JP2610870B2 - Base sequencer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) In the present invention, in the process of determining the nucleotide sequence of a nucleic acid by the Sanger method, in particular, a primer is previously labeled with a labeling dye such as a fluorescent substance or a phosphorescent substance. The present invention relates to an apparatus for reading out a sequence from gel electrophoresis at the final stage by a spectroscopic method utilizing luminescence from a labeled dye.

(Prior Art) As a conventional base sequence determination device, for example, "Nature"
Magazine, Volume 321 and Pages 674-679 (1986),
Biophysical Society of Japan Annual Meeting (October 1986, Tsukuba) 3E1130. These base sequence determination devices have been prepared in advance by the Sanger method ("Proc. Natl. Acad. Sci. USA", Vol. 74, No. 54).
The types of terminal bases (A (adenine), G (guanine),
(Temin), C (cytosine)) depending on the type of fluorescent substance, or migrated in a single migration lane, or labeled with the same fluorescent substance in separate migration lanes, The fluorescent substance is excited by laser light, and only the light of the fluorescence wavelength is received and measured, thereby detecting the migration pattern of the DNA fragment and finally determining the base sequence.

A representative example is shown in FIG. 3 in order to explain the conventional base sequence determination device in detail. An apparatus similar to the apparatus in FIG.
gh Technology ”, December 1986, page 49.

In FIG. 3, reference numeral 2 denotes a migration gel made of polyacrylamide, both ends of which are immersed in electrode baths 4 and 6.
Electrode solutions are contained in the electrode tanks 4 and 6. A migration voltage is applied between the electrode tanks 4 and 6 by a migration power supply 8. A slot 10 for injecting a sample is provided at one end of the electrophoresis gel 2, and a sample for each terminal base is injected into the slot 10, and the electrophoresis voltage from the electrophoresis power supply 8 causes the sample to flow through the electrophoresis gel 2. It is electrophoresed in 12 directions and developed.

Reference numeral 14 denotes a laser as an excitation light source. The excitation light is reflected by a half mirror or a dichroic mirror 16 and irradiates the electrophoretic gel 2 via an objective lens 18. The fluorescence from the fluorescent label of the sample that has migrated in the electrophoresis gel 2 is collected again by the objective lens 18, passes through the half mirror or dichroic mirror 16, passes through the fluorescence selection interference filter 20,
The light is received and detected by a photomultiplier tube 22 as a photoelectric element.

In the apparatus shown in FIG. 3, one objective lens 18 is commonly used for excitation light irradiation and fluorescence reception, and the objective lens 18 and the entire optical system related thereto are arranged in a sample arrangement direction 23 (a direction orthogonal to the migration direction 12; The scanning is performed mechanically in the horizontal direction in the figure.

(Problems to be Solved by the Invention) Including the base sequence determination device shown in FIG. 3, conventional base sequence determination devices excite a fluorescent substance with laser light and spectrally detect the fluorescent light. Since the fluorescent light is very weak, the Rayleigh scattering component of the excitation light due to the electrophoresis gel and the component due to the Raman scattering of water become the intensity and background light of the fluorescent light, and lower the S / N ratio.

Hereinafter, the problem will be described in detail with reference to the base sequence determination apparatus of FIG. 3 as an example.

The light received by the photomultiplier tube 22 passes through the dichroic mirror 16 and the fluorescence selection interference filter 20,
The light received by the photomultiplier tube 22 is not only fluorescent light,
Strictly speaking, Rayleigh scattered light of excitation light and Raman scattered light of water are included as background light.

When excited by laser light, the spectrum of laser light is extremely narrow, and the spectrum of Raman scattering is also extremely narrow. Rayleigh scattered light and Raman scattered light have different wavelengths from fluorescent light, so if fluorescent light can be extracted with a perfect wavelength filter (interference filter) 20, Rayleigh scattered light and Raman scattered light will be included in the light received by the photomultiplier tube 22. Scattered light and Raman scattered light are not mixed in, the background light is not increased, and the S / N ratio should not deteriorate.

