Abstract:
Although analysis can be very quickly conducted at a low cost by a method for measuring a biopolymer using a nanopore, the accuracy of distinguishing the individual monopolymers constituting the biopolymer is low. To both ends of a biopolymer through a nanopore, molecules which are larger than the nanopore are attached and then the biopolymer is reciprocated by an external force to thereby perform repeated measurements.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a technical field concerning a structure and method for performing high-precision measurement and analysis on a biopolymer such as a DNA, RNA, or protein using a nanopore structure. 
       BACKGROUND ART 
       [0002]    For diagnosis of diseases or drug discovery, it is important to analyze a biopolymer such as a nucleic acid (DNA or RNA) or a protein. In particular, since a DNA is the key substance of life, analysis thereof, that is, determination of a base sequence is quite significant to the above object. As for a principal method for analyzing the base sequence of a DNA, primarily, chemical or enzymatic reaction is utilized in order to produce a group of DNA fragments of various lengths, which have a predetermined terminal base species, with respect to a mold DNA whose sequence is wanted to be determined. The DNA fragment group is produced for each of four base species. Thereafter, gel electrophoresis is used to separate each of the DNA fragment groups in order of a molecular weight. The DNA fragment groups are introduced into a separation medium and a voltage is applied. Since a DNA fragment is a polyelectrolyte having a negative charge as a whole, electrophoresis is made in a negative-to-positive direction. In a gel, since a longer DNA fragment exhibits a smaller mobility, even DNA fragments having a difference of only one base length between them can be separated from each other. After the separation is completed, when the lengths of DNA fragments dependent on the terminal base species are measured, the positions of the base species in the mold DNA are found. Through the foregoing manipulations, the base sequence of the mold DNA is determined. In the above method, production of the DNA fragment groups or work of electrophoresis is quite labor-intensive, and an analysis time is long. Besides, a running cost is high. 
         [0003]    Patent literature 1 and non-patent literature 1 describe a biopolymer analysis method using a microscopic pore of several nanometers in diameter formed on a thin membrane of a lipid bilayer, which is an insulating membrane, using alpha-hemolysin, that is, a nanopore. A thin membrane having the nanopore is interposed between two solution vessels, a voltage gradient is brought about between the solution vessels, and a current is measured. When a DNA molecule that is a biopolymer is put in one of the solution vessels, the DNA molecule passes through the nanopore due to the voltage gradient, and thus moves to the other solution vessel. When the DNA molecule passes through the nanopore, the DNA molecule blocks a flow of ions in the nanopore. This brings about a decrease in a current (blockage current). By measuring the magnitude of the blockage current and the duration of the blockage current, the length of an individual DNA molecule passing through the nanopore can be detected. In addition, the species of individual bases constituting the DNA molecule can be theoretically distinguished from one another. 
         [0004]    In patent literature 2 and non-patent literature 2, instead of the nanopore formed in a lipid bilayer using alpha-hemolysin, a nanopore is formed in a silicon nitride (Si 3 N 4 ) membrane, which is an insulating membrane, using a technology referred to as ion-beam sculpting. A blockage current of a single-stranded DNA molecule is then measured. 
         [0005]    In non-patent literature 3, a proposal is made of means other than the foregoing blockage current as a method for measuring a DNA molecule that passes through a nanopore. A pair of metallic electrodes is disposed on the internal surface of the nanopore, and a tunneling current occurring when a DNA strand passes between the metallic electrodes is measured. Even in this method, as long as the size of the electrodes or the like is appropriately controlled, species of individual bases constituting the DNA molecule that passes through the nanopore can be distinguished from one another. 
         [0006]    Patent literature 3 describes a method for determining a base sequence of a target DNA through hybridization of a known-sequence probe to a target single-stranded DNA and detection of a position of hybridization of the known-sequence probe using a nanopore. A known-sequence probe is hybridized to a target single-stranded DNA molecule that should be determined, and the DNA molecule having undergone the hybridization is passed through a nanopore. Since a blockage current differs between a single-stranded site and a double-stranded site of the DNA molecule, when the DNA molecule passes through the nanopore, if the current is measured, the position of hybridization of the known-sequence probe can be identified. Plural kinds of known-sequence probes having different sequences are used to perform the foregoing manipulations, and data items of the positions of hybridization of the known-sequence probes to the target DNA molecule are acquired. The acquired data items are converted into sequence data using a computer algorithm. Thus, the sequence of the target DNA molecule can be determined. 
         [0007]    Non-patent literature 4 describes a consequence that when the positive and negative polarities of an applied voltage are reversed immediately after a DNA molecule passes through a nanopore, the same DNA molecule passes through the same nanopore again. 
         [0008]    In non-patent literature 5, elongation reaction of a DNA molecule is induced in a nanopore, and presence or absence of the elongation reaction is verified by measuring a blockage current. 
       CITATION LIST 
     Patent literature 
       [0009]    Patent literature 1: U.S. Pat. No. 5,795,782 
         [0010]    Patent literature 2: U.S. Pat. No. 6,627,076 
         [0011]    Patent literature 3: U.S. Patent Publication No. 2007/0190542 
       Non-Patent Literature 
       [0012]    Non-patent literature 1: PNAS 1996, Vol. 93, pp. 13770-13773 
         [0013]    Non-patent literature 2: Nature 2001, Vol. 412, pp. 166-169 
         [0014]    Non-patent literature 3: NANO Letters 2005, Vol. 5, pp. 421-424 
         [0015]    Non-patent literature 4: Nature Nanotechnology 2007, Vol. 2, pp. 775-779 
         [0016]    Non-patent literature 5: JACS 2008, Vol. 130, pp. 818-820 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0017]    As for determination of a sequence of a biopolymer, or more particularly, a DNA molecule using electrophoresis, an analysis time is long and a running cost is high. In contrast, determination of the sequence of the DNA molecule using a nanopore which is described in patent literature 1 or non-patent literature 3 has a potential of fast and inexpensive analysis but is confronted with problems described below. 
         [0018]    In patent literature 1, a difference in a blockage current, which occurs when a DNA molecule passes through a nanopore and depends on a base species, is so minute that it is hard to distinguish one base species from another. Therefore, precision in the distinction is very low. In order to improve the precision in the distinction, it is necessary to set the membranous thickness near the nanopore to a value equivalent to about one base, that is, a sub-nanometer. However, it is hard for an existing technology to attain the membranous thickness. 
