触；
    (3)检测DNA合成时所述DNA聚合酶修饰的单分子器件源漏电流随时间变化的电信号；和
    (4)分析所述电信号，获得测序结果。
  8. 根据权利要求7所述的DNA测序方法，在步骤(1)中，所述待测DNA为单链DNA或双链DNA，
    其中当待测DNA为双链DNA时，需先将双链DNA变性，再与所述测序引物和dNTP混合。
  9. 根据权利要求7或8所述的DNA测序方法，其中步骤(1)中，所述待测DNA的3’末端增加有标签序列，所述标签序列的核苷酸序列与所述测序引物的核苷酸序列反向互补；优选地，所述测序引物的核苷酸序列为SEQ ID NO.4所示的序列，所述标签序列的核苷酸序列为SEQ ID NO.4自5’末端起第51至68位所示的序列。
  10. 根据权利要求7-9中任一项所述的DNA测序方法，其中步骤(2)中，
    所述DNA聚合酶修饰的单分子器件的DNA聚合酶区域是经微腔包被的，优选地所述微腔的材质是聚二甲基硅氧烷，其中将步骤(1)的反应体系加入到所述微腔中。
  11. 根据权利要求7-10中任一项所述的DNA测序方法，其中步骤(4)包括：
    统计分析源漏电流的峰值，根据各峰值对应的碱基，获得测序结果；或者
    统计分析高态电流对应的高态时间和低态电流对应的低态时间，计算平均低态时间和平均高态时间，根据各平均低态时间和平均高态时间对应的碱基，获得测序结果。
  12. 一种用于DNA测序的试剂盒，其包括DNA聚合酶和单分子器件，其中：
    所述单分子器件是二维纳米材料单分子器件，优选石墨烯基单分子器件；或低维纳米材料单分子器件，优选一维纳米材料单分子器件，更优选硅纳米线单分子器件。
  13. 根据权利要求12所述的试剂盒，其中所述DNA聚合酶选自：
    b1)具有如SEQ ID NO.3所示的氨基酸序列的蛋白质，或其功能性突变体；或者
    b2)具有如SEQ ID NO.1所示的氨基酸序列的蛋白质，或其功能性突变体。
  14. 根据权利要求12或13所述的试剂盒，其中所述DNA聚合酶与所述单分子器件存在于分开的隔室中，其在使用时现场连接；或者
    所述DNA聚合酶连接到所述单分子器件；优选地所述DNA聚合酶具有与所述单分子器件的连接位点，且提供所述连接位点的氨基酸残基位于DNA聚合酶的构象变化区；更优选地，DNA聚合酶区域是经微腔包被的。
  15. 根据权利要求12-14中任一项所述的试剂盒，其还包括测序引物、dNTP和/或使用说明书。
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