JP4807299B2 - Electrophoresis apparatus and DNA analysis method using the apparatus - Google Patents

Description

  The present invention relates to an electrophoresis apparatus suitable for use mainly in biochemistry, molecular biology, clinical medicine, especially in the field of DNA and protein analysis, and a DNA analysis method using the apparatus.

  Capillary electrophoresis (CE) is a method suitable for separating structurally similar components, such as optical resolution and isotope separation, as well as analysis of biological components such as peptides, proteins, DNA, and sugars. Yes, it is widely used for applications such as clinical medicine, pharmaceuticals, and monitoring of environmental substances. In particular, a microchip type apparatus (microchip electrophoresis apparatus) in which microchannels are formed using photolithography technology or the like is very easy to handle, and has recently been actively used for DNA analysis and the like. (See, for example, Non-Patent Document 1).

  In a microchip electrophoresis apparatus, an electrophoresis chip in which a separation channel is formed on a substrate such as a glass plate or a quartz glass plate is used to separate components of a sample. In a conventional electrophoresis chip, as disclosed in, for example, Patent Document 1, a single separation channel is connected to a sample well (sample reservoir) to which a sample is supplied, and when passing through the separation channel. In this configuration, the sample components separated in (1) and (2) are detected by a detector. The sample injected into the sample well is introduced into the separation channel by the electrophoresis voltage applied to both ends of the separation channel and used for analysis. Even if the amount of sample actually used in the analysis is small, It is necessary to inject a certain amount of sample into the well.

  For example, when analyzing the DNA sequence using the electrophoresis apparatus as described above, only a certain region can be analyzed due to the restriction of the characteristics of the separation medium filled in the separation channel. Therefore, when it is desired to analyze the arrangement over a wide area, it is necessary to perform analysis a plurality of times using separation media having different separation characteristics. However, since it is necessary to inject the amount of the sample as described above into the sample well for each analysis, it is necessary to prepare samples for the number of times of analysis, which is a heavy burden on the analyst. In addition, when analyzing a sample such as a protein derived from a living body, the amount of the sample is limited. For this reason, a plurality of analyzes cannot be performed and a sufficiently accurate result may not be obtained.

  Furthermore, even when a sufficient amount of sample can be prepared for performing multiple analyzes, it is necessary to replace the separation medium in the separation channel or clean the inside of the separation channel for each analysis. In addition, it took time to analyze and it was difficult to improve the throughput, and the work was complicated.

JP 2002-323477 A Arai et al., “Development of Microchip Electrophoresis Device MCE-2010 and Application Examples”, [online], March 2002, Shimadzu Corporation, [March 20, 2007 Search], Internet <http : //www.shimadzu-biotech.jp/datahall/mce/sr58-101.pdf>

  The present invention has been made in view of the above problems, and the main object of the present invention is to reduce the amount of a sample necessary for analysis and to save electricity and improve work efficiency. It is to provide an electrophoresis apparatus. Another object of the present invention is to provide a DNA analysis method capable of efficiently analyzing a DNA sequence using such an electrophoresis apparatus.

The electrophoresis apparatus according to the present invention, which has been made to solve the above problems,
a) an electrophoretic member having a plurality of separation channels connected at one end to a common sample inlet, and each of the plurality of separation channels filled with a separation medium having different separation characteristics;
b) voltage application means for applying an electrophoretic voltage between the sample introduction port and the other ends of the plurality of separation channels;
c) detection means for detecting sample components separated in the plurality of separation channels by application of the electrophoresis voltage by the voltage application means;
d) Collecting detection signals obtained by electrophoresis of the same DNA sample introduced into the sample introduction port in the plurality of separation channels from the detection means, and different regions based on the detection signals A data processing unit that analyzes the base sequence of the region, searches for the overlapping portion of the region, and determines the base sequence in a wide region joined by the overlapping portion;
It is characterized by having.