         [0019]    In non-patent literature 3, the magnitude of a tunneling current occurring when a DNA molecule passes through a nanopore depends on, aside from a base species passing through the nanopore, orientation near electrodes used to measure the tunneling current of the base. Currently, it is hard to control the orientation of a passing nucleic acid. Therefore, repetitive measurement is needed in order to upgrade the precision in base species separation. 
         [0020]    By the way, according to the method described in patent literature 3, since a position of hybridization of a known-sequence probe to a target DNA molecule is detected, it is unnecessary to distinguish bases species from one another. However, it is necessary to highly precisely detect a position of hybridization on a single base level. In addition, it is necessary to hybridize plural different kinds of known-sequence probes to the same target DNA molecule in different places or at different timings and to detect the positions. Specifically, the target DNA molecule has to be amplified and introduced into different solutions in which the different known-sequence probes exist. The target DNA molecule and known-sequence probes have to be hybridized to one another. Using different nanopores or using the same nanopore at different timings, measurement has to be performed. Otherwise, after the target DNA molecule hybridized to a certain known-sequence probe is measured using the nanopore, the target DNA molecule is sampled, denatured, and returned to a single strand. The single-stranded DNA is then hybridized to a different known-sequence probe, and measured again using the nanopore. This procedure has to be repeated. In either case, quite labor-intensive work is needed. 
         [0021]    As described in non-patent literature 4,when applying a reverse voltage after a DNA molecule passes through a nanopore is repeated, the same DNA molecule can be measured plural times using the same nanopore. Therefore, although there is a possibility that measurement precision may be improved owing to repetitive measurement, application of a voltage through high-precision feedback is needed. In addition, if plural kinds of DNA molecules coexist, there is a fear of contamination. Further, the directions of the DNA molecules get randomized. 
         [0022]    In non-patent literature 5, polyethylene glycol (PEG)-biotin-mediated streptavidin is bound to a 5′ end of a target DNA molecule, and a DNA primer including several bases is hybridized to a 3′-end side thereof. The 3′ end of the target DNA molecule is thus double-stranded, and the target DNA molecule is reciprocated through a nanopore. Due to repetitive measurement using the reciprocation, there arises a possibility that high-precision nanopore measurement can be achieved. However, it is hard to control the position of hybridization of a DNA probe to the target DNA molecule. The DNA probe may be bound to various positions in the target DNA molecule, and the reciprocation may not be achieved. In addition, since the diameter of a double-stranded DNA is on the order of 2 nm, if the diameter of the nanopore is equal to or larger than 2 nm, the double-stranded part passes through the nanopore. The reciprocation becomes hard to do. 
       Solution to Problem 
       [0023]    The present invention provides a method, system, and kit making it possible to stably achieve repetitive measurement for improvement of measurement precision in nanopore measurement of a biopolymer molecule such as a nucleic acid or protein. 
         [0024]    A first solution vessel and a second solution vessel are included, and the solution vessels are partitioned by a thin membrane. The thin membrane has a pore of a nanometer size, that is, a nanopore formed therein. A molecule (first stopper molecule) having a larger size than the diameter of the aperture of the nanopore is bound to one of the ends of a biopolymer molecule, and the resultant biopolymer molecule is introduced into the first solution vessel. By applying an external force, the biopolymer is driven to pass through the nanopore, and thus moved to the second solution vessel. Using the first stopper molecule, the biopolymer molecule passing through the nanopore ceases moving halfway. After the movement of the biopolymer molecule is ceased, a molecule (second stopper molecule) that is larger than the diameter of the aperture of the nanopore and exists in the second solution vessel is bound to the other end of the biopolymer molecule. Thereafter, while the biopolymer molecule is reciprocated between the solution vessels by applying an external force, measurement is carried out in order to identify the biopolymer molecule. 
         [0025]    Binding the second stopper molecule to the biopolymer molecule may be performed while the biopolymer molecule is moving through the nanopore. When the second stopper molecule is bound to the biopolymer molecule, the external force for driving the biopolymer molecule may be stopped or may be kept applied. The second stopper molecule may be preliminarily put in the second solution vessel, or may be introduced thereinto after one end of the biopolymer molecule is moved to the second solution vessel. The stopper molecules may be the same molecule or different molecules. As means for driving the biopolymer, if the biopolymer molecule has charge, a voltage gradient may be brought about between the solution vessels, or an electrochemical gradient may be brought about by differentiating an ion composition between the solution vessels. Otherwise, a flow of a solution may be produced for driving. As means for measurement, a value of a current (blockage current) flowing through the nanopore between the solution vessels will do, or a value of a current (tunneling current) flowing between electrodes disposed in the nanopore will do. The biopolymer molecule may be labeled with a fluorescent substance, and excited near the nanopore in order to detect the emitted fluorescent light. With the stopper molecules bound to both the ends of the biopolymer molecule, a certain substance may be bound to or separated from the biopolymer molecule. For example, as described in non-patent literature 3, when a DNA molecule sequence is determined on a hybridization basis, measurement can be readily and highly precisely achieved using the aforesaid method. A stopper molecule is bound to both the ends of a DNA molecule, and a known-sequence probe is introduced into a solution vessel and hybridized to a target DNA molecule. The target DNA molecule is reciprocated between the solution vessels, and the position of hybridization is detected through blockage current measurement. Thereafter, the known-sequence probe is separated from the target DNA molecule through denaturing manipulations, and another known-sequence probe is used to repeat the same manipulations as the foregoing ones. Owing to the means, different known-sequence probes can be readily hybridized to the same target DNA molecule whatever number of times. In addition, since repetitive measurement can be performed, the position of hybridization can be highly precisely measured. 
         [0026]    A method for determining a biopolymer in accordance with the present invention is a method for determining the alignment of monomers constituting a biopolymer that is an object of measurement, using an apparatus including a first solution vessel, a second solution vessel, and a thin membrane which partitions the first solution vessel and second solution vessel and has a nanopore. The method is characterized by a step of introducing the biopolymer, which has a first molecule, which is larger than the nanopore, bound to one end thereof, into the first solution vessel, a step of moving the biopolymer from the first solution vessel to the second solution vessel through the nanopore, a step of introducing a second molecule, which is larger than the nanopore, into the second solution vessel, and binding the molecule to the other end of the biopolymer, a step of moving the biopolymer between the first solution vessel and second solution vessel through the nanopore, and measuring a temporal change in a signal generated along with the movement of the biopolymer, calculating the signal as data dependent on the species of monomers constituting the biopolymer, and determining the alignment of the monomers constituting the biopolymer. 
