JPH07190934A - Transilluminator for observing electrophoretic gel - Google Patents

Abstract

PURPOSE:To eliminate the need of protection against ultraviolet rays by irradiating an absorbing filter, which can transmit the visible light in the exciting wavelength range of a visible light exciting fluorescent dye, with a visible light at a specified incident angle and then irradiating an electrophoretic gel, subjected to dyeing with a visible light exciting fluorescent dye, with a visible light of predetermined wavelength passed through the filter. CONSTITUTION:Light from a visible light source 1 passes through a condenser lens 4, a heat absorbing filter 7, and an ND filter 5 and then reflected on a mirror 6 to enter into an exciting filter 2 at an incident angle of about 90+ or -20 deg.. The filter 2 passes a light in such wavelength region as exciting the visible light exciting fluorescent dye without lapping over the wavelength region for fluorescent observation. When the light impinges on an electrophoretic get placed on the cover glass for the filter 2, the visible light exciting fluorescent dye which has dyed the nucleic acid in the gel is excited. The nucleic acid is detected by observing and measuring the fluorescence. This constitution eliminates adverse effect on the human body because ultraviolet rays are not employed in the measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a transilluminator using visible light as excitation light for observing a fluorescently stained nucleic acid electrophoretic gel.

[0002]

2. Description of the Related Art A method of electrophoresing a nucleic acid in an agarose gel or a polyacrylamide gel to separate a plurality of nucleic acids and observing the nucleic acids by a fluorescence method is generally performed by the following procedure. (1) Nucleic acid (usually double-stranded nucleic acid) is cut to an appropriate length with a restriction enzyme or the like. (2) The cleaved nucleic acid is separated by agarose gel electrophoresis or polyacrylamide gel electrophoresis. (3) The gel is dipped in an ethidium bromide (hereinafter abbreviated as EB) solution and stained. (4) The gel is washed with an appropriate washing liquid to wash off excess EB. (5) Observe the fluorescence generated from the stained nucleic acid in the gel.

The mechanism of nucleic acid staining by EB is EB
Is said to intercalate between the base pairs of the double-stranded nucleic acid. As a result of intercalation, the fluorescence from EB is enhanced several tens of times and can be detected separately from the background.

[0004]

The prior art using the above EB has the following problems. (1) Since the absorption of EB is in the ultraviolet region (absorption maximum is 290 nm regardless of the presence or absence of nucleic acid), it is necessary to use ultraviolet light as excitation light during fluorescence observation. Therefore, the light source device for observing commercially available nucleic acid electrophoretic gels, that is, the so-called transilluminator light source is an ultraviolet lamp. However, in a transilluminator using an ultraviolet ray as a light source, the observer is exposed to the ultraviolet ray at the time of observation, so it is necessary to wear protective glasses, a protective mask, protective gloves, etc. Not only that, but it may interfere with the work such as taking pictures. Further, even if the protective equipment is worn, it is not possible to completely prevent the ultraviolet rays, so that the observer cannot completely avoid the influence of the ultraviolet rays. (2) Since EB emits fluorescence even in a free state where it is not intercalated with nucleic acid, washing operation of the gel is required. Further, the EB cannot be completely washed out from the gel even by the washing operation, so that the background cannot be completely wiped off. Therefore, when the amount of EB intercalated in the nucleic acid is small and the amount of EB intercalated in the nucleic acid is small, the difference in fluorescence between the EB intercalated in the nucleic acid and the free EB is small, and the background fluorescence is observed. In many cases, the fluorescence that should be obtained is not clear and observation with high sensitivity is difficult.

[0005] Such a problem is that visible light is used as excitation light, and a visible light-excitable fluorescent dye having a large increase in fluorescence when intercalated into a double-stranded nucleic acid and a method or apparatus for detecting nucleic acid using the same Although it is considered that the problem can be solved by the above, the present situation is that the conventional method has not yet provided a visible light-excitable fluorescent dye that uses practical visible light as excitation light, and a method or apparatus for detecting nucleic acid using the dye.

