JPS63263458A - Base sequence determining apparatus - Google Patents

Abstract

PURPOSE:To improve the sensitivity of measurement by removing an optically detected background light component from an optical detection signal. CONSTITUTION:The components below the specific wavelengths within the excitation laser light from an argon laser 14 are reflected by a dichroic mirror 16 and excite, through an objective lens 18, the migration gel 2 of the nucleic acid fragments labeled by a labeling dye. Fluorescence is generated from the gel 2. This fluorescent component and the background light component such as scattered light are detected by a photomultiplier 22 via a dichroic mirror 24, an interference filter 26, etc. The background light component is detected by an optical multiplier 28 via the mirror 24, an interference filter 26, etc. The detection outputs of these multipliers 22, 28 are processed by a differential amplifier 34, by which the background component is removed. A very weak excitation fluorescent component and phosphorescence component are thereby extracted and the sensitivity of the measurement for determining the base sequence is enhanced.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention is used in the process of determining the base sequence of a nucleic acid by Sanger's method, in which primers are labeled in advance with a labeling dye such as a fluorescent substance or a phosphorescent substance. This invention relates to an apparatus for reading sequences from the final stage of gel electrophoresis using a spectroscopic method using light emitted from the labeling dye.

(Prior art) As a conventional base sequence determination device, for example, r N at
ure J magazine, volume 321, pages 674-679 (
(1986), Japanese Biophysical Society Subcommittee (1986)
(October 2006, Tsukuba) 3El130. These base sequencing devices are equipped with the Sanger method (rPro
c, Natl, Acad, Sci, USAJ magazines.

Vol. 74, p. 5463 (1977)) The type of terminal bases (A (adenine), G (guanine), T (thymine), C (cytosine))
Depending on the situation, the fluorescent substances attached with different fluorescent substances can be run in a single migration lane, or the substances attached with the same fluorescent substance can be run in separate migration lanes, and the fluorescent substances can be excited with laser light. By receiving and measuring only light at the fluorescent wavelength, the electrophoretic pattern of the DNA fragments is detected, and the base sequence is finally determined.

In order to explain in detail a conventional base sequencing device, a representative example is shown in FIG. 3.A device similar to the device shown in FIG.
It is described in gh Technology J magazine, December 1986 issue, 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.

The electrode tanks 4 and 6 contain an electrolytic solution. Electrode tank 4,
6, a migration voltage is applied by a migration power supply 8.

At one end of the electrophoresis gel 2 there is a slot l for injecting the sample.
A sample on the terminal base side is injected into this slot IO, and the sample is electrophoresed in the direction of the arrow 12 in the electrophoresis gel 2 by the electrophoresis voltage from the electrophoresis power supply 8 and developed.

Reference numeral 14 denotes a laser as an excitation light source, and the excitation light is reflected by a half mirror or dichroic mirror 16 and irradiated onto the migration gel 2 through an objective lens 18. Fluorescence from the fluorescent label of the sample that has migrated through the electrophoresis gel 2 is again focused by the objective lens 18 and transmitted through a half mirror or dichroic mirror 16 to the interference filter 2 for fluorescence selection.
0, and is received and detected by the photomultiplier tube 22 as a photoelectric element.

In the apparatus shown in FIG. 3, one objective lens 18 is used for both excitation light irradiation and fluorescence reception, and the objective lens 18 and the entire optical system associated with it are arranged in the sample arrangement direction 23 (migration direction 1).
2 (transverse direction in the figure).

(Problems to be Solved by the Invention) Conventional base sequence determination apparatuses, including the base sequence determination apparatus shown in FIG. Since the fluorescent light is very weak, the Rayleigh scattering component of the excitation light by the electrophoretic gel and the Raman scattering component of the water become strong fluorescent light and background light, reducing the S/N ratio.

Hereinafter, the problems will be explained in detail using the base sequencing apparatus shown in 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, but the light received by the photomultiplier tube 22 is not only fluorescent light but, strictly speaking, excitation light. Rayleigh scattered light and Raman scattered light of water enter as background light.

When excitation is performed by laser light, the spectrum of the 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 complete wavelength filter (interference filter) 20, Rayleigh scattered light will be included in the light received by the photomultiplier tube 22. Scattered light and Raman scattered light will not be mixed in, background light will not be high, and therefore the S/N ratio should not deteriorate.

