JPH1068694A - Method for determining base sequence of nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for determining the base sequence of nucleic acid by detecting fluorescence having respective wavelengths by a simple mechanism. SOLUTION: A process separating nucleic acid segments labelled with fluorescent material having different light emitting wavelengths corresponding to the kind of a terminal base, a process irradiating fluorescent material with laser beam to generate fluorescence, a process condensing fluorescence in a spectrally diffracted state and a process detecting the condensed light to determine the base sequence of a sample from the relation of the wavelength of the detected light and the length of nucleic acid segments are provided. Thereby, a wavelength can be dispersed without largely changing a light path and a fluorescent image can be detected with high sensitivity without being distorted.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to DNA or RNA.
More particularly, the present invention relates to a method for determining a base sequence suitable for detecting fluorescence having different wavelengths from a nucleic acid fragment labeled with a fluorescent substance having a different emission wavelength according to the type of terminal base.

[0002]

2. Description of the Related Art Conventionally, base sequence determination on DNA has been performed by transferring a separation pattern to a photograph using autoradiography. However, in addition to the complexity of using radioactive elements, there is a problem that it takes time and effort.
Therefore, attention has been paid to a method of detecting DNA fragments during electrophoretic separation in real time by fluorescently labeling DNA (L, M, Smith, et al., Nature 321, 674 (1986)).
(LM Smithet al Nature 32
1, 674 (1986))). In this method, a specific D
Fluorescent labeling is carried out at the end or in the middle while retaining the NA end, and the base of the nucleic acid at the other end is adenine (A)
, Four types of fragment groups ending with cytosine (C), thymine (T), or guanine (G). At this time, a fluorescent substance having a different emission wavelength is used as a fluorescent substance for labeling each base group. Gel electrophoretic separation is performed on these four fragment groups together. Since the shorter the DNA fragment, the faster the electrophoresis speed, the laser is irradiated at a certain distance from the sample injection port with a laser, and the shorter fragments sequentially pass through the irradiation region and emit fluorescence. Since the emission wavelength differs depending on the type of base, the type of base can be determined from the wavelength, and the length can be determined from the migration time. In order to distinguish four types of fluorescence having different wavelengths, four types of filters having different transmission wavelengths are rotated. In addition, in order to enable measurement of multiple samples, a device using a flat gel was
Biosystems (Applied Biosystem)
ms). In this apparatus, a laser beam is narrowed down to irradiate a point on a gel plane. The fluorescent light emitted from the filter passes through the rotating filter and is detected by the light receiving unit. In the measurement of a large number of samples, information is obtained by scanning the laser irradiation position and the detection unit on a straight line in conjunction with each other.

[0003]

In the above prior art, no consideration has been given to increasing the amount of light received by the detector. That is, when trying to determine an area of 10 cm on the electrophoresis plate, the time for irradiating each point with the laser is 1/1 of one scanning time (normally about 1 second), assuming that the laser beam width is 0.5 mm. It will be 200. Furthermore, since the four filters rotate in response to the four bases, the measurement time per one base group of interest is further reduced to 1/4, and eventually, 1/800 of the total measurement time.
The degree is only used for measuring one point and one base type, and the amount of received light is small and high sensitivity cannot be obtained. An object of the present invention is to provide a fluorescence detection method for a highly sensitive fluorescence detection type electrophoresis apparatus which can receive fluorescence of each wavelength for all measurement times with a simple mechanism without using a rotating filter.

[0004]

The object of the present invention is to simultaneously irradiate the measurement area and disperse the emitted fluorescence with a direct-view prism (composite prism), and simultaneously form an image on an image amplifier with a lens system to obtain a two-dimensional detector. This is achieved by detecting using That is, this is achieved by installing a direct-view prism on the front surface of the condenser lens and simultaneously performing wavelength dispersion and image formation.