An object of the present invention is to provide a transilluminator for observing an electrophoretic gel, which is used for detecting staining of a nucleic acid developed in a gel by electrophoresis with a visible light excitation type fluorescent dye.

[0007]

The transilluminator for observing an electrophoretic gel of the present invention comprises an absorption filter capable of transmitting visible light in the excitation wavelength region of a visible light excitation type fluorescent dye for staining nucleic acid, and Optical means for irradiating visible light at an incident angle of 90 ° ± 20 °, and means for irradiating the electrophoretic gel dyed with the visible light-exciting fluorescent dye with visible light of a specific wavelength transmitted through the filter. It is characterized by

It is preferable that the transmission wavelength region of the absorption filter for excitation light (excitation light filter) that the transilluminator of the present invention has does not overlap with the observation wavelength region of fluorescence. Further, the transilluminator of the present invention may be provided with an absorption filter for fluorescence observation (fluorescence filter), if necessary.

[0009]

In the transilluminator of the present invention, visible light is used as the excitation light for the dye that performs fluorescent staining,
There is no need to take any measures to avoid UV light as with EB staining.

Further, since the wavelength region of the excitation light can be strictly controlled, it is possible to observe fluorescence with good sensitivity, which brings out the characteristics of the dye for dyeing sufficiently.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a main part of an embodiment of the transilluminator of the present invention. FIG. 1A is a plan view of the device, and is a diagram in which an exterior portion of a portion where a light source and the like are arranged is omitted. Further, FIG. 1B is a side view of the apparatus, and is a view showing an internal mechanism with an exterior panel on the side surface omitted.

This device comprises a light source 1 for generating visible light,
It has an excitation light filter 2 provided under the cover glass with visible light from the light source, and an optical system for irradiating the excitation light filter with light from the light source 1. As the light source 1,
Any light source that can generate visible light including a desired wavelength range can be used without limitation, but a light source that does not generate ultraviolet light, such as a halogen lamp or a tungsten lamp, is preferable.

As the excitation light filter 2, a filter capable of exciting a visible light excitation type fluorescent dye used for dyeing and transmitting a wavelength region which does not overlap with a fluorescence observation (observation) wavelength region is preferably used. An interference filter is preferable from the viewpoint of excellent wavelength selectivity, high transmittance in the transmission wavelength region, and high shielding property in the shielding wavelength region. It is known that the interference filter is affected by the wavelength characteristics depending on the incident angle of incident light. That is, the wavelength characteristic of the filter shifts to the short wavelength side as the angle of oblique incidence increases. The degree cannot be generally stated depending on the filter configuration, film material, etc., but generally the incident angle is θ
Is proportional to (1-cos θ). Considering the excitation efficiency, the degree of the short wavelength shift is within 5% of the fundamental wavelength, preferably within 2%, that is, the deviation of the incident angle (90 degrees) is within about 20 degrees, and preferably within 10 degrees.

The transilluminator of the present invention is provided with an optical system for irradiating the excitation filter 2 with the light from the light source at the above incident angle. In the illustrated apparatus, the incident angle of visible light from the light source 1 to the excitation light filter 2 is controlled to 90 ° ± 20 ° by the optical system having the combination of the condenser lens 4 and the mirror 6. There is. However, this optical system is not limited to the illustrated example, and various configurations can be used as long as the desired incident angle can be obtained.

In the case of a transilluminator used for observing ultraviolet excitation using EB, it is general to arrange a required number of elongated mercury lamp tubes as a light source in parallel and irradiate the electrophoresis gel. In the case of visible light excitation, this type of light source is not preferable because light having any direction reaches the filter and adversely affects the wavelength separation ability of the filter. Therefore, in the device of the present invention, the optical system having the above-mentioned configuration is used so that the characteristics of the excitation light filter can be effectively used,
In this respect, it is structurally significantly different from the transilluminator using ultraviolet light.

The brightness of the light source may be any as long as sufficient sensitivity can be obtained, but especially for optimizing the conditions of the naked eye observation, a light control circuit or an ND as shown in the optical path. It is desirable to have a structure into which the filter 5 can be inserted. In addition, depending on the type of lamp or the brightness, infrared rays may generate heat, which may adversely affect the excitation light filter, the entire device, or the electrophoretic gel placed on the cover glass. In some cases, it may be desirable to insert a heat absorption filter 7 in the optical path as shown, if desired.

