WO2017028757A1 - 固定序列的方法、芯片及应用 - Google Patents

固定序列的方法、芯片及应用 Download PDF

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WO2017028757A1
WO2017028757A1 PCT/CN2016/095048 CN2016095048W WO2017028757A1 WO 2017028757 A1 WO2017028757 A1 WO 2017028757A1 CN 2016095048 W CN2016095048 W CN 2016095048W WO 2017028757 A1 WO2017028757 A1 WO 2017028757A1
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sequence
substrate
solution
immobilizing
passivation
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PCT/CN2016/095048
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English (en)
French (fr)
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葛良进
高雁
纪道锐
邓力蔚
吴增丁
蔡金森
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深圳市瀚海基因生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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

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  • the invention belongs to the field of molecular biology, in particular to a method, a chip and an application for fixing a sequence.
  • Single-molecule sequencing technology is known as the third-generation sequencing technology. Its remarkable feature is that it can directly identify DNA fragments with high fidelity. Single-molecule sequencing technology has higher recognition than single-generation sequencing technology because it can recognize single nucleic acid molecules. Detection sensitivity.
  • the immobilization of DNA chips is the first step in single-molecule sequencing. The fixed mass and density of DNA on the chip determines whether subsequent single-molecule sequencing can proceed smoothly. To this end, researchers at home and abroad are committed to modifying the surface of the chip to introduce various functional groups on the surface, such as amino, aldehyde, carboxyl, sulfhydryl groups, etc., to form a porous three-dimensional network structure, so that DNA and the functional groups on the surface of the chip are covalently bonded. Further, it is stably cured on the surface of the substrate of the chip to increase the density of DNA immobilization.
  • the world's first single-molecule sequencing technology (tSMS) based sequencer was introduced to the market by Helicos in 2008. The principle is to perform side synthesis sequencing by detecting a single base fluorescent signal.
  • the primer used in the immobilization technique of Helicos single-molecule sequencer is a polyT sequence modified with an amino group or a DNA sequence complementary to the DNA to be tested.
  • the surface of the substrate to which the epoxy group is modified may have a fixed density of 3000 to 5000/field of view, but under the same experimental conditions, if the polyT primer modified with the amino group is replaced with the primer sequence complementary to the DNA to be tested, The fixed density is only 100 ⁇ 200 / field of view.
  • SMTS single molecule targeted sequencing technology
  • the present invention provides a sequence fixing method.
  • the method for immobilizing a sequence (preferably a targeting primer) provided by the present invention has a uniform fixed density of the sequence by adding a certain number of PolyT to the 5' end of the fixed sequence, and the fixed density can reach 2500 to 3200 points/field of view.
  • the present invention provides a method of fixing a sequence comprising the steps of:
  • a substrate is immersed in a fixing solution containing a sequence of 0.4 to 3.2 nM to obtain a sequenced substrate, which is a solid phase substrate whose surface is chemically modified, and one end of the sequence has a polyT of 10 to 30 bp.
  • the fixing solution is a phosphate solution;
  • the sequenced substrate is immersed in a passivation solution for passivation to obtain a passivated tape sequence substrate, and the passivation solution is a phosphate solution.
  • the sequence is a sequence having a polyT of 10 to 30 bp at the 5' end.
  • the sequence is such that the 5' end is attached to the surface of the substrate in such a manner that the amino group modified at the 5' end of the polyT is linked to the epoxy group on the surface of the substrate.
  • the sequence is a sequence in which a 5' to 30 bp polyT and an alkyl chain are sequentially linked at the 5' end.
  • the sequence is attached to the surface of the substrate by the alkyl chain.
  • the other end of the sequence may also be provided with an optical detection mark that can be used to verify the fixation effect of the sequence.
  • the sequence is a targeting primer
  • the method for immobilizing the targeting primer comprises the following steps:
  • the targeting primer is a primer sequence having a polyT of 10 to 30 bp at the 5' end, the primer sequence being a sequence complementary to at least a partial sequence of the template nucleic acid;
  • the washed substrate is immersed in a phosphate passivation solution for passivation; then the substrate is washed to obtain a substrate having a surface-immobilized primer attached thereto.
  • the targeting primer is a primer sequence in which a 10 to 30 bp polyT and an alkyl chain are sequentially linked at the 5' end, wherein the primer sequence is a sequence complementary to at least a partial sequence of the template nucleic acid.
  • the alkyl chain is an alkyl chain of -(CH 2 ) n -, wherein n is a natural number. According to an embodiment of the invention, the n is 6.
  • the sequence is a sequence in which the 5' end is sequentially linked with 10 bp of polyT and the alkyl chain is -(CH 2 ) 6 -.
  • the primer is attached to the surface of the substrate at the 5' end in such a manner that -(CH 2 ) 6 - is attached to the epoxy group on the surface of the substrate via an amino group.
  • the sequence is an amino group modified by a 5' end attached to a substrate having an epoxy group modified on the surface.
  • the substrate includes, but is not limited to, a glass substrate, a quartz substrate, and the like.
  • the surface of the substrate is modified with an epoxy group, an amino group, One or more of a carboxyl group, a thiol group, and an aldehyde group.
  • the fixing solution is a K 2 HPO 4 solution of 0.02 to 0.3 M.
  • the concentration of the targeting primer in the fixing solution is 0.8 to 3.2 nM, and more preferably 0.8 to 1.6 nM.
  • the strip sequence substrate is washed.
  • SSC saline sodium citrate
  • 20x SSC is the most standard imprinting and molecular hybridization treatment liquid in molecular biology.
  • 20x SSC is used for hybridization experiments to denature and clean commonly used concentrated buffers.
  • the 20x SSC was prepared by dissolving 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of water, adding a few drops of 10 mol/L NaOH solution to adjust the pH to 7.0, and adding water to a volume of 1 L.
  • the passivation time is 6 to 24 hours.
  • the passivation is passivated for 10 to 15 hours under shaking conditions.
  • the passivation is specifically: immersing the cleaned substrate in a phosphate passivation solution and shaking it on a shaker at 40 to 80 rpm/min for 10 to 15 hours.
  • the phosphate passivation solution is a 0.2 to 1.0 M K 2 HPO 4 solution.
  • the phosphate passivation solution is a K 2 HPO 4 solution having a pH of 9.0 and a concentration of 1 M.
  • the method further comprises washing the passivated strip sequence substrate.
  • the substrate is sequentially washed with a mixed solution of PBS solution, 150 mM HEPES buffer and 150 mM NaCl solution, and double distilled water.
  • the invention provides a chip comprising a sequence and a substrate, the sequence being attached to a surface of the substrate, the chip being obtained using the method of immobilizing a sequence of the first aspect of the invention.