         [0027]    A system for determining a biopolymer in accordance with the present invention is a system that uses an apparatus, which includes a thin membrane having a nanopore, to introduce a molecule that is larger than the size of the nanopore and is bound to a biopolymer that is an object of measurement. The apparatus includes first and second solution vessels that are partitioned by the thin membrane, driving means that moves the biopolymer between the first solution vessel and second solution vessel through the nanopore, means that introduces the biopolymer, which has the molecule bound to one end of the biopolymer that is the object of measurement, into the first solution vessel, means that introduces the molecule, which is bound to the other end of the biopolymer having passed through the nanopore, into the second solution vessel, detecting means that detects a signal generated along with the movement of the biopolymer made by the driving means, and calculating means that measures a temporal change in the signal detected by the detecting means, calculates the signal as data dependent on the species of monomers constituting the biopolymer, and determines the alignment of the monomers constituting the biopolymer. 
         [0028]    A kit for determining a biopolymer in accordance with the present invention is a kit including an apparatus that includes a thin membrane having a nanopore, and a molecule which is larger than the size of the nanopore and is bound to a biopolymer that is an object of measurement. The apparatus includes first and second solution vessels partitioned by the thin membrane, driving means that moves the biopolymer through the nanopore between the first solution vessel and second solution vessel, means that introduces the biopolymer, which has the molecule bound to one end of the biopolymer that is the object of measurement, into the first solution vessel, means that introduces the molecule, which is bound to the other end of the biopolymer having passed through the nanopore, into the second solution vessel, detecting means that detects a signal generated along with the movement of the biopolymer made by the driving means, and calculating means that measures a temporal change in the signal detected by the detecting means, calculates the signal as data dependent on the species of monomers constituting the biopolymer, and determines the alignment of the monomers constituting the biopolymer. The molecule is streptavidin or a bead to be bound to the biopolymer by a DIG-anti-DIG antibody bond. 
       Advantageous Effects of Invention 
       [0029]    The present invention permits stabilized repetitive measurement of a biopolymer molecule using a nanopore, and enables inexpensive, fast, and high-precision measurement. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0030]      FIG. 1A  and  FIG. 1B  include illustrative diagrams of the inside of a sample solution during sample preprocessing; 
           [0031]      FIG. 2A  and  FIG. 2B  include illustrative diagrams of a sample preprocessing method utilizing a vector; 
           [0032]      FIG. 3  is a schematic diagram of a nanopore apparatus employed in an embodiment 1; 
           [0033]      FIG. 4A  and  FIG. 4B  include enlarged diagrams of the vicinity of a nanopore in the embodiment 1; 
           [0034]      FIG. 5  is a flowchart of binding streptavidin to a 3′ end of a DNA fragment; 
           [0035]      FIG. 6A-FIG .  6 D include illustrative diagrams of a situation where streptavidin is bound to the 3′ end of the DNA fragment; 
           [0036]      FIG. 7  is a graph of a time-sequential change in a tunneling current value occurring when a DNA molecule passes through a nanopore; 
           [0037]      FIG. 8  is a schematic diagram of a nanopore apparatus employed in an embodiment 2; 
           [0038]      FIG. 9  is a diagram of an image on a CCD of light emitted through each nanopore in the embodiment 2; 
           [0039]      FIG. 10  includes graphs of time-sequential changes in a signal intensity at respective wavelengths occurring when a DNA molecule passes through a nanopore; 
           [0040]      FIG. 11  includes graphs of time-sequential changes in a signal intensity relative to respective base species occurring when the DNA molecule passes through the nanopore; 
           [0041]      FIG. 12  is a schematic diagram of a nanopore apparatus employed in an embodiment 3; 
           [0042]      FIG. 13  is a schematic diagram of a nanopore apparatus employed in an embodiment 4; and 
           [0043]      FIG. 14  includes graphs of time-sequential changes in a blockage current occurring when a DNA molecule to which respective known-sequence probes are hybridized passes through a nanopore. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0044]    Referring to the drawings, embodiments of the present invention will be described below. 
       Embodiment 1 
       [0045]    A description will be made of a method for determining a base sequence of a DNA molecule through measurement of a tunneling current in a nanopore using the present invention.  FIGS. 1A and 1B  illustratively show a state of the inside of a sample solution during sample preprocessing preceding measurement. In the solution, a target DNA molecule  101  that is double-stranded, and a double-stranded synthetic probe  103  having a 5′ end thereof labeled with biotin  102  coexist. An end face  104  of the double-stranded synthetic probe  103  that is not labeled with biotin has undergone blunt-ending reaction. Using S1 nuclease or the like, blunt-ending reaction is performed on both the ends of the target DNA molecule  101 . After the blunt-ending reaction is performed on both the ends of the target DNA molecule  101 , a ligase is used to ligate the synthetic probe  103  to both the ends of the target DNA molecule  101 . After the ligation reaction is terminated, a sample is separated in size through acrylamide gel electrophoresis. Only the target DNA molecule  101  having the synthetic probe  103  bound to both the ends thereof is cut out of an acrylamide gel and eluted to distilled water. Through the above manipulations, the synthetic probes  103  that are ligated to each other and the target DNA molecules  101  that are ligated to each other can be excluded. Thereafter, a buffer solution containing streptavidin  105  is mixed in the solution into which the target DNA molecule  101  having the synthetic probe bound to both the ends thereof is eluted. The biotin  102  with which the 5′ end of target DNA molecule  101  is labeled is bound to the streptavidin  105 , and a DNA is denatured by applying heat. Thus, a DNA fragment  106  containing the target DNA molecule  101  having streptavidin bound to the 5′ end thereof as shown in  FIG. 1B  is produced.  FIGS. 2A and 2B  illustratively show another method for producing the DNA fragment  106 . The target DNA molecule  101  is inserted to part of a multi-cloning site in a vector  107 . Thereafter, the target DNA molecule  101  is cut at restriction sites  108  and  109  in the vector through restriction enzyme digestion so that the target DNA molecule  101  can be sandwiched. A double-stranded synthetic probe  110  including the nick end of the restriction site  108  and having a 5′ end thereof labeled with biotin  102  and a double-stranded synthetic probe  111  including the nick end of the restriction site  109  and having a 5′ end thereof labeled with the biotin  102  are introduced into a fragment  112  cut from the vector. A ligation is then carried out. Thereafter, the biotin  102  with which the 5′ end is labeled and streptavidin  105  are bound to each other, and the DNA is denatured by applying heat. Eventually, a DNA fragment  106  containing the target DNA molecule  101  that has streptavidin bound to the 5′ end thereof as shown in  FIG. 1B  is produced. 
