WO2008056816A1 - Support de suspension pour une molécule d'acide nucléique linéaire, procédé d'extension d'une molécule d'acide nucléique linéaire et spécimen de molécule d'acide nucléique linéaire - Google Patents

Support de suspension pour une molécule d'acide nucléique linéaire, procédé d'extension d'une molécule d'acide nucléique linéaire et spécimen de molécule d'acide nucléique linéaire Download PDF

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Publication number
WO2008056816A1
WO2008056816A1 PCT/JP2007/072049 JP2007072049W WO2008056816A1 WO 2008056816 A1 WO2008056816 A1 WO 2008056816A1 JP 2007072049 W JP2007072049 W JP 2007072049W WO 2008056816 A1 WO2008056816 A1 WO 2008056816A1
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WO
WIPO (PCT)
Prior art keywords
linear
nucleic acid
acid molecule
linear nucleic
support
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PCT/JP2007/072049
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English (en)
Japanese (ja)
Inventor
Hiroyuki Kabata
Ariko Fuke
Toshiaki Mizuno
Original Assignee
Kyoto University
Nippon Sheet Glass Company, Limited
Japan Science And Technology Agency
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Application filed by Kyoto University, Nippon Sheet Glass Company, Limited, Japan Science And Technology Agency filed Critical Kyoto University
Priority to JP2008543157A priority Critical patent/JP5167449B2/ja
Publication of WO2008056816A1 publication Critical patent/WO2008056816A1/fr

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Classifications

    • 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
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0896—Nanoscaled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic

Definitions

  • Linear nucleic acid molecule suspension support linear nucleic acid molecule extension method, and linear nucleic acid molecule specimen
  • the present invention relates to extension or hybridization of a linear nucleic acid molecule such as a chromosome to a fiber state.
  • Chromosomes contain deoxyribonucleic acid (DNA), a bright macromolecule that retains genetic information, but in the natural state, they are several ⁇ ⁇ in diameter.
  • D is determined by fragmenting Satoshi Kajita, so the problem is that each sequence obtained by analysis cannot be written well, and the entire sequence is determined. There was a problem that it took time and money to do. If the chromosomes can be extended in the form of fibers, various analyzes of DNA will be facilitated. For this reason, reports of elongation of DNA and angular analysis of elongated DNA (DNA fiber) have been reported. In addition, the FISH method (fluorescence in situ hybridization method) using DNA fibers has also been reported.
  • the sequence (base sequence) of the bases that make up a single-stranded DNA fiber is an address that is shown in a linear form.
  • a single-stranded DNA fiber a single-stranded DNA (probe DNA) that can bind to the specified position on the fiber in a complementary manner (hybridization), and this
  • probe DNA probe DNA
  • the member group is guided to the specified sequence on the single-stranded DNA fiber according to the base sequence of each probe DNA that holds the member. It is stopped by forming a specific hydrogen bond.
  • arbitrary members can be arranged in a line as designed, joined together, and grown into an aggregate. In this case, it is easier to align the functional molecules when the single-stranded DNA fiber is suspended in the solution.
  • the DNA fiber obtained by the above-mentioned Conti and 3 ⁇ 4ensimon technology is formed by pressure-bonding a DNA double helix to the surface of a substrate.
  • double helix denaturation which is one of the steps of the FISH method (two molecules — both single-stranded DNAs dissociate together and accept another single-stranded DNA from the outside) is susceptible to inhibition. . Devising the efficiency of denaturation while avoiding inhibition is required to realize a more effective FISH method.
  • the probe is pre-hybridized after denaturation of the double helix.
  • This probe is, for example, a fluorescently labeled foreign single-stranded DNA.
  • High predization is a base sequence-specific binding to one of the two dissociated single-stranded DNAs.
  • the DNA is close to the substrate, so it suffers steric hindrance from the surface force, and the probe comes into contact with and binds to the DNA NA fiper. It is difficult to achieve hybridization. There is no effective solution for increasing the efficiency of hybridization in the conventional FISH method and nanoarchitecture method.
  • the presence or absence of a gene is analyzed based on the presence or absence of probe binding, or the distribution of genes is analyzed depending on the location of the probe on the fiber.
  • the amount of probe is typically much greater than the amount of DNA fiber.
  • the majority of the probes adsorb nonspecifically on the same surface as the substrate on which the DNA fibers are mounted, and emit a strong fluorescent background. This background is stronger than the fluorescence emitted by meaningful probes that have performed specific hybridization with DNA fibers. This makes it difficult to detect the optical signal that should be the result of the analysis, and, for example, inhibits the identification of the presence or absence of the aforementioned gene.
  • DNA can be elongated using electroosmotic flow (for example, K. Terao, H. Kabata, and M. Washizu, J. Phys, Condens. Matter 18, S654 (2006)).
  • electroosmotic flow for example, K. Terao, H. Kabata, and M. Washizu, J. Phys, Condens. Matter 18, S654 (2006).
  • electroosmotic flow for example, K. Terao, H. Kabata, and M. Washizu, J. Phys, Condens. Matter 18, S654 (2006).
  • a plurality of columnar bodies are formed on a substrate, and the surface of the columnar bodies has DNA An oligonucleotide having a sequence complementary to the sequence of the amplification target portion of the fragment is attached.
  • the columnar body is a protrusion having an arbitrary shape to which the DNA fragment can be fixed.
  • the distance between the columnar bodies is equal to or slightly shorter than the distance between the fixing parts provided on both sides of the amplification target part.
  • the DNA fragment containing the amplification target portion is introduced in an extended state, and is fixed to the columnar body by chemical bonding with the oligonucleotide on the surface of the columnar body.
