JP5391907B2 - Base sequence analyzer and program thereof - Google Patents

Description

  The present invention relates to a base sequence analysis apparatus and a program thereof.

  As one technique for determining the base sequence of a nucleic acid, a technique called the Sanger method using a DNA synthesis reaction is widely known. In this method, a DNA strand complementary to a template DNA is synthesized in the presence of four kinds of dideoxynucleoside triphosphate analogs (ddNTPs) corresponding to each base. Since DNA elongation stops at the position where ddNTP is incorporated, DNA fragments having different lengths by one base are generated by the above synthesis reaction. In the dye terminator method, which is one of the Sanger methods, the four types of ddNTPs (ddATP, ddCTP, ddGTP, and ddTTP) are labeled with different fluorescent dyes. A fluorescent label corresponding to the type of the terminal base at the end is provided. The DNA fragments are separated by electrophoresis according to the base length, and each separated DNA fragment is irradiated with excitation light to excite the fluorescent dye, and the four types of detectors corresponding to each fluorescent dye detect each fluorescent dye. The generated fluorescence is sequentially detected. Then, by processing the detection signals output from these detectors, the base sequence of the template DNA can be identified.

  The four types of detectors are set to detect each of the four types of fluorescent dye groups with the highest sensitivity. An example of such a fluorescent dye group is a dRhodamine dye in a BigDye (registered trademark) terminator kit comprising four fluorescent dyes, dR110, dR6G, dTAMRA, and dROX. FIG. 6 shows normalized emission spectra (cited from “ABIPRISM (registered trademark) BigDye Terminator Cycle Sequencing Ready Reaction Kits”) of four fluorescent dyes contained in the dRhodamine dye. The emission spectrum of each fluorescent dye is not sharp, and the bottom portion is naturally detected by a detector other than the detector corresponding to each dye. For example, the signal waveform (FIG. 7) of the base A (adenine) labeled with dR6G is detected by all four types of detectors (Da, Dt, Dg, Dc) although there is a difference in intensity. Here, the photodetectors for bases A (adenine), G (guanine), C (cytosine), and T (thymine) are referred to as photodetectors Da, Dg, Dc, and Dt, respectively. For the base A (adenine) labeled with dR6G, the signal intensity by the photodetector Da is Pa, the signal intensity by the photodetector Dt is Pt, the signal intensity by the photodetector Dg is Pg, and the signal by the photodetector Dc The strength is Pc. Since the signal intensity ratio (Pa: Pt: Pg: Pc) at this time is constant, if a reverse conversion is performed based on this value, a peak waveform of only the base A can be obtained. This is the same for the other three types of fluorescent dyes, and even when the fluorescence spectrum overlaps with the fluorescence spectrum of another base, the detected waveform is considered to be simply the addition of the spectrum. Therefore, if the signal intensity ratios for the four types of fluorescent dyes are obtained, this is represented in a matrix, and if the inverse matrix is applied to the original detected peak waveform, the peak waveform of each fluorescent dye (four types of bases) Is obtained. Obtaining the signal intensity ratio accurately is obtaining the matrix value of the fluorescent dye (see Non-Patent Document 1 and Patent Document 1).

Here, the signal intensity ratio of the peak waveform of each base, the emission intensity by each base, and the signal intensity detected by each detector are represented by the following symbols.
Signal intensity ratio of peak waveform of base A = APa (= 1.0): APg: APc: APt
Signal strength ratio of base G peak waveform = GPa: GPg (= 1.0): GPc: GPt
Signal intensity ratio of base C peak waveform = CPa: CPg: CPc (= 1.0): CPt
Signal strength ratio of base T peak waveform = TPa: TPg: TPc: TPt (= 1.0)
Luminescence intensity from base A = Ia, signal intensity detected by detector Da = Oa
Luminescence intensity by base G = Ig, signal intensity detected by detector Dg = Og
Emission intensity from base C = Ic, signal intensity detected by detector Dc = Oc
Luminescence intensity by base T = It, signal intensity detected by detector Dt = Ot
At this time, the light emission intensity (Ia, Ig, Ic, It) by each base and the signal intensity (Oa, Og, Oc, Ot) detected by the detector are expressed by the following matrix calculation.

