JP5024132B2 - Method for identifying nucleic acid base species and method for determining nucleotide sequence - Google Patents

Description

  The present invention relates to a method for identifying individual base types of nucleic acid (DNA or RNA) and a method for determining the base sequence of a nucleic acid by sequentially applying the method to the base of the nucleic acid.

  The Maxam-Gilbert method and the Sanger method have been widely used as methods for determining the base sequence of nucleic acids such as DNA and RNA. Among them, the Sanger method replicates DNA complementary strands of various chain lengths using a reaction between DNA to be sequenced and a fluorescently labeled DNA elongation inhibitor, and uses electrophoresis. The base sequence is determined. At present, the Sanger method has become the mainstream of DNA base sequence analysis, and various improvements have been added (see Patent Document 1).

  In recent years, a detection method using a DNA array has been developed and commercialized as a method for detecting a mutation in a base sequence of a specific gene (single nucleotide polymorphism: SNPs). In this method, an unknown fluorescently labeled DNA is flowed on a substrate on which a known DNA is immobilized, and hybridization is performed, thereby reading a base sequence from the known DNA bound to the fluorescently labeled unknown DNA (see Patent Document 2). .)

  Other proposed techniques include fluorescence resonance energy transfer that occurs between a fluorescently labeled DNA polymerase immobilized on a substrate and a nucleotide that is specifically fluorescently labeled for each type during complementary strand synthesis ( FRET) is optically observed to determine the original base sequence (see Patent Document 3), and the three-dimensional structure and shape of self-aggregated or hybridized DNA using a scanning probe microscope. There are methods for measuring and determining the base sequence (see Patent Documents 4 and 5).

  A scanning tunneling microscope (STM), one of the scanning probe microscopes, observes the surface of a sample using a tunnel current flowing between the probe (probe) and the surface of the sample. It is a microscope that has been actively used in recent years because it can be observed at the atomic level.

  One of the measurement modes using a scanning tunneling microscope is that the bias voltage applied between the probe and the sample is swept while the distance between the probe and the sample is fixed, and the tunnel current depending on the change in the bias voltage is swept. There is scanning tunneling spectroscopy (STS) that measures changes and observes the electronic state of the sample surface. Attempts have been made to determine the base sequence of nucleic acids using this scanning tunneling spectroscopy for observation of biomolecules (see Patent Document 6).

  As another attempt using a scanning tunneling microscope, a method for distinguishing purine bases (guanine and adenine) and pyrimidine bases (cytosine and thymine) using the difference in base height observed under the same feedback condition is reported. (See Non-Patent Document 1).

  As another attempt using scanning tunneling spectroscopy, a method for discriminating whether a purine base is guanine or adenine using the presence or absence of a peak in a specific bias voltage range has been reported (Non-Patent Document). 2).

  As another method, attempts have been made to identify bases by modifying each base in advance at the probe tip and using the difference in electrical interaction with the base species to be decoded (non-patent document). Reference 3). However, this method is limited to the identification of guanine only at present, and other bases cannot be identified.

Nucleobase identification by such a scanning probe microscope is not limited in principle to the length of the nucleic acid chain that can be decoded at one time compared to the Sanger method, which is the most commonly applied base sequence identification technology at present, Moreover, since it can be applied to the decoding of repetitive sequences, which has been considered difficult to decode, it is promising as a next-generation sequencing technology.
JP 05-038299 A Special table 2003-528626 gazette US Pat. No. 6,210,896 B1 Japanese Patent Laid-Open No. 9-299087 Japanese Patent Laid-Open No. 10-215899 WO2007 / 013370 Pamphlet Protein Nucleic Acid Enzyme Vol.48 No.5 2003 pp.614-620 Hiroyuki Tanaka, Tomoji Kawai, 53rd Symposium on Applied Physics (2006 Spring), pp.1453 Umezawa et. Al. PNAS, Vol.103, No.1, pp.10-14, 2006 J.Vac.Sci.Technol. B25 (1) 2007 pp.242-246

