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WO2020113581A1 - 纳米孔测序方法 - Google Patents

纳米孔测序方法 Download PDF

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Publication number
WO2020113581A1
WO2020113581A1 PCT/CN2018/119911 CN2018119911W WO2020113581A1 WO 2020113581 A1 WO2020113581 A1 WO 2020113581A1 CN 2018119911 W CN2018119911 W CN 2018119911W WO 2020113581 A1 WO2020113581 A1 WO 2020113581A1
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Prior art keywords
nucleic acid
nanopore
stranded
nanopore sequencing
base
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PCT/CN2018/119911
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English (en)
French (fr)
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朱国丽
沈寒婕
林建勋
江媛
曾涛
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深圳华大生命科学研究院
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Application filed by 深圳华大生命科学研究院 filed Critical 深圳华大生命科学研究院
Priority to CN201880099752.4A priority Critical patent/CN113366120B/zh
Priority to US17/292,012 priority patent/US20220002798A1/en
Priority to PCT/CN2018/119911 priority patent/WO2020113581A1/zh
Publication of WO2020113581A1 publication Critical patent/WO2020113581A1/zh

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

Definitions

  • the invention relates to the field of sequencing technology, in particular to a nanopore sequencing method.
  • Nanopore sequencing technology has the following characteristics: (1) Single-molecule DNA sequencing. There is no need to amplify and amplify sequencing objects, which can overcome sequencing bias caused by gene amplification. (2) Continuous sequencing. The reading of bases on the DNA chain is uninterrupted. This feature can not only greatly increase the speed of sequencing, but also greatly increase the read length of DNA.
  • the matching DNA library building kit mainly adopts 1D and 1D2. Because nanopore sequencing is difficult to distinguish the signal of a single base, and the difference in current signal caused by the four bases is very small, only a single detection of the base makes the sequencing accuracy lower and requires complex post-data analysis And rely on powerful hardware and software systems.
  • the existing library construction method of nanopore sequencing mainly adopts the sequencing of a single copy number base sequence. This method requires high sample starting volume and low accuracy, and has very high requirements for later data analysis.
  • the conventional double-stranded direct circularization of multi-copy nanopore library construction and Gibson-based assembly have low efficiency, and generally the circularized fragments are shorter than 1 kb, which limits sequencing applications.
  • the self-amplification method based on respiration requires high Tm values at both ends, and there may be a phenomenon that template DNA self-pairs during the amplification process.
  • the invention provides a nanopore sequencing method.
  • a multi-copy library that can be used for nanopore sequencing through various methods, multiple repeated signal measurement of long fragments can be achieved, and the accuracy of nanopore sequencing can be effectively improved.
  • a nanopore sequencing method includes: in a nanopore sequencing device, rolling nucleic acid amplification is performed using nucleic acid single-stranded loops or double-stranded loops as template nucleic acids to generate amplified single strands, and the above-mentioned expansion is performed under the action of an electric field
  • the increased single strand is introduced into the nanopore of the nanopore sequencing device, so that the amplified single strand passes through the nanopore and generates an electrical signal for each base.
  • the template Determination of base sequences on nucleic acids.
  • the template nucleic acid is a nucleic acid single-stranded loop
  • the rolling circle amplification reaction system includes: the nucleic acid single-stranded loop, an amplification primer that partially hybridizes with the nucleic acid single-stranded loop, and a chain displacement activity Polymerase, dNTPs and reaction buffer; the above methods include:
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the above reaction system further includes a probe sequence having a specific modification on the probe sequence, the specific modification can be connected to a support layer supporting the nanopore in the nanopore sequencing device, and the amplification primer includes A probe recognition site capable of binding to the above probe sequence; the above method includes:
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the above reaction system further includes a probe sequence with a specific modification on the probe sequence, the specific modification can be connected to a support layer supporting the nanopore in the nanopore sequencing device, and the amplification primer includes A probe recognition site capable of binding to the above probe sequence; the above method includes:
  • the probe sequence is added to the nanopore sequencing device to connect with the support layer, the hybridization product is added to hybridize the amplification primer to the probe sequence, and the polymerase, dNTPs and reaction buffer are added to perform rolling circle expansion. Increase to produce amplified single strands;
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the above specific modification is selected from cholesterol or biotin modification.
  • the specific modification is a cholesterol modification
  • the support layer is a phospholipid bilayer
  • the cholesterol and the phospholipid bilayer are connected by affinity bonding.
  • the specific modification is a biotin modification
  • the support layer has a streptavidin modification
  • the biotin and the streptavidin are connected by affinity binding.
  • the nucleic acid single-stranded loop is provided with a special modification that can be connected to the support layer that supports the nanopore in the nanopore sequencing device; the method includes:
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the above-mentioned special modifications are selected from biotin or polyethylene glycol.
  • the above-mentioned special modification is biotin
  • the above-mentioned support layer has a streptavidin modification
  • the above-mentioned biotin and the above-mentioned streptavidin are connected by affinity binding.
  • the template nucleic acid is a nucleic acid double-stranded loop; the nucleic acid double-stranded loop has a gap in one strand; the rolling circle amplification reaction system includes: the nucleic acid double-stranded loop, and a polymerase having strand displacement activity , DNTPs and reaction buffer; the above methods include:
  • nucleic acid double-stranded loop Adding the nucleic acid double-stranded loop, polymerase, dNTPs and reaction buffer to the nanopore sequencing device, and performing rolling circle amplification to generate an amplified single strand from the gapped single strand of the nucleic acid double-stranded loop;
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the above reaction system further includes a probe sequence having a specific modification on the probe sequence, the specific modification can be connected to a support layer supporting the nanopore in the nanopore sequencing device, and the nucleic acid double-stranded loop Contains a probe recognition site capable of binding to the above probe sequence; the above method includes:
  • the nucleic acid double-stranded loop, the probe sequence, polymerase, dNTPs, and reaction buffer are added to the nanopore sequencing device, the probe sequence is connected to the support layer through the specific modification, and rolling circle amplification is performed from The gapped single strands of the double-stranded nucleic acid loop described above produce amplified single strands;
  • the amplified single-stranded single base is gradually passed through the nanopore and an electrical signal of each base is generated.
  • an electrical signal of each base is generated.
  • the base on the template nucleic acid is realized. Sequence determination.
  • the polymerase having chain displacement activity is selected from phi29 polymerase or Bst polymerase.
  • the voltage of the above electric field is a millivolt-level voltage.
  • the above-mentioned millivolt voltage is a voltage of 10 mV or more, preferably a voltage of 50 mV or more, more preferably a voltage of 100 mV to 250 mV, and most preferably a voltage of 120 mV.
  • the above method further includes the step of preparing the nucleic acid single-stranded loop or double-stranded loop. This step includes circularizing the DNA fragment to obtain a single-stranded loop or a double-stranded loop.
  • the length of the aforementioned DNA fragment is 100 bp to 1 Mbp.
  • the aforementioned nanopores are biological nanopores and solid nanopores.
  • the biological nanopore is a protein nanopore, and the narrowest part of the protein nanopore satisfies the property of passing single-stranded nucleic acid.
  • the aforementioned solid-state nanopore is a nanopore of a silicon-based material or a graphene two-dimensional material, and the narrowest part of the silicon-based material nanopore satisfies the characteristic of passing single-stranded nucleic acid.
  • the nanopore sequencing method of the present invention is based on the preparation of multi-copy DNA nanopore libraries, which can not only make up for the problem of insufficient accuracy in the construction of conventional nanopore sequencing libraries, but also improve the efficiency of circularization, while performing rolling circle amplification Carrying out nanopore sequencing can improve the timeliness of sequencing, shorten the time for library construction and simplify the sequencing steps.
