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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an apparatus for determining the base
sequences of nucleic acids according to the Sanger method by labeling
primers with a pigment such as a fluorescent substance or phosphorescent
substance first and spectroscopically reading the sequence from fragments
as electrophoresed on a gel in the final step utilizing the luminescence
from the labeling pigment.
2. Description of the Prior Art
The bands of nucleic acid fragments as developed by gel electrophoresis can
be read utilizing fluorescence by two systems, i.e. on-line system and
off-line system.
With the on-line system, nucleic acid fragments are electrophoresed on a
gel, and during the electrophoresis, variations with time in the intensity
of fluorescence of a point on the lane are read. With the off-line system,
a gel of electrophoresed fragments is mounted after electrophoresis on a
specific reading device to read the electrophoretic pattern.
According to the Sanger method (see Proc. Natl. Acad. Sci. U.S.A., vol. 74,
p. 5463(1977)), four kinds of nucleic acid fragments wherein the terminal
base is A (adenine), G (guanine), T (thymine) or C (cytosine) are used as
a set of samples. When it is attempted to electrophorese on one lane one
kind of sample having one of the four kinds of terminal bases, or to
electrophorese many samples at the same time, the off-line system must
measure the fluorescence of a slab of electrophoretic gel in
two-dimensional directions, while even the on-line system requires
one-dimensional high-speed fluorescence measurement in the direction of
arrangement of the samples on the electrophoretic gel.
The known fluorescence measuring devices for slabs of electrophoretic gel
are all of the on-line type.
Fluorescence measurement can be realized most simply using the apparatus
shown in FIG. 9. (The apparatus described in "High Technology," December
1986, page 49 also belongs to this category.)
With reference to FIG. 9, a polyacrylamide gel 2 is immersed at its
opposite ends in an electrolyte in electrode tanks 4 and 6. A voltage is
applied across the electrode tanks 4, 6 from a power supply 8. One end of
the gel 2 is formed with slots 10 for the injection of samples. Samples of
different terminal bases are injected into the slots 10. The voltage
applied from the power supply 8 electrophoreses the samples through the
gel 2 in the direction of arrow 12 for development.
A laser 14 serving as an excitation light source emits an exciting beam,
which is reflected at a half mirror or dichroic mirror 16 and projected on
the gel 2 through an objective lens 18. The fluorescence from the
fluorescent label on the sample migrating through the gel 2 is collected
by the objective lens 18 again, transmitted through the half mirror or
dichroic mirror 16 and then through a fluorescence selecting interference
filter 20, impinges on a photomultiplier tube 22 serving as a
photoelectric device and is thereby detected.
With the apparatus of FIG. 9, the single objective lens 18 is used both for
projecting the exciting beam and for receiving the fluorescence, and the
gel 2 is mechanically scanned with the overall optical system including
the objective lens 18 and the components associated therewith in the
direction 23 of arrangement of the samples (i.e. in a transverse direction
perpendicular to the direction 12 of electrophoresis in the illustrated
case).
FIG. 10 shows another apparatus for measuring the fluorescence of a slab of
electrophoretic gel 2 (see the Proceeding of 24th Annual Meeting of the
Japanese Biophysical Society in Japan, 3E 1130, October, 1986).
The exciting beam from a laser 14 serving as an excitation light source is
made to incident by a condenser lens 24 on an end face of the gel 2 in a
direction parallel to the gel. The fluorescence is received through a lens
26 one-dimensionally or two-dimensionally at once in a direction normal to
the plane of the gel 2, passed through a fluorescence selecting
interference filter 20, amplified by an image intensifier 28 and made to
incident on a one- or two-dimensional photosensor (array type sensor) 30
for detection.
The apparatus of FIG. 9 for determining base sequences is adapted to
measure the fluorescence in the direction of reflection of the exciting
beam, so that Rayleigh scattering of the exciting beam provides intense
background light to result in an impaired S-N ratio. Rayleigh scattering
occurs intensely toward the front and rear but diminishes in a direction
at an angle of 90 degrees with the exciting beam.