  The above report on the identification of nucleobase species using scanning probe microscopy or scanning tunneling spectroscopy, together with the identification of purine and pyrimidine bases, and the specific potential of guanine bases Become. Further, in order to apply these techniques to decoding of the base sequence, it is necessary to identify whether the base is cytosine or thymine when it is identified as a pyrimidine base. In an attempt to distinguish cytosine and thymine, the current-voltage characteristics of the base were measured by scanning tunneling spectroscopy, and the logarithmic change against the voltage of the obtained current was plotted, so that the slope and the intercept at the bias voltage value were zero. A method for discriminating thymine by obtaining the value of is reported (Non-Patent Document 4). However, in this method, in order to obtain reliable logarithmic change data of the current, it is necessary to sweep the bias voltage over a wide range, and the measurement time becomes long. The distance between the probe tip and the base changes due to the steady temperature drift, and the tunnel current value may change. It is hard to say that cytosine and thymine are completely identified. Thus, until now, there is no known effective means for distinguishing cytosine and thymine using a scanning probe microscope or scanning tunneling spectroscopy.

  In order to cope with such a problem, the present invention distinguishes cytosine and thymine, which are pyrimidine bases, using scanning tunneling spectroscopy (claims 1 to 3), and a nucleic acid base using the method. It is an object of the present invention to provide a method for determining a sequence (claims 4 to 7).

  For the pyrimidine bases cytosine and thymine, the surface is fixed on the conductive substrate surface, and the bias voltage applied between the probe and the substrate is changed with the distance between the probe and the base of the scanning probe microscope fixed. When the tunnel current flowing between the probe and the base was measured, and the behavior of the change in the tunnel current with respect to the bias voltage was compared, it was found that there was a great difference. In Timin, the rate of change of the logarithmically converted tunneling current with respect to the bias voltage changes with a specific bias voltage (this specific bias voltage is called an inflection bias voltage), whereas cytosine has such an inflection bias voltage. Does not have The pyrimidine base species identification method of the present invention is a method for identifying whether the target pyrimidine base is cytosine or thymine by utilizing the difference in behavior of the tunnel current change before and after the inflection bias voltage. It is.

That is, the identification method of the present invention for identifying a pyrimidine base species in a sample nucleic acid includes the following steps (A) to (C).
(A) A thymine is fixed on the surface of a substrate having a conductive surface, and the probe and thymine are changed by changing a bias voltage applied between the probe and the substrate in a state where the distance between the probe and the thymine of the scanning probe microscope is fixed. Measuring a tunnel current flowing between and a bias voltage that changes the rate of change of the logarithmically converted tunnel current with respect to the bias voltage,
(B) The surface having a pyrimidine base in the sample nucleic acid to be identified is extended on the surface of the substrate having conductivity, and fixed in a state in which each base is in direct contact with the substrate, and between the probe and the pyrimidine base. The change of the tunnel current flowing between the probe and the base is measured by changing the bias voltage applied between the probe and the substrate in a specific voltage range including the inflection bias voltage between them with a fixed distance. And (C) whether the pyrimidine base is cytosine or thymine based on the rate of change before and after the inflection bias voltage based on the change in tunneling current with respect to the change in bias voltage obtained in step (B). Identifying.

  Although the change in the bias voltage in the step (B) may be continuously performed in the specific range, in order to shorten the measurement time, the change in the bias voltage in the step (B) The inflection bias voltage and the maximum bias voltage may be used at three points. In that case, the identification in the step (C) is based on the ratio between the change rate of the tunnel current between the lowest bias voltage and the inflection bias voltage and the change rate of the tunnel current between the inflection bias voltage and the highest bias voltage. Preferably based on.

  When the change of the tunnel current when the bias voltage is changed is large, it is preferable to log the tunnel current for processing. In that case, if the identification in the step (C) is performed based on the change rate of the logarithmically converted tunnel current, the identification becomes easy.

  There is a known method for identifying whether a base in a nucleic acid is a purine base or a pyrimidine base using a scanning probe microscope (see Non-Patent Document 1). In addition, as for a purine base, there is a known method for identifying whether it is guanine or adenine using a scanning probe microscope (see Non-Patent Document 2). When these known methods are combined with the pyrimidine base species identification method of the present invention, all base species of nucleic acids can be identified.