Therefore, in order to obtain the original signal, that is, the emission intensity (Ia, Ig, Ic, It) of each base purely from the signal intensity (Oa, Og, Oc, Ot) obtained by each detector, a matrix is formed on both sides. You can apply an inverse matrix of M. This inverse matrix is a matrix value.

  The dye terminator method is used to obtain the matrix value of the fluorescent dye by four types of detectors that selectively detect four types of fluorescence wavelengths by migrating one type of base modified with a different fluorescent color for each base. In general, the intensity (height) of the signal peak obtained in step 1 is measured.

  However, since a reagent kit in which four types of fluorescent dyes are mixed is used to determine the base sequence of an actual sample, in order to calibrate the matrix values as described above, a base kit determination reagent kit Apart from this, a special reagent kit with separate fluorescent dyes is required. The reason why the bases are migrated one by one in this way is because it is not possible to explicitly distinguish which base the obtained peak waveform is. Next, it is assumed that the detector having the highest signal intensity for the peak waveform is the base (for example, base A in the case of FIG. 7). This is because there is no guarantee that is composed of only one base and never overlaps with another base. If they partially overlap, the signal intensity ratio changes and an accurate matrix value cannot be obtained.

  As described above, the conventional method has a problem in that it takes a lot of time and labor to calculate the matrix value because it is necessary to migrate the bases one by one. Moreover, since a special reagent kit is required separately from the reagent kit for determining the base sequence as described above, there is a problem that the cost increases.

  In order to solve this problem, in recent years, matrix values have been calculated by automatically detecting a pure peak consisting of only one base from the electrophoresis waveform obtained by actual sample migration without using a dedicated reagent kit. A base sequence analyzing apparatus having a function of determining a position suitable for the above (that is, a position for obtaining the above signal intensity ratio) has been developed. In this base sequence analyzer, for example, a matrix value is calculated by automatically calculating the portion where the same base is continuous on the electrophoresis waveform for each type of base, and determining the signal intensity ratio of each detector at that position. ing.

Japanese Patent No. 3690271

Nakamura, Sakuma, Harada, Yamamoto, Nishine, “Development Technology in RISA384 System”, Shimazu Review, Shimazu Review Editorial Department, March 2002, Vol. 58, No. 3, No. 4, pp.111-124

  However, in the method for determining the position on the electrophoresis waveform suitable for the calculation of the matrix value by automatic calculation as described above, the position suitable for the calculation of the matrix value is not necessarily accurately determined depending on the type of sample and the reaction state. There were cases where it was not possible.

  The present invention has been made in view of such problems, and the object of the present invention is to perform matrix values using electrophoresis waveforms obtained by actual sample migration or the like without performing migration of each type of base one by one. Provides a base sequence analyzer that can appropriately set the position used to calculate the matrix value on the electrophoretic waveform (that is, the position where the ratio of the signal intensity by each detector should be obtained), and a program therefor There is to do.

In order to solve the above problems, the base sequence analyzer according to the present invention separates DNA fragment groups labeled with different fluorescent dyes for each type of terminal base according to the base length, and makes them correspond to the respective fluorescent dyes. A base sequence analyzer used for determining the base sequence of a nucleic acid based on the fluorescence intensity waveform signals obtained from the four detection means,
a) signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means;
b) A base sequence for performing a matrix transformation on the fluorescence intensity waveform signal acquired by the signal acquisition means to obtain a signal waveform for each base type, and determining a nucleic acid base sequence based on the signal waveform for each base type A determination means;
c) matrix value deriving means for deriving matrix values for the matrix transformation;
With
The matrix value deriving means comprises:
d) Electrophoretic waveform display means for displaying the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detecting means on the display device;
e) On the electrophoresis waveform displayed by the electrophoresis waveform display means, designation input acceptance means for accepting designation of a position for obtaining a signal intensity ratio described later for each type of base from a user;
f) Matrix value determining means for determining the signal intensity ratio of the four detecting means at each position specified by the specified input receiving means, and determining the matrix value from the signal intensity ratio;
It is characterized by having.