  • the invention is suitable for all base sequences that can be subjected to nanopore sequencing, and there is no phenomenon of joint contamination and self-pairing.
  • FIG. 1 is a schematic diagram of a DNA linker sequence structure used in the construction of a Chinese library according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of a method for sequencing a single-stranded DNA loop hybridized with a linker and placed in a nanopore device according to an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a method for sequencing a single-stranded DNA loop hybridized with a surface-fixed sequence in a nanopore system according to an embodiment of the present invention
  • FIG. 4 is a schematic diagram of a method for performing sequencing on a single-stranded DNA loop with a special modification fixed on the surface of a support layer in an embodiment of the present invention
  • FIG. 5 is a schematic diagram of a method for constructing and sequencing a DNA double-stranded library without a probe recognition site in an embodiment of the present invention
  • FIG. 6 is a schematic diagram of a method for constructing and sequencing a DNA double-stranded library with a probe recognition site in an embodiment of the present invention
  • FIG. 7 is a diagram of the results of the double-stranded circular DNA agarose gel electrophoresis experiment prepared in the examples of the present invention.
  • FIG. 9 is a graph of current change results obtained by performing nanopore sequencing based on single-stranded loops in an embodiment of the present invention.
  • FIG. 10 is a graph of current change results obtained by performing nanopore sequencing based on double-stranded loops in an embodiment of the present invention.
  • the invention is mainly based on the method of rolling circle amplification to complete the construction and sequencing of a multi-copy library based on nanopores.
  • the present invention relates in detail to the technical solution for the construction of multi-copy libraries and the required oligonucleotide sequence composition, special modifications, reaction conditions, method steps, etc.
  • the basic principle of the present invention is: after breaking the nucleic acid to be tested (such as DNA) into a long fragment, a single-stranded loop or a double-stranded loop is constructed by a specific method, a rolling circle amplification method and a nanopore sequencing device are used in the electric field. Under the action, the template nucleic acid to be tested will pass through the nanopore in a single-stranded mode and generate electrical signals of different bases. By judging the differences in electrical signals caused by different bases, the template nucleic acids of different sequences can be identified.
  • a nanopore sequencing method includes: in a nanopore sequencing device, rolling nucleic acid amplification using nucleic acid single-stranded loops or double-stranded loops as a template nucleic acid generates amplified single-stranded DNA in an electric field
  • the amplified single strand is introduced into the nanopore of the nanopore sequencing device, so that the amplified single strand passes through the nanopore and generates an electrical signal for each base, by judging the electricity caused by different bases The signal difference enables the determination of the base sequence on the template nucleic acid.
  • the nanopore sequencing device may be implemented according to the existing devices, such as the nanopore sequencing device disclosed in the international application WO2014013262A1.
  • a nanopore sequencing device can be a device based on biological nanopores or solid nanopores.
  • the biological nanopores can be protein nanopores, and the solid nanopores can be silicon-based nanopores.
  • the narrowest part of the nanopores can satisfy single-stranded nucleic acids. Passing characteristics.
  • the supporting layer supporting the nanopores may be a phospholipid bilayer (phospholipid membrane), a polymer membrane, or other surface-modified (eg, biotin-modified) support.
  • the template nucleic acid includes two types, that is, a single-stranded nucleic acid loop or a double-stranded nucleic acid loop. They can all be obtained by circularizing nucleic acid (eg DNA) fragments. Such DNA fragments, that is, DNA samples used for circularization and library construction, generally need to be suitable for the construction of single-stranded loops or double-stranded loops, such as 100bp to 1Mbp.
  • the nucleic acid (eg DNA) fragments can be derived from Any biological samples of interest, such as animals, plants, microorganisms, etc.
  • a single-stranded nucleic acid loop such as a single-stranded DNA loop, generally uses a DNA fragment as a template, and a closed DNA single-stranded loop obtained by connecting a designed adaptor and performing circularization may also be referred to as a library.
  • Double-stranded nucleic acid loops such as double-stranded DNA loops, are generally double-stranded circular DNA with gaps in one strand obtained by using DNA fragments as templates, specially designed linkers and other suitable technical solutions.
  • a DNA linker sequence is used for library construction.
  • the DNA linker sequence is composed of two base sequences of different lengths. As shown in FIG. 1, the longer base sequence is at 3′
  • the extended portion of the terminal has a sequence capable of specifically binding to the single-stranded loop.
  • the DNA linker sequence has a specific modified base sequence on one strand, such as the modification of the 3′ end of the short chain in FIG. 1, for example, it may be cholesterol or biotin modification, and the specific modification can be matched with the nanopore sequencing device that supports the nanopore
  • the support layer is connected to fix the entire DNA linker sequence on the support layer.
  • the linker sequence necessary for nanopore sequencing is the extended portion at the 3′ end shown in FIG.
  • the sequence that the single-stranded loop specifically binds to serves as a primer for rolling circle amplification, and this sequence is also referred to as "amplification primer” in the present invention.
  • the short chain with a modification at the 3′ end in FIG. 1 is a preferred sequence for connecting to the support layer of the nanopore, so that the amplified single strand is close to the nanopore and enters the nanopore.
  • the short chain It is also called “probe sequence” in the present invention.
  • the reaction system of rolling circle amplification in the nanopore sequencing of the present invention includes the following components: a nucleic acid single-stranded loop, an amplification primer partially hybridizing to the nucleic acid single-stranded loop, and a strand having Replace active polymerases, dNTPs, reaction buffers, etc.
  • the nanopore sequencing method of the present invention includes: hybridizing a nucleic acid single-stranded loop with an amplification primer to form a hybridization product; adding the hybridization product to a nanopore sequencing device, adding polymerase, dNTPs, and a reaction buffer to perform rolling circle amplification to produce expansion Increased single-strand; under the action of an electric field, the amplified single-stranded single base gradually passes through the nanopore and generates an electrical signal for each base.
  • the template nucleic acid Determination of base sequence.
  • a method for constructing and sequencing a nanopore multicopy library based on a single-stranded DNA loop shows a hybridization of a single-stranded DNA loop with a linker (including long and short chains) It is then placed in a nanopore device for sequencing.
  • the reaction system includes a nucleic acid single-stranded loop, an amplification primer (i.e., long chain) that hybridizes with the nucleic acid single-stranded loop portion, a probe sequence (i.e., short chain), and polymerases, dNTPs, and reaction buffers that have strand displacement activity.
  • the needle sequence has a specific modification (for example, cholesterol or biotin modification), which can be connected to the support layer supporting the nanopore in the nanopore sequencing device, and the amplification primer includes a probe recognition site capable of binding to the probe sequence
  • the method includes: hybridizing amplification primers, probe sequences and nucleic acid single-stranded loops to form a hybrid product; adding the hybrid product to the nanopore sequencing device, connecting the probe sequence to the support layer, adding polymerase, dNTPs and reaction buffer
  • the liquid is subjected to rolling circle amplification to produce an amplified single-strand; under the action of an electric field, the single base of the amplified single-strand is gradually passed through the nanopore and an electrical signal for each base is generated, which is caused by judging different bases.
  • the difference in electrical signals enables the determination of the base sequence on the template nucleic acid.