  • PCR polymerase chain reaction
  • DNA fibers When strictly determining the electrical conductivity of DNA, originally isolated DNA fibers A single substance should be measured, but in reality, multiple DNA molecules aggregated in a mass or film are measured. At this time, the aggregates of DNA molecules are randomly attached on the My plate, for example, so that the measurement environment (hydration near the plate, gas-liquid atmosphere, presence of conductive impurities such as ions, DNA The concentration was not uniform and the DNA structure was inconsistent. To solve this problem, DNA is fixed in a fiber shape and measured in a large amount of solution that can provide a constant measurement environment. In other words, the DNA fiber is preferably suspended at a position away from the surface. In order to use double-stranded DNA as an example of a linear polymer for electrical circuit wiring, it is necessary to handle longer DNA molecules.
  • An object of the present invention is to provide a novel extension technique such as a chromosome without the above-mentioned problems.
  • the support according to the present invention comprises a surface comprising a plurality of linear convex portions and a bottom portion between the linear convex portions, and the linear convex portion does not chemically react with the linear nucleic acid molecule.
  • This support is used to suspend the elongated linear nucleic acid molecule in the air between the tops of the plurality of linear protrusions.
  • a linear nucleic acid molecule is a force that can be made into a fiber.
  • S A linear compound that is not in a fiber state in the natural state. For example, a chromosome containing a double-stranded DNA molecule or a single-stranded DNA molecule is labeled.
  • the width of the bottom has a value that does not come into contact with the bottom even if the linear nucleic acid molecule passed between the tops of the linear protrusions on both sides of the bottom extends elastically.
  • the plurality of linear protrusions are, for example, concentric circles or concentric polygons. Further, the plurality of linear convex portions may form a mesh.
  • the support may include at least one of the linear protrusion and the bottom. Made of porous material.
  • the height of the plurality of linear convex portions may not be constant.
  • the support further includes a lid material facing the surface.
  • a surface corresponding to the surface of the lid member includes a protruding portion that can contact the linear protrusion of the structure while being covered with the structure.
  • a linear nucleic acid molecule specimen according to the present invention comprises a fiber-like linear nuclear acid that is spanned and fixed in a hollow state between the above-mentioned support and the plurality of linear collar portions of the support. It consists of molecules. Further, the linear nucleic acid molecule may be further coated with a polymeric plastic material that can be dissolved or embrittled by heating, decompression, or soaking.
  • the bottom portion between the plurality of linear protrusions is a surface that generates an electroosmotic flow or an electrophoretic flow made of a charged material or an artificially charged material, Between the linear convex portions is the flow path of the electroosmotic flow or the electrophoretic flow. Linear nucleic acid molecules are transported in the channel.
  • a liquid containing a linear nucleic acid molecule that can be fiberized but is not fiberized is allowed to stand on the surface of the support.
  • the support includes the surface including a plurality of linear convex portions and a bottom portion between the linear convex portions, and is made of a material that does not chemically react with a linear nucleic acid molecule.
  • a shear stress is applied to the linear nucleic acid molecule on the support, and as a result, the linear nucleic acid molecule is in a fiber state and is spanned between the plurality of linear protrusions. Fixed.
  • the shear stress is generated, for example, by a centrifugal force generated by rotating the support body around a rotation axis perpendicular to the surface.
  • the linear nucleic acid molecule in a fiber state is hybridized with a probe.
  • linear nucleic acid molecule extension method for example, after fixing a linear nucleic acid molecule to the support, a fading-colored IJ is sealed between the surface of the support and a cover glass for visualization. Visualizing the linear nucleic acid molecule.
  • the linear nucleic acid molecule is a single-stranded DNA or a multi-stranded DNA.
  • a liquid containing linear nucleic acid molecules is added to the support.
  • a visualization agent is further added to visualize the linear nucleic acid molecule.
  • a bottom portion between the linear convex portions is filled with a polymer plastic material that can be dissolved after curing, and the polymer plastic material is cured.
  • the support is used.
  • the support is a microchannel
  • the plurality of linear recesses are arranged in a flow direction
  • the shear stress is generated by electroosmotic flow.
  • the circuit board according to the present invention includes a plurality of linear protrusions made of a material that does not chemically react with a linear nucleic acid molecule, a surface including a bottom portion between the linear protrusions, and the plurality of linear protrusions. It is equipped with a linear nucleic acid molecule in a fiber state that is spanned and fixed between the convex portions in a hollow state.
  • the fiber in which the linear nucleic acid molecule in the fiber state is a conductive substance is coated with a metal.
  • the shearing stress acts on the linear nucleic acid molecule in the liquid, and the linear nucleic acid molecule is elongated longer than before. Since a support having linear protrusions is used, a straight-chain nucleic acid molecule in a fiber state that is not crimped can be easily produced. In addition, it has become possible to improve the detection efficiency of the probe and clarify the positional relationship of the binding sites, which are development issues that were desired to be achieved with the current FISH technology. This technique is also suitable for chromosome diagnosis and polymorphism analysis. Useful for optical mapping of genes.
  • nanowire substrate in electrical and electronic engineering, or as a bottom-up assembly saddle for nanotechnology.
  • FIG. 1 is a diagram for schematically explaining elongation of a linear nucleic acid molecule.
  • FIG. 2 is an example of a concentric polygonal support.
  • FIG. 3 is an example of a support having a mesh structure.
  • FIG. 4 is a diagram of an example of a support made of a porous material.
  • FIG. 5 is a diagram of an example of a support made of a porous material.
  • FIG. 6 is a diagram of an example of a support made of a porous material.
  • FIG. 7 is a diagram of an example of a support body having a linear convex portion composed of / lines.
  • FIG. 8 is a diagram of an example of a support body having linear protrusions made of line segments.
  • FIG. 9 is a diagram of another example of the support.
  • FIG. 10 is a light micrograph of a substrate with concentric linear protrusions on the left side, and a DNA fluorescence micrograph of HeLa cells stretched on the substrate on the right side.
  • FIG. 11 is a partially enlarged view of FIG.
  • Figure 12 shows the photomicrograph of the left side of the substrate with concentric linear protrusions and the right side of the FISH of chromosome 7 using the EGFR gene marker, and the right side, stretched on the substrate. This is a fluorescence micrograph of FISH on DNA fiber.