         [0046]      FIG. 3  is a schematic diagram of a nanopore apparatus employed in the present embodiment. The nanopore apparatus includes a first solution vessel  202 , a second solution vessel  203 , and a nanopore thin membrane  204  that separates the solution vessels from each other. The solution vessels are provided with introduction ports  206  and  207  respectively through which a solution is introduced, and discharge ports  208  and  209  respectively through which the solution is discharged. In order to bring about a voltage gradient between the solution vessels via the nanopore thin membrane  204 , the solution vessels  202  and  203  are provided with electrodes  210  and  211  respectively. The electrodes  210  and  211  are connected to a voltage source  212  capable of changing polarities and an ammeter  213 . The nanopore thin membrane  204  is formed with a thin membrane of an insulator having a nanopore  205  of 1 nm in diameter formed therein. Herein, Si 3 N 4  is adopted as the material of the insulator thin membrane. Alternatively, a plastic material such as SiO 2  or asphaltene will do. Further, a thin membrane produced by coating an insulating material over the surface of a metallic membrane made of Al or the like. Herein, the diameter of the nanopore  205  is 1 nm. Alternatively, the diameter may range about 0.5 nm to about 50 nm. Incidentally, the size of streptavidin used as a stopper molecule is on the order of 5 nm and much larger than the size of the nanopore. As the size of the stopper molecule relative to the diameter of the nanopore, any size capable of hindering advancement of a DNA fragment will do. For improvement of precision, the size is preferably 1.2 times to 50 times larger. 
         [0047]      FIG. 4A  is an enlarged view of the vicinity of the nanopore  205 .  FIG. 4B  is an a-a′ sectional view of  FIG. 4A . A pair of electrodes  216  and  217  is disposed on the internal surface of the nanopore  205 . The electrodes  216  and  217  are connected to a voltage source  215  and an ammeter  214 . 
         [0048]    Referring to the flowchart of  FIG. 5 , a description will be made of a method for binding streptavidin to the 3′ end of the DNA fragment  106 . Incidentally,  FIG. 6  illustratively shows a procedure of binding streptavidin to the 3′ end of the DNA fragment  106 . 
         [0049]    To begin with, the DNA fragment  106  obtained with the foregoing method is mixed in a buffer solution, and introduced into the first solution vessel  202  through the introduction port  206 , and the buffer solution alone is introduced into the second solution vessel  203  through the introduction port  207  ( 401 ) ( FIG. 6A ). A voltage is applied from the voltage source  212  so that the electrode  210  can behave as a cathode and the electrode  211  can behave as an anode. This causes the DNA fragment  106  to migrate from the first solution vessel  202  to the second solution vessel  203  ( 402 ) ( FIG. 6B ). Concurrently with the voltage application, the ammeter  213  is used to measure a flow of ions through the nanopore  205 . Since the size of streptavidin  105  is on the order of 5 nm, the streptavidin cannot pass through the nanopore of 1 nm in diameter. The DNA fragment  106  has therefore the 3′ end (end that is not labeled by the streptavidin  105 ) thereof first introduced into the nanopore  205 . When the DNA fragment  106  is introduced into the nanopore  205 , a current value decreases. As mentioned previously, the size of the streptavidin  105  is larger than the diameter of the nanopore  205 . Therefore, immediately before the streptavidin  105  passes through the nanopore  205 , the movement of the DNA fragment  106  to the second solution vessel  203  is ceased. After the decrease in a current is verified, a biotin-3′ end-DNA-labeling kit is inserted to the second solution vessel  203  through the introduction port  207  in order to labeled the 3′ end of the DNA fragment  106  with biotin  113  ( 403 ) ( FIG. 6C ). 
         [0050]    After biotin labeling is completed, the buffer alone is introduced through the introduction port  207  in order to remove biotin, which has not reacted, from the second solution vessel  203  ( 404 ). Thereafter, a solution containing streptavidin is introduced through the introduction port  207  so that the streptavidin  114  can be bound to the biotin  113  with which the 3′ end of the DNA fragment  106  is labeled. Thus, an arrayed DNA fragment  115  is produced ( 405 ) ( FIG. 6D ). The buffer alone is introduced through the introduction port  207  in order to remove streptavidin, which has not reacted, from the second solution vessel  203  ( 406 ). 
         [0051]    As described above, streptavidin that is one and the same substance is used as the first and second stopper molecules. Alternatively, different substances may be used. For example, as another method for producing the arrayed DNA fragment  115  using different substances as the first and second stopper molecules, a method to be described below is available. In the method shown in  FIG. 2 , the 3′ ends of the synthetic probes  110  and  111  other than the nick ends thereof are labeled with digoxigein (DIG). Aside from this, the aforesaid method is followed to produce the DNA fragment  106 . At this time, the 3′ end of the DNA fragment  106  is labeled with DIG. Thereafter, according to the same method as the aforesaid one, the DNA fragment  106  having the 3′ end thereof labeled with DIG is migrated from the first solution vessel  202  to the second solution vessel  203 . Since the size of DIG itself is much smaller than the diameter of 1 nm of the nanopore  205 , the DNA fragment  106  has therefore the 3′ end thereof first introduced into the nanopore  205 . When the movement of the DNA fragment  106  is ceased by streptavidin  105 , an anti-digoxigenin (DIG) antibody labeled with a bead whose diameter is larger than 1 nm is introduced into the second solution vessel  203 , and bound to DIG with which the 3′ end of the DNA fragment  106  is labeled. Thereby, an arrayed DNA fragment  115  formed using streptavidin as the first stopper molecule and the bead mediated by a DIG-anti-DIG antibody bond as the second stopper molecule is produced. Herein, an example in which the DIG-anti-DIG bond bead is used as a stopper molecule other than biotin and streptavidin has been described so far. Alternatively, a method of binding a gold particle to a thiolated end of a DNA, a method of modifying an end of a DNA with an amino group, and binding the DNA end to a bead, which is modified with a carboxyl group, through dehydration reaction, or the like may be adopted. 