  • the amplification primer (ie, long chain) and the probe sequence (ie, short chain) have partial sequence complementarity, and the 3'end of the short chain is modified (eg, cholesterol or biotin, etc.). Hybridize with the short chain, and the hybridization product will hybridize with the single-stranded loop. Then, the hybridized product is put into the sequencing device where the nanopore has been formed, and the 3'-end modifier of the short fragment will interact with the surface of the support layer and be adsorbed on the surface, thereby realizing the enrichment of the hybridized product on the surface of the nanopore.
  • the 3'-end modifier of the short fragment will interact with the surface of the support layer and be adsorbed on the surface, thereby realizing the enrichment of the hybridized product on the surface of the nanopore.
  • a method for constructing and sequencing a multi-copy library of nanopores based on single-stranded DNA loops shows that a single-stranded DNA loop hybridizes to a surface-fixed sequence in a nanopore system.
  • the reaction system includes a nucleic acid single-stranded loop, an amplification primer (i.e., long chain) that hybridizes with the nucleic acid single-stranded loop portion, a probe sequence (i.e., short chain), and polymerases, dNTPs, and reaction buffers that have strand displacement activity
  • the needle sequence has a specific modification (for example, cholesterol or biotin modification), which can be connected to the support layer supporting the nanopore in the nanopore sequencing device, and the amplification primer includes a probe recognition site capable of binding to the probe sequence
  • the method includes: hybridizing the amplification primer and the nucleic acid single-stranded loop to form a hybridization product; adding a probe sequence to the nanopore sequencing device to connect it with the support layer, adding a hybridization product to hybridize the amplification primer and the probe sequence, adding Rolling circle amplification of polymerase, dNTPs and reaction buffer produces amplified single strands; under the action of an electric field, the single bases
  • two oligo sequences are designed, in which one strand is a short strand with a modification at the 3'end (such as cholesterol, biotin, etc.), which is called a probe sequence; the other strand is both A long chain with a probe recognition site and capable of hybridizing with a single-stranded DNA loop is called an amplification primer.
  • the amplified primer is hybridized with a single-stranded DNA loop to form a primer-ssDNA hybrid product .
  • the modified short chain (probe sequence) at the 3'end to the surface of the support layer form a high-density probe sequence on the surface of the support layer, and place the primer-single-stranded DNA hybridization product into a sequencing device that forms a nanopore
  • the probe recognition site on the hybridization product and the probe sequence on the surface of the support layer are hybridized with each other.
  • Add polymerase and dNTP to the nanopore sequencing device, and apply a voltage across the nanopore, the 5'end of the long chain (amplification primer) will enter the nanopore under the action of the electric field, and under the action of the polymerase Passing through the bases one by one generates a corresponding current signal.
  • the modification on the short chain may be a cholesterol modification
  • the support layer may be a phospholipid bimolecular layer
  • cholesterol and the phospholipid bimolecular layer are connected by affinity bonding.
  • the modification on the short chain may be a biotin modification, the streptavidin modification on the support layer, and the biotin and streptavidin are connected by affinity binding.
  • a method for constructing and sequencing a nanopore multicopy library based on a single-stranded DNA loop shows a method of performing special modifications on the single-stranded DNA loop fixed on the surface of the support layer for sequencing Methods.
  • the reaction system includes a single-stranded nucleic acid loop, an amplification primer (ie, a long chain) that hybridizes with a single-stranded nucleic acid loop, and a polymerase, dNTPs, and reaction buffer with strand displacement activity.
  • the nucleic acid single-stranded loop is provided with a special modification (such as biotin or polyethylene glycol, etc.), and this special modification can be connected to the support layer supporting the above-mentioned nanopore in the nanopore sequencing device.
  • the method includes: adding an amplification primer and a single-stranded nucleic acid loop to a nanopore sequencing device, hybridizing the amplification primer and a single-stranded nucleic acid loop to form a hybridization product, and connecting the single-stranded nucleic acid loop to the support layer through special modification and adding polymerization Enzymes, dNTPs and reaction buffers undergo rolling circle amplification to produce amplified single strands; under the action of an electric field, the single bases of the amplified single strands are gradually passed through the nanopore and an electrical signal for each base is generated. Judge the difference of electric signal caused by different bases to realize the determination of the base sequence on the template nucleic acid.
  • a specific base on a single-stranded loop of a nucleic acid is labeled and modified.
  • the labeling modification may be biotin or other similar chemical substances (such as polyethylene glycol). It has normal polymerization and will not be replaced from the surface of the bilayer under the action of polymerase. After the reaction starts, the single-stranded DNA amplified by the rolling circle will pass through the nanopore under the action of an electric field and generate a corresponding electrical signal.
  • the special modification may be biotin, the streptavidin modification on the support layer, and the biotin and streptavidin are connected by affinity binding.
  • a method for constructing and sequencing a nanopore multicopy library based on double-stranded DNA loops shows a DNA double-stranded loop library construction and sequencing without probe recognition sites
  • the template nucleic acid is a nucleic acid double-stranded loop, and one strand of the nucleic acid double-stranded loop has a gap; its reaction system includes: a nucleic acid double-stranded loop, and polymerases, dNTPs, and reaction buffers having strand displacement activity.
  • the method includes: adding nucleic acid double-stranded loop, polymerase, dNTPs and reaction buffer to a nanopore sequencing device, and performing rolling circle amplification to generate an amplified single strand from the gapped single strand of the nucleic acid double-stranded loop ; Under the action of the electric field, the amplified single-stranded single base gradually passes through the nanopore and generates an electrical signal for each base. By judging the difference in electrical signals caused by different bases, the base sequence on the template nucleic acid is realized. Determination.
  • the method for preparing a double-stranded nucleic acid loop with a gap can be performed according to the existing conventional methods.
  • the DNA double-stranded loop is prepared as follows: the end of the DNA fragment is repaired and A is added to the end; a specially designed linker is added (for example, the two linkers are asymmetric, one long, one relatively Short junctions forming a bifurcated structure); using specially designed primers (for example, with U bases) for PCR amplification; using the User enzyme treatment to obtain double-stranded DNA with complementary sequences at both ends; circularization treatment, Due to the specially designed primers, a double-stranded DNA loop with gaps will be formed during circularization.
  • a method for constructing and sequencing a nanopore multicopy library based on double-stranded DNA loops shows a DNA double-stranded loop library construction and sequencing with a probe recognition site
  • the template nucleic acid is a nucleic acid double-stranded loop, and one strand of the nucleic acid double-stranded loop has a gap
  • its reaction system includes: a nucleic acid double-stranded loop, polymerases having strand displacement activity, dNTPs and reaction buffer, and probe sequences
  • the probe sequence has a specific modification, and the specific modification can be connected to the support layer supporting the nanopore in the nanopore sequencing device, and the nucleic acid double-stranded loop includes a probe recognition site that can be combined with the probe sequence.
  • the method includes: adding nucleic acid double-stranded loop, probe sequence, polymerase, dNTPs and reaction buffer to a nanopore sequencing device, connecting the probe sequence to the support layer through specific modification, and performing rolling circle amplification to remove
  • the gapped single strand of the chain ring produces an amplified single strand; under the action of an electric field, the single base of the amplified single strand is gradually passed through the nanopore and an electrical signal for each base is generated, by judging different bases The difference in the electrical signal caused by the base allows the determination of the base sequence on the template nucleic acid.
  • the polymerase having chain displacement activity may be selected from phi29 polymerase or Bst polymerase.
  • the voltage of the electric field is generally a millivolt-level voltage, for example, a voltage of 10 mV or more, preferably a voltage of 50 mV or more, more preferably a voltage of 100 mV to 250 mV, and most preferably a voltage of 120 mV.
  • DNA was extracted using E. coli standard strains as raw materials, and single-stranded circular DNA was prepared according to the instructions of the kit.