  • Figure 13 is a photomicrograph of the provided substrate, and a fluorescence micrograph of DNA fiber ⁇ ISH using the MYC gene label stretched on the substrate on the right and top sides.
  • Figure 14 is a fluorescent micrograph on chromosome 7 to which a centromeric gene marker has been added.
  • Figure 15 is a diagram for explaining the detection of the copy number of oncogenes on the chromosome and the detection of the spatial position of oncogenes.
  • Fig. 16 is a photograph of an example of normal and abnormal (amplification) detection using EGFR and centromere gene labeling.
  • Figure 17 is a photograph of suspended chromosome fiber using EGFR and centromere gene markers.
  • Figure 18 is a picture of a suspended chromosome with a MYC gene marker.
  • FIG. 19 is a photograph of an example of detection of translocation of DNA fibers by FISH.
  • FIG. 20 is a diagram for explaining selective polymerase chain reaction.
  • FIG. 21 is a cross-sectional view of the microphone port channel.
  • Figure 22 is a diagram of a chromosome stretched by electroosmotic flow.
  • Figure 23 shows an example of a nanoarchitecture.
  • FIG. 24 shows the transfer of a linear nucleic acid molecule from a linear nucleic acid molecule suspended support to obtain a partially presented film.
  • FIG. 25 is a diagram in which a linear nucleic acid molecule is transferred from a linear nucleic acid molecule suspension support to obtain a film in which all of the linear nucleic acid molecules are presented.
  • FIG. 26 is a diagram for explaining the protection and distribution of linear nucleic acid molecules suspended over the tops of the linear protrusions.
  • Fig. 27 is a diagram for explaining a method of suspending linear nucleic acid molecules when there is a significant difference in height between the saddle-shaped convex part of the structure and the bottom part between them.
  • Figure 28 shows an example of nanowire wiring.
  • the elongated linear nucleic acid molecule is immobilized on a glass plate.
  • a polymeric plastic material containing a linear nucleic acid molecule is layered by centrifugal force, and the linear nucleic acid molecule is extended into a fiber state therein. Then, the layer of the polymeric plastic material is solidified, and the linear nucleic acid molecule in the fiber state is embedded in the polymer film.
  • Fiber FISH technology is useful for FISH by extending DNA on a substrate such as a glass slide, and is useful for human chromosome genetic diagnosis and large-scale structural polymorphism analysis.
  • the stretched DNA fiber may be deformed because it is crimped to the substrate.
  • the signal of the probe eg probe DNA
  • the stretched state on the substrate When the DNA fiber is hybridized (crossed) with the probe DNA, the fiber structure changes due to the crimping to the substrate. Probe DNA cannot access the extended DNA fiber, leading to a decrease in probe binding capacity and FISH efficiency. In that case, it is difficult to grasp the spatial relationship of genes.
  • Figure 1 shows schematically an example of the elongation of a linear nucleic acid molecule (chromosome).
  • Linear nucleic acid Suspension support (hereinafter simply referred to as “support”), which is a support that supports linear nucleic acid molecules in a hollow state.
  • 10 is a plurality of linear protrusions 1 2 and the bottom between them 1 4 and has a surface to be a force.
  • the plurality of linear protrusions 12 are arranged concentrically.
  • the stage is rotated for an appropriate time (for example, 30 seconds) at a rotational speed of, for example, 300,000 rpm.
  • a rotational speed for example, 300,000 rpm.
  • the liquid on the support 10 expands due to the centrifugal force generated by the rotation, and shear stress acts on the chromosomes in the liquid.
  • the chromosome 16 extends substantially linearly across the plurality of linear protrusions 12 and is crimped to the top of the protrusions 12.
  • the chromosome 16 that has been fiberized on the support is held between the linear convex portions 12 without being in contact with the bottom portion 14 and separated from the hollow portion.
  • a “linear nucleic acid molecule” is a linear compound that can be made into a fiber but not in a fiber state in the natural state.
  • a chromosome containing a double-stranded DNA molecule or a single-stranded DNA molecule And a hybrid compound of a labeled complementary molecule.
  • DNA may be denatured and entangled by the hybridization process, making it difficult to relax and difficult to stretch (hard to unravel). For example, if the single-stranded DNA molecule is stretched first before the crossing treatment, for example, the single-stranded DNA molecule is stretched first, this inconvenience is avoided.
  • the present invention can eliminate this drawback.
  • the linear nucleic acid molecule can be first stretched at a time when it is easy to be unwound and then subjected to a hybridization treatment.
  • the purpose of the cross process is, for example, visualization.
  • the linear nucleic acid molecule preferably has a visualization site.
  • visualization include, for example, dyeing Addition of colorant and subsequent microscopic observation, or radioisotope labeling followed by autoradiography, or fluorescent substance labeling and subsequent scanner reading.
  • the visualization agent is further visualized and visualized.
  • the support 10 is a glass plate having a plurality of linear protrusions 12, a surface that is lower than the linear protrusions 12, and has a bottom 14 and a force.
  • the material of the support 10 is not limited to glass, but a material that does not chemically bond to a linear nucleic acid molecule is used.
  • “Linear” means an elongated shape, and convex 1 2 has an appropriate width capable of supporting a linear nucleic acid molecule.
  • the top of the linear protrusion 1 2 is preferably flat. In Fig.
  • the force that only two linear protrusions 1 2 are drawn schematically is actually the width of the bottom 14 and the number of linear protrusions 1 2 It is set appropriately (for example, 20 pieces with a width of 0.1 mm).
  • the height of the linear convex part 12 ie, the depth of the bottom part 14
  • the elasticity of the DNA NA chain up to 120% of its own length.
  • the height of the convex portion 1 2 is higher than 1/3 of the width of the bottom portion 14 or higher than 1 Z 2 of the width of the bottom portion 14.
  • the depth between the convex portions 12 be deeper than the depth of focus of the microscope.