  In the electrophoresis apparatus according to the present invention, when the sample to be analyzed is injected into the sample introduction port in the electrophoresis member, the amount is appropriately set in each of the plurality of separation channels by the electrophoresis voltage applied by the voltage applying means. Samples are introduced. Samples may be introduced into the plurality of separation channels simultaneously or not. After that, the sample is moved in the opposite direction from the sample introduction port side in the separation channel by the electrophoresis voltage, and the sample components are separated by the action of the separation medium along with the movement. In the plurality of separation channels, the sample components may or may not be separated at the same time. Since the separation characteristics of the separation media filled in the plurality of separation channels are different from each other, even if components of the same sample are separated, the separation mode is different. As a result, the signals detected by the detection means are different.

  In addition, as a detection means, a fluorescence detector, an electrochemical detector, etc. can be used.

  As described above, according to the electrophoresis apparatus according to the present invention, a plurality of different detection results can be obtained in parallel using the sample injected into the sample inlet only once. Specifically, by using this detection result, for example, in the DNA sequence analysis, different regions can be analyzed. For example, in protein identification, the types of proteins that can be identified can be increased by identifying different types of proteins. As described above, since it is possible to perform analysis for a single sample injection in the case where a plurality of analyzes had been required in the prior art, the amount of the prepared sample can be reduced. Thereby, labor saving can be achieved, and the extra cost for it can be reduced. In addition, more accurate results can be obtained for precious samples whose amount is originally limited, such as biological samples. Further, even when a plurality of analyzes with different separation characteristics are performed, operations such as replacement of the separation medium and washing can be omitted, so that throughput can be improved.

  As one aspect of the electrophoresis apparatus according to the present invention, the other ends of the plurality of separation channels can be connected to a common liquid reservoir. According to this configuration, the buffer liquid is stored in the common liquid reservoir, and a predetermined electrophoresis voltage is applied between the liquid reservoir and the sample inlet by the voltage applying means, so that the plurality of separation channels can be separated. Separation analysis can be performed in parallel by applying the same electrophoresis voltage between both ends.

  On the other hand, by separating the liquid reservoirs connected to the other ends of the plurality of separation channels, different migration voltages are applied between both ends of each separation channel, that is, application of migration voltages having different voltage values. Alternatively, the electrophoresis voltage can be applied at different timings.

  Further, in the electrophoresis apparatus according to the present invention, the electrophoresis member can be a capillary, but in particular, integration can be achieved by using a microchip (electrophoresis chip).

  Furthermore, the DNA analysis method according to the present invention is a DNA analysis method using the electrophoresis apparatus according to the present invention as described above, and the separation characteristics of the same DNA sample introduced into the introduction port are different. Electrophoreses in multiple separation channels, collects detection signals, analyzes base sequences of different regions based on the detection signals, searches for overlapping portions of the regions, and joins at the overlapping portions It is characterized by determining a base sequence in a wide region.

  According to this DNA analysis method, it is possible to analyze a base sequence in a wide region from a short chain to a long chain by analysis according to one sample injection without repeating a plurality of analyzes as in the prior art. . Thereby, it is possible to expand the analysis target while shortening the time required for analysis.

  A microchip electrophoresis apparatus will be described as an example of the electrophoresis apparatus according to the present invention. First, the structure of an electrophoresis chip used in the microchip electrophoresis apparatus of this embodiment will be described with reference to FIG.

  FIG. 1A is a top plan view of the electrophoresis chip 10 (electrophoretic member in the present invention), and FIG. 1B is a cross-sectional view taken along line A-A ′ in FIG. The electrophoresis chip 10 includes an elongated flat rectangular parallelepiped substrate 11 formed by bonding a pair of transparent flat plates 11a and 11b made of a glass plate, a quartz glass plate, or the like. On the upper surface of the lower transparent flat plate 11b, two grooves extending in parallel are formed by etching, for example, and the upper transparent flat plate 11a is formed at a position corresponding to one end of the two grooves. A common substantially elliptical through hole is drilled, and another substantially circular through hole is drilled at a position corresponding to the other end of the two grooves. The width of the groove portion is about 10 to 100 μm and the depth is about 5 to 50 μm.