  According to such a configuration, the user refers to the waveform displayed on the electrophoretic waveform display means, and the designated input accepting means accepts the position determined by the user that is appropriate as the position where the signal intensity ratio should be obtained. Can do. In general, even if the above-described conventional automatic calculation method cannot accurately determine the position where the signal intensity ratio is to be obtained, an appropriate position may be known if the user refers to the electrophoretic waveform. Therefore, according to the base sequence analysis apparatus of the present invention, it is possible to more accurately determine a position suitable for calculating the matrix value.

  In the base sequence analysis apparatus according to the present invention, the position where the signal intensity ratio should be obtained may be designated by the user for all four types of bases, or the user designates only a part of the bases. It may be. In the latter case, first, the apparatus side automatically calculates the position on the waveform for which the signal intensity ratio should be obtained for all the bases, and displays the position on the display device together with the waveform. It is conceivable to refer to the positions so that the positions can be changed as necessary.

That is, the base sequence analysis apparatus according to the present invention is obtained from four detection means that separate DNA fragment groups labeled with different fluorescent dyes for each type of terminal base according to the base length and correspond to the respective fluorescent dyes. A base sequence analyzer used for determining the base sequence of a nucleic acid based on the fluorescence intensity waveform signal,
a) signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means;
b) A base sequence for performing a matrix transformation on the fluorescence intensity waveform signal acquired by the signal acquisition means to obtain a signal waveform for each base type, and determining a nucleic acid base sequence based on the signal waveform for each base type A determination means;
c) matrix value deriving means for deriving matrix values for the matrix transformation;
With
The matrix value deriving means comprises:
d) designated position automatic decision means for deciding a designated position for each type of base to determine a signal intensity ratio described later based on the fluorescence intensity waveform signals of the four detection means;
e) designated position display means for displaying on the display device the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means and the designated position determined by the designated position automatic decision means;
f) designated position changing means for accepting a change of the designated position displayed on the display device from a user;
g) Matrix value determining means for determining a signal intensity ratio of the four detection means at the designated position and determining the matrix value from the signal intensity ratio;
It may be characterized by having.

In addition, the program according to the present invention, which has been made to solve the above problems, separates DNA fragment groups labeled with different fluorescent dyes for each type of terminal base according to the base length, and makes them correspond to the respective fluorescent dyes. A base sequence analyzer used for determining a base sequence of a nucleic acid based on fluorescence intensity waveform signals obtained from four detection means, wherein the signal acquisition is performed for obtaining fluorescence intensity waveform signals from the four detection means. Means, an electrophoretic waveform display means for displaying an electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means on a display device, and a base type by performing matrix conversion on the fluorescent intensity waveform signal acquired by the signal acquiring means A program for controlling a base sequence analyzer comprising a base sequence determination means for determining a signal waveform for each base and determining a base sequence of a nucleic acid based on the signal waveform for each base type A is, the base sequence analysis apparatus,
Means for deriving a matrix value for the matrix transformation,
a) designated input receiving means for receiving a position designated by the user for each type of base on the electrophoresis waveform displayed on the display device by the electrophoresis waveform display means, as a position for obtaining a signal intensity ratio described later;
b) Matrix value determining means for determining the signal intensity ratio of the four detectors at each position received by the designated input receiving means, and determining the matrix value from the signal intensity ratio;
It is characterized by functioning as matrix value deriving means having