  • the linear nucleic acid molecule in the Fino state is hollow and maintains the original fiber shape. It is desirable that the width of the collar 1 2 is narrow.
  • the plurality of # spring-like convex portions 1 2 are arranged concentrically in the above example, and the intervals are constant.
  • the central part of the support 10 is flat, and the liquid containing the linear nucleic acid molecule is allowed to stand at the central part (flat part).
  • the support 10 is placed on the stage of the rotating device 18 and is rotated at a high speed around a rotation axis perpendicular to the surface of the support 10. The rotation causes a centrifugal force to act on the liquid on the support 10 to generate a shearing stress on the linear nucleic acid molecule, and the linear nucleic acid molecule in the liquid is elongated in a fiber shape (fiber shape).
  • the linear nucleic acid molecule 16 is expanded into a plurality of linear protrusions. It is fixed by being crimped to the top of 1 2, and is kept floating between the linear protrusions 1 2. Therefore, the elongated linear nucleic acid molecule (eg, DNA fiber) is in a state of being separated from the support 10 except for the linear convex portion 12 and is hollow between the linear convex portions 12. It is floating and not crimped to the support 10. This technology makes it easy to create DNA fibers that include parts that are not constrained to the support 10. When using FISH technology for DNA fibers, the spacing between the linear protrusions 12 should be set so that probe DNA can hybridize.
  • FISH FISH
  • the difference in height (or depth, or aspect ratio) between multiple linear protrusions and the bottom below the linear protrusions is, for example, the characteristics of the support material (crystal structure, etc.) and the height difference It can be set freely by combining with the processing method to provide
  • a processing method wet etching
  • the obtained aspect ratio is about 0.5.
  • a convex part with a height of about 1 Z 2 of the width of the bottom part is produced.
  • an ultraviolet light exposure application using SU-8 gives an aspect ratio of 15 or, alternatively, a PMMA resist and synchrotron radiation exposure. Then, an aspect ratio of 100 or more can be obtained.
  • the operator determines the desired hollow part length, and then considers the elongation rate of the linear nucleic acid molecule (for example, from 120% to 170% for DNA).
  • Set the aspect ratio Appropriate supports can be manufactured by selecting and combining materials and processing methods suitable for the set values. For this purpose, the aspect ratio is preferably in a range larger than 1/2.
  • the higher the aspect ratio (the greater the depth from the top of the plurality of protrusions to the bottom between the plurality of protrusions), the more the hollow portion of the linear nucleic acid molecule is in contact with the bottom, Alternatively, it is possible to eliminate the problem that the bottom surface is visualized by being mixed with impurities adhering to the convex surface.
  • the convex portion having a large aspect ratio hinders the process of stretching the linear nucleic acid molecule into a fiber, that is, the liquid containing the linear nucleic acid molecule is placed on the surface of the support. There is a possibility of blocking the spreading and spreading.
  • This blockage has low viscosity and low surface tension (high spreadability)
  • a liquid such as chlorofluorocarbon that can get over the convex part
  • the corner can be determined.
  • the liquid containing the linear nucleic acid molecule is water, for example, the bottom upper surface and the convex side surfaces are hydrophobized (for example, with silicone) to prevent water from entering the bottom and blocking the convex portions. By doing so, water selectively passes through the top of the convex part and spreads.
  • the upper limit of the aspect ratio between the convex part and the bottom part of the support is provided so as not to inhibit the spread of the liquid containing the linear nucleic acid molecule.
  • linear protrusions 12 Various shapes can be adopted for the linear protrusions 12 provided on the support 10.
  • the structures of the linear protrusions 12 and the bottom 14 in the support 10 are not limited to concentric circles.
  • the elongated linear nucleic acid molecules can be supported at the apexes of the plurality of linear protrusions 12 and can be held hollow between them.
  • the plurality of linear protrusions 12 may be concentric ellipses. In this case, the interval between the II-shaped convex portions 12 changes within a certain range.
  • the shape of the linear protrusions 12 need not be limited to a circle, but may be a structure that surrounds a liquid containing a linear nucleic acid molecule, such as a triangle. Therefore, it may generally be a concentric polygon.
  • the # spring-like convex portion 12 is a triangle, and the plurality of linear convex portions 12 are arranged as concentric triangles.
  • the linear convex portion may have a shape in which line segments are combined.
  • Fig. 3 shows an example of a structure in which a bottom 14 is provided in a mesh shape.
  • the linear convex portion 12 2 ′ is formed such that a plurality of parallel upright plates are orthogonal to a plurality of other parallel upright plates.
  • the distance between the linear protrusions 1 2 "varies within a certain range. This range is the length of the linear polymer to be stretched and fixed, and the linear height desired by the operator.
  • the chromosome located at the bottom 14 'in a partition is elongated by centrifugal force and has two linear protrusions. Part 1 2 'is supported hollowly.
  • the material of the linear convex group is not limited to a smooth filling (nonporous) structure such as a glass plate.
  • the linear protrusion 10 may be composed of a three-dimensional porous structure (such as silica gel).
  • the three-dimensional porous structure is made of a porous material through which liquid can pass.
  • the bottom 14 between the linear protrusions 1 2 or As shown, both the bottom 14 and the linear protrusion 12 may be composed of a three-dimensional porous structure.
  • probes and visualization agents can pass through the 3D porous mesh freely, so that injection and discharge can be performed from a position where force is applied to the entire support. It becomes.
  • the elongated shape of the linear convex portion is not limited to a polygonal column or a circular column having the same height (height difference from the bottom), but may be a polygonal column or a cylinder (for example, FIG. 9) having a changed height.
  • the lower limit is that the linear polymer 16 (for example, chromosomal fiber) fixed in a hollow on the support 10 is the bottom The height is high enough not to touch.
  • the upper limit of the change in the height of the linear convex portion is that the solution containing the linear polymer provided to the support 10 gets over the linear convex portion and spreads on the surface of the support 10.
  • the chromosome is extended by centrifugal force and supported in a hollow space between the two linear protrusions 1 2 '' '.