  • Hybridize the YJ-1058 and YJ-1092 sequences in Table 1 to obtain a 25 ⁇ M hybrid adapter product Take the above product 13 ⁇ L, add 1.6 ⁇ L 25 ⁇ M adapter, 0.55 ⁇ L 600 U/ ⁇ L T4 DNA ligase (BGI), add water to make up 30 ⁇ L, mixed by vortex and placed in the PCR instrument.
  • the reaction procedure is as follows: 20°C, 30 minutes, 4°C. 0.5X AM and Xpure magnetic beads were used for purification, and 60 ⁇ L of TE was used for dissolution.
  • Lane M is 1kb plus plus ladder.
  • Lane 1 is the PCR product, and the triangle corresponds to the linear phiX174 band.
  • Lane 2 is the circularization product, the lower triangle corresponds to the linear phiX174 band, and the upper triangle corresponds to the circularized double-stranded DNA loop.
  • Lane 3 is the circularization product after digested by the Plasmid-safe enzyme, and the triangle corresponds to the circularized double-stranded DNA circle.
  • Lane 4 is the product band after the digestion of the Plasmid-safe enzyme and then treated with polymerase I. The triangle corresponds to the circularized double-stranded DNA circle.
  • the comparison of lanes 1 and 2 shows that the cyclization efficiency is close to 30%; the comparison of lanes 2 and 3 shows that the triangle above corresponds to a double-stranded ring; the comparison of lane 3 and lane 4 shows that the formed double-stranded ring contains a closed The complete ring.
  • the result of this example shows that the above double-stranded DNA library can be prepared and subjected to rolling circle amplification for sequencing.
  • oligo design two oligonucleotide sequences (oligo), one of which is a short segment with a special modification (cholesterol) at the 3'end as a probe sequence (sequence: SEQ ID NO: 5: CCGCGTGAGATCTGATGATGTTTTTAAATTTGGGTTT/iSpC3/-Cholesterol , Where /iSpC3/ represents -CH2CH2CH2-, used to block the polymerization of DNA, Cholesterol refers to cholesterol), and the other is a long-chain sequence that has both a probe recognition site and hybridization with a single-stranded DNA loop (the sequence is : SEQ ID NO: 6: /iSpC3/AAAAAAACCTTCC/iSpC3/CCTTCCCATCATCATCATCAGATCTCACGCGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

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Abstract

一种纳米孔测序方法，包括：在纳米孔测序装置中，以核酸单链环或双链环为模板进行滚环扩增，产生出扩增的单链，在电场的作用下，将扩增的单链引入纳米孔测序装置的纳米孔中，使扩增的单链通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。

Description

纳米孔测序方法 技术领域
本发明涉及测序技术领域，具体涉及一种纳米孔测序方法。
背景技术
目前二代测序的读长较短，难以对相似基因、SNP位点以及转录异构体等进行准确检测；纳米孔测序技术有如下特点：(1)单分子DNA测序。无需对测序对象进行扩增和放大，能够克服由基因扩增引起的测序偏向性。(2)连续测序。DNA链上碱基的阅读是不间断的，这一特性不仅能够大幅度提高测序速度，也会使DNA的读长大幅度增加。
目前英国牛津纳米孔技术公司(Oxford Nanopore Technologies)基于该技术已经推出商业化产品MinION、GridION和PromethION。配套的DNA建库试剂盒主要采用1D和1D2两种方式。由于纳米孔测序难以对单个碱基的信号进行区分，并且四种碱基引起的电流信号差别很小，仅靠单次对碱基的检测使得测序的准确率较低，需要复杂的后期数据分析并且依赖强大的硬件和软件系统。
目前基于牛津纳米孔技术公司公司的MinION平台，有公司推出1D、2D、1D2的建库方式，但是这两种方式都存在准确率不足以及需要的样本起始量高的缺点。一些其他相关科研机构也先后开发了基于多拷贝数文库构建方法，包括分子内结合纳米孔一致性测序方法(INC-seq)(Li et al.GigaScience(2016)5:34 DOI 10.1186/s13742-016-0140-7)，滚环扩增多联一致性测序的方法(R2C2)(doi:https://doi.org/10.1101/338020)，以及基于呼吸作用的自扩增建库方法(专利申请号201610420179.5)。
现有纳米孔测序的文库构建方式主要采用对单个拷贝数的碱基序列进行测序，这种方式对样本起始量要求较高且准确率较低，而且对后期数据分析有着非常高的要求。而多拷贝纳米孔文库构建的常规双链直接环化以及基于Gibson组装的效率较低，并且一般环化的片段短于1kb，限制了测序应用。基于呼吸作用的自扩增方法对于两端的Tm值要求比较高，并且可能存在模板DNA在扩增过程中自配对的现象。
发明内容
本发明提供一种纳米孔测序方法。通过多种方法构建可用于纳米孔测序的多拷贝文库，可以实现长片段的多次重复信号测定，能够有效提高纳米孔测序的准确率。
本发明通过如下技术方案实现：
一种纳米孔测序方法，包括：在纳米孔测序装置中，以核酸单链环或双链环为模板核酸进行滚环扩增产生出扩增的单链，在电场的作用下，将上述扩增的单链引入纳米孔测序装置的纳米孔中，使上述扩增的单链通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述模板核酸为核酸单链环；上述滚环扩增的反应体系包括：上述核酸单链环，与上述核酸单链环部分杂交的扩增引物，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液；上述方法包括：