The excitation light filter 2 excites a visible light excitation type fluorescent dye that stains (intercalates with nucleic acid) nucleic acid in an electrophoretic gel (including nucleic acid developed by electrophoresis). It transmits visible light of a required wavelength, and the range of transmission wavelength is set according to the optical characteristics of the visible light excitation type fluorescent dye used for dyeing. This transmission wavelength range can effectively excite the dye that stains the nucleic acid,
In addition, the excitation light itself is set so as not to obstruct the observation of the fluorescence generated by the excitation. For example, in FIG.
When a visible light excitation type fluorescent dye having optical characteristics as shown in (b) is used and a wavelength range including a wavelength y showing the maximum of the fluorescence spectrum is selected as a wavelength range for fluorescence observation, the absorption spectrum of the excitation light Preferably, the wavelength range that includes the maximum absorption wavelength x and does not overlap with the wavelength range is set as the transmission wavelength range of the excitation light filter.

For highly sensitive fluorescence observation, the transmittance of the excitation light filter in the transmission wavelength region is 80% or more, preferably 90% or more, and the transmittance in the shielding wavelength region is 0.01 to
0.001%, preferably 0.001-0.0001
% Or less is suitable.

When the electrophoretic gel placed on the cover glass is irradiated with visible light in a specific wavelength region through the excitation light filter 2, the visible light excitation type fluorescent dye intercalated in the nucleic acid in the gel is excited and fluorescence is emitted. Occurs. Nucleic acid is detected by observing or measuring this fluorescence.

For example, in fluorescence observation in nucleic acid staining with EB, since the excitation light is ultraviolet light, it is invisible to the human eye, and the observer can observe fluorescence in the visible region as it is. Further, for example, a film for photography such as Polaroid 667 (manufactured by Polaroid Co., Ltd.) also has no spectral sensitivity in the ultraviolet region, and therefore fluorescence can be photographed as it is. On the other hand, when visible light is used as the excitation light, it becomes difficult to observe the fluorescence as it is as described above due to the influence of the excitation light.

Therefore, the observation of fluorescence in the apparatus of the present invention is carried out through the fluorescence filter. The transmission wavelength range of this fluorescence filter is set according to the optical characteristics of the visible light excitation type fluorescent dye used for staining the nucleic acid in the electrophoretic gel. It is preferable that these transmission wavelength regions are set so that the regions and the transmission wavelength regions of this fluorescence filter do not overlap.

An interference filter is also suitably used for this fluorescent filter because of its strict wavelength selectivity, high transparency in the transmission wavelength region, and high shielding property in the shielding wavelength region.

The position where the fluorescence filter is set is preferably immediately after the gel (with respect to the direction of the light beam from the light source toward the observer) when the fluorescently stained nucleic acid is observed with the naked eye. In that case, it is necessary to provide a cover for preventing the leakage of the excitation light so that the excitation light does not adversely affect the observation. Further, when taking a picture, basically, a filter may be set on the taking lens, but even in that case, it is preferable that another filter is set immediately after the gel as described above.

The fluorescent filter may be mounted as a part of the transilluminator, or may be used at any time during observation.

The transmittance of the fluorescent filter is 80% or more, preferably 90% or more, as in the case of the excitation light filter, in order to observe fluorescence with high sensitivity.
The transmittance in the shielding wavelength region is 0.01 to 0.001%, preferably 0.001 to 0.0001% or less.

The fluorescent image of the nucleic acid developed in the electrophoretic gel through the fluorescent filter can be visually observed. In addition, it is possible to take a picture with a camera, and it is also possible to detect the intensity of the light transmitted through the fluorescence filter at an appropriate wavelength with a sensor and convert it into an electrical signal or the like for measurement.