  • the invention provides the use of a chip as provided by the second aspect of the invention in capturing a target region and/or nucleic acid sequencing.
  • the nucleic acid sequencing includes DNA and/or RNA sequencing.
  • the capture target region is a chip immobilized with a probe (primer) to capture a target nucleic acid to be tested (in the field of nucleic acid sequencing technology, also referred to as "template nucleic acid").
  • targeting primers and “sequences”, “primers” or “probes” as used herein may be used interchangeably to refer to a (oligo)nucleotide sequence.
  • the sequence is a targeting primer, 5' of the targeting primer
  • the primer sequences of 10 to 30 bp polyT and an alkyl chain are sequentially connected, wherein the primer sequence is a sequence complementary to at least a partial sequence of the template nucleic acid.
  • the invention provides a kit comprising the chip and reagent of the second aspect of the invention.
  • the reagents used can be selected depending on the use of the kit.
  • the invention provides the use of the kit of the fourth aspect in capturing a target region and/or nucleic acid sequencing.
  • the reagents used may be selected depending on the use of the kit, and the reagents may include one or more.
  • the reagents required may include immobilization reagents, extension reaction reagents, imaging reagents, and reagents for excising optical detection label molecules.
  • the kit also includes a buffer or other sequencing necessary reagent.
  • the immobilization reagent, the extension reaction reagent, the imaging reagent, and the reagent for excising the optical detection label molecule are not particularly limited. It is generally used in the art. For example, a person skilled in the art separately configures a buffer solution for different processes such as a fixed reaction reagent, an extension reaction reagent, and an optical detection label molecule, as needed.
  • the method for immobilization of the sequence provided by the first aspect of the present invention when used for single-molecule sequencing, does not need to add polyA to the 3' end of the template nucleic acid, and the template nucleic acid can be captured by targeting the primers.
  • the nucleic acid is measured to achieve real-time sequencing, and the fixed density can reach 2500-3200 points/field of view.
  • FIG. 1 is a schematic flow chart of a method for immobilizing a targeting primer in single molecule sequencing according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram of fixing a targeting primer according to an embodiment of the present invention.
  • 3 to 6 show the results of the immobilization reaction of the nucleic acid chip according to the embodiment of the present invention.
  • the method for immobilizing a targeting primer in single molecule targeted sequencing comprises the following steps:
  • the targeting primer is a primer sequence having a polyT of 10 to 30 bp at the 5' end, the primer sequence being a sequence complementary to at least a partial sequence of the template nucleic acid;
  • the washed substrate is immersed in a phosphate passivation solution for passivation; then the substrate is washed to obtain a substrate having a surface-immobilized primer attached thereto.
  • the targeting primer (partial sequence of the nucleic acid fragment to be tested) is complementary to cover the length of the gene fragment to be sequenced as much as possible, and the stable and efficient capture of the targeted DNA fragment, such as sequencing of the HBV virus, needs to cover all gene mutation points and Drug resistance sites.
  • Targeting primers are typically immobilized on an optically clear modified substrate.
  • the surface of the substrate needs to be chemically modified so that the targeting primer is immobilized on the surface of the substrate by chemical bonding or physical adsorption.
  • the substrate is any suitable carrier that has low natural fluorescence or is substantially non-fluorescent.
  • the substrate used in the present invention may be two-dimensional or three-dimensional, and may include a flat surface (eg, a glass slide), or may be other shapes.
  • the substrate of the present invention may comprise glass (for example, controlled pore glass (CPG)), quartz, plastic (such as polystyrene, not limited to low crosslinked and highly crosslinked polystyrene), polycarbonate, polypropylene. And polymethymethacrylate (methyl methacrylate), acrylic copolymer, polyamide, silicon, metal (eg, alkanethiolate-derived gold), cellulose, nylon, latex, dextran, gel matrix (eg, silica gel), Polyacrylaldehyde, or composite.
  • CPG controlled pore glass
  • plastic such as polystyrene, not limited to low crosslinked and highly crosslinked polystyrene
  • polycarbonate polypropylene.
  • polymethymethacrylate methyl methacrylate
  • acrylic copolymer polyamide
  • silicon metal
  • Suitable three-dimensional substrates include, for example, spheres, microparticles, beads, membranes, slides, plates, micromachined chips, tubes (eg, capillaries), microwells, microfluidic devices, channels, filters, or any other Suitable for anchoring the structure of a nucleic acid.
  • the substrate can include a planar array or matrix with targeted primer regions, such as nucleoside-derived CPG and polystyrene substrate sheets; derivatized magnetic substrate sheets, and the like.
  • the targeting primers are immobilized on the surface of the substrate by conventional chemical bonding or physical adsorption in the industry, and can be directly or indirectly (e.g., by biotin) immobilized on the surface of the substrate.
  • biotin e.g., by biotin immobilized on the surface of the substrate.
  • FIG. 2 a schematic diagram of fixing a targeting primer in the embodiment of the present invention.
  • Figure 2 includes ab, a is a schematic diagram of immobilization of targeting primer DNA on an epoxy slide, and b is a schematic diagram of a reaction scheme for immobilizing a targeting primer on a slide modified with other groups.
  • the technique utilizes the advantage that the epoxy group itself is unstable, has a large tension, and can chemically react with the DNA modified with -NH 2 , and the DNA to be immobilized by the new -CH 2 -NH- covalent bond The chain is immobilized on the surface of the chip modified with epoxy groups.
  • This immobilization technique is only one specific embodiment of the invention, and optimization of the method of the invention is effective for all immobilized vectors and DNA sequences.
  • the 5' end of the DNA sequence is linked with a group that can chemically react with the chip, such as an amino group, an aldehyde group, a carboxyl group, a thiol group, etc.; the 3' end is modified with a single molecule fluorescent substance, such as Cy3, Cy5, for the purpose of counting the fixed number. It is convenient to investigate the fixation effect of the targeting primer.
  • the 3' end of the oligonucleic acid strand of the 5'-linked polythymidine nucleotide (polydT) of the primer molecule is targeted, and the 5' of the polydT oligonucleic acid chain is bonded to the surface of the substrate by an epoxy bond. Hehe.
  • the 5' of the targeting primer molecule is ligated to the oligonucleic acid chain of the polythymidine nucleotide (polydT) and the alkyl chain (e.g., the alkyl group of -(CH 2 ) n - a chain, wherein n is a natural number, preferably an alkyl chain of -(CH 2 ) 6 -), wherein the 3' end of the poly dT oligonucleic acid strand is linked to the 5' of the targeting primer molecule, and the poly dT oligonucleic acid strand is The 5 terminal is attached to an alkyl chain which is bonded to the surface of the substrate by an epoxy bond.