  The pair of transparent flat plates 11a and 11b are bonded together with the grooves on the inside. As a result, the upper open surface of the groove is closed by the lower surface of the upper transparent flat plate 11a, and two parallel separation channels, the first channel 15 and the second channel 16, are formed. The first flow path 15 and the second flow path 16 are connected to the lower open surface of the substantially elliptical through-hole to which one end is connected in common and the other ends of the first flow path 15 and the second flow path 16, respectively. The lower open surfaces of the substantially circular through-holes are respectively closed by the upper surface of the transparent flat plate 11b, the former being the sample well 12 as the sample introduction port, and the latter being the separation medium filling ports 13 and. A first buffer reservoir 17 capable of storing a buffer (electrophoretic solution) is provided on the sample well 12, and a common second buffer reservoir (similarly capable of storing a buffer above the separation medium filling ports 13 and 14). A liquid reservoir 18 in the present invention is provided. In addition, a detector, which will be described later, for detecting the sample components separated in the flow paths 15 and 16 is disposed at the position of reference numeral 26a (26b) in FIG.

  FIG. 2 is a configuration diagram of the main part of the entire analysis system including the electrophoresis apparatus of this embodiment. The sample injection unit 20 injects a sample to be analyzed into the sample well 12 of the electrophoresis chip 10, and the electrophoresis voltage application unit (voltage application means in the present invention) 21 includes the first channel 15 and the second channel 16. The buffer injection unit 23 injects a buffer (electrophoretic solution) into the first and second buffer reservoirs 17 and 18, and the separation medium injection unit 24 separates the separation medium injection unit 24. The medium is injected from the separation medium filling ports 13 and 14, and the cleaning unit 25 is for cleaning the sample well 12, the buffer reservoirs 17 and 18, and the like. Further, the fluorescence detector (detection means in the present invention) 26 includes an excitation light irradiation unit 26a and a light receiving unit 26b, which are respectively arranged with respect to the first channel 15 and the second channel 16, and the first channel. 15. The sample components separated in the second flow path 16 are sequentially detected over time, and detection signals are output respectively. The data processing unit 27 receives this detection signal and executes predetermined data processing. The control unit 28 enables automatic analysis by comprehensively controlling the above-described units. The data processing function included in the data processing unit 27 can be realized by operating dedicated software on a general-purpose computer.

  Next, an example of the analysis procedure by the electrophoresis apparatus according to the present embodiment will be described with reference to the flowchart of FIG. First, the separation medium injection unit 24 injects the separation medium A from the first separation medium filling port 13 of the electrophoresis chip 10 and fills the first flow path 15 with the separation medium A (step S1). If necessary, the sample well 12 may be washed by the washing unit 25 to remove the separation medium A overflowing from the first flow path 15. Next, the separation medium injection unit 24 injects the separation medium B from the second separation medium filling port 14 of the electrophoresis chip 10 and fills the second flow path 16 with the separation medium B (step S2). Separation medium A and separation medium B have different separation characteristics. Thereafter, the separation medium A and B overflowing the sample well 12 is removed by the cleaning unit 25 to clean the inside of the sample well 12 (step S3).

  If there is a possibility that the separation media A and B contain unnecessary components that are inconvenient for analysis, in order to remove them, a buffer is injected into the first and second buffer reservoirs 17 and 18 by the buffer injection unit 23. In this state, a predetermined voltage is applied between the pair of electrodes immersed in the buffers stored in both the reservoirs 17 and 18 by the electrophoresis voltage application unit 21, thereby existing in the channels 15 and 16. Perform a pre-run to move unwanted components. Then, after pre-run for a predetermined time, the cleaning unit 25 removes the buffers in both the reservoirs 17 and 18 and performs cleaning (step S4). However, if there is no fear that unnecessary components are mixed in the separation media A and B, the process of step S4 can be omitted.