That is, the identification method of the present invention for identifying a base species in a sample nucleic acid includes the following steps (A) to (F).
(A) A thymine is fixed on the surface of a substrate having a conductive surface, and the probe and thymine are changed by changing a bias voltage applied between the probe and the substrate in a state where the distance between the probe and the thymine of the scanning probe microscope is fixed. Measuring a tunnel current flowing between and a bias voltage that changes the rate of change of the logarithmically converted tunnel current with respect to the bias voltage,
(B) The guanine is fixed on the surface of the substrate having conductivity, and the bias voltage applied between the probe and the substrate is changed with the distance between the probe and the guanine of the scanning probe microscope fixed, thereby changing the probe and the guanine. Measuring the tunnel current flowing between the two, detecting the peak of guanine on the differential curve due to the bias voltage of the tunnel current, and obtaining the peak detection bias voltage,
(C) a step of extending a portion having a base sequence that is a target of identification of a sample nucleic acid on the surface of a substrate having conductivity, and fixing the base in a state in which each base is in direct contact with the substrate;
(D) Using a scanning probe microscope, the topography image is measured by feedback control so that the tunnel current flowing between the probe and the base becomes constant, and the base is a purine base based on the height of the base image. Identifying whether it is a pyrimidine base;
(E) For the base identified as the purine base in step (D), the distance between the probe and the base is fixed and applied between the probe and the substrate within a specific range including the peak detection bias voltage therebetween. The tunnel current flowing between the probe and the base is measured by changing the bias voltage, and the purine base is determined depending on whether or not a peak is detected on the differential curve by the bias voltage of the tunnel current at the peak detection bias voltage. Identifying whether it is guanine or adenine,
(F) For the base identified as the pyrimidine base in step (D), the distance between the probe and the substrate is within a specific voltage range including the inflection bias voltage with the distance between the probe and the base fixed. Measuring the change in the tunnel current flowing between the probe and the base by changing the bias voltage applied to the base, and (G) the pyrimidine base is cytosine or thymine based on the rate of change in the tunnel current The process of identifying

  In step (E), the bias voltage is changed at three points of the minimum bias voltage, the inflection bias voltage, and the maximum bias voltage within a specific range, and the pyrimidine base is identified by the tunnel between the minimum bias voltage and the inflection bias voltage. If the measurement is performed based on the ratio between the current change rate and the tunnel current change rate between the inflection bias voltage and the maximum bias voltage, the measurement time can be shortened.

  Further, if the identification in the step (E) is performed based on the logarithmically converted change rate of the tunnel current, the identification becomes easy.

  Determination of the base sequence of the sample nucleic acid can be realized by sequentially executing steps (D) to (G) along the base of the sample nucleic acid fixed on the substrate.

  The method for discriminating between purine bases and pyrimidine bases and the method for discriminating between purine base species are not limited to the above-mentioned known methods, and other methods can be used as long as they are methods using a scanning probe microscope. In combination with the pyrimidine base species identification method, a sample nucleic acid base species identification method can be used, and further, a sample nucleic acid base sequence determination method can be configured.

  Since the pyrimidine base species identification method of the present invention discriminates cytosine and thymine from the change in tunnel current before and after the inflection bias voltage, it is only necessary to measure a narrow range of bias voltage change, and the measurement time can be shortened.

  According to the base sequence determination method of the present invention, high-speed sequencing using a scanning probe microscope is possible.

  Various types of scanning probe microscopes such as an atomic force microscope and a scanning near-field light microscope can be used as the scanning probe microscope for carrying out the present invention. A preferable example is a scanning tunneling microscope (STM). In the following examples, a scanning tunneling microscope is used.

An outline of a scanning tunneling microscope is schematically shown in FIG. The scanning tunnel microscope includes a microprobe 2 having a probe at the tip and a control device 4 as its constituent elements. The sample nucleic acid 6 is placed on a stage in a scanning probe microscope with at least the surface fixed on the surface of the conductive substrate 8. The controller 4 applies a bias voltage V _s between the surface of the probe 2 and the substrate 8, and detects a tunneling current I _t flowing from the probe 2 to the sample nucleic 6, controls the position of the probe 2.