  • polydT polythymidine nucleotide
  • the fixed primer density is required to be as high as possible in ensuring the dispersion at the single molecule level, thus ensuring the highest possible sequencing throughput.
  • the substrate can be subjected to other processing in the industry to improve nucleic acid attachment efficiency.
  • the substrates used in the present invention can be treated to reduce background noise.
  • the epoxide used to modify the substrate may also be a derivative of an epoxide.
  • the fixative is a 0.02-0.3 M K 2 HPO 4 solution.
  • the concentration of the targeting primer in the fixing solution is 0.8 to 3.2 nM, and more preferably 0.8 to 1.6 nM.
  • chemical passivation treatment is also required to eliminate unblocked epoxy groups and prevent noise caused by non-specific adsorption during sequencing.
  • surface passivation Since the fluorescently labeled base molecules in the sequencing are negatively charged, in our specific experiments, high concentrations of phosphate are used to block the epoxy groups on the surface of the glass substrate and generate negative charges. Layer to reduce negatively charged base adsorption.
  • the passivation time is from 6 to 24 hours.
  • step (2) the passivation is passivated for 10 to 15 hours under shaking conditions.
  • the passivation is specifically: immersing the cleaned substrate in a phosphate passivation solution and shaking it on a shaker at 40-80 rpm/min for 10-15 hour.
  • the phosphate passivation solution is a 0.2 to 0.8 M K 2 HPO 4 solution.
  • the phosphate passivation solution is a K 2 HPO 4 solution having a pH of 9.0 and a concentration of 1 M.
  • the invention also provides a single molecule targeted sequencing kit comprising a substrate, a targeting primer and a fixed reaction reagent.
  • the targeting primer is a primer sequence having a polyT of 10 to 30 bp at the 5' end, the primer sequence being a sequence complementary to at least a partial sequence of the template nucleic acid.
  • the single molecule targeted sequencing kit also includes a buffer or other sequencing necessary reagent.
  • a buffer or other sequencing necessary reagent For example, A person skilled in the art separately configures a buffer solution for different processes such as a fixed reaction reagent, an extension reaction reagent, and an optical detection label molecule, as needed.
  • the invention also provides the use of a method of immobilizing a targeting primer in single molecule targeted sequencing as described in the first aspect in DNA or RNA sequencing.
  • the method for immobilizing the targeting primer provided by the invention is used for single molecule sequencing, and the single molecule targeted sequencing scheme does not need to add polyA to the 3' end of the template nucleic acid, and the template nucleic acid can be captured by targeting the primer to realize real time. Sequencing allows the fixed density to reach 2500-3200 points/field of view.
  • the reagents used in the embodiments of the present invention are all commercially available products, and the databases used in the embodiments of the present invention are all public online databases.
  • 20x SSC Dissolve 175.3g NaCl and 88.2g sodium citrate in 800ml water, add a few drops of 10mol/l NaOH solution to adjust the pH to 7.0, add water to 1L, and autoclave after packing.
  • SSC is the most standard imprinting and molecular hybridization solution in molecular biology. 20xSSC is used in hybridization experiments to denature and clean commonly used concentrated buffers.
  • This embodiment provides a method for immobilizing a targeting primer in single molecule targeted sequencing, which specifically includes the following steps:
  • the substrate to be immobilized was purged with a nitrogen gas gun having an epoxy group on the surface, and the base glass slide was obtained from an epoxy-modified slide of SCHOTT Co.;
  • the target primer was diluted to a concentration of 0.8 nM with a 0.2 M K 2 HPO 4 fixative solution, and the substrate was immersed in the above fixed solution containing 0.8 nM targeting primer, and immersed for 60 min;
  • a phosphate passivation solution which is a K 2 HPO 4 solution having a pH of 9 and a concentration of 1 M, and is shaken at a temperature of 80 rpm/min on a shaker at room temperature. Passivation for 15h;
  • the substrate is sequentially washed with a mixed solution of PBS solution, 150 mM HEPES buffer and 150 mM NaCl solution, and double distilled water to obtain a substrate on which the surface-immobilized primer is immobilized;
  • the present invention was carried out by following the DNA (underlined portion -NH 2 (CH 2 ) 6 -):
  • TTTTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • T50-Cy3, C50-Cy3, and T10C50-Cy3 were respectively fixed on the substrate, and the operation was repeated in the above-described fixing step, and the number of the fixed sequences was counted six times. The result is shown in Figure 3.
  • the fixed density will be reduced from 2800 to 150; when in C50 When the 10 bp polyT was added to the 5' end, the fixed density was increased to 2800, which was basically equivalent to the fixed density of Helicos directly using polyT as a primer, indicating that the presence of a base T at the 5' end of the DNA greatly increased the fixed density.
  • 10 to 20 bp polyT can increase the fixed density to about 2800, and the fixed density is preferably 2500 to 3200.
  • the fixed density is higher than 4000, the fixed density of the targeting primer is too high, which will cause subsequent treatment.
  • the steric hindrance of the hybridized DNA molecule is too large, which in turn causes the hybridization density to be too low. Therefore, the present invention preferably binds 10 to 20 bp (preferably 10 bp) of polyT at the 5' end of the targeting primer to be immobilized.
  • the present invention is carried out as follows (underlined portion is -NH 2 (CH 2 ) 6 -):
  • TTTTTTTTTTTTCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • CAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • TTTTTTTTTTAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • CAGACACATCCAGCGATAAC is shown as SEQUENCE NO.11.
  • TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC is shown as SEQUENCE NO.
  • the DNA chip immobilization method was used for the immobilization.
  • the results of the experiment showed that the above DNA obtained a similar fixation effect as T10C50-Cy3, and a similar fixation rule of "T10C50-Cy3 and C50-Cy3": DNA consisting of polyT-(CH 2 )n
  • T10C50-Cy3 and C50-Cy3 DNA consisting of polyT-(CH 2 )n
  • the fixed density will be reduced from 2000-3500 to 150; when 10-30 bp polyT is added to the 5' end of -(CH 2 )n-, The fixed density is increased again to 2000-3500, indicating that the presence of a base T at the 5' end of the DNA greatly increases the fixed density.
  • step 1) the targeting primer to be immobilized is T10C50-Cy3, and the concentrations thereof are 0.4 nM, 0.8 nM, 1.6 nM, and 3.2 nM, respectively.
  • step 2) in passivation, the two batches are parallelized. For the test, the two sets of experiments were passivated for 15 h at 0 rpm/min and 80 rpm/min shaking, respectively.
  • Figure 4 shows the results of fluorescence microscopy photographing at 0 rpm/min during passivation, including a-d, fixed effects of 0.4 nM, 0.8 nM, 1.6 nM, 3.2 nM T10C50-Cy3.