将上述核酸单链环与上述扩增引物杂交形成杂交产物；
将上述杂交产物加入上述纳米孔测序装置中，加入上述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述反应体系还包括探针序列，上述探针序列上具有特定修饰，该特定修饰能够与上述纳米孔测序装置中支撑上述纳米孔的支撑层连接，上述扩增引物上包含能够与上述探针序列结合的探针识别位点；上述方法包括：
将上述扩增引物、上述探针序列和上述核酸单链环杂交形成杂交产物；
将上述杂交产物加入上述纳米孔测序装置中，使上述探针序列与上述支撑层连接，加入上述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述反应体系还包括探针序列，上述探针序列上具有特定修饰，该特定修饰能够与上述纳米孔测序装置中支撑上述纳米孔的支撑层连接，上述扩增引物上包含能够与上述探针序列结合的探针识别位点；上述方法包括：
将上述扩增引物和上述核酸单链环杂交形成杂交产物；
向上述纳米孔测序装置中加入上述探针序列使其与上述支撑层连接，加入上述杂交产物 使上述扩增引物与上述探针序列杂交，加入上述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述特定修饰选自胆固醇或生物素修饰。
在优选实施方案中，上述特定修饰是胆固醇修饰，上述支撑层是磷脂双分子层，上述胆固醇与上述磷脂双分子层通过亲和结合实现连接。
在优选实施方案中，上述特定修饰是生物素修饰，上述支撑层上具有链霉亲和素修饰，上述生物素与上述链霉亲和素通过亲和结合实现连接。
在优选实施方案中，上述核酸单链环上带有特殊修饰，该特殊修饰能够与上述纳米孔测序装置中支撑上述纳米孔的支撑层连接；上述方法包括：
将上述扩增引物、上述核酸单链环加入上述纳米孔测序装置中，使上述扩增引物、上述核酸单链环杂交形成杂交产物，且使上述核酸单链环通过上述特殊修饰与上述支撑层连接，加入上述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述特殊修饰选自生物素或聚乙二醇。
在优选实施方案中，上述特殊修饰是生物素，上述支撑层上具有链霉亲和素修饰，上述生物素与上述链霉亲和素通过亲和结合实现连接。
在优选实施方案中，上述模板核酸为核酸双链环；上述核酸双链环一条链带有缺口；上述滚环扩增的反应体系包括：上述核酸双链环，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液；上述方法包括：
将上述核酸双链环、聚合酶、dNTPs和反应缓冲液加入上述纳米孔测序装置中，进行滚环扩增以从上述核酸双链环的带有缺口的单链产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述反应体系还包括探针序列，上述探针序列上具有特定修饰，该特定修饰能够与上述纳米孔测序装置中支撑上述纳米孔的支撑层连接，上述核酸双链环上包 含能够与上述探针序列结合的探针识别位点；上述方法包括：
将上述核酸双链环、上述探针序列、聚合酶、dNTPs和反应缓冲液加入上述纳米孔测序装置中，使上述探针序列通过上述特定修饰与上述支撑层连接，进行滚环扩增以从上述核酸双链环的带有缺口的单链产生出扩增的单链；
在电场的作用下，使上述扩增的单链单个碱基逐步通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
在优选实施方案中，上述具有链置换活性的聚合酶选自phi29聚合酶或Bst聚合酶。
在优选实施方案中，上述电场的电压是毫伏级电压。
在优选实施方案中，上述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV-250mV的电压，最优选120mV的电压。
在优选实施方案中，上述方法还包括制备上述核酸单链环或双链环的步骤，该步骤包括对DNA片段进行环化得到单链环或双链环。
在优选实施方案中，上述DNA片段的长度是100bp至1Mbp。
在优选实施方案中，上述纳米孔是生物纳米孔和固态纳米孔。
在优选实施方案中，上述生物纳米孔是蛋白纳米孔，该蛋白纳米孔的最窄处满足使单链核酸通过的特性。
在优选实施方案中，上述固态纳米孔是硅基材料或者石墨烯二维材料的纳米孔，该硅基材料纳米孔的最窄处满足使单链核酸通过的特性。
本发明的纳米孔测序方法，基于多拷贝DNA纳米孔文库制备，不仅可以弥补常规纳米孔测序建库中的准确率不足的问题，还可以提高环化效率，并且在进行滚环扩增的同时进行纳米孔测序可以提高测序的时效性，缩短建库的时间并且简化测序步骤。同时本发明对所有可以进行纳米孔测序的碱基序列都适应，而且不会存在接头污染以及自配对的现象。
附图说明
图1为本发明实施例中文库构建使用的DNA接头序列结构示意图；
图2为本发明实施例中单链DNA环与接头杂交后置于纳米孔装置进行测序的方法示意图；
图3为本发明实施例中单链DNA环在纳米孔体系内与表面固定序列杂交进行测序的方法示意图；
图4为本发明实施例中对单链DNA环进行特殊修饰固定于支撑层表面进行测序的方法示意图；
图5为本发明实施例中无探针识别位点的DNA双链环文库构建和测序方法示意图；
图6为本发明实施例中有探针识别位点的DNA双链环文库构建和测序方法示意图；
图7为本发明实施例中制备的双链环状DNA琼脂糖凝胶电泳实验结果图；
图8为本发明实施例中双链环状DNA滚环扩增得到的DNA纳米球正向测序的结果图；
图9为本发明实施例中基于单链环进行纳米孔测序得到的电流变化结果图；
图10为本发明实施例中基于双链环进行纳米孔测序得到的电流变化结果图。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明主要基于滚环扩增的方法，完成基于纳米孔的多拷贝文库的构建及测序。本发明详细涉及多拷贝文库构建的技术方案以及其所需要的寡核苷酸序列组成、特殊修饰、反应条件、方法步骤等。
本发明的基本原理是：将待测的核酸(如DNA)打断成长片段后，通过特定的方法构建单链环或双链环，通过滚环扩增方法以及纳米孔测序装置，在电场的作用下，待测模板核酸会以单链的模式通过纳米孔并产生不同碱基的电信号，通过判断不同碱基引起的电信号差异，从而实现对不同序列模板核酸进行识别。
本发明一个实施例中，一种纳米孔测序方法，包括：在纳米孔测序装置中，以核酸单链环或双链环为模板核酸进行滚环扩增产生出扩增的单链，在电场的作用下，将上述扩增的单 链引入纳米孔测序装置的纳米孔中，使上述扩增的单链通过上述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对上述模板核酸上碱基序列的测定。
本发明实施例中，纳米孔测序装置可以按照现有该类装置实施，例如国际申请WO2014013262A1中公开的纳米孔测序装置。这种纳米孔测序装置可以是基于生物纳米孔或固态纳米孔的装置，生物纳米孔可以是蛋白纳米孔，固态纳米孔可以是硅基材料纳米孔，纳米孔的最窄处满足使单链核酸通过的特性。支撑纳米孔的支撑层可以是磷脂双分子层(磷脂膜)、多聚物膜或其它做了表面修饰(例如生物素修饰)的支撑物。
本发明实施例中，模板核酸包括两种类型，即核酸单链环或双链环。它们都可以通过对核酸(如DNA)片段进行环化而得到。这样的DNA片段，即用于进行环化建库的DNA样本，片段大小一般是需要适合构建单链环或双链环的长度，例如100bp至1Mbp，该核酸(如DNA)片段，可以来源于任何感兴趣的生物样品，如动物、植物、微生物等。核酸单链环，例如单链DNA环，一般是以DNA片段为模板，通过连接设计的接头并进行环化后得到的封闭DNA单链环，也可称为文库。核酸双链环，例如双链DNA环，一般是以DNA片段为模板，经过特殊设计的接头连接以及其他合适的技术方案得到的一条链上具有缺口的双链环状DNA。
如图1所示，本发明一个实施例中，文库构建使用DNA接头序列，该DNA接头序列由两条不同长度的碱基序列组成，如图1所示，较长的碱基序列在3’端的延长部分具有一段能够与单链环特异性结合的序列。该DNA接头序列一条链上带有特定修饰的碱基序列，如图1中短链3’端的修饰，例如可以是胆固醇或生物素修饰，该特定修饰能够与纳米孔测序装置中支撑纳米孔的支撑层连接而将整个DNA接头序列固定在支撑层上。
需要说明的是，在本发明实施例中，在模板核酸是核酸单链环的情况下，纳米孔测序必需的接头序列是如图1所示的那条在3’端的延长部分具有一段能够与单链环特异性结合的序列，其作为滚环扩增的引物，该序列在本发明中也称“扩增引物”。而图1中3’端带有修饰的短链是作为优选存在的序列，用于连接到纳米孔的支撑层上，以使扩增的单链靠近纳米孔并进入纳米孔中，该短链在本发明中也称“探针序列”。