The method for determining the nucleotide sequence of a nucleic acid according to the present invention comprises the steps of: separating a nucleic acid fragment labeled with a fluorescent substance having a different emission wavelength according to the type of a terminal base by electrophoresis; A step of exciting to generate fluorescence, a step of refracting the fluorescence a plurality of times to disperse light of a wavelength corresponding to the type of the phosphor in different directions, and a step of collecting the dispersed light. Detecting the illuminated light;
Determining the base sequence of the sample from the relationship between the wavelength of the detected light and the length of the nucleic acid fragment.

[0006]

In a direct-view prism (composite prism) in which a plurality of prisms are combined, unlike an ordinary single prism, the optical path after emission is not much different from that before incidence, and only dispersion by wavelength is caused. For this reason, it is possible to form an image on an image amplifier or a high-sensitivity two-dimensional detector in a wavelength-dispersed type using a lens system without distorting the fluorescent ray image. If the fluorescent image is taken in the horizontal direction, fluorescent images having wavelength dispersion in the vertical direction will be arranged. The wavelength is identified by the position in the vertical direction, and the position of the irradiated part is identified from the coordinates in the horizontal direction. The vertical dispersion indicates the difference in the type of bases labeled with different phosphors, and the left and right reflect the difference in the loading position, indicating the difference in the sample.

[0007]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIGS. FIG. 1 shows the entire apparatus configuration.
FIG. 2 shows the principle of base sequence determination, and FIG. 3 shows the operation principle of the combination prism. A fluorescent label (F) is applied to one end of the DNA (sample) 16 whose sequence is to be determined, and a fragment group ({A} family) whose other end ends with an adenine (A) base is prepared. A similar fragment group (family) is prepared for other bases, cytosine (C), guanine (G), and thymine (T). However, the type of the labeled phosphor is changed for each fragment group. These fragment groups are combined into one, loaded on an electrophoresis gel, and subjected to electrophoresis. One migration path is used for one sample (analyte). In this embodiment, since a plurality of migration paths can be secured, many samples (samples) can be measured simultaneously. DNA
The shorter the fragment, the faster it migrates, so illuminating it at a certain distance from the starting point with light and observing the fluorescence emitted from the DNA fragment that passes through it allows you to determine the base length from the migration time and the terminal base type from the fluorescence wavelength. .

[0008] A fluorescence image emitted from a gel, a phosphor, or the like of a linear irradiation portion of excitation light including a plurality of electrophoresis paths passes through a filter 4 for cutting the excitation light, is wavelength-dispersed by a direct-view prism 14, and is condensed. An image is formed as a wavelength dispersion image on the image amplifier 6 by the image lens 5. The wavelength-dispersed amplified image is received by the two-dimensional detector 7. The position in the horizontal direction (the direction perpendicular to the migration direction) on the two-dimensional detector 7 indicates the irradiation part coordinates, that is, the distinction of the migration path, and the vertical direction (the migration direction) corresponds to the direction in which the wavelength is dispersed. 1 on the two-dimensional detector 7
The fluorescent image dispersed in wavelength is received by one horizontal light receiving element line or a plurality of horizontal light receiving element lines.
Therefore, the wavelength-dispersed fluorescent images are obtained at four different positions on the two-dimensional detector 7 in the vertical direction. The signal is input to the detection circuit 9, and the state of signal detection can be seen on the monitor 8. Also, the memory 1 of the signal from the detection circuit 9
0, can be processed and analyzed by the computer 11 and the output device 12. Note that, as the two-dimensional detector 7, an array of a plurality of line sensors may be used.

Using an excitation wavelength of 488 nm, FITC (emission wavelength: 515 nm) and its isomer (emission wavelength: 535 n)
(555 nm, 575 nm, and 575 nm) will be described. As shown in FIG. 3, the refractive index n ₂
When the prisms of n and n ₃ are used in combination, the dispersion of the refraction angle depending on the wavelength ∂u _out / ∂λ is given by (Equation 1).