  • Figure 5 shows the results of fluorescence microscopy at a rotation speed of 80 rpm/min during passivation, including a-d, 0.4nM, 0.8nM, 1.6nM, 3.2nM T10C50-Cy3.
  • Figure 6 shows the fixed density of DNA immobilized at different concentrations at a rotational speed of 80 rpm/min.
  • the fixed density of the targeting primer will increase as the concentration of the targeted primer to be immobilized increases, thereby being able to be based on the fixed concentration we need. Choose the best fixed concentration. It is verified by subsequent hybridization experiments that when the fixed density is higher than 4000, the fixed density of the targeting primer is too high, which will cause the spatial steric hindrance of the DNA molecule to be hybridized to be too large, resulting in too low hybridization density. As a result of the hybridization experiment, we chose a suitable fixed concentration of 0.8-1.6 nM.
  • a method for immobilizing a targeting primer in single molecule targeted sequencing includes the following steps:
  • the substrate to be immobilized is blown off with a nitrogen gas gun, and the surface of the substrate is provided with an epoxy group;
  • the targeting primer was diluted to a concentration of 0.8 nM with a 0.3 M K 2 HPO 4 fixing solution, and the substrate was immersed in the above fixed solution containing 0.8 nM targeting primer, and immersed for 120 min.
  • the targeting primer was T20C20-Cy3. ;
  • phosphate passivation solution which is a K 2 HPO 4 solution having a pH of 9 and a concentration of 0.8 M, and shaken at a temperature of 80 rpm/min at room temperature. Passivation on the bed for 24h;
  • the substrate is sequentially washed with a mixed solution of PBS, 150 mM HEPES buffer and 150 mM NaCl solution, and double distilled water to obtain a substrate on which the surface-immobilized primer is immobilized;
  • the number of sequences fixed on the substrate was repeated three times and the calculated average density of T20C20-Cy3 was 2716, and the standard deviation was 128.16.
  • the fixed result of T20C20-Cy3 was similar to that of T10C50-Cy3 (see Figure 3).
  • a method for immobilizing a targeting primer in single molecule targeted sequencing includes the following steps:
  • the substrate to be immobilized is blown off with a nitrogen gas gun, and the surface of the substrate is provided with an epoxy group;
  • the targeting primer was diluted to a concentration of 0.8 nM with a 0.02 M solution of K 2 HPO 4 , and the substrate was immersed in the above fixed solution containing 0.8 nM of the targeting primer, and immersed for 60 min.
  • the targeting primer was T20C50-Cy3. ;
  • a phosphate passivation solution which is a K 2 HPO 4 solution having a pH of 9 and a concentration of 0.2 M, and shaken at a temperature of 80 rpm/min at room temperature. Passivation on the bed for 6h;
  • the substrate was washed successively with a mixed solution of PBS solution, 150 mM HEPES buffer and 150 mM NaCl solution, and double distilled water to obtain a substrate on which the surface-immobilized primer was immobilized.
  • a method for immobilizing a targeting primer in single molecule targeted sequencing includes the following steps:
  • the substrate to be immobilized is blown off with a nitrogen gas gun, and the surface of the substrate is provided with an epoxy group;