  • the above-mentioned various microstructures of the support 10 can be produced by using a well-known photolithography technique, but preferably the method described in Japanese Patent Application Laid-Open No. 2 005 2 3 0 6 4 7 is used. It is done. In the latter manufacturing method, when a stress-affected residue is formed on the surface of a glass plate such as an ano-reminosilicate, and then wet etching is performed, the etching rate between the stress-affected residue and other portions is reduced. Due to the difference, the microstructure is formed It is. Specifically, in the production of the glass plate used in the present embodiment, the stress-affected residual portion is formed in a portion where the linear convex portion 12 of the support 10 is to be formed.
  • the stress-affected residual portion can be formed by pressing with, for example, an indenter having a sharp tip or a desired pressing portion (so-called mold).
  • a mask for wet etching may be opened by applying a surface protective coating agent. Next, wet etching is performed. When a normal mask is applied, the etching force is applied in a non-directional direction. In a state including a portion where a stress is applied, etching is performed with directionality. As a result, due to the difference in the etching rate, a fine structure composed of the linear convex portion 12 and the bottom portion 14 is formed.
  • the linear nucleic acid molecule suspension support preferably has the following properties. This is because the support does not emit light (low autofluorescence) due to the excitation light (for example, ultraviolet light) required for the probe to emit fluorescence.
  • the probe diffuses freely in order to efficiently bind to the chromosome (the plurality of protrusions and the bottom part between the plurality of protrusions are rich in substance permeability). Therefore, it is desirable to be a porous glass body.
  • the convex part of the support is a substance with high affinity (polycation, etc.) that makes it easy to fix the chromosome, and conversely, the bottom part is a substance with low affinity (polyaion that does not touch the chromosome). Etc.) and surface modification.
  • the fiberization that is, the elongation of the linear nucleic acid molecule will be further explained. This is considered to be due to shear stress in the liquid.
  • the stage on which the liquid is placed is rotated, the liquid containing the linear nucleic acid molecule is also rotated on the support, and the centrifugal force due to the rotation acts in the direction parallel to the support with respect to the liquid. .
  • Centrifugal force is proportional to the distance from the center of rotation and the square of the rotational speed.
  • the liquid thins in layers as it rotates.
  • liquids are viscous, so the magnitude of the fluid flow velocity changes in a direction perpendicular to the surface of the support. The flow velocity increases as you move away from the support and becomes constant when you leave it sufficiently.
  • a force called shear stress is generated in the direction of flow depending on the viscosity. The shear stress is zero on the surface of the support, but increases as it moves away from the surface, and becomes constant when it is sufficiently away from the surface.
  • the shear stress acting on that linear nucleic acid molecule is not supported.
  • the size is different between the side closer to the holder and the side farther, and acts as a shear force. For this reason, along with the rotation, the linear nucleic acid molecule that was folded in the natural state is loosened and is elongated in the fiber direction in the direction of the centrifugal force.
  • the position of the linear nucleic acid molecule is preferably in the vicinity of the center of rotation before the rotation.
  • the shear stress is small near the center of rotation and increases with distance from the center of rotation.
  • the linear nucleic acid molecule has one end near the center of rotation and receives a small shear stress, and the other end moves away from the center of rotation and receives a greater shear stress.
  • the centrifugal force due to the rotation is greater, and thus the distance from the center of rotation is more likely. As a result, the linear nucleic acid molecule is elongated.
  • linear nucleic acid molecule extended in this way is presented as if it were polar coordinates with the origin near the center of rotation and the vector itself, so it is also effective for mapping genetic loci.
  • the linear nucleic acid molecule does not necessarily have to be partially fixed to the support 10 before the rotation.
  • the elongation of a linear nucleic acid molecule does not depend much on the rotation speed and rotation time.
  • rotation conditions rotation speed, rotation time, etc.
  • rotation control is easy. Therefore, elongation of linear nucleic acid molecules can be performed with high reproducibility.
  • the rotation conditions can be set within a wide range, a spin coater used for semiconductor manufacturing can be used as the rotation device 18, and a simple rotation device for rotating at a desired rotation speed can be used. That is, the elongation of the linear nucleic acid molecule can be realized using a general-purpose rotating device.
  • the shear stress generated by rotation is relatively mild, and linear nucleic acid molecules are not easily fragmented during elongation.
  • the linear nucleic acid molecule is contained together with a relaxation solution containing a surfactant or the like in a solvent such as ethanol.
  • a surfactant or the like facilitates the relaxation of the folded linear nucleic acid molecule
  • the relaxation liquid is a liquid used to relax the linear nucleic acid molecule.
  • a visualizing agent such as a staining agent for visualizing the linear nucleic acid molecule is further included in the liquid.
  • Linear nucleic acid molecules stained with a stain are visualized, for example, with a microscope.
  • Other examples of visible light include, for example, radioisotope labeling followed by autoradiography, or fluorescent labeling followed by scanner reading.
  • the In the in-situ hybridization (I SH) method a label for visualization is added to the side of the “neutral phase” [• compound, so no staining agent is used. If the label is derived from a radioisotope, the linear nucleic acid molecule is visualized with an autoradiograph. If the linear nucleic acid molecule is a chromosome visualized by the FI SH method, genetic diagnosis and mapping are possible. In addition to the DNA synthase chain reaction (PCR), fixed chromosomes can be used as substrates for enzyme reactions such as restriction, transcription, and replication. It is also widely used as a landmark to do this.
  • PCR DNA synthase chain reaction
  • the linear nucleic acid molecule is contained in a solvent such as ethanol together with a relaxation solution and a staining agent.
  • a solvent such as ethanol
  • the linear nucleic acid molecules in the liquid are fixed to the support 10 in a fiber state, and other components with low viscosity and high volatility are obtained.
  • the components are scattered out of the support 10 by centrifugal force. As a result, a preparation for microscopic observation is obtained.
  • the chromosome was fixed on the support.