         [0052]    After the arrayed DNA fragment  115  is produced, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as an anode and the electrode  211  can behave as a cathode. This causes the arrayed DNA fragment  115  to migrate from the second solution vessel  203  to the first solution vessel  202  for a certain time. During the migration, a tunneling current is measured using the electrodes  216  and  217  in order to identify base species constituting the arrayed DNA fragment  115 . Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as the cathode and the electrode  211  can behave as the anode. This causes the arrayed DNA fragment  115  to migrate from the first solution vessel  202  to the second solution vessel  203  for the certain time. During the migration, the tunneling current is measured using the electrodes  216  and  217 . By repeating the migration between the solution vessels and the measurement of the tunneling current, the measurement of the tunneling current of the same target DNA molecule can be performed plural times. This permits high-precision determination of base species. 
         [0053]    Now, a method for determining base species will be described below. Bases fall into four species of adenine (A), thymine (T), guanine (G), and cytosine (C). An inherent current value is observed for each of the base species, and sent to data processing means  400 .  FIG. 7  shows an example. In advance, tunneling currents occurring when polymers each of which has bases of one species concatenated pass through a nanopore are measured, and current values associated with the respective base species are obtained and stored in a memory in the data processing means. The data processing means  400  then compares a current value, which is obtained at the time of measuring a tunneling current of a target DNA, with the current values that are associated with the respective base species and that are measured in advance, and thus determines the base species of the target DNA. 
         [0054]    As for switching the polarities of a voltage in reciprocation measurement, the voltage source  212  is controlled so that automatic switching can be achieved at intervals of a certain time. A control unit is included in the data processing means  400 . The certain time may be variably set. When a stopper molecule approaches a nanopore, a decrease in a current passing through the nanopore can be measured. Therefore, the decrease in the current may be used as a trigger to switch the voltage polarities. 
         [0055]    In the present embodiment, measurement of a tunneling current using one nanopore is performed. Alternatively, when plural nanopores are used to concurrently measure tunneling currents of numerous different target DNA molecules, a throughput can be improved. 
         [0056]    In the present embodiment, for an explanatory purpose, a description has been made of an example in which after a DNA having a stopper molecule bound to a 5′ end thereof is introduced into the first solution vessel and passed through a nanopore, a stopper molecule is bound to a 3′ end thereof. In a similar method, a DNA fragment having a stopper molecule bound to a 3′ end thereof may be used. After the DNA fragment is introduced into the nanopore, a stopper molecule may be bound to a 5′ end thereof. 
       Embodiment 2 
       [0057]    A description will be made of a method for determining a base sequence of a DNA molecule through fluorescence detection measurement which utilizes fluorescence resonance energy transfer (FRET) of a nanopore. A target DNA molecule labeled with a fluorescent substance Cy5 serving as an acceptor is produced according to a method described below. As described in conjunction with  FIG. 2B  of the embodiment 1, the DNA fragment  106  having the 5′ end thereof labeled with biotin-mediated streptavidin and the 3′ end thereof labeled with DIG is produced. Thereafter, a reaction solution containing dCTP, dGTP, dTTP, Cy5-labeled dATP, DNA polymerase, and a primer that has a complementary sequence with respect to a synthetic probe portion (either the probe  110  or  111 ) of the DNA fragment  106 , and the DNA fragment  106  having the 3′ end thereof labeled with DIG are mixed in order to induce elongation reaction. During the elongation reaction, heat denaturation is not induced. Using different reaction tubes, the same manipulations are performed on a reaction solution in which dCTP alone is labeled with Cy5, a reaction solution in which dGTP alone is labeled, and a reaction solution in which dTTP alone is labeled. Thus, a double-stranded DNA fragment  117  that contains the target DNA fragment  101  and is labeled with a Cy5 fluorescent substance  116  is produced. 
         [0058]      FIG. 8  is a schematic diagram of a nanopore apparatus employed in the present embodiment. The nanopore apparatus includes a first solution vessel  202 , a second solution vessel  203 , and a nanopore thin membrane  218  that partitions the solution vessels. The solution vessels  202  and  203  are provided with introduction ports  206  and  207  respectively through which a solution is introduced, and discharge ports  208  and  209  respectively through which the solution is discharged. In order to bring about a voltage gradient between the solution vessels via the nanopore thin membrane  218 , the solution vessels  202  and  203  are provided with electrodes  210  and  211  respectively. The electrodes  210  and  211  are connected to a voltage source  212  capable of changing polarities. The first solution vessel  202  has an optically transparent irradiation window  305  and a detection window  306 . The nanopore thin membrane  218  is formed with a Si 3 N 4  thin membrane having nanopores  205  of 3 nm in diameter formed therein. The plural nanopores  205  are formed like a grid at intervals of 1 μm. In the vicinity of the nanopores  205 , Qdots (605)  219  that is excited with blue light and emits fluorescent light of 605 nm are immobilized as donors. 
         [0059]    Laser light  302  oscillated by a laser light source (wavelength of 488 nm)  301  has the angle thereof adjusted by a mirror  303 , concentrated by a condenser lens  304 , and irradiated to all the Qdots  219  through the irradiation window  305 . Fluorescent light emitted near each of the nanopores  205  is concentrated by an objective lens  307  through the detection window  306 , has light other than light, which has a wavelength ranging from 550 nm to 700 nm, cut by a filter  308 , and is spectroscopically diffracted by a prism  309 . An image is, as shown in  FIG. 9 , formed on a CCD  311  by an image formation lens  310 . Data of the CCD  311  is stored in the data processing means  400 .  FIG. 9  shows the image formed on the CCD  311  and stored in the data processing means  400 . Spots represent luminescent points of the respective nanopores. The axis of abscissas is associated with a wavelength direction. 