在模板核酸是核酸单链环的情况下，本发明的纳米孔测序中滚环扩增的反应体系包括以下成分：核酸单链环，与核酸单链环部分杂交的扩增引物，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液等。本发明的纳米孔测序方法包括：将核酸单链环与扩增引物杂交形成杂交产物；将杂交产物加入纳米孔测序装置中，加入聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每 个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
在优选实施方案中，一种基于单链DNA环的纳米孔多拷贝文库构建和测序方法，如图2所示，示出了一种单链DNA环与接头(包括长链和短链)杂交后置于纳米孔装置进行测序的方法。反应体系包括核酸单链环，与核酸单链环部分杂交的扩增引物(即长链)，探针序列(即短链)，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液，探针序列上具有特定修饰(例如胆固醇或生物素修饰)，该特定修饰能够与纳米孔测序装置中支撑纳米孔的支撑层连接，扩增引物上包含能够与探针序列结合的探针识别位点；该方法包括：将扩增引物、探针序列和核酸单链环杂交形成杂交产物；将杂交产物加入纳米孔测序装置中，使探针序列与支撑层连接，加入聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
具体实施例中，扩增引物(即长链)、探针序列(即短链)二者具有部分序列互补，短链的3’末端进行修饰(例如胆固醇或生物素等)，先将长链和短链进行杂交，杂交产物再与单链环杂交。然后，将杂交后产物投放至已形成纳米孔的测序装置中，短片段3’端修饰物会与支撑层表面相互作用进而吸附在表面，实现杂交产物在纳米孔表面的富集。向测序装置中加入聚合酶以及dNTP，并对整套测序装置施加电场，使接头序列中的长链5’端进入到纳米孔中并在聚合酶和电场的作用下实现单个碱基逐步通过纳米孔，记录产生的电流信号。
在优选实施方案中，一种基于单链DNA环的纳米孔多拷贝文库构建和测序方法，如图3所示，示出了一种单链DNA环在纳米孔体系内与表面固定序列杂交。反应体系包括核酸单链环，与核酸单链环部分杂交的扩增引物(即长链)，探针序列(即短链)，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液，探针序列上具有特定修饰(例如胆固醇或生物素修饰)，该特定修饰能够与纳米孔测序装置中支撑纳米孔的支撑层连接，扩增引物上包含能够与探针序列结合的探针识别位点；该方法包括：将扩增引物和核酸单链环杂交形成杂交产物；向纳米孔测序装置中加入探针序列使其与支撑层连接，加入杂交产物使扩增引物与探针序列杂交，加入聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
具体实施例中，设计两段寡核苷酸序列(oligo)，其中一条链是3’端带有修饰(例如胆固醇、生物素等)的短链，称为探针序列；另一条链是既带有探针识别位点又能够与DNA单链环杂交的长链，称为扩增引物，将该扩增引物与DNA单链环杂交，形成引物-单链DNA (primer-ssDNA)杂交产物。将3’端带有修饰的短链(探针序列)固定于支撑层表面，在支撑层表面形成高密度的探针序列，并将引物-单链DNA杂交产物投放至形成纳米孔的测序装置中，使杂交产物上的探针识别位点与支撑层表面的探针序列相互杂交。向纳米孔测序装置中加入聚合酶和dNTP，并在纳米孔两端施加电压，长链(扩增引物)的5’端会在电场作用下进入到纳米孔中，并在聚合酶的作用下逐个碱基的通过，产生相应的电流信号。
上述实施例中，短链上的修饰可以是胆固醇修饰，支撑层可以是磷脂双分子层，胆固醇与磷脂双分子层通过亲和结合实现连接。其它实施例中，短链上的修饰可以是生物素修饰，支撑层上具有链霉亲和素修饰，生物素与链霉亲和素通过亲和结合实现连接。
在优选实施方案中，一种基于单链DNA环的纳米孔多拷贝文库构建和测序方法，如图4所示，示出了一种对单链DNA环进行特殊修饰固定于支撑层表面进行测序的方法。反应体系包括核酸单链环，与核酸单链环部分杂交的扩增引物(即长链)，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液。核酸单链环上带有特殊修饰(例如生物素或聚乙二醇等)，该特殊修饰能够与纳米孔测序装置中支撑上述纳米孔的支撑层连接。该方法包括：将扩增引物、核酸单链环加入纳米孔测序装置中，使扩增引物、核酸单链环杂交形成杂交产物，且使核酸单链环通过特殊修饰与支撑层连接，加入聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
具体实施例中，对核酸单链环上特定的碱基进行标记修饰，该标记修饰可以为生物素或者其他类似的化学物质(如聚乙二醇)，带有该标记修饰的碱基需要既具有正常的聚合作用又不会在聚合酶的作用下从双分子层表面被替换。在反应开始后，滚环扩增后的单链DNA会在电场作用下穿过纳米孔并产生相应的电信号。
上述实施例中，特殊修饰可以是生物素，支撑层上具有链霉亲和素修饰，生物素与链霉亲和素通过亲和结合实现连接。
在优选实施方案中，一种基于双链DNA环的纳米孔多拷贝文库构建和测序方法，如图5所示，示出了一种无探针识别位点的DNA双链环文库构建和测序方法，其模板核酸为核酸双链环，该核酸双链环一条链带有缺口；其反应体系包括：核酸双链环，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液。该方法包括：将核酸双链环、聚合酶、dNTPs和反应缓冲液加入纳米孔测序装置中，进行滚环扩增以从核酸双链环的带有缺口的单链产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
本发明实施例中，制备一条链带有缺口的核酸双链环的方法可以按照现有常规方法进行。例如，在一个实施例中，按照如下方法制备DNA双链环：对DNA片段进行末端修复并在末端加A；加特殊设计的接头(例如，两段接头为不对称、一条较长、一条较短的形成分叉结构的接头)；使用特殊设计的引物(例如，带有U碱基)进行PCR扩增；使用User酶进行处理，获得两端具有互补序列的双链DNA；环化处理，由于特殊设计的引物，在环化时会形成带有缺口的双链DNA环。
在优选实施方案中，一种基于双链DNA环的纳米孔多拷贝文库构建和测序方法，如图6所示，示出了一种有探针识别位点的DNA双链环文库构建和测序方法，其模板核酸为核酸双链环，该核酸双链环一条链带有缺口；其反应体系包括：核酸双链环，具有链置换活性的聚合酶、dNTPs和反应缓冲液，以及探针序列，该探针序列上具有特定修饰，该特定修饰能够与纳米孔测序装置中支撑上述纳米孔的支撑层连接，核酸双链环上包含能够与探针序列结合的探针识别位点。该方法包括：将核酸双链环、探针序列、聚合酶、dNTPs和反应缓冲液加入纳米孔测序装置中，使探针序列通过特定修饰与支撑层连接，进行滚环扩增以从核酸双链环的带有缺口的单链产生出扩增的单链；在电场的作用下，使扩增的单链单个碱基逐步通过纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对模板核酸上碱基序列的测定。
本发明实施例中，具有链置换活性的聚合酶可以选自phi29聚合酶或Bst聚合酶等。电场的电压一般是毫伏级电压，例如10mV以上的电压，优选50mV以上的电压，更优选100mV-250mV的电压，最优选120mV的电压。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例1：单链环状DNA的制备
根据MGIEasy TMDNA文库制备试剂盒(深圳华大智造科技有限公司)以大肠杆菌标准菌株为原料提取DNA，按照试剂盒说明书操作制备单链环状DNA。
实施例2：双链环状DNA的制备
1、phiX174质粒线性化
取1ug phiX174(1μL，NEB公司)，加入1μL ssPI酶(NEB公司)，5μL 10X Smart buffer，补加43μL水至50μL。涡旋混匀后放入PCR仪中，反应程序如下所示：37℃反应30分钟，65℃反应20分钟，4℃保存。
2、线性化DNA末端加A