The program according to the present invention separates a group of DNA fragments labeled with different fluorescent dyes for each type of terminal base according to the base length, and obtains fluorescence intensities obtained from the four detection means corresponding to the respective fluorescent dyes. A base sequence analyzing apparatus used for determining a base sequence of a nucleic acid based on a waveform signal, comprising: a signal acquisition unit for acquiring fluorescence intensity waveform signals from the four detection units; and a fluorescence of the four detection units An electrophoretic waveform display means for displaying an electrophoretic waveform based on an intensity waveform signal on a display device, and performing a matrix conversion on the fluorescence intensity waveform signal acquired by the signal acquisition means to obtain a signal waveform for each type of base, A program for controlling a base sequence analyzer comprising base sequence determination means for determining a base sequence of a nucleic acid based on a signal waveform for each type, the base sequence analyzer ,
Means for deriving a matrix value for the matrix transformation,
a) designated position automatic determination means for determining a designated position for each type of base based on the fluorescence intensity waveform signals of the four detection means;
b) designated position display means for displaying on the display device the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means and the designated position determined by the designated position automatic decision means;
c) designated position changing means for changing the designated position displayed on the display device in accordance with a user operation;
d) Matrix value determining means for determining a signal intensity ratio of the four detection means at the designated position and determining the matrix value from the signal intensity ratio;
It may function as a matrix value deriving unit having

  According to the base sequence analyzing apparatus and program according to the present invention having the above-described configuration, the matrix value is calculated more easily by using the electrophoresis waveform obtained by actual sample migration or the like, without migrating the bases one by one. At this time, the position where the signal intensity ratio should be obtained can be set more appropriately and reliably on the electrophoretic waveform. As a result, an accurate matrix value can be obtained, and as a result, more accurate base sequence determination can be realized.

The block diagram which shows the principal part structure of the base sequence analysis system containing the base sequence analyzer which concerns on one Example of this invention. The flowchart explaining the procedure at the time of calculating a matrix value in the base sequence analysis system of the Example. The figure which shows an example of the screen display in the base sequence analysis system of the Example. The figure which shows another example of the screen display in the base sequence analysis system of the Example. The flowchart explaining the procedure at the time of calculating a matrix value in the base sequence analysis system which concerns on the other Example of this invention. The figure which shows the emission spectrum of dRhodamin pigment | dye. The figure which shows the signal peak waveform of the base A detected by each detector.

  Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. FIG. 1 is a block diagram showing a configuration of a base sequence analysis system to which a base sequence analysis apparatus according to an embodiment of the present invention is applied. This base sequence analysis system mainly includes an electrophoresis apparatus 10 and an analysis apparatus 20.

  The electrophoresis apparatus 10 includes an electrophoresis unit 11 that separates a sample by capillary electrophoresis, and a fluorescence detection unit 12 that detects the separated sample. Further, the fluorescence detection unit 12 is provided with an excitation optical system 13 for irradiating the sample flowing in the capillary with excitation light and a photodetector corresponding to each base. Here, the bases A (adenine) and G (guanine) are provided. ), C (cytosine), and T (thymine) photodetectors are referred to as photodetectors Da, Dg, Dc, and Dt, respectively.

  The electrophoresis unit 11 is provided with voltage application means (not shown). A capillary (glass capillary) filled with a separation medium such as an electrophoresis gel or polymer is set in the electrophoresis unit 11, and the voltage application means By applying a voltage to both ends of the capillary, a sample (a group of DNA fragments) is introduced from one end of the capillary and migrates toward the other end of the capillary. Each DNA fragment in the sample is separated according to the base length in the process of moving in the capillary, and is irradiated with excitation light by the excitation optical system 13 near the end of the capillary. The fluorescent dye imparted to the DNA fragment is excited by the excitation light to generate fluorescence having a specific wavelength, and the fluorescence is detected by the photodetectors Da, Dg, Dc, and Dt.

  Detection signals from the photodetectors Da, Dg, Dc, and Dt are input to the analysis device 20. This analyzer 20 corresponds to the base sequence analyzer of the present invention. The analysis device 20 stores various data analysis programs. By executing analysis processing according to the acquired data for the acquired data, the base sequence is determined and the matrix value is calculated. To do. The result is displayed on a monitor 60 attached to the analysis device 20 or printed out on a printer (not shown). The analysis device 20 has not only data processing but also a function of controlling the operation of each part of the electrophoresis device 10.