One measurement mode scanning tunneling microscope is a mode tunneling current I _t flowing between the probe 2 and the base is a feedback control (FB) the height of the probe 2 to be constant, the base high You can get information about

Other operation modes are probes 2 and the distance between the base and fixed by changing the bias voltage V _s scanning tunneling spectroscopy to measure changes in tunneling current I _t (STS).

  In the present invention, the topographic image is first measured by feedback control on the sample nucleic acid 6 fixed on the surface of the substrate 8 to specify the position of the base and based on the difference in the height of the base image constituting the nucleic acid. To identify whether the base is a purine base or a pyrimidine base. For example, when two types of bases having different measured base heights are measured, the higher base is identified as a purine base and the lower base is identified as a pyrimidine base.

  Next, the probe 2 is moved onto a specific base, the feedback circuit is turned off, and the distance between the probe 2 and the base is fixed. Scanning tunneling spectroscopy is applied to identify whether the purine base is guanine or adenine, and for the pyrimidine base, whether it is cytosine or thymine.

When discriminating the purine base species, the bias voltage V _s applied between the probe and the base is changed in the range of −2.5 V to 1 V, for example, and the tunnel current flowing between the probe and the base is measured, If a peak peculiar to the differential value of the tunnel current bias voltage V _s is detected, the purine base is identified as guanine, and if not detected, it is identified as adenine.

When identifying the pyrimidine base species, the bias voltage V _s applied between the probe and the base is changed within a specific range C as shown in FIG. By measuring the change in the tunnel current flowing between the probe and the base with respect to the change in the bias voltage, the difference in the rate of change in the tunnel current with respect to the bias voltage before and after the inflection bias voltage can be used to determine the cytosine of the pyrimidine base. And identify thymine.

  The specific range C is an appropriate range set in advance so as to sandwich the inflection bias voltage obtained in advance, and is a bias voltage range in which the most significant difference is observed in the current-voltage characteristics of both cytosine and thymine. is there. Information regarding the voltage range is acquired and set in advance. Data is acquired only within the set voltage range, and the difference in change rate before and after the inflection bias voltage is calculated. Thereby, highly reliable data can be acquired in a short measurement time.

  Hereinafter, the method for identifying the pyrimidine bases cytosine and thymine will be described in more detail with reference to FIGS.

  FIG. 2 shows a method for preparing a DNA sample. A solution obtained by suspending a synthetic DNA having a thiolated 3 ′ end (with a protecting group: manufactured by Operon Biotechnologies) in an aqueous dithiothreitol solution is used as a solid phase extraction column (Sep-Pak cartridge: manufactured by Waters) 10. The DNA solution 12 was prepared by carrying out deprotection treatment. Gold thin film substrate (gold thin film was formed on the surface of the sapphire substrate) was subjected to Piranha cleaning (resist stripper SH-303: 30 minutes immersion in Kanto Chemical Co., Ltd., followed by 30 minutes ultrapure water cleaning) in the resulting DNA solution 12 No. 14) was immersed for 3 hours to adsorb DNA onto the gold thin film surface. Thereafter, the gold thin film substrate 14 on which the DNA was adsorbed was washed in ultrapure water for 30 minutes and dried to obtain a sample for STM observation.

Figure 3 is 1V applied bias voltage is a STM image of the cytosine base on the gold thin film substrate obtained by feedback condition to 1pA the tunneling current I _t.

  FIG. 4 shows current-voltage characteristics obtained by moving the probe over the base in the image to cut the feedback circuit and fixing the distance between the probe and the base to about 1 nm. Further, among the obtained current-voltage characteristics, the logarithmic value is calculated for the current value measured in the range of the bias voltage of 1V to 3V, and the result of replotting with respect to the voltage value is shown in FIG. Since the graph of FIG. 5 shows a straight line shape, the current-voltage characteristic of the cytosine base in the bias voltage range of 1 V to 3 V has a characteristic that the tunnel current varies exponentially with the applied bias voltage. Recognize.