  • the targeting primers were diluted to a concentration of 0.8 nM with a 0.05 M solution of K 2 HPO 4 , and the substrate was immersed in the above fixed solution containing 0.8 nM of targeting primers, and immersed for 45 minutes, and the targeting primers were respectively T10C20- Cy3, T10C30-Cy3, T10C40-Cy3;
  • a phosphate passivation solution which is a K 2 HPO 4 solution having a pH of 9 and a concentration of 0.6 M, and shaken at a temperature of 80 rpm/min at room temperature. Passivation on the bed for 12h;
  • the substrate was washed successively with a mixed solution of PBS solution, 150 mM HEPES buffer and 150 mM NaCl solution, and double distilled water to obtain a substrate on which the surface-immobilized primer was immobilized.

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Abstract

本发明提供了一种固定序列的方法、芯片及应用。所述固定序列的方法包括：(1)将一基底浸泡在含0.4～3.2nM序列的固定液中，获得带序列基底，所述基底为表面进行化学修饰的固相基质，所述序列的一个末端具有10～30bp的polyT，所述固定液为磷酸盐缓冲液；(2)将所述带序列基底浸入钝化液中进行钝化，获得钝化后的带序列基底，所述钝化液为磷酸盐溶液。本发明提供的固定序列密度均匀，约为3000点/视野。

Description

固定序列的方法、芯片及应用
本申请要求了2015年08月14日提交中国专利局的，申请号201510501968.7，发明名称为“一种单分子靶向测序中靶向引物的固定方法、单分子靶向测序试剂盒及应用”的中国专利申请的优先权，上述在先申请的内容以引入的方式并入本申请中。
技术领域
本发明属于分子生物学领域，特别是涉及一种固定序列的方法、芯片及应用。
背景技术
单分子测序技术被誉为第三代测序技术，其显著特征是可以高保真地对DNA片段直接进行识别，单分子测序技术由于能识别到单个核酸分子，其具有比第二代测序技术更高的检测灵敏度。DNA芯片的固定是单分子测序的第一步，DNA在芯片上的固定质量及密度决定着后续的单分子测序能否顺利进行。为此国内外研究人员致力于对芯片表面进行改性在表面引入各种官能团，如氨基、醛基、羧基、巯基等形成孔状的立体网架结构，使DNA与芯片表面的官能团共价结合进而稳定地固化于芯片的基底表面，提高DNA固定的密度。
世界上首个基于单分子测序技术(tSMS)的测序仪是由Helicos公司于2008年推向市场的。其原理是通过检测单个碱基荧光信号来实现边合成边测序。其中Helicos公司的单分子测序仪的固定技术中所用的引物为修饰有氨基的polyT序列或与待测DNA互补的DNA序列，然而，发明人通过实验表明，修饰有氨基的polyT的DNA很容易固定到表面修饰有环氧基团的基底表面，固定密度可以达到3000～5000/视野，但在同样的实验条件下，若将修饰有氨基的polyT引物更换为待测DNA互补的引物序列后，其固定密度只有100～200/视野。
为此，针对单分子靶向测序中与待测DNA序列互补的引物，需要进一步改进其固定技术，以推动单分子靶向测序技术(SMTS)的发展。
发明内容
有鉴于此，本发明提供了一种序列固定方法。本发明提供的序列(优选为靶向引物)的固定方法，通过在固定的序列的5’端加了一定数目的PolyT，对序列的固定密度均匀，固定密度可达2500～3200点/视野。
第一方面，本发明提供了一种固定序列的方法，包括以下步骤：
(1)将一基底浸泡在含0.4～3.2nM序列的固定液中，获得带序列基底，所述基底为表面进行化学修饰的固相基质，所述序列的一个末端具有10～30bp的polyT，所述固定液为磷酸盐溶液；
(2)将所述带序列基底浸入钝化液中进行钝化，获得钝化后的带序列基底，所述钝化液为磷酸盐溶液。
如本发明所述的，所述序列为5’端具有10～30bp的polyT的序列。在该实施方式中，序列为5’端连接到基底表面的方式为：polyT的5’端修饰的氨基与基底表面的环氧基连接。
优选地，所述序列为5’端顺次连接有10～30bp的polyT以及烷基链的序列。
在本发明实施方式中，所述序列通过所述烷基链连接到所述基底表面。
在本发明的另一实施方式中，所述序列的另一端还可以带有光学检测标记，可以用于检验所述序列的固定效果。
在本发明一实施方式中，所述序列为靶向引物，所述靶向引物的固定方法，包括以下步骤：
(1)将一基底浸泡在含0.4～3.2nM靶向引物的固定液中，之后清洗基底，
其中，所述靶向引物为5’端具有10～30bp的polyT的引物序列，所述引物序列为与模板核酸的至少部分序列互补的序列；
(2)将清洗后的基底浸入磷酸盐钝化液中进行钝化；之后再清洗基底，获得表面固定有靶向引物的基底。
优选地，所述靶向引物为5’端顺次连接有10～30bp的polyT以及烷基链的引物序列，其中，所述引物序列为与模板核酸的至少部分序列互补的序列。
本发明实施方式中，所述烷基链为-(CH2)n-的烷基链，其中，n为自然数。根据本发明一实施例，所述n为6。
更优选地，所述序列为5’端顺次连接有10bp的polyT以及烷基链为-(CH2)6-的序列。
在该实施方式中，引物为5’端连接到基底表面的方式为：-(CH2)6-通过氨基与基底表面的环氧基连接。
根据本发明一实施例，所述序列为通过5’端修饰的氨基连接到表面修饰有环氧基的基底。所述基底包括但不限于玻璃基底、石英基底等。所述基底表面修饰有环氧基、氨基、 羧基、巯基和醛基中的一种或多种。
本发明实施方式中，步骤(1)中，所述固定液为0.02～0.3M的K2HPO4溶液。
本发明实施方式中，步骤(1)中，所述固定液中靶向引物的浓度为0.8～3.2nM，进一步优选为0.8～1.6nM。
本发明实施方式中，步骤(1)中，在获得所述带序列基底之后，清洗所述带序列基底。
根据本发明一实施例，步骤(1)中，依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、pH＝8.5的K2HPO4来清洗基底。
SSC(saline sodium citrate)是分子生物学上最为标准的印迹及分子杂交处理液，20x SSC用于杂交实验变性及清洗常用浓缩型缓冲液。其中，20x SSC的配制方法为：在800ml水中溶解175.3g NaCl和88.2g柠檬酸钠，加入数滴10mol/L的NaOH溶液调节pH值至7.0，加水定容至1L。
本发明实施方式中，步骤(2)中，所述钝化的时间为6～24小时。
优选地，步骤(2)中，所述钝化是在摇晃条件下钝化10～15小时。
根据本发明一实施例，步骤(2)中，所述钝化具体为：将清洗后的基底浸入磷酸盐钝化液中，并置于40～80rpm/min的摇床上摇晃10～15小时。
本发明实施方式中，步骤(2)中，所述磷酸盐钝化液为0.2～1.0M的K2HPO4溶液。