However, the dichroic mirror 16 and the interference filter 20
Although the excitation light component and the Raman scattered light component of water are incident on the photomultiplier tube 22 in spite of using, the dichroic mirror 16 and the interference filter 20 cannot completely separate the wavelength. It is. For example, the interference filter 20
Is an ideal interference filter, the wavelength can be separated only when the incident light enters the interference filter 20 at a right angle. Actually, since the fluorescent light is incoherent and has the size of the fluorescent light source, even if the objective lens 18
However, this condition is not satisfied even if parallel light is obtained.

Even if a diffraction grating is used instead of the interference filter 20,
Since the entrance slit has a certain size, the number of diffraction gratings is not infinite, and the fluorescent light is also incoherent, the diffraction grating similarly cannot be an ideal wavelength filter.

That is, it is impossible to completely separate only the fluorescence wavelength with a physical system that can be experimentally performed, and the mixing of the excitation wavelength component due to Rayleigh scattering and the Raman scattering wavelength component of water as background light into the photomultiplier tube 22 is avoided. Absent.

As shown in FIG. 3, in the method of observing the time change of fluorescence at a specific place on the electrophoresis gel while electrophoresis is performed (online method), a method of measuring a pattern which has been electrophoresed and developed by another electrophoresis apparatus (offline method) Advantageously, there is no difference in the optical properties depending on the location of the electrophoretic gel itself as compared with the method (1), but after all, the passage of nucleic acid fragments, the time variation of the optical properties of the electrophoretic gel 2 itself, the generation of bubbles, or the laser light itself The fluctuation of the output causes the background light intensity to fluctuate greatly due to the Rayleigh scattered light of the excitation light and the Raman scattered light of the water, and wraps up the fluctuation of the fluorescent output, which is the original signal, and the measurement sensitivity decreases.

The above problem is exactly the same when a phosphorescent substance is used instead of a fluorescent substance as a labeling dye.

An object of the present invention is to provide a base sequence determination apparatus having high measurement sensitivity by removing a variation in background light from a signal detected by irradiating an electrophoresis gel with excitation light.

(Means for Solving the Problems) In the present invention, when the fluorescence measurement or the phosphorescence measurement is performed from the electrophoretic gel, the change in the optical characteristics of the electrophoretic gel itself, which is the background light of the detection signal, the change in the Rayleigh scattered light intensity, At least a part of the change in the Raman scattered light is measured at the same time, and the fluctuation of the background light is removed from the detection signal.

Referring to FIG. 1 and FIG. 2 showing the embodiment, the base sequence determination apparatus of the present invention is a mechanism (4) for electrophoresing a nucleic acid fragment labeled with a labeling dye on a slab-like electrophoresis gel (2). , 6,8), a mechanism (14,16,18,44) for irradiating the electrophoresis gel (2) with an excitation light beam, and a first light receiving unit for receiving light from a portion irradiated by the excitation light beam (20, 22, 46, 52) and an optical system (24, 26, 46, 52) for extracting at least one of an excitation wavelength component and a Raman scattering wavelength component of water from a part of the light received by the first light receiving unit. 54)
A second light receiving unit (28, 52) for receiving the light extracted by the optical system; a variable resistor (36) for adjusting the level of the second light receiving unit; The data processing unit (30, 32, 34, 34) calculates the luminescence intensity of the labeled dye by subtracting an amount proportional to the output of the second light receiving unit and determines the base sequence of the nucleic acid based on the luminescence intensity. 38, 40).

(Example) FIG. 1 shows an example in which the present invention is applied to the base sequence determination device shown in FIG. The same parts as those in FIG. 3 are denoted by the same reference numerals.

As the slab-like electrophoresis gel 2, a gel made of 8% polyacrylamide is used. Electrode tanks 4 and 6 containing an electrolytic solution are provided above and below the electrophoresis gel 2, and an electrophoresis current is applied by an electrophoresis power supply 8.