         [0060]    The double-stranded DNA fragment  117  obtained according to the foregoing method is mixed in a buffer solution and introduced into the first solution vessel  202  through the introduction port  206 . The buffer solution alone is introduced into the second solution vessel  203  through the introduction port  207 . A voltage is applied from the voltage source  212  so that the electrode  210  can behave as a cathode and the electrode  211  can behave as an anode. This causes the double-stranded DNA fragment  117  to migrate from the first solution vessel  202  to the second solution vessel  203 . Since the size of streptavidin  105  is on the order of 5 nm, the streptavidin  105  cannot pass through the nanopore of 3 nm in diameter. The double-stranded DNA fragment  117  has an end thereof, which is not labeled with the streptavidin  105 , first introduced into the nanopore  205 . With the movement of the double-stranded DNA fragment  117  ceased by the streptavidin  105 , an anti-DIG antibody  118  labeled with a bead whose diameter is larger than 3 nm is introduced into the second solution vessel  203 . The anti-DIG antibody  118  is bound to DIG with which the 3′ end of the double-stranded DNA fragment  117  which is not labeled with the fluorescent substance  116  is labeled. After labeling with the anti-DIG antibody  118  is completed, the buffer alone is introduced through the introduction port  207  in order to remove an anti-DIG antibody, which has not reacted, from the second solution vessel  203 . Meanwhile, the voltage is kept applied. Incidentally, a target DNA molecule included in the double-stranded DNA fragment  117  introduced into each of the nanopores may be the same one or may be a DNA molecule different from the others. By statistically processing detected light waves, respective base species can be identified. When one and the same DNA molecule is employed, a time can be shortened by decreasing the reciprocating frequency of the DNA molecule, and precision in identifying base species can be improved. In contrast, when different molecules are employed, many molecules can be concurrently measured and a throughput can be improved. 
         [0061]    After the double-stranded DNA fragment  117  is labeled with the anti-DIG antibody  118 , the laser light  302  is oscillated from the laser light source  301  in order to excite the Qdots  219 . Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as an anode and the electrode  211  can behave as a cathode. This causes the anti-DIG antibody  118 -labeled double-stranded DNA fragment  117  to migrate from the second solution vessel  203  to the first solution vessel  202  for a certain time. During the migration, when the fluorescent substance  118  with which the double-stranded DNA fragment  117  is labeled passes near the Qdot  219 , transfer of excitation energy due to resonance takes place and causes the fluorescent substance  118  to emit light. The emitted light is detected by the CCD  311 .  FIG. 10  shows temporal variations in the intensities of pixels, which are associated with the wavelengths of 605 nm and 670 nm respectively, in the spots on the CCD  311  representing the emitted light waves. The wavelength of 605 nm corresponds to light emitted from the Qdot  219 , while the wavelength of 670 nm corresponds to light emitted from the fluorescent substance  118 . Based on the detected temporal variation in a signal intensity associated with the wavelength of 670 nm, the position of labeling of the double-stranded DNA fragment  117  with the fluorescent substance  118  can be calculated. Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as the cathode and the electrode  211  can behave as the anode. This causes the double-stranded DNA fragment  117  to migrate from the first solution vessel  202  to the second solution vessel  203  for the certain time. During the migration, fluorescent light is, as mentioned above, detected by the CCD  311 . By repeating the migration and the detection of the fluorescent light, the position of labeling of the double-stranded DNA fragment  117  with the fluorescent substance  118  can be measured plural times. The position can therefore be highly precisely calculated.  FIG. 11  shows temporal variations in signal intensities obtained at respective wavelengths after performing repetitive work of migration and detection of fluorescent light similar to the foregoing ones on the double-stranded DNA fragment  117  having four different kinds of dNTPs thereof labeled with a fluorescent substance. Based on the temporal variation in the signal intensity associated with the wavelength of 670 nm, the position of each base species in the target DNA molecule  101  can be distinguished. By combining data items of respective base species, the base sequence of the target DNA molecule  101  is determined. 
         [0062]    For the switching of voltage polarities in reciprocation measurement, the voltage source  212  is controlled so that automatic switching can be achieved at intervals of a certain time. The certain time can be variably set. A control unit is incorporated in the data processing means  400 . When a stopper molecule approaches a nanopore, a decrease in a current passing through the nanopore is measured. Therefore, the voltage polarities may be switched with the decrease in the current as a trigger. 
         [0063]    In the present embodiment, only one kind of fluorescent substance is employed. Alternatively, four kinds of dNTPs may be labeled with different fluorescent substances, and a double-stranded DNA fragment  117  having all the dNTPs thereof labeled may be produced, and fluorescent light may be detected by performing the same manipulations as the aforesaid ones. Thus, the base sequence of the target DNA molecule  101  may be determined. By using a high-viscosity buffer solution or decreasing a voltage to be applied for migration, the migration speed of the double-stranded DNA fragment  117  can be lowered, and fluorescent light can be highly sensitively detected. In the present embodiment, Qdots are adopted as donors for FRET. Alternatively, a fluorescent substance will do. In addition, although  FIG. 8  shows an example in which two DNA fragments are concurrently measured, one DNA fragment may be measured or three or more DNA fragments may be concurrently measured. 
       Embodiment 3 
       [0064]    A description will be made of a method for determining a base sequence of a DNA molecule through detection and measurement of fluorescent light through a nanopore.  FIG. 12  is a schematic diagram of a nanopore apparatus employed in the present embodiment. Constituent features other than a laser light source  301 , a filter  308 , a position of irradiation of laser light to the nanopore apparatus, and the construction of a nanopore thin membrane are identical to those of the embodiment 2. A nanopore thin membrane  218  is made of quartz glass, and the surface of the nanopore thin membrane  218  is coated with a resin whose refractive index is lower than that of quartz (for example, Fluorinert). At this time, a resin is peeled off from sharp distal ends  220  near nanopores. Laser light  302  emitted from a laser light source (wavelength of 633 nm)  301  is concentrated by a condenser lens  304  and irradiated to the flank  312  of the nanopore thin membrane. At this time, the laser light  302  propagates through the nanopore thin membrane  218  while totally reflecting. However, since the distal ends  220  are not resin-coated, the laser light slightly oozes as near-field light to a buffer solution with which the solution vessels are filled. Since the laser light  302  propagates through the entire nanopore thin membrane  218  while totally reflecting, a near field occurs at each of the distal ends  220 . Fluorescent light emitted near each of nanopores  205  is concentrated by an objective lens  307  via a detection window  306 . Light other than light whose wavelength ranges from 660 nm to 700 nm is cut by a filter  308 , and the remaining light is spectroscopically diffracted by a prism  309 . An image is then formed on a CCD  311  by an image formation lens  310 . Data of the CCD  311  is stored in data processing means  400 .  FIG. 12  shows an example in which two DNA fragments are concurrently measured. Alternatively, one DNA fragment may be measured or three or more DNA fragment may be concurrently measured. 
         [0065]    According to the same method as the one in the embodiment 2, manipulations are performed for producing a double-stranded DNA fragment  117 , and binding an anti-DIG antibody  118 , which is labeled with a bead whose diameter is larger than 3 nm, to DIG with which a 3′ end of a strand that is not labeled with a fluorescent substance  116 . 