优选地，步骤(2)中，所述磷酸盐钝化液为pH＝9.0、浓度为1M的K2HPO4溶液。
本发明实施方式中，步骤(2)中，在获得钝化后的带序列基底，之后，还包括清洗所述钝化后的带序列基底。
根据本发明一实施例，步骤(2)中，依次用PBS溶液、150mM的HEPES缓冲液与150mM的NaCl溶液组成的混合溶液及双蒸水来清洗基底。
第二方面，本发明提供了一种芯片，所述芯片包括序列和基底，所述序列与所述基底的表面连接，所述芯片利用本发明第一方面固定序列的方法所获得。
第三方面，本发明提供了如本发明第二方面提供的芯片在捕获目标区域和/或核酸测序中的用途。所述核酸测序包括DNA和/或RNA测序。所述捕获目标区域是采用固定有探针(引物)的芯片来捕获待测目标核酸(在核酸测序技术领域，也可称“模板核酸”)。
如无特殊说明，文中所称的“靶向引物”与“序列”、“引物”或“探针”可替换使用，指一段(寡)核苷酸序列。
例如，当所述芯片用于捕获目标区域时，所述序列为靶向引物，所述靶向引物的5’ 端顺次连接有10～30bp的polyT以及烷基链的引物序列，其中，所述引物序列为与模板核酸的至少部分序列互补的序列。
第四方面，本发明提供了一种试剂盒，包括本发明第二方面所述的芯片和试剂。可以根据所述试剂盒的用途，来选择其所用的试剂。
第五方面，本发明提供了第四方面所述的试剂盒在在捕获目标区域和/或核酸测序中的用途。
可以根据所述试剂盒的用途，来选择其所用的试剂，所述试剂可以包括一种或多种。例如，当所述芯片用于核酸测序(例如单分子测序)时，所需要的试剂可包括固定反应试剂、延伸反应试剂、成像试剂、切除光学检测标记分子的试剂。
可以理解的是，所述试剂盒还包括缓冲液或其他测序必要试剂。在本发明实施例中，所述固定反应试剂、延伸反应试剂、成像试剂、切除光学检测标记分子的试剂均没有特别限制。本领域现有普通常用的即可。比如，本领域技术人员根据需要分别配置针对的固定反应试剂、延伸反应试剂、切除光学检测标记分子等不同过程的缓冲液。
本发明第一方面提供的序列固定的方法在用于单分子测序时，单分子靶向测序方案无需在模板核酸的3'末端加上polyA，只需通过靶向引物即可捕获模板核酸(待测核酸)，实现实时测序，可使固定密度达到2500-3200点/视野以上。
附图说明
图1为本发明实施例提供的单分子测序中靶向引物的固定方法的流程示意图；
图2为本发明实施例对靶向引物进行固定的原理图；
图3～6为本发明实施例提供的核酸芯片的固定反应结果。
具体实施方式
结合图1，本发明提供的单分子靶向测序中靶向引物的固定方法，包括以下步骤：
(1)将一基底浸泡在含0.4～3.2nM靶向引物的固定液中，之后清洗基底，
其中，所述靶向引物为5’端具有10～30bp的polyT的引物序列，所述引物序列为与模板核酸的至少部分序列互补的序列；
(2)将清洗后的基底浸入磷酸盐钝化液中进行钝化；之后再清洗基底，获得表面固定有靶向引物的基底。
靶向引物(与待测核酸片段的部分序列)互补尽可能覆盖所需待测序基因片段的长度，稳定高效的捕获靶向DNA片段，比如针对HBV病毒的测序，需要覆盖所有的基因突变点和耐药位点。靶向引物一般固定在光学透明的经过修饰的基底上面。基底表面需要经过化学修饰以便于靶向引物通过化学键或者物理吸附作用固定在基底表面。基底为具有低的天然荧光或基本上不发荧光的任何合适的载体。
在一个优选的实施方案中，用于本发明的基底可以是二维或三维的，并且可以包括一个平坦表面(例如，玻璃载玻片)，或者可以是其他形状。本发明的基底可以包括玻璃(例如，可控孔度玻璃(CPG))，石英，塑料(如聚苯乙烯，不限于低交联和高交联的聚苯乙烯)，聚碳酸酯，聚丙烯和聚methymethacrylate(甲基丙烯酸甲酯)，丙烯酸共聚物，聚酰胺，硅，金属(例如，alkanethiolate-衍生金)，纤维素，尼龙，乳胶，葡聚糖，凝胶基质(例如，硅胶)，聚丙烯醛，或复合材料。
合适的三维基底包括，例如，球，微粒，珠，膜，载玻片，平板，微机械加工的芯片，管(例如，毛细管)，微孔，微流体装置，通道，过滤器，或任何其它合适用于锚定核酸的结构。基底可包括具有靶向引物区域的平面阵列或矩阵，比如包括核苷衍生的CPG和聚苯乙烯基底片；衍生的磁基底片等。
靶向引物通过行业内常规的化学键或者物理吸附作用固定在基底表面，可以直接或间接(如通过生物素)固定在基底表面。在一个具体实施方案中，我们使用的是经环氧硅烷处理的低荧光玻璃表面，其表面的环氧键可以和靶向引物末端的氨基进行化学键合。
如图2所示，本发明实施例对靶向引物进行固定的原理图。图2包括a-b，a为在环氧基玻片上固定靶向引物DNA示意图，b为在修饰有其他基团的玻片上固定靶向引物的反应流程示意图。该技术利用了环氧基团本身不稳定，张力较大，能和修饰有-NH2的DNA发生化学反应的优点，通过新的-CH2-NH-这个共价键将待固定的DNA单链固定在修饰有环氧基团的芯片表面。该固定技术仅是本发明的一种具体实施例，本发明方法的优化对所有的固定载体和DNA序列均有效。DNA序列5’端连有可以和芯片发生化学反应的基团，如氨基、醛基、羧基、巯基等；3’端用单分子荧光物质修饰，如Cy3、Cy5，目的在于统计固定的数目，便于考察靶向引物的固定效果。
在一些实施方案中，靶向引物分子的5’连接聚胸腺嘌呤核苷酸(聚dT)的寡核酸链的3端，聚dT寡核酸链的5’通过氨基与基底表面的环氧基键合。在一个优选的实施方案中，靶向引物分子的5’顺次连接有聚胸腺嘌呤核苷酸(聚dT)的寡核酸链和烷基链(比 如，-(CH2)n-的烷基链，其中，n为自然数，优选为-(CH2)6-的烷基链)，其中，聚dT寡核酸链的3’端与靶向引物分子的5’相连，聚dT寡核酸链的5端与烷基链相连，烷基链通过氨基与基底表面的环氧键键合。
固定好的引物密度要求为在保证单分子级别的分散度上，尽可能的密度高一些，这样可以保证尽可能高的测序通量。在一实施方案中，基底可以采用行业内其他处理方式，以改善核酸附着效率。在其他实施方案中，可以对本发明中使用的基片进行处理，以降低背景噪音。用于对基底进行修饰的环氧化物还可以为环氧化物的衍生物。
在一些实施方案中，步骤(1)中，所述固定液为0.02～0.3M的K2HPO4溶液。
在一些实施方案中，步骤(1)中，所述固定液中靶向引物的浓度为0.8～3.2nM，进一步优选为0.8～1.6nM。
在一些实施方案中，步骤(1)中，依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、pH＝8.5的K2HPO4来清洗基底。
在基底固定了靶向引物之后，还需要经过化学钝化处理以消除未封闭的环氧基团和防止测序过程中的产生的非特异性吸附带来的噪声。表面钝化处理有很多种方法，由于测序中的荧光标记的碱基分子是负电的，在我们的具体实验中，使用高浓度的磷酸盐来封闭玻璃基底表面的环氧基团，并产生负电层，以减小负电性的碱基吸附。
在本发明一些实施方案中，步骤(2)中，所述钝化的时间为6～24小时。
在本发明一些优选实施方案中，步骤(2)中，所述钝化是在摇晃条件下钝化10～15小时。
在本发明一些优选实施方案中，步骤(2)中，所述钝化具体为：将清洗后的基底浸入磷酸盐钝化液中，并置于40～80rpm/min的摇床上摇晃10～15小时。
在本发明一些实施方案中，步骤(2)中，所述磷酸盐钝化液为0.2～0.8M的K2HPO4溶液。
在本发明一些优选实施方案中，步骤(2)中，所述磷酸盐钝化液为pH＝9.0、浓度为1M的K2HPO4溶液。
本发明还提供了一种单分子靶向测序试剂盒，包括基底、靶向引物和固定反应试剂。
所述靶向引物为5’端具有10～30bp的polyT的引物序列，所述引物序列为与模板核酸的至少部分序列互补的序列。
可以理解的是，所述单分子靶向测序试剂盒还包括缓冲液或其他测序必要试剂。比如， 本领域技术人员根据需要分别配置针对的固定反应试剂、延伸反应试剂、切除光学检测标记分子等不同过程的缓冲液。