However, the dichroic mirror 16 and the interference filter 2
The reason why the excitation light component and the Raman scattered light component of water enter the photomultiplier tube 22 even though 0 is used is that the dichroic mirror 16 and interference filter 20 cannot completely separate the wavelengths. For example, even if the interference filter 20 is an ideal interference filter, wavelengths can be separated only when the incident light enters the interference filter 20 at right angles. In reality, fluorescent light is incoherent and has the size of a fluorescent light source, so even if parallel light is obtained with the objective lens 18, this condition is not met.

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 incoherent, the diffraction grating is also not an ideal wavelength filter.

In other words, in an experimentally possible physical system, it is not possible to completely separate only the fluorescence wavelength, and it is impossible to avoid the excitation wavelength component due to Rayleigh scattering and the Raman scattering wavelength component of water from entering the photomultiplier tube 22 as background light. do not have.

As shown in Figure 3, in the method (online method) that observes the temporal change in fluorescence at a specific location on the electrophoresis gel without electrophoresis, the method (offline method) that measures the developed pattern after electrophoresis is performed in a separate electrophoresis device (offline method). Although this method is advantageous in that there are no differences in optical properties depending on the location of the electrophoresis gel itself, it still results in the passage of nucleic acid fragments, temporal fluctuations in the optical properties of the electrophoresis gel itself, generation of bubbles, or laser light. Due to fluctuations in the output of the detector itself, the background light intensity due to the Rayleigh scattered light of the excitation light and the Raman scattered light of the water fluctuates greatly, which engulfs the fluctuations in the fluorescence output, which is the original signal, and reduces measurement sensitivity.

The above problem is exactly the same even if 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 device with high measurement sensitivity by removing fluctuations in background light from a signal detected by irradiating a migration gel with excitation light.

(Means for Solving the Problems) In the present invention, when fluorescence measurement or phosphorescence measurement is performed from a migration gel, changes in the optical properties of the migration gel itself, which become the background light of the detection signal, changes in the intensity of Rayleigh scattered light, At least a portion of the changes in the Raman scattered light are simultaneously measured, and the fluctuations in the background light are removed from the detection signal.

To explain with reference to FIGS. 1 and 2 showing examples,
The base sequencing device of the present invention includes a mechanism (4, 6, 8) for electrophoresing a nucleic acid fragment labeled with a labeling dye onto a slab-like electrophoresis gel (2).

A mechanism (14,
16, 18, 44), and a first light receiving section (20, 22, 4) that receives light from the part irradiated with the excitation light beam.
6, 52), and an optical system (24, 26, 46, 54) that extracts at least one of the excitation wavelength component and the Raman scattering wavelength component of water from a portion of the light received by the first light receiving section.
and a second light receiving section (28, 52) that receives the light extracted by this optical system, and subtracting an amount proportional to the output of the second light receiving section from the output of the first light receiving section. The data processing unit (30, 32, 34) calculates the luminescence intensity of the labeling dye and determines the base sequence of the nucleic acid based on this luminescence intensity.
, 38, 40).

(Example) FIG. 1 shows an example in which the present invention is applied to the base sequencing apparatus shown in FIG. 3. The same parts as in FIG. 3 are given the same reference numerals.

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

The upper end of the electrophoresis gel 2 has a slot l for injecting the sample.
0, and each slot 10 is treated with the Sanger method and the primer is labeled with a fluorescent substance gtT.
A DNA fragment pre-labeled with RITc was inserted into its terminal bases A, T, C, and G separately, and the motive direction 1
2. The fluorescent substance TRITC has an absorption peak around 545 nm, and a peak of emitted fluorescence around 570 nm.

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

The dichroic mirror 16 is one that transmits light of 540 nm or more and reflects light of wavelengths shorter than 540 nm. The dichroic mirror 16 is provided at a position to reflect the laser beam from the laser 14.

The objective lens 18 focuses the light reflected by the dichroic mirror 16 onto the surface of the electrophoretic gel 2.

The objective lens 18 also collects the light from the irradiated portion of the electrophoretic gel 2 and makes it enter the dichroic mirror 16 .

A dichroic mirror 24 is provided at a position to receive the light transmitted through the dichroic mirror 16. As the dichroic mirror 24, one that transmits light of 540 nm or more and reflects light of wavelengths shorter than that is used.