Slot for injecting sample at the upper end of electrophoresis gel 2
Each slot 10 is treated by the Sanger method and the primer is labeled with a fluorescent substance TRITC which is a labeling dye.
The DNA fragments labeled in advance are separately placed in their terminal bases A, T, C, and G, respectively, and are allowed to migrate in the migration direction 12.
The fluorescent substance TRITC has an absorption peak near 545 nm, and a peak of emitted fluorescence near 570 nm.

An argon laser having an oscillation wavelength of 514 nm is used as the laser 14 as an excitation light source.

A dichroic mirror 16 that transmits light of 540 nm or more and reflects light of a wavelength shorter than 540 nm is used. The dichroic mirror 16 is provided at a position where the laser light from the laser 14 is reflected.

The objective lens 18 condenses the light reflected by the dichroic mirror 16 on the surface of the electrophoresis gel 2. The objective lens 18 also condenses the light from the irradiated portion of the electrophoresis gel 2 and causes the light to enter the dichroic mirror 16.

A dichroic mirror 24 is provided at a position where the light transmitted through the dichroic mirror 16 is received. The dichroic mirror 24 also transmits light of 540 nm or more,
One that reflects light of a wavelength less than that is used.

A photomultiplier tube 22 is provided at a position for receiving the light transmitted through the dichroic mirror 24 via an interference filter 20. The interference filter 20 has a narrow transmission band at a fluorescence peak wavelength of 570 nm.

At a position where the light reflected by the dichroic mirror 24 is received, a photomultiplier tube 28 is provided via an interference filter 26. As the interference filter 26, a filter having a narrow transmission band at 514 nm is used. Dichroic mirror 24
The light reflected by is a light having a wavelength shorter than 540 nm. The reflected light is further extracted by the interference filter 26 of 514 nm and detected by the photomultiplier tube 28. What is detected by the photomultiplier tube 28 is an excitation light component due to Rayleigh scattering.

Reference numeral 30 denotes an amplifier for amplifying the detection signal of the photomultiplier tube 22 for detecting fluorescence, and reference numeral 32 denotes an amplifier for amplifying the detection signal of the photomultiplier tube 28 for detecting the excitation light component. The output of the amplifier 30 is input to the non-inverting input terminal of the differential amplifier 34, and the output of the amplifier 32 is input to the inverting input terminal of the differential amplifier 34 via the variable resistor 36 for level adjustment.

The output of the differential amplifier 34 is converted into a digital signal by an A / D converter 38 and input to a microcomputer 40.

The entire optical system including the dichroic mirror 16 and the objective lens 18 is driven by a driving mechanism 42 in a direction 23 orthogonal to the electrophoresis direction 12.
Is mechanically scanned at high speed. By controlling the drive mechanism 42, the microcomputer 40 calculates the DNA migration pattern from the input fluorescence pattern and determines the target base sequence.

To explain the operation of the present embodiment, the light received by the photomultiplier tube 22 includes not only the fluorescent light from the fluorescent label of the DNA fragment, but also the Rayleigh scattered light and the Raman scattered light, which are dichroic mirrors 16 and 24 and interference filters. Contaminated by 20 imperfect separation properties.

Considering the luminous flux received by the photomultiplier tube 28,
Since the light beam 25 before being incident on the dichroic mirror 24 is the same as the light beam received by the photomultiplier tube 22, the light beam received by the photomultiplier tube 28 is the signal of the signal detected by the photomultiplier tube 22. Reflects background light. Strictly speaking, the output of the photomultiplier tube 28 includes a fluorescent component, but most of the output is the background light itself.

The background light consists of Rayleigh scattered light and Raman scattered light,
Rayleigh scattering is much stronger than Raman scattering, and in this case, the wavelength is also closer to fluorescence than Rayleigh scattering. Therefore, it can be considered that the background light is almost due to Rayleigh scattering.

Even if the light receiving signal of the photomultiplier tube 22 fluctuates, if the fluctuation is caused by a fluctuation of the background light for some reason, the output of the photomultiplier tube 28 also fluctuates, and if the fluctuation is a fluorescent light, If the signal is a true signal due to light fluctuation, only the output of the photomultiplier tube 22 fluctuates, and the output of the photomultiplier tube 28 does not fluctuate. Therefore, if the difference between the two outputs is obtained by the differential amplifier 34, the output represents only the fluorescence signal.