[0010]

１u _out / ∂λ = 2α ₂ / (λ _I -λ _C ) ｛(nd ₂ -1) / (νd ₂ )-[(nd ₃ -1) / (νd ₃ )]］ ( n ₂ −n ₁ ) / (n ₃ −n ₁ )｝ (Formula 1) α ₂ is the apex angle of the first prism, n ₂ is the refractive index, n ₁ is the refractive index of air, and n ₃ is the second prism. Is the refractive index of nd ₂ , n
d ₃ is the nd of the glass constituting the first and second prisms
Νd ₂ and νd ₃ are νd values. λ _F =
0.4861 μm and λ _C = 0.6563 μm. α ₂
When SFS1 is used for the first prism material and FK5 is used for the second prism material, the apex angle α of the second prism
₃ becomes (Equation 2).

[0011]

Α ₂ · (n ₂ −n ₁ ) / (n ₃ −n ₁ ) ≒ 34.5 ° (Expression ₂ ) Further, νd ₂ = 21, nd ₂ = 1.92, and νd ₃ = 70 ,
Since nd ₃ = 1.4, the dispersion of the refraction angle depending on the wavelength ∂
u _out / ∂λ is given by (Equation 3).

[0012]

∂u _out /∂λ=(−2×0.262/0.1702)｛0.92/21−0.92/70｝=−0.095 (Equation 3) Here, assuming that the wavelength difference Δλ = 20 nm and the image forming position L = 60 mm, the variance Δδ becomes (Equation 4).

[0013]

Δδ = 0.095 · Δλ · L = 0.11 (mm) (Equation 4) That is, a dispersion of 0.11 mm is obtained on the image amplifier. In embodiments where greater dispersion is required, α ₂ may be 30 °
Assuming that L = 100 mm, the dispersion was increased to 0.36 mm so that wavelength discrimination could be sufficiently performed. The position resolution of the image amplifier is about 0.03 mm.

[0014]

According to the present invention, it is possible to perform wavelength dispersion without greatly changing the optical path, and therefore, it is possible to perform wavelength dispersion measurement without distorting the fluorescent image. This method eliminates the need to alternately receive different wavelengths with a rotating filter,
Very high sensitivity can be obtained because fluorescence of each wavelength can be received over the entire measurement time with a simple mechanism.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of an apparatus according to an embodiment of the present invention.

FIG. 2 is a diagram showing the principle of DNA base sequence determination in an example of the present invention.

FIG. 3 is a diagram showing an example of a direct-view prism according to the embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Gel plate, 2 ... Laser, 3 ... Mirror, 4 ... Reflection filter, 5 ... Lens, 6 ... Image amplifier, 7 ... 2D detector, 8 ... Monitor, 9 ... Detection circuit, 10 ... Memory,
11: Computer, 12: Output device, 13: Laser beam, 1
4 ... direct-view prism, 15 ... DNA band, 16 ... target DNA, 17 ... electrophoresis plate, 18 ... sample well, 1
9: incident light, 20: first prism, 21: second prism, 22: third prism.

Claims (3)

[Claims] 1. A step of electrophoretically separating nucleic acid fragments labeled with a fluorescent substance having a different emission wavelength according to the type of a terminal base, and a step of irradiating a laser beam to excite the fluorescent substance to generate fluorescence. And the step of collecting the fluorescence while keeping the fluorescence, and the step of detecting the collected light, and determining the base sequence of the sample from the relationship between the wavelength of the detected light and the length of the nucleic acid fragment. A method for determining a nucleotide sequence of a nucleic acid, comprising: 2. A step of separating, by electrophoresis, nucleic acid fragments labeled with a fluorescent substance having a different emission wavelength according to the type of a terminal base, and a step of irradiating a laser beam to excite the fluorescent substance to generate fluorescence. And spectrally condensing the fluorescence,
Detecting the condensed light, and determining the base sequence of the sample from the relationship between the wavelength of the detected light and the length of the nucleic acid fragment. 3. A step of electrophoretically separating nucleic acid fragments labeled with a fluorescent substance having a different emission wavelength according to the type of terminal base, and a step of irradiating a laser beam to excite the fluorescent substance to generate fluorescence. And refracting the fluorescence light a plurality of times to disperse light of a wavelength corresponding to the type of the phosphor in different directions, condensing the dispersed light, and detecting the condensed light. And determining the nucleotide sequence of the nucleic acid.