  Next, a sample is injected into the sample well 12 from the sample injection unit 20, while a buffer is injected into the second buffer reservoir 18 from the buffer injection unit 23 (step S5). Then, a predetermined electrophoresis voltage is applied between the electrode immersed in the sample in the sample well 12 and the electrode immersed in the buffer in the second buffer reservoir 18 by the electrophoresis voltage application unit 21, thereby It introduce | transduces into the 1st flow path 15 and the 2nd flow path 16, respectively (step S6). That is, the sample held in the sample well 12 by one sample injection operation is moved in parallel along the two paths of the first flow path 15 and the second flow path 16 by a predetermined amount. Thereafter, if necessary, the cleaning unit 25 removes and cleans the sample remaining in the sample well 12 (step S7).

  Then, a buffer is injected and stored in the first buffer reservoir 17 by the buffer injection unit 23 (step S8). At this time, the buffer injected in step S5 is stored in the second buffer reservoir 18. Then, a predetermined electrophoresis voltage is applied between the electrode immersed in the buffer in the first buffer reservoir 17 and the electrode immersed in the buffer in the second buffer reservoir 18 by the electrophoresis voltage application unit 21. The sample previously held in the flow paths 15 and 16 by the electric field formed by this electrophoresis voltage is moved from the sample well 12 side toward the separation medium filling ports 13 and 14, and the separation medium A, The components are separated in the longitudinal direction of the channels 15 and 16 by the interaction between B and various components in the sample. However, since the separation characteristics are different between the separation medium A and the separation medium B, for example, different types of sample components are favorably separated.

  When the separated sample components pass through the measurement positions of the excitation light irradiation unit 26a and the light receiving unit 26b of the fluorescence detector 26, the difference in the fluorescence characteristics is detected by the fluorescence detector 26, and this is reflected in the detection. A signal is obtained (step S9). Since sample components are detected independently in parallel in the first flow path 15 and the second flow path 16, two detection signals corresponding to the respective flow paths 15 and 16 are sent to the data processing unit 27. Based on the data obtained by converting the detection signal into a digital value, the data processing unit 27 creates an electropherogram with the horizontal axis as the migration time, for example.

  As described above, according to the microchip electrophoresis apparatus according to the present embodiment, what has conventionally been necessary to perform the analysis twice by replacing the separation medium filled in the separation channel is performed once. Results can be obtained by a single concurrent analysis after injection of a sample. Thereby, since it is sufficient to prepare a sample for the amount of one sample injection, labor and labor can be saved and the efficiency of analysis can be improved when preparation of the sample takes time and labor. Moreover, even when the sample is expensive, the cost can be reduced. Furthermore, even when only a very small amount of sample can be prepared originally, a good analysis using different separation characteristics can be performed.

  Next, an analysis method for analyzing the length of a DNA sequence by an analysis system using the microchip electrophoresis apparatus will be described. When DNA is separated for each chain length by electrophoresis as described above, in general, short-chain DNA is well separated in a high concentration separation medium, but separation of long-chain DNA is poor. On the other hand, in a low concentration separation medium, on the contrary, long DNA is well separated, but short DNA is poorly separated. For this reason, it is difficult to acquire data that can analyze the chain length of a wide region from a short chain to a long chain with only one kind of separation medium.

  Therefore, in the microchip electrophoresis apparatus, the first flow path 15 of the electrophoresis chip 10 is filled with a high concentration separation medium, and the second flow path 16 is filled with a low concentration separation medium. Then, the DNA sample is separated and analyzed by the above-described method, components separated by the two flow paths 15 and 16 are detected by the fluorescence detector 26, and the data processing unit 27 collects the data.

  Now, as shown in FIG. 4, when the length of DNA that needs to be decoded is spread over a very wide region, for example, the sequence analysis of the region of the chain length L1 to L3 from the data collected for the first channel 15 From the data collected for the second flow path 16, the sequence analysis of the region of chain lengths L2 to L4 can be performed. In both analysis results, since the sequences analyzed in a partial region (L2 to L3 region) overlap, the data processing unit 27 searches for this overlap portion, and if an overlap portion is found. The short chain side region and the long chain side region are combined with this in between, and a sequence analysis result of a wide region extending over the chain lengths L1 to L4 is obtained.