Further with the apparatus of FIG. 9, the objective lens 18, as well as the
excitation optical system and the light-receiving optical system must be
mechanically moved wholly or partly for scanning. For on-line measurement,
it is required that all the lanes be scanned within a period of time which
is sufficiently short relative to the speed of electrophoresis, whereas
such a precision optical system is generally heavy, great in inertia and
in no way adapted for high-speed scanning. Even if so adapted, the system
will then be very costly.
In the case of the apparatus of FIG. 10, the electrophoretic gel 2 is
exceedingly great relative to the diameter of the fluorescence receiving
lens 26 which is usually usable, with the result that the solid angle of
the fluorescence received is extremely small, giving a feeble fluorescence
detection signal, which must be compensated for by using a one- or
two-dimensional sensor and amplifying the output greatly. For this
purpose, there arises a need to use, for example, the image intensifier
28, which nevertheless is very expensive.
Further if the gel 2 is thin, it is likely that the laser beam will not be
confined in the gel. Another problem is also encountered in that unless
the gel is accurately planar, the exciting beam will be bent upon
incidence thereon, failing to afford any measurement.
SUMMARY OF THE INVENTION
The main object cf the invention is to overcome the foregoing problems and
to provide an apparatus for determining a base sequence wherein the
luminescence from a labeling pigment is received from a direction at an
angle of 90 degrees with an exciting beam in which direction the influence
of scattering of the beam is diminished, without the necessity of using a
light receiving unit which itself needs to be moved or of using any
array-type sensor.
The invention provides an apparatus for determining a base sequence
comprising an assembly for making an exciting beam incident on a slab of
electrophoretic gel in a direction normal to the plane thereof, the gel
containing nucleic acid fragments labeled with a labeling pigment and
developed or being developed therein by electrophoresis, and a light
receiving unit for receiving at an end face of the gel the luminescence
emitted by the labeling pigment on the fragments developed in the gel.
With this apparatus, an exciting beam is made incident on a slab of
electrophoretic gel in a direction normal to the plane thereof, the gel
containing nucleic acid fragments labeled with a labeling pigment, such as
a fluorescent substance, and developed or being developed therein by
electrophoresis. The labeling pigment on the nucleic acid fragments
developed in the gel emits fluorescence, which is propagated through the
gel by total reflection and emerges from an end face of the gel. The
emergent fluorescence is detected at the end face for the determination of
the base sequence of the fragments.
The gel material to be used in the invention for developing nucleic acid
fragments is one which is usually used for the Sanger method, for example,
8% polyacrylamide gel.
Examples of useful exciting beams are laser beams, among which argon laser
beam is desirable.
The primers of nucleic acid to be used in the invention for the
determination are labeled with a labeling pigment before use. Examples of
useful labeling pigments are as follows.
______________________________________
Max.
absorption Exciting Ar
Max. wavelength
wavelength laser wave-
of fluorescence
Name (symbol)
(nm) length (nm)
emitted (nm)
______________________________________
Fluoresein 489 488 520
isothiocyanate
(FiTC)
Tetramethyl
554 514 573
rhodamine iso-
thiocyanate
(TRiTC)
4-Fluoro-7-
475 488 540
nitrobenzo-
furazen (NBD-F)
______________________________________
According to the present invention, the exciting beam is made incident on a
slab of electrophoretic gel in a direction normal to the plane of the gel.
The means therefor can be one selected from among those known for use in
the art.
When the exciting beam is to be made incident on an electrophoretic gel
which has already been developed by electrophoresis, incidence means is
used for scanning the gel in the direction of electrophoresis and in a
direction perpendicular to this direction. An example of such means is one
including a pair of galvanomirrors. Further when an electrophoretic gel
being developed by electrophoresis is to be handled, means is used which
is adapted to scan the gel along a straight line perpendicular to the
direction of electrophoresis, such as one incorporating a regular
polyhedral mirror and an f.theta. lens system.
The fluorescence emitted by the labeled nucleic acid fragment exposed to
the exciting beam is propagated through the gel by total reflection and
emerges from an end face of the gel, so that the emergent fluorescence is
received at the end face for detection. The fluorescence is received by a
unit which comprises a plurality of optical fibers in the form of a
bundle. This bundle has a rectangular end face of a small width and
conforming to the shape of the end face of the gel. The other end face of
the bundle is opposed to the sensitive surface of a photoelectric device.