  The photodetectors Da, Dg, Dc, and Dt collect a fluorescence signal from a DNA fragment that passes through a predetermined position of the capillary sequentially. Therefore, in the analysis device 20, as shown in FIG. 3, when the signal intensity is on the vertical axis and the migration time is taken on the horizontal axis, a fluorescence intensity waveform signal that becomes a fluorescence intensity waveform is acquired for each photodetector. . The fluorescence intensity waveform shown in FIG. 3 is also referred to as an “electrophoresis waveform” in this specification. In FIG. 3, the electrophoretic waveforms obtained by the respective photodetectors are superimposed and displayed, but these may be displayed side by side on the screen.

  The analysis device 20 is a general-purpose personal computer, and a central control unit 30 mainly composed of a CPU functionally includes an electrophoretic waveform generation unit 31, a specified point determination unit 32, a matrix value calculation unit 33, a matrix conversion. Part 34 and base sequence determination part 35. The central control unit 30 includes an input unit 70 including a pointing device such as a keyboard and a mouse, a display control unit 50 that outputs a display signal to be displayed on the screen of the monitor 60, a storage unit 40 including a hard disk device, and the like. Is connected. The storage unit 40 stores various analysis results obtained by the central control unit 30 and includes a matrix value storage unit 41 for storing the matrix values calculated by the matrix value calculation unit 33.

  The photodetectors Da, Dg, Dc, Dt of the electrophoresis apparatus 10 are set so as to detect each of the four types of fluorescent dyes used for labeling DNA fragments with the highest sensitivity. However, since the emission spectrum of each fluorescent dye has a certain width, for example, the fluorescent dye attached to the DNA fragment having the terminal base A is the photodetector Da associated with the fluorescent dye. The other photodetectors Dg, Dc, and Dt are also detected at a constant rate. Therefore, if the signal intensity ratios of the four types of photodetectors Da, Dg, Dc, and Dt obtained by the emission of each fluorescent dye are obtained in advance, it is represented by a matrix, and its inverse matrix, that is, the matrix value is represented by each photodetector. By multiplying the detected peak waveform, a peak waveform derived purely from only one kind of base can be obtained.

  Hereinafter, the operation of the analyzing apparatus of this embodiment at the time of calculating such matrix values will be described with reference to the flowchart of FIG.

  First, a predetermined sample is electrophoresed in the electrophoresis apparatus 10. Here, the sample is a mixture of DNA fragments labeled with four types of fluorescent dyes depending on the type of terminal base, and is generated by a Sanger reaction using a predetermined template DNA and a general reagent for base sequence analysis. Is done. Here, the sequence of the template DNA is not particularly limited as long as it contains four types of bases. In addition, as a sample to be subjected to electrophoresis, a sample prepared for calculating a matrix value may be used, or an actual sample for which a base sequence is to be determined may be used.

  The analysis device 20 acquires fluorescence intensity waveform signals that are sequentially output from the photodetectors Da, Dg, Dc, and Dt of the electrophoresis apparatus 10 (step S11), and the electrophoresis waveform generation unit 31 monitors these waveform signals. Drawing data to be displayed on the 60 screen is generated. The drawing data is output to the monitor 60 via the display control unit 50. As a result, an image in which four electrophoretic waveforms based on the outputs of the photodetectors Da, Dg, Dc, and Dt as shown in FIG. 3 are superimposed is displayed on the screen of the monitor 60 (step S12).

  The user refers to the electrophoretic waveform displayed on the monitor 60, searches for an appropriate position as the position where the signal intensity ratio regarding the base A is to be obtained, and performs a predetermined operation (for example, at the same time as a mouse click operation) with the input unit 70. The position is designated as a position where the signal intensity ratio is to be obtained (hereinafter referred to as a designated point) (step S13). As a result, the instruction signal from the input unit 70 is input to the central control unit 30, and the designated point determination unit 32 sends an instruction to the display control unit 50 to perform a predetermined image display based on the instruction signal. Thereby, the designated point mark 81 indicating the designated point for the base A is displayed at the position designated by the user on the screen.