Further, the cutoff wavelength of the excitation light or fluorescence filter is set according to the optical characteristics of the ultraviolet light excitation type fluorescent dye to be used, but when it is intercalated with nucleic acid, the absorption wavelength (excitation wavelength) ), The cutoff wavelength is set in consideration of the width of this shift.

As the visible light excitation type fluorescent dye used for the observation by fluorescence staining of the electrophoretic gel of nucleic acid using the device of the present invention, the excitation light is visible light, and the wavelength region of the hot excitation light is the long region of the fluorescent field. It is possible to use without particular limitation as long as it does not overlap with, or even if it overlaps, the wavelength range of excitation light and the wavelength range of fluorescence observation can be separated by optical filters for excitation light and fluorescence.

As such a dye, for example, a pyrylium salt or a pyrylium-similar salt described in claims 1 to 12 of Japanese Patent Application No. 227204/1993 by the present applicant can be used. The representative examples are listed. The dye of Compound 1 of Reference Example 1 has an absorption maximum of 4
The cut-off wavelength of the filter was set so that it would shift to the 0 nm long wavelength side. Also, this dye has a large Stokes shift of 65 nm,
There is little overlap between the excitation light and the fluorescence, and the transmission wavelength region of the filter and the transmission wavelength region of the fluorescence filter can be easily set, which is particularly useful for fluorescence observation. Also, this dye, when free, does not fluoresce at all, or
It emits only weak fluorescence as compared with EB. further,
Even if weak fluorescence is generated, the maximum increase in fluorescence when intercalated with nucleic acid is about 400 times, so
It enables highly sensitive observation.

An example of observing a fluorescent image of nucleic acid in an electrophoretic gel using the transilluminator of the present invention will be described below with reference to a reference example.

Reference Example 1 (Synthesis of Dye) 100 ml of acetic anhydride and 30 ml of concentrated sulfuric acid were mixed while being cooled, and the resulting mixed liquid was heated for 3 hours while being kept at 80 ° C. in a water bath. 20 ml of acetic anhydride, p
-Dimethylaminoacetophenone (30 ml) was added at room temperature, the temperature was then raised to 45 ° C, and the mixture was stirred for 24 hours for reaction. To this reaction solution, an equal amount of ethanol was added, cooled, and an aqueous potassium iodide solution was further added to precipitate crude crystals. The crude crystals were collected by filtration and recrystallized with an ethanol / ether mixture (volume ratio, 1: 4) to give 2,
Green crystals of 4-bis (p-methylaminophenyl) -6-methylpyrylium iodide (Compound 1) were obtained. Structure of compound 1

[0032]

[Chemical 1] Analysis result of compound 1: Melting point: 254-257 ° C. UV / visible (CH ₃ CN ε × 10 ⁻⁴ ) λ max: 444
nm (2.43), 540 nm (8.24) NMR ( ¹ H, DMSO) δ ppm: 8.3737 (1 H,
s), 8.2729 (1H, d, J = 9.0 Hz),
8.1795 (1H, d, J = 9.0Hz), 7.88
64 (1H, s), 6.9117 (4H, t, J _AB = J
_BC = 9.77), 3.1829 (6H, s), 3.13
40 (6H, s), 2.6809 (3H, s) FAB mass m / z 333 IR (KBr) νcm ⁻¹ : 1645, 1610 (s
h), 1580 (s), 1490 (s), 1270, 1
Reference example 2 (detection example) A 300 W halogen lamp is used as a light source, an interference filter having optical characteristics shown in FIGS. 3 and 4 is used as an excitation light filter, and a light source for the excitation filter is provided by an optical system in the apparatus. An apparatus having the configuration shown in FIG. 1 was prepared in which the incident angle of the light beam from was controlled to be 90 degrees (the deviation of the incident angle was within 7 degrees).

Then, as a double-stranded nucleic acid, a DNA size marker λ / HindIII-φX174 / HaeIII
Visible light excitation type fluorescent dye (Compound 1) prepared by performing agarose electrophoresis of a digested product (manufactured by TOYOBO Co., Ltd.) by a conventional method and producing a gel in which nucleic acid was developed according to the method of Reference Example 1.
It was dipped in a 0.1 mg / l solution of 10 minutes for 10 minutes for staining. After that, wash the gel with water for about 5 seconds, wipe the surface of the gel gently, and place it on the cover glass of the transilluminator,
The light from the light source was applied through the excitation light filter.