取20μL线性化的phiX174，加3μL 10X NEB buffer2(NEB公司)，0.075μL 100mM dATP(Enzymatics公司)，6U Klenow Exo-(Enzymatics公司)，用水补到30μL，涡旋混匀后放入PCR仪中，反应程序如下所示：37℃反应30分钟，4℃保存。
3、加接头连接
将表1中YJ-1058与YJ-1092序列杂交得到25μM的杂交接头产物，取上述产物13μL，加入1.6μL 25μM的接头，0.55μL 600U/μL的T4 DNA连接酶(BGI公司)，加水补到30μL，涡旋混匀后放入PCR仪中，反应程序如下所示：20℃，30分钟，4℃保存。使用0.5X的AM Xpure磁珠进行纯化，并用60μL的TE进行溶解。
4、对连接产物进行PCR
取320ng的上述产物，加入10U Pfu Curbo Tur(Promega公司)、100μL PfuCx mix3(Promega公司)、1.6μL 25mM dNTP(Enzymatics)、5μL 20μM YJ-1054引物(表1)、5μL 20μM YJ-1055引物(表1)，用水补到200μL。混匀后使用如下反应程序：95℃，3min；9个循环的92℃，30secs+56℃，30secs+72℃，20min，68℃，18min，以0.1秒的速率降温至4℃。
5、使用User酶处理PCR产物
取4.5ug PCR产物，加入11μL 10X Taq buffer(NEB公司)、12μL User酶(NEB公司)，加水至110μL，混匀后在PCR仪上反应60min，再加入180μL TA buffer(BGI公司)，用水补至1810μL，在70℃水浴锅中反应30min后放置室温冷却20min。
6、环化
在上述产物中加入200μL 10X smart buffer，2μL T4 DNA连接酶(NEB公司)，用水补到2ml，室温反应2小时，使用0.5×的1:1的AM Xpure磁珠和HBB混合液进行纯化，并使用60μL TE溶解。
7、消化未环化产物
取59.1μL环化后产物，加入8.9μL 9X PS mix、9μL Plasmid-Safe酶、3μL ATP，混匀后置于PCR仪器中37℃反应1小时。使用1X AM Xpure磁珠进行纯化，并用60μL TE溶解。
8、聚合酶I(Polymerase I)处理
取10μL上述纯化后的产物加入1μL聚合酶I，9μL NEBuffer 2，混匀后放入PCR仪中反应，反应程序：14℃，30min。
9、琼脂糖凝胶电泳
分别取20ng的PCR产物、环化产物、消化后的环化产物、以及使用聚合酶I处理后的产物，使用0.8％的胶进行凝胶电泳实验，结果如图7所示。
M泳道为1kb plus ladder。泳道1为PCR产物，三角对应的是线性phiX174的条带。泳道2为环化产物，下面三角对应的是线性phiX174的条带，上面三角对应环化后的双链DNA环。泳道3为经过Plasmid-safe酶消化后的环化产物，三角对应环化后的双链DNA环。泳道4为经过Plasmid-safe酶消化后的环化产物再使用聚合酶I处理后的产物条带，三角对应环化后的双链DNA环。泳道1和2对比，可以看出环化效率接近于30％；泳道2和3对比可以说明上面三角对应的为双链环；泳道3和泳道4对比，可以说明形成的双链环包含一条封闭完整的环。
表1
Figure PCTCN2018119911-appb-000001
实施例3：双链环状DNA的滚环扩增
取出环化后的双链DNA文库、2×phi29 buffer(Enzymatics公司)、phi29酶(Enzymatics公司)，100mM EDTA pH8.0(Invitrogen公司)置于冰盒上约0.5h，待融化后，使用漩涡振荡器震荡混匀5s后，短暂离心置于冰盒上备用。
1、加入20μL的40fmol浓度的双链DNA链环。
2、加入20μL的2X phi29 buffer到PCR管中，混匀后置于PCR设备中，30℃孵化，5分钟。
3、在每个反应管中加入40μL的2x phi29，4μL的phi29酶(Enzymatics公司)用枪头上下搅拌均匀，置于PCR仪中，反应条件为30℃，2hr。
4、待反应完成后，在每个反应管中加入20μL 100mM EDTA pH 8.0，进行混合。
5、取出BGISEQ-500测序试剂盒，根据试剂盒说明书，将测序芯片装载至BGISEQ-500测序仪上，将上述反应产物DNA纳米球(DNB)加载到BGISEQ-500平台。根据测序试剂盒说 明书进行测序，设置参数进行拍照，第一轮正向测序的结果如图8所示。图8显示了在BGISEQ-500平台上进行正向测序，其中一个循环的结果，图中的亮点表明有多个拷贝数的DNB，方框突出显示出了出现DNB位置。
该实施例结果说明上述双链DNA文库能够制备并进行滚环扩增用于测序。
实施例4：基于单链环进行纳米孔测序
1、设计两条寡核苷酸序列(oligo)，其中一条是3’端带有特殊修饰(胆固醇)的短片段作为探针序列(序列为：SEQ ID NO：5：CCGCGTGAGATCTGATGATGTTTTTAAATTTGGGTTT/iSpC3/-Cholesterol，其中/iSpC3/表示-CH2CH2CH2-，用于阻断DNA的聚合，Cholesterol是指胆固醇)，另一条是既带有探针识别位点又可以与DNA单链环杂交的长链序列(序列为：SEQ ID NO：6：/iSpC3/AAAAAAACCTTCC/iSpC3/CCTTCCCATCATCATCAGATCTCACGCGGAAAAAAAAAAAAAAAAAAAAAAA/iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3/ GAACGACATGGCTA CGA，下划线序列部分与环状DNA互补，/iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3//iSpC3/起识别作用，当检测到该处电流信号意味着开始检测待测序列)，将该长链序列与实施例1中的单链环状DNA杂交，形成引物-单链DNA(primer-ssDNA)杂交产物。
2、搭建纳米孔测试装置：使用Axopatch 200B Patch Clam(Axon公司)与数据采集卡1550A(Axon公司)搭建信号采集平台，使用带有100μM的孔的槽(warner，BCH-M13)进行纳米孔实验成膜实验。
3、向进行实验的槽两边的池子中都加入1mL 300mM的KCl溶液，对槽的另一边(trans)施加120mV电压，形成双层磷脂膜，并加入MspA蛋白，形成蛋白纳米孔。在一边槽(cis)中加入5μL 10μM的primer-ssDNA杂交产物到cis端，并加入10μL 10mg/ml的野生型(WT)phi29聚合酶，以及10μL 25mM的dNTP(enzymatics公司)，在常温下进行反应并记录电流变化，如图9所示。
实施例5：基于双链环进行纳米孔测序
1、取1ug phiX174(1μL，NEB公司)，加入1μL ssPI酶(NEB公司)，5μL 10X Smart buffer，补加43μL水至50μL。涡旋混匀后放入PCR仪中，反应程序如下所示：37℃，30分钟,65℃反应20分钟，4℃保存。
2、将表2中GL-25与GL-26序列杂交得到25μM的杂交接头产物，取上述产物13μL，加入1.6μL 25μM的接头，9μL 3×HB buffer(BGI公司)，0.55μL 600U/μL的T4 DNA连接酶(BGI公司)，加水补到30μL，涡旋混匀后放入PCR仪中，反应程序如下所示：20℃，30分钟，4℃保 存。使用0.5×的AM Xpure磁珠进行纯化，并用60μL的TE进行溶解。
3、对连接产物进行PCR
取320ng的上述产物，加入10U Pfu Curbo Tur(Promega公司)、100μL PfuCx mix3(Promega公司)、1.6μL 25mM dNTP(Enzymatics公司)、5μL 20μM YJ-1054引物(表2)、5μL 20μM YJ-1055引物(表2)，用水补到200μL。混匀后使用如下反应程序：95℃，3min，9个循环的92℃，30secs+56℃，30secs+72℃，20min，68℃，18min，以0.1秒的速率降温至4℃。
4、使用User酶处理PCR产物
取4.5ug PCR产物，加入11μL 10X Taq buffer(NEB公司)、12μL User酶(NEB公司)，加水至110μL，混匀后在PCR仪上反应60min，再加入180μL TA buffer(BGI公司)，用水补至1810μL，在70℃水浴锅中反应30min后放置室温冷却20min。