  In order to accurately calculate the matrix value, it is desirable to obtain the signal intensity ratio at the time when only one type of base is detected. Therefore, when the user designates the designated point as described above, for example, a location where the same type of base appears continuously on the electrophoresis waveform is designated as the designated point for the base. In particular, since it is unlikely that peaks other than the first and last peaks at locations where three or more identical bases are continuous, it is desirable to designate such positions as designated points. In addition, the mobility of each base is also different due to the difference in molecular weight of each fluorescent dye, and the peak interval becomes wide at a location where a base having a high mobility and a base having a low mobility are continuous. Therefore, in such a place, the peak may be independent without overlapping with other peaks, so the appearance position of such a peak may be designated as the designated point.

  The same operation is performed for the other bases G, C, and T (step S13), and when the designation of designated points for all the bases is completed, the user operates the input unit 70 and presses the OK button 82 on the screen. . As a result, the position to which the designated point mark 81 is assigned is determined as the designated point for each base, and the designated point determining unit 32 determines the fluorescence intensity corresponding to each designated point from the horizontal position coordinates of each designated point mark 81. A position on the waveform signal (that is, migration time) is obtained.

  The position information of each designated point obtained by the designated point determination unit 32 is input to the matrix value calculation unit 33. The matrix value calculation unit 33 calculates the intensity ratios of the fluorescence intensity waveform signals of the photodetectors Da, Dg, Dc, and Dt at the time corresponding to each designated point based on the position information (step S14). Then, a matrix is created from these signal intensity ratios, and its inverse matrix is obtained as a matrix value (step S15). As described above, the obtained matrix value is stored in the matrix value storage unit 41 (step S16 and end).

  The matrix value obtained as described above is automatically read from the matrix value storage unit 41 and used in the subsequent base sequence analysis. At that time, fluorescence intensity waveform signals from the respective photodetectors Da, Dg, Dc, and Dt obtained by electrophoresis of the sample whose base sequence is to be obtained by the electrophoresis apparatus 10 are input to the matrix conversion unit 34. Then, by multiplying these fluorescence intensity waveform signals by the matrix value read from the matrix value storage unit 41, a waveform signal for each type of base is obtained. Then, the base sequence is determined by processing the waveform signal for each base type obtained by the matrix converter 34 according to a predetermined algorithm in the base sequence determination unit 35, and the result is output to the monitor 60. An example of the screen display at this time is shown in FIG. This is based on matrix conversion of the fluorescence intensity waveform signal shown in FIG. 3 using the matrix value obtained from the signal intensity ratio at the position designated by each designated point mark 81 in FIG. The results are shown.

  The matrix value is calculated once when the apparatus is started up, and if the obtained matrix value is stored in the matrix value storage unit 41, the stored matrix value can be used for subsequent base sequence determination. . However, due to changes in the optical system of the electrophoresis apparatus 10 over time and subtle differences in fluorescence emission due to changes in the reaction reagent over time, the matrix values obtained in the past do not always match and correct analysis results may not be obtained. is there. In such a case, the matrix value can be calculated using an actual sample to be sequenced.

  In addition, although the example which the user designates all the designation | designated points regarding each base was shown above, it is good also as a structure which a user can change suitably the designation | designated point calculated | required by the apparatus side by automatic calculation. In this case, it is assumed that the designated point determination unit 32 has a function of calculating a designated point by automatic calculation in addition to the above-described function.

  The operation of the analysis device 20 at the time of calculating the matrix value in such an example will be described with reference to the flowchart of FIG. First, fluorescence intensity waveform signals from each of the photodetectors Da, Dg, Dc, and Dt by electrophoresis using the same sample as described above are input to the electrophoresis waveform generation unit 31 and the designated point determination unit 32 (step S21). ). The designated point determination unit 32 determines a position suitable as the designated point from the waveform signal by automatic calculation according to a predetermined algorithm (step S22). That is, as described above, a location where the same base appears continuously or a peak that does not overlap with another peak is detected for each type of base, and such a position is set as a temporary designated point.