最后，本发明还提供了一种如第一方面所述的单分子靶向测序中靶向引物的固定方法在DNA或RNA测序中的用途。
本发明提供的靶向引物的固定方法在用于单分子测序时，单分子靶向测序方案无需在模板核酸的3'末端加上polyA，只需通过靶向引物即可捕获模板核酸，实现实时测序，可使固定密度达到2500-3200点/视野以上。
具体实施例
材料及试剂说明：
非特殊说明，本发明实施例采用的试剂均为市售商品，本发明实施例采用的数据库均为公开的在线数据库。
20x的SSC:在800ml水中溶解175.3g NaCl和88.2g柠檬酸钠，加入数滴10mol/l NaOH溶液调节pH值至7.0，加水定容至1L，分装后高压灭菌。SSC是分子生物学上最为标准的印迹及分子杂交处理液，20xSSC用于杂交实验变性及清洗常用浓缩型缓冲液。
实施例1
本实施例提供了一种单分子靶向测序中靶向引物的固定方法，具体包括如下步骤：
(1)将待固定序列的基底用氮气枪吹净待用，所述基底表面带有环氧基，基底玻片来自于为SCHOTT公司的环氧基修饰的玻片；
用0.2M的K2HPO4的固定液将靶向引物稀释成浓度为0.8nM，将基底浸泡在上述含0.8nM靶向引物的固定液中，浸泡60min；
之后依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、pH＝8.5的K2HPO4来清洗基底；
(2)将清洗后的基底浸入磷酸盐钝化液，所述磷酸盐钝化液为pH＝9、浓度为1M的K2HPO4溶液，并于室温下以80rpm/min的转速在摇床上进行钝化15h；
之后再依次用PBS溶液、150mM的HEPES缓冲液与150mM的NaCl溶液组成的混合溶液及双蒸水来清洗基底，获得所述表面固定有靶向引物的基底；
(3)通过荧光显微镜对固定好的芯片进行照相，每个芯片拍摄连续的20个视野，然后统计每张照片的单分子荧光点数，计算每个视野下荧光点数的平均值。
具体地，为观察引物尾部polyT对固定密度的影响，本发明如下DNA进行(下划线部 分为-NH2(CH2)6-)固定：
T50-Cy3(5’→3’)：
AmMC6TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-Cy3，
具体地，所述
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT为SEQUENCE NO.1所示。
T10C50-Cy3(5’→3’)：
AmMC6TTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.2所示。
C50-Cy3(5’→3’)：
AmMC6CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3
具体地，所述
CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.3所示。
在本实施例中，将T50-Cy3、C50-Cy3、T10C50-Cy3分别固定在基底上，按上述的固定步骤进行操作，重复六次并统计固定上的序列数目。结果如图3所示。由图3可知，当靶向引物由T50-Cy3换为C50-Cy3时(即将修饰有氨基的polyT引物更换为待测DNA互补的引物序列)，固定密度会由2800降为150；当在C50的5’端加入10bp的polyT时，固定密度又增加至2800，基本上与Helicos直接采用polyT作引物的固定密度相当，这说明DNA的5’端存在一段碱基T会大大提高固定的密度。(经后续实验验证，10～20bp的polyT均可将固定密度提高至2800左右，固定密度优选为2500～3200)，当固定密度高于4000时，靶向引物的固定密度过高会造成后续待杂交的DNA分子的空间位阻过大，进而会导致杂交密度过低。因此，本发明优选在待固定靶向引物的5’端连接10～20bp(优选10bp)的polyT。
具体地，为进一步观察引物尾部polyT的数量以及不同靶向引物序列对固定密度的影响，本发明如下DNA进行(下划线部分为-NH2(CH2)6-)固定：
T10C20-Cy3(5’→3’)：
AmMC6TTTTTTTTTTCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTCAGACACATCCAGCGATAAC为SEQUENCE NO.4所示。
C20-Cy3(5’→3’)：
AmMC6CAGACACATCCAGCGATAAC-Cy3，
具体地，所述
CAGACACATCCAGCGATAAC为SEQUENCE NO.5所示。
T10C30-Cy3(5’→3’)：
AmMC6TTTTTTTTTTTAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTTAAAACGCCGCAGACACATCCAGCGATAAC为
SEQUENCE NO.6所示。
C30-Cy3(5’→3’)：
AmMC6AAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
AAAACGCCGCAGACACATCCAGCGATAAC为
SEQUENCE NO.7所示。
T10C40-Cy3(5’→3’)：
AmMC6TTTTTTTTTTAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.8所示。
C40-Cy3(5’→3’)：
AmMC6AGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
AGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.9所示。
T20C20-Cy3(5’→3’)：
AmMC6TTTTTTTTTTTTTTTTTTTTCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTTTTTTTTTTTCAGACACATCCAGCGATAAC为SEQUENCE NO.10所示。
C20-Cy3(5’→3’)：
AmMC6CAGACACATCCAGCGATAAC-Cy3，
具体地，所述
CAGACACATCCAGCGATAAC为SEQUENCE NO.11所示。
T20C50-Cy3(5’→3’)：
AmMC6TTTTTTTTTTTTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
TTTTTTTTTTTTTTTTTTTTCAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.12所示。
C50-Cy3(5’→3’)：
AmMC6CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC-Cy3，
具体地，所述
CAGGATGCAGAGGAAGATGATAAAACGCCGCAGACACATCCAGCGATAAC为SEQUENCE NO.13所示。
采用前述DNA芯片固定方法进行固定，实验结果显示，上述DNA均获得与T10C50-Cy3类似的固定效果，以及“T10C50-Cy3和C50-Cy3”类似的固定规律：DNA由polyT-(CH2)n-DNA-Cy3换为-(CH2)n-DNA-Cy3时，固定密度会由2000-3500降为150左右；在-(CH2)n-的5’端加入10-30bp的polyT时，固定密度又增加至2000-3500，这说明DNA的5’端存在一段碱基T会大大提高固定的密度。本实施方式中的部分实验结果如图3所示。
实施例2
钝化中摇晃转速对固定照片中荧光斑点的影响及不同浓度的靶向引物对固定效果的影 响