         [0066]    After the double-stranded DNA fragment  117  is labeled with the anti-DIG antibody  118 , laser light  302  is oscillated from the laser light source  301  in order to produce near-field light at each of the distal ends  220 . Thereafter, a voltage is applied from a voltage source  212  so that an electrode  210  can behave as an anode and an electrode  211  can behave as a cathode. This causes the anti-DIG antibody  118 -labeled double-stranded DNA fragment  117  to migrate from the second solution vessel  203  to the first solution vessel  202  for a certain time. During the migration, when a fluorescent substance  118  with which the double-stranded DNA fragment  117  is labeled passes through the near-field light in the vicinity of each of the distal ends  220 , the fluorescent substance  118  emits light. The emitted light is detected by a CCD  311 . Based on a temporal variation in a detected signal intensity, a position of labeling of the double-stranded DNA fragment  117  with the fluorescent substance  118  can be calculated. Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as the cathode and the electrode  211  can behave as the anode. This causes the double-stranded DNA fragment  117  to migrate from the first solution vessel  202  to the second solution vessel  203  for the certain time. During the migration, the emitted light is detected by the CCD  311 . By repeating the migration and the detection of emitted light, the position of labeling of the double-stranded DNA fragment  117  with the fluorescent substance  118  can be measured plural times. This permits high-precision position calculation. Manipulations for detecting a fluorescent-substance labeled position are performed on the double-stranded DNA fragment  117 , which has four different kinds of dNTPs thereof labeled with a fluorescent substance, in order to determine the base sequence of the target DNA molecule  101 . 
         [0067]    For switching of voltage polarities in reciprocation measurement, the voltage source  212  is controlled so that automatic switching can be achieved at intervals of a certain time. The certain time can be variably set. A control unit may be incorporated in the data processing means  400 . When a stopper molecule approaches a nanopore, a decrease in a current passing through the nanopore can be measured. Therefore, the switching of the voltage polarities may be performed with the decrease in the current as a trigger. 
         [0068]    In the present embodiment, only one kind of fluorescent substance is used. Alternatively, four kinds of dNTPs may be labeled with different fluorescent substances. The double-stranded DNA fragment  117  having all the dNTPs thereof labeled may be produced, and fluorescent light may be detected by performing the same manipulations as the foregoing ones. Thus, the base sequence of the target DNA molecule  101  may be determined. In addition, by using a high-viscosity buffer solution or decreasing a voltage to be applied during migration, the migration speed of the double-stranded DNA fragment  117  can be lowered. Eventually, the fluorescence light can be detected highly sensitively. 
       Embodiment 4 
       [0069]    A description will be made of a method for determining a base sequence of a DNA molecule on a hybridization basis. A DNA fragment  106  containing a target DNA molecule  101  that has streptavidin bound to a 5′ end thereof is produced according to the same method as the one in the embodiment 1. 
         [0070]      FIG. 13  shows a schematic diagram of a nanopore apparatus employed in the present embodiment. The nanopore apparatus includes a first solution vessel  202 , a second solution vessel  203 , and a nanopore thin membrane  221  that partitions the solution vessels. The solution vessels are provided with introduction ports  206  and  207  respectively through which a solution is introduced, and discharge ports  208  and  209  respectively through which the solution is discharged. In order to bring about a voltage gradient between the solution vessels via the nanopore thin membrane  221 , the solution vessels are provided with electrodes  210  and  211  respectively. The electrodes  210  and  211  are connected to a voltage source  212  capable of changing polarities and an ammeter  213 . The nanopore thin membrane  221  is constructed with a Si 3 N 4  thin membrane in which a nanopore  205  of 3 nm in diameter is formed. Using a temperature adjustment unit  313 , the temperature of the solution in the first solution vessel  202  and second solution vessel  203  can be adjusted to range from 20° C. to 100° C. Control of the voltage source  212 , control of the temperature adjustment unit  313 , acquisition of a current value of the ammeter  213 , and processing of obtained data are carried out by data processing means  400 . 
         [0071]    The DNA fragment  106  obtained according to the aforesaid method is mixed in a buffer solution and introduced into the first solution vessel  202  through the introduction port  206 . The buffer solution alone is introduced into the second solution vessel  203  through the introduction port  207 . A voltage is applied from the voltage source  212  so that the electrode  210  can behave as a cathode and the electrode  211  can behave as an anode. This allows the DNA fragment  106  to migrate from the first solution vessel  202  to the second solution vessel  203 . Concurrently with the voltage application, a current is measured using the ammeter  213 . Since the size of streptavidin  105  is on the order of 5 nm, the streptavidin cannot pass through the nanopore of 3 nm in diameter. The DNA fragment  106  has the 3′ end thereof (end that is not labeled with the streptavidin  105 ) first introduced into the nanopore  205 . When the DNA fragment  106  is introduced into the nanopore  205 , a current value decreases. As mentioned previously, the size of the streptavidin  105  is larger than the diameter of the nanopore  205 . Therefore, immediately before the streptavidin  105  passes through the nanopore  205 , the movement of the DNA fragment  106  to the second solution vessel  203  is ceased. After the decrease in the current is verified, a biotin-3′ end-DNA labeling kit is inserted into the second solution vessel  203  through the introduction port  207  in order to label the 3′ end of the DNA fragment  106  with biotin  113 . After labeling with biotin is completed, the buffer alone is introduced through the introduction port  207  in order to remove biotin, which has not reacted, from the second solution vessel  203 . A solution containing streptavidin is introduced through the introduction port  207  in order to bind the streptavidin  114  to the biotin  113 , with which the 3′ end of the DNA fragment  106  is labeled, whereby an arrayed DNA fragment  115  is produced. The buffer alone is introduced through the introduction port  207  in order to remove streptavidin, which has not reacted, from the second solution vessel  203 . 
         [0072]    After the arrayed DNA fragment  115  is produced, a known-sequence probe  119  including six bases is introduced into the solution vessels through the introduction ports  206  and  207  respectively, and hybridized to the arrayed DNA fragment  115 . After hybridization reaction is completed, a buffer alone is introduced through the introduction ports  206  and  207  in order to remove the known-sequence probe  119 , which has not reacted, from the solution vessels  203 . A voltage is applied from the voltage source  212  so that the electrode  210  can behave as the anode and the electrode  211  can behave as the cathode. This allows the arrayed DNA fragment  115  to migrate from the second solution vessel  203  to the first solution vessel  202  for a certain time. Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as the anode and the electrode  211  can behave as the cathode. This allows the arrayed DNA fragment  115  to migrate from the first solution vessel  202  to the second solution vessel  203  for the certain time. As for the timing of switching the voltage polarities, the voltage source  212  is controlled so that automatic switching can be achieved at intervals of the certain time. The certain time can be variably set. When a stopper molecule approaches the nanopore, a decrease in a current passing through the nanopore can be measured. Therefore, the switching of the voltage polarities may be performed with the decrease in the current as a trigger. During the migration of the arrayed DNA fragment  115 , the ammeter  213  is used to measure a blockage current. Based on a temporal variation in the measured blockage current, a position at which the known-sequence probe  119  is hybridized to the arrayed DNA fragment  115  can be calculated. 