  On the other hand, the electrophoretic waveform generation unit 31 generates electrophoretic waveform drawing data to be displayed on the screen of the monitor 60 from the input fluorescence intensity waveform signal. Information on the provisional designated point determined by the designated point determination unit 32 and drawing data of the migration waveform generated by the migration waveform generation unit 31 are output to the display control unit 50, and the display control unit 50 displays the drawing data on the drawing data. A composite image in which designated point marks 81 representing temporary designated points are superimposed is output to the monitor 60. Thereby, on the screen of the monitor 60, as shown in FIG. 3, four electrophoretic waveforms based on the outputs of the respective photodetectors Da, Dg, Dc, and Dt and designated point marks 81 for the respective bases are displayed. (Step S23).

  Here, each designated point mark 81 can be freely moved in the horizontal direction by a user operation. The user refers to these electrophoretic waveforms and designated point marks to determine whether or not the position of the designated point mark 81 for each base is appropriate, and performs a predetermined operation (for example, for example, using the input unit 70 as necessary). The designated point mark 81 is moved to an appropriate position by performing a mouse drag operation (step S24). When the user determines that all the designated point marks 81 are arranged at appropriate positions, the user operates the input unit 70 and presses an OK button 82 on the screen. As a result, the position to which the designated point mark 81 is assigned is determined as the designated point, and the designated point determination unit 32 determines the position on the fluorescence intensity waveform signal corresponding to each designated point from the horizontal position coordinates of each designated point mark. (Ie, migration time) is determined. Thereafter, a matrix value is obtained from the signal intensity ratio of each of the photodetectors Da, Dg, Dc, Dt at each position on the fluorescence intensity waveform signal in the matrix value calculation unit 33 (steps S25, S26), and the obtained matrix is obtained. The value is stored in the matrix value storage unit 41 (step S27).

  Although the best mode for carrying out the present invention has been described above, the present invention is not limited to the above-described embodiments, and appropriate modifications are allowed within the scope of the gist of the present invention. For example, in the above example, a base sequence analysis system in which the electrophoresis apparatus 10 and the analysis apparatus 20 are configured separately is shown. However, the base sequence analysis apparatus of the present invention is configured integrally with the electrophoresis apparatus. May be. In the above description, a capillary electrophoresis apparatus using a capillary tube is shown as the electrophoresis apparatus. However, a so-called microchip capillary electrophoresis apparatus using a flat plate in which a fine channel is formed may be used. The present invention can be similarly applied to an electrophoresis apparatus using a slab gel in addition to a capillary electrophoresis apparatus.

DESCRIPTION OF SYMBOLS 10 ... Electrophoresis apparatus 11 ... Electrophoresis part 12 ... Fluorescence detection part 13 ... Excitation optical system
Da, Dg, Dc, Dt ... photodetector 20 ... analysis device 30 ... central control unit 31 ... migration waveform generation unit 32 ... designated point determination unit 33 ... matrix value calculation unit 34 ... matrix conversion unit 35 ... base sequence determination unit 40 ... Storage unit 41 ... Matrix value storage unit 50 ... Display control unit 60 ... Monitor 70 ... Input unit 81 ... Specified point mark

Claims (4)