The light receiving unit may be in the form of a molded product prepared
by molding the bundle of optical fibers into a desired shape using glass,
acrylic resin or the like.
The fluorescence emerging from the end face of the light receiving unit is
passed, for example, through a fluorescence selecting interference filter
first and then converted to an electric signal by a photomutiplier tube.
The signal is further converted by an A/D converter to a digital signal,
which is fed to a computer. On the other hand, data representing the
position of incidence of the exciting beam is fed from a beam position
sensor to the microcomputer. In this way, the base sequence of the DNA
checked is determined.
The intensity of the fluorescence emitted from the location of projection
of the exciting beam and emergent from the end face of the electrophoretic
gel is thought to be dependent on the solid angle as the light is seen at
the gel end face and actually varies with the condition of the end face,
etc. Accordingly, the intensity of the fluorescence appearing at the end
face can be corrected by actual measurements to assure determination with
improved accuracy. The apparatus of the invention may be provided with a
device for effecting such correction. Such a device would, for example,
have a means for receiving an input expressed as the intensity of the
singal of the photomultiplier tube and an input expressed as the intensity
of fluorescence at the position of projection of the exciting beam to
correct the intensity of fluorescence on an experimental basis. The DNA
base sequence is determined according to the intensity of fluorescence
thus corrected.
To utilize the fluorescence with improved efficiency and to measure the
intensity thereof with higher sensitivity by intensifying the exciting
beam, it is useful according to the invention to cover with a mirror or
like reflector at least one of the end faces, front surface and rear
surface of the electrophoretic gel other than the measuring end face of
the gel and the surface portion thereof to be exposed to the exciting
beam.
For example, when an electrophoretic gel already developed is to be scanned
over a surface in its entirety, the reflector may be provided over the
entire surface opposite to the surface to be exposed to the exciting beam
and over the end face opposite to the end face where the light receiving
unit is disposed. Further when an electrophoretic gel being developed is
to be scanned along a straight line perpendicular to the direction of
electrophoresis, the reflector may be provided over all the surfaces of
the gel other than the measuring end face thereof and the striplike
portion to be exposed to the exciting beam.
The apparatus of the invention for use in determining the base sequences of
nucleic acids will be usable for other purposes, for example, for the
fluorescence determination of usual electrophoretic gels and thin layer
chromatography.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view schematically showing an embodiment;
FIG. 2 is a schematic view in section taken along the line C--C in FIG. 1;
FIG. 3 is a perspective view schematically showing another embodiment;
FIG. 4 is a schematic plan view showing another embodiment;
FIG. 5 is a schematic plan view showing another embodiment;
FIG. 6 is a view in section taken along the line A--A in FIG. 5;
FIG. 7 is a schematic plan view showing another embodiment;
FIG. 8 is a view in section taken along the line B--B in FIG. 7; and
FIGS. 9 and 10 are perspective views schematically showing different
conventional apparatus for determining base sequences.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention will be described below with reference to the
embodiments shown in the drawings. However, these embodiments in no way
limit the invention.
FIG. 1 shows a base sequence determining apparatus of the off-line type
embodying the invention, and FIG. 2 is a view in section taken along the
line C--C in FIG. 1.
Indicated at 2 is an electrophoretic polyacrylamide gel prepared according
to the Sanger method by labeling DNA fragment primers with FiTC (listed
above), injecting the samples individually in the order of the terminal
bases A, G, T and C into one end of the gel 2 and electrophoresing the
samples by an electrophoretic apparatus. Indicated at 32 are DNA fragments
developed by the electrophoresis.
An argon laser 14 serving as an excitation light source emits an exciting
beam 15, which is concentrated by a condenser lens 24. The argon laser
emits a beam at 488 nm. Indicated at 34 is a galvanomirror for scanning
the surface of the gel 2 with the beam 15 in the direction 12 of
electrophoresis. The exciting beam 15 reflected from the galvanomirror 34
is directed by a galvanomirror 36 toward the gel 2 to scan the surface of
the gel at a high speed in a zone direction 23 perpendicular to the
direction 12. The angles of rotation of the mirrors 34 and 36 are fed to a
microcomputer 42 as data representing the position of projection of the
exciting beam on the gel 2. These mirrors 34 and 36 are rotated by drive
means (not shown) in response to signals from the microcomputer 42.