按照上述实施例1中记载的实验方法进行操作，不同的是：
步骤1)中，待固定的靶向引物为T10C50-Cy3，其浓度分别为0.4nM、0.8nM、1.6nM、3.2nM；此外，步骤2)中，在钝化时，分为两批进行平行试验，两组实验分别在0rpm/min和80rpm/min摇晃条件下钝化15h。
重复三次并统计基底上固定的序列数目，荧光显微镜的拍照结果如图4和图5所示。图4为钝化过程中转速为0rpm/min时荧光显微镜拍照结果，包括a-d，分别0.4nM、0.8nM、1.6nM、3.2nM的T10C50-Cy3的固定效果。图5为钝化过程中转速为80rpm/min时荧光显微镜拍照结果，包括a-d，分别0.4nM、0.8nM、1.6nM、3.2nM的T10C50-Cy3的固定效果。
图6为转速设置为80rpm/min，不同浓度固定DNA的固定密度。
由图4-6可知：
1.当靶向引物在固定液中的浓度相同的情况下，在Cy3的激发波长为532nm时，通过对比拍摄到的荧光照片可以看出，在钝化过程中将摇床的转速设置为40～80rpm/min(在一些实施例中，优选为80rpm/min)可以显著消除掉固定照片中的大荧光斑点，而在钝化过程中不进行摇晃时，在拍摄到的荧光照片观察到大块的光斑，这可能是环氧基基底对不带氨基的DNA的非特异性吸附，也可能是DNA分子的团聚，总之，这将会影响固定的效果；
2.在钝化过程中转速一定的条件下(如同为80rpm/min)，靶向引物的固定密度会随着待固定靶向引物浓度的提高而提高，由此可以根据我们需要的固定浓度来选择最佳的固定浓度。经后续的杂交实验验证，当固定密度高于4000以上时，即靶向引物的固定密度过高，这会造成后续待杂交的DNA分子空间位阻过大，会导致杂交密度过低，故根据杂交实验结果，我们选择较为适宜的固定浓度为0.8～1.6nM。
实施例3
一种单分子靶向测序中靶向引物的固定方法，包括如下步骤：
(1)将待固定序列的基底用氮气枪吹净待用，所述基底表面带有环氧基；
用0.3M的K2HPO4的固定液将靶向引物稀释成浓度为0.8nM，将基底浸泡在上述含0.8nM靶向引物的固定液中，浸泡120min，所述靶向引物为T20C20-Cy3；
之后依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、 pH＝8.5的K2HPO4来清洗基底；
(2)将清洗后的基底浸入磷酸盐钝化液，所述磷酸盐钝化液为pH＝9、浓度为0.8M的K2HPO4溶液，并于室温下以80rpm/min的转速在摇床上进行钝化24h；
之后再依次用PBS、150mM的HEPES缓冲液与150mM的NaCl溶液组成的混合溶液及双蒸水来清洗基底，获得所述表面固定有靶向引物的基底；
(3)通过荧光显微镜对固定好的芯片进行照相，每个芯片拍摄连续的20个视野，然后统计每张照片的单分子荧光点数，计算每个视野下荧光点数的平均值。
重复三次并统计基底上固定的序列数目，通过计算发现，T20C20-Cy3固定平均密度为2716，标准方差是128.16，T20C20-Cy3的固定结果与T10C50-Cy3的固定结果类似(见图3)。
实施例4
一种单分子靶向测序中靶向引物的固定方法，包括如下步骤：
(1)将待固定序列的基底用氮气枪吹净待用，所述基底表面带有环氧基；
用0.02M的K2HPO4的固定液将靶向引物稀释成浓度为0.8nM，将基底浸泡在上述含0.8nM靶向引物的固定液中，浸泡60min，所述靶向引物为T20C50-Cy3；
之后依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、pH＝8.5的K2HPO4来清洗基底；
(2)将清洗后的基底浸入磷酸盐钝化液，所述磷酸盐钝化液为pH＝9、浓度为0.2M的K2HPO4溶液，并于室温下以80rpm/min的转速在摇床上进行钝化6h；
之后再依次用PBS溶液、150mM的HEPES缓冲液与150mM的NaCl溶液组成的混合溶液及双蒸水来清洗基底，获得所述表面固定有靶向引物的基底。
实施例5
一种单分子靶向测序中靶向引物的固定方法，包括如下步骤：
(1)将待固定序列的基底用氮气枪吹净待用，所述基底表面带有环氧基；
用0.05M的K2HPO4的固定液将靶向引物稀释成浓度为0.8nM，将基底浸泡在上述含0.8nM靶向引物的固定液中，浸泡45min，所述靶向引物分别为T10C20-Cy3、T10C30-Cy3、T10C40-Cy3；
之后依次采用3x SSC与0.1％的Triton组成的混合溶液、3x的SSC及浓度为150mM、pH＝8.5的K2HPO4来清洗基底；
(2)将清洗后的基底浸入磷酸盐钝化液，所述磷酸盐钝化液为pH＝9、浓度为0.6M的K2HPO4溶液，并于室温下以80rpm/min的转速在摇床上进行钝化12h；
之后再依次用PBS溶液、150mM的HEPES缓冲液与150mM的NaCl溶液组成的混合溶液及双蒸水来清洗基底，获得所述表面固定有靶向引物的基底。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (17)

  1. 一种固定序列的方法，其特征在于，包括以下步骤：
    (1)将一基底浸泡在含0.4～3.2nM序列的固定液中，获得带序列基底，所述基底为表面进行化学修饰的固相基质，所述序列的一个末端具有10～30bp的polyT，所述固定液为磷酸盐溶液；
    (2)将所述带序列基底浸入钝化液中进行钝化，获得钝化后的带序列基底，所述钝化液为磷酸盐溶液。
  2. 如权利要求1所述的固定序列的方法，其特征在于，所述序列为5’端顺次连接有10～30bp的polyT以及烷基链的序列。
  3. 如权利要求1所述的固定序列的方法，其特征在于，所述基底的表面修饰有环氧基、氨基、羧基、巯基和醛基中的一种或多种。
  4. 如权利要求2所述的固定序列的方法，其特征在于，所述序列通过所述烷基链连接到所述基底表面。
  5. 如权利要求1-4任一项所述的固定序列的方法，其特征在于，所述序列通过5’端修饰的氨基连接到表面修饰有环氧基的基底。
  6. 如权利要求1所述的固定序列的方法，其特征在于，所述基底选自玻璃，石英，塑料，聚碳酸酯，聚丙烯，聚甲基丙烯酸甲酯，丙烯酸共聚物，聚酰胺，硅，金属，纤维素，尼龙，乳胶，葡聚糖，凝胶基质，聚丙烯醛和其他复合材料中的至少一种。
  7. 如权利要求1所述的固定序列的方法，其特征在于，步骤(1)中，所述固定液为0.02～0.3M的K2HPO4溶液。
  8. 如权利要求1所述的固定序列的方法，其特征在于，步骤(1)中，所述浸泡的时 间为45min～120min。
  9. 如权利要求1所述的固定序列的方法，其特征在于，步骤(1)中，在获得所述带序列基底之后，清洗所述带序列基底。
  10. 如权利要求1所述的固定序列的方法，其特征在于，步骤(2)中，所述钝化液为0.2～1.0M的K2HPO4溶液。
  11. 如权利要求1所述的固定序列的方法，其特征在于，步骤(2)中，所述钝化的时间为6～24小时。
  12. 如权利要求1所述的固定序列的方法，其特征在于，步骤(2)中，所述钝化是在摇晃条件下钝化10～15小时。
  13. 如权利要求1所述的固定序列的方法，其特征在于，步骤(2)中，在获得钝化后的带序列基底之后，还包括清洗所述钝化后的带序列基底。
  14. 一种芯片，所述芯片包括序列和基底，所述序列与所述基底的表面连接，所述芯片利用权利要求1-13任一项所述的方法获得。
  15. 如权利要求14所述的芯片在捕获目标区域和/或核酸测序中的用途。
  16. 一种试剂盒，其特征在于，包括权利要求14所述的芯片和试剂。
  17. 如权利要求16所述的试剂盒在捕获目标区域和/或核酸测序中的用途。
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