  When trying to obtain a sequence analysis result in such a wide area in one analysis, the resolution becomes low and it takes time for long chain sequence analysis. However, in the microchip electrophoresis apparatus of the above embodiment, 2 is required. Since it is sufficient that only the regions assigned to the respective flow paths 15 and 16 can be separated, high resolution can be easily obtained. Furthermore, the sequence analysis time can be shortened by optimizing the separation conditions.

  In addition, in the said Example, although the flow path for separation is two, three or more may be sufficient. Further, although the second buffer reservoir 18 is made common to the first and second flow paths 15 and 16, it may be divided for each flow path. By separating the second buffer reservoir 18, different migration voltages can be applied to both ends of the first flow path 15 and both ends of the second flow path 16. That is, it is possible to simultaneously apply electrophoresis voltages of different voltage values, or to separate analysis by applying the electrophoresis voltage to both ends of the first channel 15 in a time division manner, for example, not both at the same time. After performing the above, it is also possible to perform an analysis with a shifted timing, such as performing separation analysis by applying an electrophoresis voltage to both ends of the second flow path 16.

  Moreover, in the said Example, although the flow path length of the two flow paths 15 and 16 is the same, it can also be set as a different flow path length. Thereby, it is possible to adjust the time so that electrophoresis can be performed in each channel in the shortest time, and to adjust the electric field strength in the channel. Further, the adjustment of the migration time can also be realized by adjusting the detection position by the detector 26 to the optimum position for each flow path.

  Moreover, although the said Example applies this invention to a microchip electrophoresis apparatus, this invention is applicable also to a capillary electrophoresis apparatus. Further, since the above embodiment is merely an example of the present invention, in addition to the above-described various modifications, any appropriate changes, modifications, additions, etc. within the spirit of the present invention are included in the scope of the claims of the present application. Obviously it will be done.

The top view (a) of the electrophoresis chip used for the electrophoresis apparatus of one Example of this invention, and A-A 'arrow line sectional drawing (b). The block diagram of the principal part of the whole analysis system containing the electrophoresis apparatus of a present Example. The flowchart which shows an example of the analysis procedure by the electrophoresis apparatus by a present Example. Explanatory drawing of the DNA chain length analysis method using the electrophoresis apparatus by a present Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Electrophoresis chip 11 ... Board | substrate 11a, 11b ... Transparent flat plate 12 ... Sample well 13 ... 1st separation medium filling port 14 ... 2nd separation medium filling port 15 ... 1st flow path 16 ... 2nd flow path 17 ... 1st Buffer reservoir 18 ... second buffer reservoir 20 ... sample injection unit 21 ... electrophoresis voltage application unit 23 ... buffer injection unit 24 ... separation medium injection unit 25 ... washing unit 26 ... fluorescence detector 26a ... excitation light irradiation unit 26b ... light reception unit 27 ... Data processing unit 28 ... Control unit

Claims (4)

a) an electrophoretic member having a plurality of separation channels connected at one end to a common sample inlet, and each of the plurality of separation channels filled with a separation medium having different separation characteristics;
b) voltage application means for applying an electrophoretic voltage between the sample introduction port and the other ends of the plurality of separation channels;
c) detection means for detecting sample components separated in the plurality of separation channels by application of the electrophoresis voltage by the voltage application means;
d) Collecting detection signals obtained by electrophoresis of the same DNA sample introduced into the sample introduction port in the plurality of separation channels from the detection means, and different regions based on the detection signals A data processing unit that analyzes the base sequence of the region, searches for the overlapping portion of the region, and determines the base sequence in a wide region joined by the overlapping portion;
An electrophoretic device comprising:   2. The electrophoresis apparatus according to claim 1, wherein the other ends of the plurality of separation channels are connected to a common liquid reservoir.   The electrophoresis apparatus according to claim 1, wherein the electrophoresis member is a microchip. A DNA analysis method using the electrophoresis apparatus according to any one of claims 1 to 3, wherein the same DNA sample introduced into the sample introduction port is passed through a plurality of separation channels having different separation characteristics. Collect each detection signal by electrophoresis, analyze the base sequence of each different region based on the detection signal, search for the overlapping part of the region and determine the base sequence in the wide region joined by the overlapping part DNA analysis method characterized by performing.

Images