A bundle of optical fibers 44 is provided at one side of the gel 2. The
optical fibers are so arranged into the bundle 44 that the bundle has a
rectangular end face with a small width and shaped in conformity with the
shape of an end face of the gel 2 to which it is opposed. The other end of
the bundle 44 is small-sized. The light emanating from this end face is
led through a fluorescence selecting interference filter 20 to a
photomultiplier tube 22. The detection signal from the tube 22 is
converted by an A/D converter 41 to a digital signal, which is then fed to
the microcomputer 42.
The microcomputer 42 receives from the galvanomirrors 34, 36 the signals
representing the beam projection position on the gel 2 and accepts the
fluorescence at this position in terms of the detection signal from the
photomultiplier tube 22. In this way, by scanning the entire surface of
the gel 2 by the galvanomirrors 34, 36, the pattern of the DNA fragment
samples developed on the gel 2 by electrophoresis can be obtained as the
fluorescence detection signal from the tube 22.
When the exciting beam 15 is projected on the DNA fragment sample developed
in the gel 2, fluorescence 3 occurs as shown in FIG. 2.
We have conducted experiments and research and found that when a DNA
fragment labeled with a fluorescent substance is present at the position
where the exciting beam impinges on the gel, the fluorescence emitted by
the substance is propagated through the gel by total reflection and
emerges from an end face of the gel. The emergent fluorescence is due to
total reflection because even if the gel end face is viewed at widely
varying angles, the fluorescence is observable and further because the
fluorescence is still observable even if the gel is bent. Since the light
detected at the end face of the gel is transmitted by total reflection,
the solid angle, in the direction of thickness of gel, of the light which
can reach the end face is dependent on the total reflection critical angle
of the gel and of the neighboring optical material) and is very great.
(The solid angle is indicated at .alpha. in FIG. 2.) Furthermore, the
direction from which the fluorescence is received is at an angle of 90
degrees with the exciting beam, and in this direction, the propagation of
Rayleigh scattered light is minimized. Our experiments have revealed that
it was totally unlikely that the exciting beam would scatter in directions
within the plane of the electrophoretic gel, propagate and excite the
fluorescent substance at locations where no exciting beam was projected.
The attenuation of the fluorescence due to the scattering of light in the
gel 2 at this time is as slight as about 3%, as measured using 8%
polyacrylamide gel generally used in the Sanger method.
The fluorescence emergent at the end face is received by the optical fiber
bundle 44, propagated through the bundle 44 and through the fluorescence
selecting interference filter 20 and converted to an electric signal in
the photomultiplier tube 22. The optical fiber bundle 44 to be used is,
for example, ESKA (commercial product of Mitsubishi Rayon Co., Ltd.).
As well known, the DNA fragments in the present case have already been
developed (i.e., separated) by electrophoresis in the order of decreasing
length in the migration direction of electrophoresis, so that the sequence
can be determined by reading the pattern from zone to zone (as grouped by
the difference in the terminal base). From the angles of rotation of the
galvanomirrors 34, 36, the location where the exciting beam impinges is
calculated by the microcomputer 42, and the intensity of fluorescence then
emitted is detectable, revealing an electrophoretic pattern as
contemplated.
FIG. 3 shows a base sequence determining apparatus of the on-line type
embodying the invention.