As a result of taking a photograph with a camera equipped with the fluorescent filter shown in FIGS.
Up to a band was calculated where ~ 0.1 ng of double stranded nucleic acid was present. This is more sensitive than the case of using EB, which will be described later, by two orders of magnitude or more, although it is dyed with a low-concentration solution, dyed for a short time, and substantially unwashed.

The absorption maximum and the fluorescence maximum of the fluorescent dye used in this example are 58 and 58% respectively in the presence of the double-stranded nucleic acid.
The wavelengths are 0 nm and 645 nm, and an excitation filter and a fluorescence filter were manufactured so as to be compatible with these wavelengths.

When the above dye is intercalated in the double-stranded nucleic acid, the absorption maximum shifts to the long wavelength side of 40 nm, and the cutoff wavelength of the filter was set to match it.

Comparative Example Up to electrophoresis, an agarose gel treated in exactly the same manner as in Example was immersed in a 0.5 mg / l solution of EB for 45 minutes for staining. Then, after immersing in water and washing for 20 minutes, the surface water was lightly wiped and then a photograph was taken using a commercially available transilluminator for EB. As a result, it was calculated that a minimum of 10 ng of double-stranded nucleic acid was present. The band was only observed.

The method using this EB is the same as the method described in the molecular cloning which is a typical instruction manual. The results were also almost the same as those described in the same book.

[0039]

EFFECTS OF THE INVENTION By using the transilluminator for exciting visible light of the present invention, it becomes possible to observe nucleic acid with high sensitivity as compared with the case of using the conventional method and apparatus.

Further, since it is excited by visible light, it is possible to avoid the adverse effect of ultraviolet rays on the human body, which has been a problem in the conventional method, and it is possible to eliminate the troublesomeness of using a protector or the like.

[Brief description of drawings]

FIG. 1 is a diagram showing a structure of a transilluminator of the present invention which uses visible light as excitation light, (a) is a plan view showing an internal mechanism with a part of an exterior panel omitted, and (b).
FIG. 3 is a side view showing an internal mechanism with a side exterior panel omitted.

FIG. 2 is a diagram for explaining a method of setting a transmission wavelength region of a filter for excitation light, where (a) shows a case where the excitation wavelength region and the fluorescence wavelength region do not overlap, and (b) shows an excitation wavelength region and the fluorescence wavelength region. The case where wavelength regions overlap is shown.

FIG. 3 is a diagram showing a transmission wavelength region of an excitation light filter used in Reference Example 2.

FIG. 4 is a diagram showing a shield wavelength region of a filter for excitation light used in Reference Example 2.

5 is a diagram showing a transmission wavelength region of a fluorescence filter used in Reference Example 2. FIG.

FIG. 6 is a diagram showing a shielding wavelength region of a fluorescence filter used in Reference Example 2.

[Explanation of symbols]

 1 Light source 2 Excitation filter with cover glass 3 Exhaust fan 4 Condenser lens 5 ND filter 6 Mirror 7 Heat absorption filter

Claims (3)

[Claims] 1. An absorption filter capable of transmitting visible light in the excitation wavelength region of a visible light excitation type fluorescent dye for staining nucleic acid, and irradiating the filter with visible light at an incident angle of 90 ° ± 20 °. A transilluminator for observing an electrophoretic gel, comprising: an optical means for irradiating the electrophoretic gel stained with the visible light excitation fluorescent dye with visible light of a specific wavelength that has passed through the filter. 2. The transmission wavelength range of the absorption filter is
The transilluminator for observing an electrophoretic gel according to claim 1, which does not overlap with a fluorescence wavelength region used when observing fluorescence of the visible light excitation type fluorescent dye. 3. The transilluminator for observing an electrophoretic gel according to claim 1, which has an absorption filter for fluorescence that transmits visible light in a fluorescence wavelength region used for the fluorescence observation at an observation position of the electrophoretic gel. .