  • the chromosome can be extended by rotating the support without necessarily fixing.
  • the extended linear nucleic acid molecule is fixed to the linear convex part of the support.
  • a rotating device for example, a spin coater
  • rotate the glass plate and extend the chromosome on the glass plate.
  • Rotation speed and rotation time The rotation conditions including (for example, 5 00 rpm, 30 seconds) may be set appropriately. For example, at a speed between 3 00 Orpra and 6 0 0 O rpm, a similar extension is performed, and at a rotation time between 5 seconds and 30 seconds, a similar extension is performed. .
  • the low viscosity component in the liquid is lost by scattering from the glass plate.
  • an anti-fading agent is sealed between this glass plate and another glass plate.
  • the prepared slide is observed with a fluorescence microscope (B excitation light), for example.
  • FIG. 10 shows an example of decompression using the procedure described above.
  • the left side is a light micrograph of a glass plate 10 having concentric linear protrusions 12, and the right side is a HeLa cell (a human eclampsia is stretched on the glass plate).
  • This is a fluorescence micrograph (B excitation light) of DNA of cells derived from cancer.
  • This is the state after many chromosomes in the normal aggregation state (colored with fluorescent dye Y o -Pro) are relaxed with a relaxation solution and rotated at 300 O rpm with a subcoin.
  • Fig. 11 is an enlarged view of the photograph shown in Fig. 10.
  • the left side shows an enlarged photomicrograph of a glass plate with concentric linear projections
  • the right side shows This is an enlarged fluorescence micrograph (B excitation light) of a DNA fiber stretched on a glass plate.
  • B excitation light a DNA fiber stretched on a glass plate.
  • both ends of the fountain-shaped convex part appear bright in parallel. This is due to secondary light (stray light from outside and diffusely reflected light at both ends) commonly seen in fluorescence observation. is there.
  • the other bright lines are DNA fiber visualization probes. It can be seen that many of the chromosomes are all elongated in the form of fibers. Therefore, this method is effective for optical mapping of genes (high spatial resolution detection of visible probe molecules).
  • the fiber-like linear nucleic acid molecule which is fixed in a hollow state between the linear convex portions, can be provided as a linear nucleic acid molecule specimen, but the linear convex portion 1 2
  • a hybrid compound (complex) of a single-stranded DNA molecule and a complementary labeled molecule can be obtained.
  • hybrid compounds with complementary molecules with fluorescent labels can be obtained.
  • DNA is not subject to steric hindrance from the substrate surface, is not deformed by surface pressure bonding, and further, denaturation from double helix to single-stranded DNA is not inhibited. Has the effect of being easy to combine. Therefore, the detection efficiency with the probe DNA is improved, and the positional relationship between the binding sites becomes clear.
  • This technique is suitable for chromosome analysis and polymorphism analysis.
  • a rotating device for example, a spin coater
  • rotate the glass plate and extend the chromosome on the glass plate.
  • the rotation conditions including the rotation speed and rotation time may be set appropriately. For example, at a rotational speed between 300 Orpm and 600 Orpm, a similar extension occurs, and at a rotation time between 5 seconds and 30 seconds, a similar extension occurs.
  • the low-viscosity component in the liquid is lost by scattering from the glass plate.
  • an anti-fading agent is sealed between this glass plate and another glass plate.
  • the prepared slide is observed with a fluorescence microscope (G excitation light), for example.
  • the molecular hybrid formation site is detected directly on the chromosome as a fluorescent signal.
  • FI SH adds an EG FR gene marker to chromosome 7.
  • the left side shows a photomicrograph of a support with concentric linear protrusions
  • the right side shows two results of FI SH on a DNA fiber stretched on the support.
  • a fluorescence micrograph (B excitation light) is shown.
  • the bar is 5 m long (150,000 base pairs).
  • the width of the convex part 12 is about 10 m, and the width of the bottom part 14 is about 90 // m.
  • the upper fluorescence micrograph on the right side shows a flat surface portion (that is, a portion not including the linear convex portion shown by the broken-line circle on the left side).
  • the FI SH image of the flat part is taken with a considerably higher sensitivity than the FI SH image of the hollow part (that is, between the plurality of linear protrusions or the upper part of the bottom). That is, it is visualized under conditions where the brightness setting and contrast setting of the imaging camera are further enhanced. This is because, as described above, the hyperprecipitation of the fluorescently labeled probe is naturally obstructed in the flat part, so that it is much darker than the hollow part from the beginning of observation.
  • the plane part in addition to the bright linear part, there are many point-like parts, which indicate a probe that is non-specifically adsorbed.
  • the plane part has two meanings that are different from the target label and the unnecessary label, that is, the meaning of analysis specifically hybridized to DNA.
  • the lower right fluorescence micrograph shows a hollow region including the upper portion of the bottom (groove), that is, the linear convex portion (that is, the portion indicated by the broken-line circle on the left).
  • the nonspecific probe does not enter the field of view because it is located far below. Since only the DNA spanned between the two linear protrusions is in focus, only the sign looks beautiful. In other words, non-specific adsorption and specific adsorption can be distinguished.
  • FIG. 13 shows a photomicrograph of a support with concentric linear protrusions on the left, and two FISH fluorescence microscopes for DNA fibers stretched on the support on the right.
  • the photograph (G excitation light) is shown above and below.
  • the width of the convex portion 12 is about 10 m, and the width of the bottom portion 14 is about 90 m.
  • the bar indicates the length of 5 ⁇ .
  • the upper right fluorescence micrograph shows the flat surface (that is, the portion not including the linear convex portion shown by the broken-line circle on the left).
  • the signal of the DNA fiber bonded to the flat plate is crushed, and the spatial relationship is unclear. Dyeing is insufficient and no bright spots are visible.
  • the lower right fluorescence micrograph shows a hollow portion (above the bottom or above the groove), that is, a region including a linear convex portion (that is, a portion indicated by a broken circle on the left side). .