A photomultiplier tube 22 is provided through an interference filter 20 at a position where the light transmitted through the dichroic mirror 24 is received. The interference filter 2o has a fluorescence peak wavelength of 570
Use one with a narrow transmission band in the nm range.

A photomultiplier tube 28 is provided at a position to receive the light reflected by the dichroic mirror 24 via an interference filter 26. 514 nm as the interference filter 26
Use one with a narrow transmission band. What is reflected by the dichroic mirror 24 is light having a wavelength shorter than 540 nm, and this reflected light is further extracted by a 514 nm interference filter 26 and detected by a photomultiplier tube 28 . What is detected by the photomultiplier tube 28 is the excitation light component due to Rayleigh scattering.

30 is an amplifier for amplifying the detection signal of the photomultiplier tube 22 for fluorescence detection, and 32 is the photomultiplier tube 2 for detecting the excitation light component.
This is an amplifier that amplifies the detection signal of 8. 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 a variable resistor 36 for level adjustment.

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

The entire optical system including the dichroic mirror 16 and the objective lens 18 is mechanically scanned at high speed in a direction 23 perpendicular to the migration direction 12 by a drive mechanism 42 . The microcomputer 40 calculates the migration pattern of DNA from the input fluorescence pattern by controlling the driver groove 42,
Determine the target base sequence.

To explain the operation of this embodiment, the light received by the photomultiplier tube 22 includes not only fluorescent light from the fluorescent label of the DNA fragment, but also Rayleigh scattered light and Raman scattered light, which are transmitted through the dichroic mirror 16, 24 and the interference filter. Contaminated by incomplete separation characteristics of 20.

Considering the light flux received by the photomultiplier tube 28,
The light beam 25 that reaches the dichroic mirror 24 passes through the photomultiplier tube 22. The light flux received by the photomultiplier tube 28 reflects the background light of the signal detected by the photomultiplier tube 22. Strictly speaking, the output of the photomultiplier tube 28 includes a fluorescent component, but most of it is background light itself.

Background light consists of Rayleigh scattered light and Raman scattered light, but Rayleigh scattering is much stronger than Raman scattering, and in this case, the wavelength of Rayleigh scattering is closer to that of fluorescence, so it is assumed that most of the background light is due to Rayleigh scattering. There's no harm in thinking about it.

Even if the light reception signal of the photomultiplier tube 22 fluctuates, if the fluctuation is due to a fluctuation in the background light for some reason, the output of the photomultiplier tube 28 will similarly fluctuate, and if the fluctuation is caused by a fluctuation in the background light If the signal is a true signal due to fluctuations in light, only the output of the photomultiplier tube 22 will fluctuate, and the output of the photomultiplier tube 28 will not fluctuate.

Therefore, if the differential amplifier 34 takes the difference between the surface outputs, the output will represent only the fluorescent signal.

A variable resistor 36 for level adjustment is used to amplify l! so that the output of the differential amplifier 34 becomes 0 when there is no fluorescent substance, that is, before electrophoresis. This is to adjust the amplification degree of s32.

The base sequence is determined by inputting the 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 represents another embodiment.

Electrode vessels 4 and 6 are provided at both 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 transferred to the electrophoresis gel 2 by the electrophoresis current from the electrophoresis charge g8.
The electrophoresis process is the same as in the embodiment shown in FIG.

This embodiment also uses the argon laser 14 as the excitation light source. 44 is a regular polygonal mirror that reflects the laser light from the argon laser 14 and irradiates it in the normal direction of the surface of the electrophoretic gel 2. By rotating the regular polygonal mirror 44, the laser beam serving as excitation light is caused to scan the electrophoretic gel 2 in a direction perpendicular to the electrophoretic direction.

One end surface of an optical fiber bundle 46 is provided on one end surface of the electrophoresis gel 2 so as to face the end surface of the electrophoresis gel 2 in order to receive fluorescent light from the fluorescent label of the excited nucleic acid fragment. The other end surface of the electrophoretic gel 2, that is, the optical fiber 4
A mirror 48 is provided on the end surface opposite to the end surface where 6 is provided 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 six-rotator 50 is provided between the optical fiber bundle 46 and the photomultiplier tube 52, and a rotor 50 for switching filters is provided.
is provided with an interference filter 20 for selecting fluorescence and an interference filter 54 for selecting Raman scattered light of water.
ral Density (attenuation) filters 56 are provided in an overlapping manner.