         [0073]    Thereafter, voltage application is ceased. The temperature adjustment unit  313  is used to raise the temperature of the solution in the first solution vessel  202  and second solution vessel  203  up to 95° C. for a certain time. The known-sequence probe  119  is separated from the arrayed DNA fragment  115  through heat denaturation. A buffer alone is introduced through the introduction ports  206  and  207  in order to remove the known-sequence probe  119  from the solution vessels. The temperatures of the solution vessels are lowered to 40° C. A known-sequence probe having a different sequence from the known-sequence probe  119  is introduced into the solution vessels through the introduction ports  206  and  207  respectively, and hybridized to the arrayed DNA fragment  115 . After the hybridization reaction is completed, the buffer alone is introduced through the introduction ports  206  and  207  in order to remove the known-sequence probe, which has not reacted, from the solution vessels. A voltage is applied from the voltage source  212  so that the electrode  210  can behave as the anode and the electrode  211  can behave as the cathode. This causes the arrayed DNA fragment  115  to migrate from the second solution vessel  203  to the first solution vessel  202  for a certain time. Thereafter, a voltage is applied from the voltage source  212  so that the electrode  210  can behave as the cathode and the electrode  211  can behave as the anode. This causes the arrayed DNA fragment  115  to migrate from the first solution vessel  202  to the second solution vessel  203  for the certain time. During the migration, the ammeter  213  is used to measure a blockage current. 
         [0074]    Hybridization of a known-sequence probe to an arrayed DNA fragment, migration of the arrayed DNA fragment, measurement of a blockage current, and separation of the known-sequence probe from the arrayed DNA fragment through heat denaturation are repeated 4 n  times (where n denotes the base length of the known-sequence probe, that is, 6 in the present embodiment) using known-sequence probes having different sequences. Data items of the positions of hybridization of the respective known-sequence probes can be converted into the base sequence data of the target DNA molecule  101  using a computer algorithm. A concrete method will be described below in conjunction with  FIG. 14 . After the known-sequence probe  119  is hybridized to the target DNA  101 , and passed through a nanopore, when a blockage current is measured, a waveform  123  of block current values is observed. The measurement is repeated according to the method described previously in order to estimate to what position in the target DNA  101  the known-sequence probe  119  is hybridized. The foregoing manipulations are performed using known-sequence probes  120 ,  121 , and  120 . The obtained positions of hybridization of the respective known-sequence probes to the target DNA  101  are superposed on one another, whereby a sequence complementary to that of the target DNA  101  can be drawn out. Eventually, sequence data of the target DNA  101  can be obtained. 
         [0075]    By utilizing the present invention, determination of a base sequence of a target DNA molecule on a hybridization basis can be achieved without amplification of the target DNA molecule and without use of plural nanopores. 
         [0076]    In the present embodiment, the length of a known-sequence probe is six bases. If the length of the known-sequence probe is long, a rise in a cost of probe production or an increase in erroneous hybridization takes place. In contrast, if the length of the known-sequence probe is short, unless a measurement resolution is raised, an accurate position of hybridization of a probe cannot be measured. Therefore, the length of the known-sequence probe preferably ranges from about three bases to about ten bases. 
         [0077]    In order to highly precisely detect a position of hybridization of a known-sequence probe to a target DNA molecule, when a blockage current is measured, the polarities of the electrodes  210  and  211  may be repeatedly changed in order to measure the blockage current plural times. 
         [0078]    After measurement is completed, a nuclease or an acid may be used to cut the arrayed DNA fragment  115  so as to remove the arrayed DNA fragment  115  from the solution vessels. Thus, a nanopore thin membrane may be reused. 
         [0079]    For detection of a position of hybridization of a known-sequence probe, part of the known-sequence probe may be labeled with a fluorescent substance, and fluorescent-light detection described in relation to the embodiment 2 or 3 may be employed. 
         [0080]    For detection of a position of hybridization of a known-sequence probe, if a tunneling current or fluorescent light is detected instead of a blockage current, concurrent measurement of plural target molecules can be readily achieved. Eventually, a throughput can be improved. 
       INDUSTRIAL APPLICABILITY 
       [0081]    DNA sequencer 
       REFERENCE SIGNS LIST 
       [0082]      101 : target DNA molecule,  102 : biotin,  103 : synthetic probe,  104 : end face,  105 : streptavidin,  106 : DNA fragment,  107 : vector,  108 : restriction site,  109 : restriction site,  110 : synthetic probe,  111 : synthetic probe,  112 : fragment,  113 : biotin,  114 : streptavidin,  115 : arrayed DNA fragment,  116 : fluorescent substance,  117 : double-stranded DNA fragment,  118 : bead-labeled anti-DIG antibody,  119 : known-sequence probe,  120 : known-sequence probe,  121 : known-sequence probe,  122 : known-sequence probe,  123 : waveform of blockage current values,  124 : waveform of blockage current values,  125 : waveform of blockage current values,  126 : waveform of blockage current values,  202 : first solution vessel,  203 : second solution vessel,  204 : nanopore thin membrane,  205 : nanopore,  206 : introduction port,  207 : introduction port,  208 : discharge port,  209 : discharge port,  210 : electrode,  211 : electrode,  212 : voltage source,  213 : ammeter,  214 : ammeter,  215 : voltage source,  216 : electrode,  217 : electrode,  218 : nanopore thin membrane,  219 : Qdot,  220 : distal end,  221 : nanopore thin membrane,  301 : laser light source,  302 : laser light,  303 : mirror,  304 : condenser lens,  305 : irradiation window,  306 : detection window,  307 : objective lens,  308 : filter,  309 : prism,  310 : image formation lens,  311 : CCD,  312 : flank of a nanopore thin film,  313 : temperature adjustment unit,  400 : data processing unit