The variable resistor 36 for adjusting the level adjusts the amplification degree of the amplifier 32 so that the output of the differential amplifier 34 becomes 0 when there is no fluorescent substance, that is, before the migration.

The base sequence is determined by inputting a fluorescence output signal from which background light has been removed to the microcomputer 40 while scanning the entire optical system including the objective lens 23 through the drive mechanism 42.

 FIG. 2 shows another embodiment.

Electrode tanks 4 and 6 are provided at the upper and lower ends of the electrophoresis gel 2, and the nucleic acid fragment sample injected into the sample injection slot 10 at the upper end of the electrophoresis gel 2 is electrophoresed in the electrophoresis gel 2 by electrophoresis current from the electrophoresis power supply 8. The points are the same as the embodiment of FIG.

Also in this embodiment, an argon laser 14 is used as an excitation light source. Reference numeral 44 denotes a regular polygon mirror that reflects the laser beam from the argon laser 14 and irradiates the laser beam in the normal direction of the surface of the electrophoresis gel 2. The rotation of the regular polygon mirror 44 causes the laser light, which is the excitation light, to scan the electrophoresis gel 2 in a direction orthogonal to the electrophoresis direction.

One end face of the electrophoresis gel 2 is provided so that one end face of the optical fiber bundle 46 faces the end face of the electrophoresis gel 2 in order to receive fluorescent light from the fluorescent label of the excited nucleic acid fragment. The other end face of the electrophoresis gel 2, that is, the optical fiber 46
A mirror 48 is provided on the end face opposite to the end face provided with the mark 48 in order to increase the sensitivity of receiving fluorescent light.

The other end of the optical fiber bundle 46 is bundled so that light emitted from the other end is incident on the photomultiplier tube 52.

A rotator 50 for switching the filter is provided between the optical fiber bundle 46 and the photomultiplier tube 52. The rotator 50 is provided with an interference filter 20 for selecting fluorescence and an interference filter 54 for selecting Raman scattered light of water. The interference filter 54 has a neutral density (ND) filter for adjusting the incident intensity. 56 are provided in an overlapping manner.

In this embodiment, the DNA fragment sample to be injected into the slot 10 at the upper end of the electrophoresis gel 2 uses a primer whose label is labeled with a fluorescent substance XRITC. XRITC has an absorption peak at 582 nm and a generated fluorescence peak at 601 nm. In this case, the XRITC fluorescence wavelength 601 nm is Raman scattering of water 619n
Near m, the background of the fluorescence reception signal is mainly due to Raman scattering of the water.

In this embodiment, the Raman scattered light of the water is mixed into the same photomultiplier tube 52 together with the fluorescent light, and the Raman scattered light component of the water is extracted by the interference filter 54 and measured.

Reference numeral 58 denotes an amplifier for amplifying the output signal of the photomultiplier tube 52. The output of the amplifier 58 is converted into a digital signal by the A / D converter 38 and taken into the microcomputer 40. The microcomputer 40 also rotates the rotating body 50 of the filter by the rotating mechanism 60.

Half mirror between regular polygon mirror 44 and running gel 2
62 is provided, a part of the excitation light applied to the electrophoresis gel 2 is taken out by the half mirror 62 and input to the position sensor 64, and the detection signal of the position sensor 64 is taken into the microcomputer 40, The irradiation position of the excitation light is detected. As the position sensor 64, for example, a semiconductor position sensor S-1352 (a product of Hamamatsu Photonics) or the like can be used.

As the optical fiber bundle 46, for example, ESKA (a product of Mitsubishi Rayon Co., Ltd.) can be used.

 Next, the operation of the present embodiment will be described.

The DNA fragment sample is electrophoresed in the electrophoresis gel 2, the excitation light from the argon laser 14 is scanned by the rotation of the regular polygon mirror 44, and the signal is detected by the photomultiplier 52 through the optical fiber bundle 46, and the fluorescence is detected. Raman scattering of water is much stronger than light. So by selecting the ND filter 56,
When there is no fluorescent substance in the electrophoresis gel 2, the photomultiplier 52
Is adjusted so that the signal obtained through the interference filter for fluorescence 20 and the signal obtained through the interference filter for Raman wavelength 54 of water have the same strength.