5、环化
在上述产物中加入200μL 10X smart buffer，2μL T4连接酶(NEB公司)，用水补到2ml，室温反应2小时，使用0.5×的1:1的AM Xpure磁珠和HBB混合液进行纯化，并使用60μL TE溶解。
6、消化未环化产物
取59.1μL环化后产物，加入8.9μL 9X PS mix、9μL Plasmid-Safe酶、3μL ATP，混匀后置于PCR仪器中37℃反应1小时。使用1X AM Xpure磁珠进行纯化，并用60μLTE溶解，浓度为20ng/μL。
7、搭建纳米孔测试装置，使用Axopatch 200B Patch Clam(Axon公司)与数据采集卡1550A(Axon公司)搭建信号采集平台，使用带有100μM的孔的槽(warner，BCH-M13)进行纳米孔实验成膜实验。
8、向进行实验的槽两边的池子中都加入1mL 300mM的KCl溶液，并在一边槽(cis)中加入5μL 10μM寡核苷酸序列(序列为：SEQ ID NO：7：AAAAAAAAAAAAAAAAA-chelesetrol)；对槽的另一边(trans)施加120mV电压，用升降法形成双层磷脂膜，并加入MspA蛋白，形成蛋白纳米孔。
9、加入400ng纯化后的双链环到cis端，并加入10μL 10mg/ml的野生型phi29聚合酶，以10μL 25mM的dNTP(enzymatics公司)，在常温下进行反应并记录电流变化，如图10所示。
表2
Figure PCTCN2018119911-appb-000002
Figure PCTCN2018119911-appb-000003
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (20)

  1. 一种纳米孔测序方法，其特征在于，所述方法包括：在纳米孔测序装置中，以核酸单链环或双链环为模板核酸进行滚环扩增产生出扩增的单链，在电场的作用下，将所述扩增的单链引入纳米孔测序装置的纳米孔中，使所述扩增的单链通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  2. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述模板核酸为核酸单链环；所述滚环扩增的反应体系包括：所述核酸单链环，与所述核酸单链环部分杂交的扩增引物，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液；所述方法包括：
    将所述核酸单链环与所述扩增引物杂交形成杂交产物；
    将所述杂交产物加入所述纳米孔测序装置中，加入所述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  3. 根据权利要求2所述的纳米孔测序方法，其特征在于，所述反应体系还包括探针序列，所述探针序列上具有特定修饰，该特定修饰能够与所述纳米孔测序装置中支撑所述纳米孔的支撑层连接，所述扩增引物上包含能够与所述探针序列结合的探针识别位点；所述方法包括：
    将所述扩增引物、所述探针序列和所述核酸单链环杂交形成杂交产物；
    将所述杂交产物加入所述纳米孔测序装置中，使所述探针序列与所述支撑层连接，加入所述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  4. 根据权利要求2所述的纳米孔测序方法，其特征在于，所述反应体系还包括探针序列，所述探针序列上具有特定修饰，该特定修饰能够与所述纳米孔测序装置中支撑所述纳米孔的支撑层连接，所述扩增引物上包含能够与所述探针序列结合的探针识别位点；所述方法包括：
    将所述扩增引物和所述核酸单链环杂交形成杂交产物；
    向所述纳米孔测序装置中加入所述探针序列使其与所述支撑层连接，加入所述杂交产物使所述扩增引物与所述探针序列杂交，加入所述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电 信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  5. 根据权利要求3或4所述的纳米孔测序方法，其特征在于，所述特定修饰选自胆固醇或生物素修饰。
  6. 根据权利要求5所述的纳米孔测序方法，其特征在于，所述特定修饰是胆固醇修饰，所述支撑层是磷脂双分子层，所述胆固醇与所述磷脂双分子层通过亲和结合实现连接。
  7. 根据权利要求5所述的纳米孔测序方法，其特征在于，所述特定修饰是生物素修饰，所述支撑层上具有链霉亲和素修饰，所述生物素与所述链霉亲和素通过亲和结合实现连接。
  8. 根据权利要求2所述的纳米孔测序方法，其特征在于，所述核酸单链环上带有特殊修饰，该特殊修饰能够与所述纳米孔测序装置中支撑所述纳米孔的支撑层连接；所述方法包括：
    将所述扩增引物、所述核酸单链环加入所述纳米孔测序装置中，使所述扩增引物、所述核酸单链环杂交形成杂交产物，且使所述核酸单链环通过所述特殊修饰与所述支撑层连接，加入所述聚合酶、dNTPs和反应缓冲液进行滚环扩增产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  9. 根据权利要求8所述的纳米孔测序方法，其特征在于，所述特殊修饰选自生物素或聚乙二醇。
  10. 根据权利要求9所述的纳米孔测序方法，其特征在于，所述特殊修饰是生物素，所述支撑层上具有链霉亲和素修饰，所述生物素与所述链霉亲和素通过亲和结合实现连接。
  11. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述模板核酸为核酸双链环；所述核酸双链环一条链带有缺口；所述滚环扩增的反应体系包括：所述核酸双链环，以及具有链置换活性的聚合酶、dNTPs和反应缓冲液；所述方法包括：
    将所述核酸双链环、聚合酶、dNTPs和反应缓冲液加入所述纳米孔测序装置中，进行滚环扩增以从所述核酸双链环的带有缺口的单链产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  12. 根据权利要求11所述的纳米孔测序方法，其特征在于，所述反应体系还包括探针序列，所述探针序列上具有特定修饰，该特定修饰能够与所述纳米孔测序装置中支撑所述纳米孔的支撑层连接，所述核酸双链环上包含能够与所述探针序列结合的探针识别位点；所述方 法包括：
    将所述核酸双链环、所述探针序列、聚合酶、dNTPs和反应缓冲液加入所述纳米孔测序装置中，使所述探针序列通过所述特定修饰与所述支撑层连接，进行滚环扩增以从所述核酸双链环的带有缺口的单链产生出扩增的单链；
    在电场的作用下，使所述扩增的单链单个碱基逐步通过所述纳米孔并产生每个碱基的电信号，通过判断不同碱基引起的电信号差异，实现对所述模板核酸上碱基序列的测定。
  13. 根据权利要求2或11所述的纳米孔测序方法，其特征在于，所述具有链置换活性的聚合酶选自phi29聚合酶或Bst聚合酶。
  14. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述电场的电压是毫伏级电压。
  15. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述毫伏级电压是10mV以上的电压，优选50mV以上的电压，更优选100mV-250mV的电压，最优选120mV的电压。
  16. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述方法还包括制备所述核酸单链环或双链环的步骤，该步骤包括对DNA片段进行环化得到单链环或双链环。
  17. 根据权利要求16所述的纳米孔测序方法，其特征在于，所述DNA片段的长度是100bp至1Mbp。
  18. 根据权利要求1所述的纳米孔测序方法，其特征在于，所述纳米孔是生物纳米孔和固态纳米孔。
  19. 根据权利要求18所述的纳米孔测序方法，其特征在于，所述生物纳米孔是蛋白纳米孔，该蛋白纳米孔的最窄处满足使单链核酸通过的特性。
  20. 根据权利要求18所述的纳米孔测序方法，其特征在于，所述固态纳米孔是硅基材料或者石墨烯二维材料的纳米孔，该硅基材料纳米孔的最窄处满足使单链核酸通过的特性。
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