6, the applied bias voltage 1V, indicating the STM image of thymine bases on the gold thin film substrate measured in feedback condition to 1pA the tunneling current I _t.

Similarly to the case of cytosine base, the probe was moved onto the base in the image of FIG. 6 to cut the feedback circuit, and the current-voltage characteristics were acquired by fixing the distance between the probe and the base to about 1 nm. The acquired current-voltage characteristics are shown in FIG. Moreover, the logarithm of the current value measured in the range of the bias voltage of 1 V to 3 V among the obtained current-voltage characteristics was calculated in the same manner as in the case of cytosine base, and the result plotted against the voltage value is shown in FIG. Show. As can be seen from this figure, the current-voltage characteristics of thymine bases show a continuous exponential function observed with cytosine bases, with the slope increasing discontinuously at the point where the applied bias voltage is about 2V. It can be seen that it has characteristics that are different from those of the change. Thus, current tunneling current I _t in the measurement of the cytosine bases were log transformed - in slope of the voltage characteristic present invention discontinuously varying the bias voltage (approximately 2V) is called inflection bias voltage.

  To identify the pyrimidine base species, it is only necessary to change the bias voltage within a narrow voltage range sandwiching the inflection bias voltage. An example of the change in the bias voltage is a continuous change in the range of 1 V to 3 V with 2 V in between, for example.

  Another example of the change in the bias voltage is a discontinuous change in the voltage value of a total of three points including the inflection bias voltage and two points before and after the inflection bias voltage. For example, from the results of FIGS. 3 to 8, the value of the tunnel current measured when the bias voltage applied between the probe and the base is around 1 V, 2 V, and 3 V is measured. By plotting the logarithmic values of the measured tunnel currents against the applied bias voltage, the average rate of change in the bias voltage range between 1V and 2V and the bias voltage range of 2V and 3V can be approximated. Ask. By comparing the ratio of the average rate of change between cytosine and thymine, it is possible to identify whether the target pyrimidine base is cytosine or thymine.

Based on such knowledge, the most effective bias voltage values V ₁ , V ₂ , V ₃ (in this case (V ₁ , V ₂ , V ₃ ) = (1V, 2V) for discrimination between cytosine and thymine , 3V)) in advance, and by measuring only the tunnel current value at the voltage value, highly reliable data can be acquired in a short measurement time.

An example of data processing required for the above-described identification method is shown in FIG. At the time of STS measurement, the tunnel current (I ₁ , I ₂ , I ₃ ) obtained when a bias voltage Vs (V ₁ , V ₂ , V ₃ ) previously stored in a database such as a computer is applied is the computer 20. Is taken in. The computer 20 converts the tunnel currents (I ₁ , I ₂ , I ₃ ) into logarithmic values (LogI ₁ , LogI ₂ , LogI ₃ ), respectively. Next, average change rates a ₁ and a ₂ between V _{1 and} V _{2 and} between V _{2 and} V ₃ are obtained as follows based on the respective voltages and logarithmic current values.
a ₁ = (Log I ₂ −Log I ₁ ) / (V ₂ −V ₁ )
a ₂ = (Log I ₃ -Log I ₂ ) / (V ₃ -V ₂ )

Finally, the ratio value (a ₂ / a ₁ ) of the _two average rate of change is calculated, and the magnitude relationship with a constant P whose ratio value is greater than 1 is determined, and (a ₂ / a ₁ ) is If it is P or more, the pyrimidine base is thymine, and if (a ₂ / a ₁ ) is smaller than P, the pyrimidine base is cytosine. Thereby, a series of processing from current-voltage characteristic acquisition to cytosine-thymine discrimination can be automated.

  By combining this cytosine-thymine discrimination with a method for discriminating between purine bases and pyrimidine bases using a scanning tunneling microscope, and a method for discriminating purine base species using a scanning tunneling microscope, as shown in FIG. The base sequence of DNA fixed on the substrate can be automatically determined using a scanning tunneling microscope.