In this example, the DNA fragment sample to be injected into the slot IO at the upper end of the electrophoresis gel 2 is
Use the one labeled with TC.

XRITC has an absorption peak at 582 nm and a generated fluorescence peak at 601 nm. In this case, XRIT
The fluorescence wavelength of 601 nm of C is Raman scattering of water619
The background of the fluorescence reception signal is mainly due to Raman scattering of this water.

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

Reference numeral 58 denotes an amplifier that amplifies the output signal of the photomultiplier tube 52 , and the output of the amplifier 58 is converted into a digital signal by the A/D converter 38 and input to the microcomputer 40 . The microcomputer 40 also operates a rotating machine 4i1.
The rotor 50 of the filter is rotated by J60.

A half mirror is provided between the regular polygonal mirror 44 and the electrophoretic gel 2.
A part of the excitation light irradiated onto the electrophoresis gel 2 is extracted by the half mirror 62 and sent to the position sensor 6.
4 and the detection signal of the position sensor 64 is taken into the microcomputer 4o to detect the irradiation position of the excitation light. products) etc. can be used.

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

Next, the operation of this embodiment will be explained.

- Electrophores the DNA fragment sample in electrophoresis gel 2,
The excitation light from the argon laser 14 is transferred to a regular polygon mirror 44.
When the electrophoretic gel 2 is scanned by the rotation of the electrophoretic gel 2 and the signal is detected by the photomultiplier tube 52 via the optical fiber bundle 46, the Raman scattering of water is very strong compared to the fluorescent light. so that the signal detected from the photomultiplier tube 52 when there is no fluorescent substance at all has the same intensity as the signal obtained through the fluorescence interference filter 20 and the signal obtained through the water Raman wavelength interference filter Adjust to.

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

Next, the regular polygon mirror 44 is sequentially rotated and the same operation is repeated to calculate the electrophoresis pattern. Once the electrophoresis pattern is known, the base sequence can be determined and the objective can be achieved.

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

According to this embodiment, fluorescence can be received in the 90 degree direction where Rayleigh scattering of excitation light, which is a major hindrance to fluorescence measurement, is minimal.

The optical system including the rotating body 5° of the filter and the photomultiplier tube 52 and the data processing unit in the embodiment shown in FIG.
The embodiment shown in FIG. 1 and the embodiment shown in FIG. 2 can be used in combination as appropriate for other mechanisms, such as replacing the optical system including photomultiplier tubes 22 and 28 with the data processing section.

In the embodiment shown in FIG. 1, the excitation light component is detected in order to remove fluctuations in background light, and in the embodiment shown in FIG. 2, the Raman scattered light component of water is detected.

Even if both components are extracted and detected together with the fluorescent light using a half mirror, the purpose can be sufficiently achieved because the fluorescent intensity is very small compared to the excitation light and the Raman scattered light.

Furthermore, although a fluorescent substance is used as the labeling dye in the examples, a phosphorescent substance may be used instead of the fluorescent substance (see Japanese Patent Application No. 862,230/1986).

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

[Brief explanation of the drawing]

FIG. 1 is a schematic perspective view showing a base sequencing device. 2...Migration gel, 4.6...Electrode tank. 8...Migration power supply, 14...Argon laser, 16.24...Dichroic mirror, 18.
...Objective lens, 20.26,54...Interference filter, 22.2
8,52...Photomultiplier tube. 30.32.58...Amplifier, 34...
・Differential amplifier, 40...Microcomputer, 42...
...Drive mechanism, 44...Regular polygon mirror. 46... Optical fiber bundle. 60...Rotation mechanism. 62...Half mirror 1 64...Position sensor.

Claims (1)

[Claims] (1) A mechanism for electrophoresing nucleic acid fragments labeled with a labeling dye onto a migration gel, a mechanism for irradiating the migration gel with an excitation light beam, and a first mechanism for receiving light from the area irradiated with the excitation light beam. a light receiving section; an optical system that extracts at least one of an excitation wavelength component and a Raman scattering wavelength component of water from a portion of the light received by the first light receiving section; and a light receiving section that receives the light extracted by the optical system. The luminescence intensity of the labeling dye is calculated by subtracting an amount proportional to the output of the second light-receiving part from the output of the second light-receiving part and the first light-receiving part, and based on this luminescence intensity, A base sequence determination device comprising a data processing unit that determines the base sequence of a nucleic acid.