  • the signal of the DNA fiber suspended between the linear protrusions is clearly dyed and the distribution of bright spots is clear. For this reason, the three-dimensional structure and the spatial positional relationship are clear.
  • FISH adds a centromeric gene marker to chromosome 7.
  • the fiberized DNA and centromeric label are visible between the two linear protrusions.
  • the lower photomicrograph of Fig. 14 shows a fiber that is laid in a hollow space on the right and left of the right-side linear convex part and a fiber that is in contact with the flat part.
  • the brightness of the label (indicated by the arrow mark) in the hollow part (left side) is brighter and clearer than the probe label (indicated by the arrow) on the planar part (right side) of the support.
  • the bar indicates a length of 5 ⁇ .
  • the spatial position of the oncogene can be determined by determining the oncogene distance.
  • the number of copies of oncogenes on the chromosome can be detected. Therefore, the glass slide described above can be provided as a clinical gene detection kit. It can also be used as a slide glass for microscopic examination of chromosomes in basic biology research (for example, chromosome polymorphism analysis in human genetics research).
  • PCR DNA synthase chain reaction
  • FI SH failure in the FI SH method
  • PCR also has a high efficiency for binding enzymes (polymerases) to DNA fibers that have been crimped to the surface.
  • the efficiency of synthesizing DNA was very low.
  • these problems can be solved by using a support. After adding a PCR reagent to the chromosome held in a hollow space on the support, repeat the specified heating and cooling on the support, and PCR (in this case, not the test tube PC R, Slide PC R) is possible. At this time, the chromosome need not be visualized.
  • Figure 16 shows an example of normal and abnormal (in this case amplification) detection.
  • the red signal indicates the EGFR (epidermal growth factor receptor) gene
  • the green indicates the chromosome 7 centromere.
  • the photo on the left shows the chromosomes identified by the EGFR gene
  • the photo on the right shows the chromosomes identified by the EGFR gene and the chromosome 7 centromere. Both pictures are clearly labeled, and on the left side, amplification of the EGFR gene is observed, indicating that it is abnormal.
  • the chromosome can be identified by FISH, a suspended chromosome fiber.
  • the red signole indicates the EGFR (epidermal growth factor receptor) gene
  • the green indicates the chromosome 7 centromere.
  • the M phase chromosome of Hela cells is shown in an unstretched state. Since the chromosomes are not elongated, red and green are present in one place.
  • the red signal indicating the EGFR gene and the centromere of chromosome 7 are labeled in the same suspended fiber. The green signal to recognize is localized. Therefore, in this example, the suspended chromosome fiber 1 was identified as chromosome 7 by the fiber FISH method.
  • Figure 18 shows an example of a suspended chromosome labeled with MYC.
  • the photograph on the left side of the figure shows the hybridized chromosomes suspended above the bottom between the support convex parts, and the right side shows the chromosomes hyperhybridized in the flat part as a comparative example.
  • the chromosomal fiber at the suspended site is specifically labeled with the probe (MYC) (higher brightness).
  • Figure 19 shows an example in which chromosome translocation was identified when the MYC gene marker was added to chromosome 8 by FISH.
  • the labeling site of the probe is shown at the bottom of the figure.
  • the upstream side of the cleavage site during MYC translocation is labeled with a green probe, and the downstream side is labeled with a red probe.
  • the upper photo shows an example of a normal chromosome.
  • both a red marker and a green marker are observed on one chromosome, indicating that it is a normal chromosome.
  • the red-labeled chromosome lacks the green-labeled part, and the green-labeled part is bound to another chromosome.
  • the translocation t (8; 14) (MYC; IgH) of the chromophore could be discriminated.
  • the target product (target amplified DNA molecule) amplified by the PCR method has so far been devised by covering the glass slide (support 10) with a lid 20 as shown in Fig. 20.
  • a lid 20 As described above, it can be obtained selectively and with a high degree of purification.
  • the conventional PCR method it was inevitable that the target amplified DNA molecule derived from the target site was mixed with other non-specific amplified DNA molecules (Fig. 20 (a)).
  • high purity cannot be expected by PCR on a slide in a general test tube, but purity can be improved, for example, in the structures shown in (b) and (c) of FIG.
  • the lid as shown in Fig.
  • linear nucleic acid molecules can be transported using electroosmotic flow.
  • electroosmotic flow this occurs when ions in the electric double layer generated at the solid Z liquid interface move by an externally applied electric field.
  • Producing electroosmotic flow by configuring the bottom of the microchannel with a charged or artificially charged material (eg glass substrate, Nafion membrane or plasma-washed silicon) Is known. If the surface of the glass substrate under the solution is negatively charged, the cations in the solution are attracted to it, and an electric double layer is formed at the solid Z liquid interface.
  • a charged or artificially charged material eg glass substrate, Nafion membrane or plasma-washed silicon
  • a structure is provided in which a plurality of bottom portions 1 4 between the plurality of linear convex portions 1 2 ′′ communicate with each other.
  • the bottom portions 1 4, Other parts such as the linear convex part are insulators.
  • the DC constant voltage power supply device is connected to the electrode pair previously provided on the bottom 14 '((a) in FIG. 21).
  • a constant voltage is applied to the electrode to generate an electroosmotic flow that flows in a uniform direction.
  • the chromosome fiber 16 is fixed on the spring-like convex portion 1 2 "as schematically shown in Fig. 22.
  • the chromosome fiber 16 is irradiated with, for example, femtosecond or laser. By doing so (not shown in the figure), both sides of the target DNA portion 16 on the fiber are selectively burned out ((b) in Fig. 21).
  • the target DN A portion 16 ' is transported along the electroosmotic flow and collected, for example, on the negative electrode side (Fig. 21 (c)).
  • the micro-analysis system can be configured similarly using the structures shown in FIGS.
  • the linear nucleic acid molecule 16 extended over the top of the linear cage 12 has complementarity to a foreign molecule, it can be applied as a cage-type nanoarchitecture cage.
  • DN ⁇ with a known base sequence.