For this purpose, a base height threshold value for distinguishing purine bases and pyrimidine bases from the height of the topography image is set and stored in the database. Also, the inflection bias voltage for discrimination of pyrimidine base species is obtained, and the most effective bias voltage values V ₁ , V ₂ (inflection bias voltage) and V ₃ for the discrimination between cytosine and thymine are stored in the database. To do. For determining whether it is cytosine or thymine from the ratio value ratio (a ₂ / a ₁ ) based on the logarithmic value of the tunnel current at the bias voltage values V ₁ , V ₂ and V ₃ . A constant P is also set and stored in the database. Furthermore, a peak detection bias voltage for identifying the purine base species is obtained, a change range of the bias voltage for detecting a peak specific to guanine is set, and these are also stored in the database.

  In measurement, sample DNA is fixed on the surface of a conductive substrate, and a topography image is measured using a scanning tunneling microscope with feedback control so that the tunnel current flowing between the probe and the base is constant. The position of the base is specified.

Next, the probe is positioned at the base at the end of the base sequence portion whose base sequence is to be determined. For the base, it is determined whether the base is a purine base or a pyrimidine base based on the difference in height of base images that have already been obtained. If the base is identified as a purine base, the tunnel current that flows between the probe and the base by changing the bias voltage within the bias voltage range held in the database with the distance between the probe and the base fixed. And whether the purine base is guanine or adenine is discriminated based on whether or not a peak at the peak detection bias voltage is detected. If the base is identified as a pyrimidine base, the tunnel current flowing between the probe and the base is measured with a bias voltage of 3 points held in the database with the distance between the probe and the base fixed. The ratio value (a ₂ / a ₁ ) of the above average change rate is calculated, and it is discriminated whether the pyrimidine base is cytosine or thymine by comparing the ratio value with the constant P held in the database. To do.

In this way, the base sequence of the sample DNA is determined by automatically advancing the work of positioning the probe in order from the base at the end of the base sequence portion whose base sequence is to be determined and identifying the base type. .
The examples show how to implement the present invention using a scanning tunneling microscope, but the same can be achieved using an atomic force microscope or a scanning near-field light microscope, which is another scanning probe microscope. can do. Further, the present invention can be similarly applied to RNA as well as DNA.

  INDUSTRIAL APPLICABILITY The present invention can be used to identify pyrimidine base species in nucleic acids using a scanning probe microscope, and further used for a nucleic acid base sequencing method using a scanning probe microscope. be able to.

(A) is a schematic block diagram which shows the scanning tunnel microscope which implements this invention, (B) is a graph which shows the bias voltage-tunnel current characteristic of cytosine and thymine by a scanning tunneling spectroscopy. It is a figure which shows the preparation method of a DNA sample in order of a process. It is the STM image of the cytosine base on the gold | metal thin film board | substrate acquired on feedback conditions. It is a graph which shows the tunnel current-voltage characteristic acquired by fixing the distance of a probe and cytosine. FIG. 5 is a graph showing logarithmic conversion of the tunnel current based on the result of FIG. It is an STM image of a thymine base on a gold thin film substrate acquired under feedback conditions. It is a graph which shows the tunnel current-voltage characteristic acquired by fixing the distance of a probe and thymine. FIG. 8 is a graph showing logarithmic conversion of the tunnel current based on the result of FIG. It is a flowchart which shows an example of the data processing which implement | achieves the identification method of a pyrimidine base species. It is a flowchart which shows the base sequence determination method of DNA.