First, the regular polygon mirror 44 is fixed at a certain angle for a short period of time, during which the interference filter 20 and the interference filter 54 are switched to measure the fluorescence component and the Raman scattered light component, and the result is subtracted by the microcomputer 40. Then, the calculation result is used as a fluorescence signal.

Next, the regular polygon mirror 44 is sequentially turned, and the same operation is repeated to calculate a migration pattern. If the migration pattern is known, the base sequence can be determined, and the object can be achieved.

In FIG. 2, the optical fiber bundle 46 is used as a device for guiding the fluorescent light from the end surface of the electrophoresis gel 2 to the photomultiplier tube 52, but this device simply receives the fluorescent light at the end surface of the electrophoresis gel 2 and Since it is for guiding to the photocathode of the multiplier 52, an optical molded product may be used as long as it fulfills such a function.

According to this embodiment, it is possible to receive the fluorescence in the 90-degree direction where the Rayleigh scattering of the excitation light, which greatly hinders the measurement of the fluorescence, is minimized.

The optical system including the rotating body 50 and the photomultiplier tube 52 of the filter in the embodiment of FIG. 2 and the data processing unit include the dichroic mirrors 24 and 26, the interference filter 20, and the photomultiplier tubes 22 and 28 in FIG. Other mechanisms, such as replacing the optical system and the data processing unit, can be used by appropriately combining the embodiment of FIG. 1 and the embodiment of FIG.

In the embodiment of FIG. 1, the excitation light component is detected in order to remove the fluctuation of the background light, and in the embodiment of FIG. 2, the Raman scattering light component of the water is detected. In addition, even if the fluorescent light is taken out and detected by the half mirror, the purpose can be sufficiently achieved because the fluorescence intensity is much smaller than the excitation light or the Raman scattered light.

In the examples, a fluorescent substance is used as the labeling dye, but a phosphorescent substance can be used instead of the fluorescent substance (see Japanese Patent Application No. 62-862230).

(Effects of the Invention) In the present invention, since the fluctuation of the background light is removed from the signal obtained by detecting the emission intensity from the labeling dye, most of the fluctuation of the background light which is a problem when detecting weak fluorescence or phosphorescence is detected. Most components are removed, the S / N ratio is improved, and the nucleotide sequence can be determined accurately.

[Brief description of the drawings]

1 and 2 are schematic perspective views of an embodiment showing a data processing section and the like as block diagrams, respectively, and FIG. 3 is a schematic perspective view showing a conventional base sequence determination device. 2 ... electrophoresis gel, 4,6 ... electrode tank, 8 ... electrophoresis power supply, 14
…… Argon laser, 16,24 …… Dichroic mirror, 18 …… Objective lens, 20,26,54 …… Interference filter, 2
2,28,52 …… Photomultiplier tube, 30,32,58 …… Amplifier, 34…
... Differential amplifier, 40 ... Microcomputer, 42 ... Drive mechanism, 44 ... Polygonal mirror, 46 ... Optical fiber bundle,
60 rotation mechanism, 62 half mirror, 64 position sensor.

Claims (1)

(57) [Claims] 1. A mechanism for electrophoresing a nucleic acid fragment labeled with a labeling dye on a migration gel, a mechanism for irradiating the migration gel with an excitation light beam, and receiving light from a portion irradiated by the excitation light beam. A first light receiving unit, an optical system that extracts at least one of an excitation wavelength component and a Raman scattering wavelength component of water from a part of the light received by the first light receiving unit, and a light that is extracted by the optical system. A second light receiving section for receiving light, a variable resistor for adjusting the level of the second light receiving section, and a sign obtained by subtracting an amount proportional to the output of the second light receiving section from the output of the first light receiving section. A base sequence determination device comprising: a data processing unit that calculates the emission intensity of the dye and determines the base sequence of the nucleic acid based on the emission intensity.