A group of DNA fragments labeled with different fluorescent dyes for each type of terminal base is separated according to the base length, and the nucleic acid base sequence is based on the fluorescence intensity waveform signals obtained from the four detection means corresponding to the respective fluorescent dyes. A base sequence analyzer used for determining
a) signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means;
b) A base sequence for performing a matrix transformation on the fluorescence intensity waveform signal acquired by the signal acquisition means to obtain a signal waveform for each base type, and determining a nucleic acid base sequence based on the signal waveform for each base type A determination means;
c) matrix value deriving means for deriving matrix values for the matrix transformation;
With
The matrix value deriving means comprises:
d) Electrophoretic waveform display means for displaying the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detecting means on the display device;
e) On the electrophoresis waveform displayed by the electrophoresis waveform display means, designation input acceptance means for accepting designation of a position for obtaining a signal intensity ratio described later for each type of base from a user;
f) Matrix value determining means for determining the signal intensity ratio of the four detecting means at each position specified by the specified input receiving means, and determining the matrix value from the signal intensity ratio;
A base sequence analyzing apparatus characterized by comprising: A group of DNA fragments labeled with different fluorescent dyes for each type of terminal base is separated according to the base length, and the nucleic acid base sequence is based on the fluorescence intensity waveform signals obtained from the four detection means corresponding to the respective fluorescent dyes. A base sequence analyzer used for determining
a) signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means;
b) A base sequence for performing a matrix transformation on the fluorescence intensity waveform signal acquired by the signal acquisition means to obtain a signal waveform for each base type, and determining a nucleic acid base sequence based on the signal waveform for each base type A determination means;
c) matrix value deriving means for deriving matrix values for the matrix transformation;
With
The matrix value deriving means comprises:
d) designated position automatic decision means for deciding a designated position for each type of base to determine a signal intensity ratio described later based on the fluorescence intensity waveform signals of the four detection means;
e) designated position display means for displaying on the display device the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means and the designated position determined by the designated position automatic decision means;
f) designated position changing means for accepting a change of the designated position displayed on the display device from a user;
g) Matrix value determining means for determining a signal intensity ratio of the four detection means at the designated position and determining the matrix value from the signal intensity ratio;
A base sequence analyzing apparatus characterized by comprising: A group of DNA fragments labeled with different fluorescent dyes for each type of terminal base is separated according to the base length, and the nucleic acid base sequence is based on the fluorescence intensity waveform signals obtained from the four detection means corresponding to the respective fluorescent dyes. A base sequence analyzing apparatus used for determining a signal, a signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means, and an electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means Electrophoretic waveform display means to be displayed on the apparatus, and the fluorescence intensity waveform signal acquired by the signal acquisition means are subjected to matrix conversion to obtain a signal waveform for each base type, and nucleic acid based on the signal waveform for each base type A base sequence analyzing device comprising a base sequence determining means for determining the base sequence of the base sequence analyzing device, the base sequence analyzing device,
Means for deriving a matrix value for the matrix transformation,
a) designated input receiving means for receiving a position designated by the user for each type of base on the electrophoresis waveform displayed on the display device by the electrophoresis waveform display means, as a position for obtaining a signal intensity ratio described later;
b) Matrix value determining means for determining the signal intensity ratio of the four detectors at each position received by the designated input receiving means, and determining the matrix value from the signal intensity ratio;
A program characterized in that it functions as a matrix value deriving means. A group of DNA fragments labeled with different fluorescent dyes for each type of terminal base is separated according to the base length, and the nucleic acid base sequence is based on the fluorescence intensity waveform signals obtained from the four detection means corresponding to the respective fluorescent dyes. A base sequence analyzing apparatus used for determining a signal, a signal acquisition means for acquiring fluorescence intensity waveform signals from the four detection means, and an electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means Electrophoretic waveform display means to be displayed on the apparatus, and the fluorescence intensity waveform signal acquired by the signal acquisition means are subjected to matrix conversion to obtain a signal waveform for each base type, and nucleic acid based on the signal waveform for each base type A base sequence analyzing device comprising a base sequence determining means for determining the base sequence of the base sequence analyzing device, the base sequence analyzing device,
Means for deriving a matrix value for the matrix transformation,
a) designated position automatic determination means for determining a designated position for each type of base based on the fluorescence intensity waveform signals of the four detection means;
b) designated position display means for displaying on the display device the electrophoretic waveform based on the fluorescence intensity waveform signals of the four detection means and the designated position determined by the designated position automatic decision means;
c) designated position changing means for changing the designated position displayed on the display device in accordance with a user operation;
d) Matrix value determining means for determining a signal intensity ratio of the four detection means at the designated position and determining the matrix value from the signal intensity ratio;
A program characterized in that it functions as a matrix value deriving means.

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