  • a surface structure including a linear convex portion 12 and a bottom portion 14 therebetween is produced on the substrate.
  • the single-stranded DNA16 is stretched and fixed in the sky above the bottom (between the convex parts) (Fig. 23 (b)).
  • double-helix DNA is stretched and fixed, only one molecule of two single-stranded DNAs is degraded.
  • This single-strand can be achieved, for example, by using an enzyme (such as Exonucleaselll) that liberates mononucleotides 3 and 5 from the hydroxyl end of the double-stranded DNA by catalyzing the hydrolysis of phosphate ester bonds. It is.
  • an enzyme such as Exonucleaselll
  • For the obtained single-stranded DNA fiber 16 prepare a probe having a ⁇ ⁇ -sequence complementary to the self-sequence of the base from which this DNA is exposed (Fig. 23 (c)).
  • a nano member for example, a plurality of colloid particles having different functions or each subunit of a multi-subunit protein
  • an operator wants to assemble such as a polypeptide oligonucleotide is used. Fix with linking agent.
  • Each nano member is aligned and suspended on the single-stranded DNA fiber according to the probe sequence (Fig. 23 (d)).
  • P-contacted nano members are linked through their respective chemical properties (for example, antigen-antibody affinity or radical polymerization) ((d) in FIG. 23).
  • the nano members are assembled on the single-stranded DNA fiber 16 in the order designed by the operator.
  • the assembled nano member assembly 30 is separated from the single-stranded DNA fiber 16 by excising the linking agent (for example, addition of an amino acid degrading enzyme or nucleolytic enzyme) ((e) of FIG. 23), For example, it is transported and collected by electroosmotic flow (Fig. 21).
  • all or all of the suspended DN A fiber 16 Can be transferred and stored and distributed.
  • photocuring resin 32 such as SCR701 is overlaid on the entire upper surface of support 10 ((a) in FIG. 24), and then cured to film-like resin 3 2 ′ by irradiation with UV light. By peeling this film, the DNA fiber can be easily transferred from the linear protrusion 12 to the filter surface ((b) in FIG. 24). At this time, only the portion of the total length of the DNA fiber molecule that was in contact with the linear convex portion 12 is presented on the film surface ((c) in FIG. 24). In the example shown in Figure 25, two different cured resins are used properly.
  • the bottom is filled with the first cured resin 3 2 a, which is a high molecular weight plastic material that can be dissolved after curing.
  • the second cured resin 3 2 b is added to the DNA fiber. Cure on top.
  • the second cured resin 3 2 b is removed from the linear nucleic acid molecular suspension support. As a result, as shown in (b), it is possible to acquire a state in which the full length of DNA fiber 16 is exposed on the film surface and provide it as a specimen.
  • the suspended DNA fiber is unstable because it is suspended from the linear convex part and the bottom part between it, and is unstable. For example, it is cut by excessive external force during transportation, or foreign impurities May be contaminated or modified by Therefore, as shown in Fig. 26, the polymer fiber having a plastic fluidity that can be melted after curing toward the bottom 14 between the plurality of linear projections 1 2 with the DNA fiber 16 suspended.
  • Embedding protection can be achieved by filling with plastic material 34 (eg agarose).
  • plastic material 34 eg agarose
  • the linear nucleic acid molecules are elongated on the plate-like surface (b), and the polymer plasticizer is dissolved in a state suspended between the linear projections (c) to form the linear projections 12.
  • a linear nucleic acid molecule 16 (d) which is suspended and held in a hollow state can be obtained.
  • the linear nucleic acid molecule 16 stretched across the top of the linear protrusions 12, 2 is also applied as a wiring on a nanowire substrate in electrical and electronic engineering. Is possible.
  • a surface structure 10 including a linear convex portion and a bottom portion therebetween is produced on the substrate.
  • a thin film electrode 40 is provided only on the top of the convex portion, and further, for example, a metal is attached to D NA 16 (Fig. 28 (b)) extending and fixed across the bottom.
  • the colloid coating 4 2 Fig. 28 (c)
  • it can be used as a nanowire self-wire in both DC and AC circuits.
  • Nanowire wiring refers to conductive polymer wiring of about one molecule width.
  • FIG. 28 only the DC circuit is shown as an example. It is also possible to use a DNA part without a metal colloid coating, for example as a resistor, without a metal colloid coating.
  • the wiring by the linear nucleic acid molecule is not limited between the linear convex parts, and can be formed between the convex part and the bottom part ((a) in FIG. 28).
  • a simple circuit is constructed.
  • the discussion of whether the DNA itself is a conductor or an insulator is actually undecided, and it is applied as ⁇ for measuring electrical conductivity to determine this approval or disapproval.

Abstract

L'invention concerne une nouvelle technique d'extension d'une molécule d'acide nucléique linéaire telle qu'un chromosome. Un support pour suspendre des molécules d'acide nucléique linéaires étendues, dans l'air, sur des parties supérieures de multiples parties convexes linéaires est doté des multiples parties convexes linéaires telles que décrites ci-dessus qui sont faites à partir d'une matière ne subissant pas de réaction chimique avec les molécules d'acide nucléique linéaires et d'un plan constitué par la partie inférieure des multiples parties convexes linéaires telles que décrites ci-dessus. Lors de l'extension, un liquide contenant les molécules d'acide nucléique linéaires est amené à se maintenir sur le plan décrit ci-dessus du support. Ensuite, une force de cisaillement est appliquée aux molécules d'acide nucléique linéaires contenues dans le liquide. Il en résulte que les molécules d'acide nucléique linéaires s'étendent dans un état de type fibre, pontées sur les multiples parties convexes linéaires, et sont ainsi fixées.
PCT/JP2007/072049 2006-11-07 2007-11-07 Support de suspension pour une molécule d'acide nucléique linéaire, procédé d'extension d'une molécule d'acide nucléique linéaire et spécimen de molécule d'acide nucléique linéaire WO2008056816A1 (fr)

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