Explanation of symbols

2 Probe 4 Control device 6 Sample nucleic acid 8 Substrate 12 DNA solution 14 Gold thin film substrate

Claims (7)

An identification method comprising the following steps (A) to (C) for identifying a pyrimidine base species in a sample nucleic acid.
(A) A thymine is fixed on the surface of a substrate having a conductive surface, and the probe and the thymine are changed by changing a bias voltage applied between the probe and the substrate in a state where the distance between the probe and the thymine of the scanning probe microscope is fixed. Measuring a tunnel current flowing between and a bias voltage that changes the rate of change of the logarithmically converted tunnel current with respect to the bias voltage,
(B) The portion having the pyrimidine base in the sample nucleic acid to be identified is extended on the surface of the substrate having conductivity on the surface and fixed in a state where each base is in direct contact with the substrate, and the probe and the pyrimidine base are fixed. Measure the change of the tunnel current flowing between the probe and the base by changing the bias voltage applied between the probe and the substrate in a specific voltage range including the inflection bias voltage with the distance between them fixed And (C) based on the change of the tunnel current with respect to the change of the bias voltage obtained in the step (B), from the ratio of the change before and after the inflection bias voltage, whether the pyrimidine base is cytosine or thymine The process of identifying whether there is. The change of the bias voltage in the step (B) is performed at three points of the minimum bias voltage in the specific range, the inflection bias voltage, and the maximum bias voltage,
The identification in the step (C) is based on the ratio between the change rate of the tunnel current between the lowest bias voltage and the inflection bias voltage and the change rate of the tunnel current between the inflection bias voltage and the highest bias voltage. The pyrimidine base species identification method according to claim 1 performed.   The pyrimidine base species identification method according to claim 1 or 2, wherein the identification in the step (C) is performed based on a logarithmically converted rate of change of the tunnel current. An identification method comprising the following steps (A) to (G) for identifying a base species in a sample nucleic acid.
(A) A thymine is fixed on the surface of a substrate having a conductive surface, and the probe and thymine are changed by changing a bias voltage applied between the probe and the substrate in a state where the distance between the probe and the thymine of the scanning probe microscope is fixed. Measuring a tunnel current flowing between and a bias voltage that changes the rate of change of the logarithmically converted tunnel current with respect to the bias voltage,
(B) The guanine is fixed on the surface of the substrate having conductivity, and the bias voltage applied between the probe and the substrate is changed with the distance between the probe and the guanine of the scanning probe microscope fixed, thereby changing the probe and the guanine. Measuring the tunnel current flowing between the two, detecting the peak of guanine on the differential curve due to the bias voltage of the tunnel current, and obtaining the peak detection bias voltage,
(C) a step of extending a portion having a base sequence that is a target of identification of a sample nucleic acid on the surface of a substrate having conductivity, and fixing the base in a state in which each base is in direct contact with the substrate;
(D) Using a scanning probe microscope, the topography image is measured by feedback control so that the tunnel current flowing between the probe and the base becomes constant, and the base is a purine base based on the height of the base image. Identifying whether it is a pyrimidine base;
(E) For the base identified as the purine base in step (D), the distance between the probe and the base is fixed and applied between the probe and the substrate within a specific range including the peak detection bias voltage therebetween. The tunnel current flowing between the probe and the base is measured by changing the bias voltage, and the purine base is determined depending on whether or not a peak is detected on the differential curve by the bias voltage of the tunnel current at the peak detection bias voltage. Identifying whether it is guanine or adenine,
(F) For the base identified as the pyrimidine base in step (D), the distance between the probe and the substrate is within a specific voltage range including the inflection bias voltage with the distance between the probe and the base fixed. Measuring the change in the tunnel current flowing between the probe and the base by changing the bias voltage applied to the base, and (G) the pyrimidine base is cytosine or thymine based on the rate of change in the tunnel current The process of identifying The change of the bias voltage in the step (E) is performed at three points of the minimum bias voltage in the specific range, the inflection bias voltage, and the maximum bias voltage,
The pyrimidine base is identified based on the ratio of the rate of change of the tunnel current between the minimum bias voltage and the inflection bias voltage and the rate of change of the tunnel current between the inflection bias voltage and the maximum bias voltage. The base species identification method according to claim 4.   6. The base species identification method according to claim 4 or 5, wherein the identification in the step (E) is performed based on a logarithmically converted rate of change of the tunnel current.   The base species identification according to any one of claims 4 to 6, wherein the base sequence of the sample nucleic acid is determined by sequentially performing steps (D) to (G) along the base of the sample nucleic acid fixed to the substrate. Method.