An electrophoretic polyacrylamide gel 2 is placed at its opposite ends in
electrode tanks 4, 6, with a voltage applied across the tanks by a power
supply 8. DNA fragments prepared by the Sanger method already described
and having primers labeled with FiTC are injected into slots 10 at one end
of the gel 2 in the order of terminal bases A, G, T and C. The fragments
are electrophoresed as bands 32 by the application of the voltage. An
argon laser 14 emits an exciting beam 15 at 488 nm. Indicated at 46 is a
regular polyhedral mirror for projecting the exciting beam 15 along a
straight line for scanning. An f.theta. lens system 48 converges the beam
15 on the straight line. The mirror 46 and the f.theta. lens system 48
cause a spot of the exciting beam 15 to scan the gel 2 on the straight
line 49 perpendicular to the direction 12 of electrophoresis at a high
speed. The scanning of a plane by such regular polyhedral mirror and
f.theta. lens system is well known (see for example, "Hikari Gijyutsu Oyo
System (Technology Application Systems)" pages 107 to 110 edited by the
Japanese Precision Mechanical Society, published by Shoko-do, in 1983).
A half mirror 50 is disposed in the optical path between the f.theta. lens
system 48 and the gel 2. The exciting beam 15 is partially reflected from
the half mirror 50 for a beam position sensor 52 to detect the position of
the beam 15. For example, S-1352 (product of Hamamatsu Photonics Inc.,
Hamamatsu, Japan) can be used as the sensor 52.
The same optical fiber bundle 44 as shown in FIG. 1 is disposed with its
rectangular end face opposed to an end face of the gel 2. The other end of
the bundle 44 is opposed to a photomultiplier tube 22 with a fluorescence
selecting interference filter 20 interposed therebetween.
The detection signal from the photomultiplier tube 22 is converted by an
A/D converter 41 to a digital signal, which is then fed to a microcomputer
42. The microcomputer 42 also receives from the sensor 52 data indicating
the position of projection of the exciting beam 15.
The operation of the embodiment of FIG. 3 will now be described.
When there is a band of DNA fragment at the position of projection of the
exciting beam 15 at a certain moment, the band emits fluorescence, which
is propagated through the electrophoretic gel 2 and incident on the
optical fiber bundle 44. The light then impinges on the photomultiplier
tube 22 through the interference filter 20 as already described. The band
pattern of the DNA is detected by the microcomputer 42 from the data given
by the sensor 52 and indicating the position of projection of the beam 15
and the fluorescence detection signal from the tube 22. Since the DNA
fragments are developed in the order of decreasing molecular length in the
direction of electrophoresis as already mentioned, the base sequence can
be determined from the detected pattern by a well-known method.
FIG. 4 shows another embodiment which is adapted to correct the intensity
of fluorescence emitted from the position of projection of an exciting
beam 15 and emanating from an end face of an electrophoretic gel 2.
It is thought that the intensity of the fluorescence 3 reaching the end
face of the gel 2 is dependent on the solid angle .theta. through which
the gel end face is viewed from the position of projection of the exciting
beam 15. In actuality, however, the intensity varies, for example, with
the condition of the end face. Accordingly, the intensity of the
fluorescence emerging from the end face is corrected with the intensity of
the fluorescence 3 measured at the position (X, Y) where the beam 15 is
incident on the gel. Indicated at 54 is a unit for carrying out
calculation for intensity correction. The intensity of the signal from a
photomultiplier tube 22 and the intensity of fluorescence at the position
(X, Y) are fed to this unit to correct the intensity of fluorescence at
the gel end face on an experimental basis. A data processing unit 56
determines the base sequence of DNA based on the corrected fluorescence
intensity.
FIG. 5 is a schematic plan view showing another embodiment, and FIG. 6 is a
view in section taken along the line A--A in FIG. 5.
With the embodiment of FIG. 5, an exciting beam 15 is projected on a gel 2
from the front toward the rear side perpendicular to the plane of the
drawing.
An optical fiber bundle 44 is disposed at one end face of the gel 2, and a
mirror 58 at the other end face thereof, whereby the fluorescence
propagated in a direction away from the fiber bundle 44 is reflected at
the mirror 58 and directed toward the bundle 44.
A mirror 60 for reflecting the exciting beam 15 is also provided as opposed
to the rear surface of the gel 2 opposite to the other surface thereof to
be exposed to the beam 15.
The mirror 58 serves to utilize the fluorescence efficiently, while the
mirror 60 intensifies the exciting beam 15, hence improved sensitivity.
FIGS. 7 and 8 show another embodiment of the invention designed as an
on-line system. FIG. 8 is a view in section taken along the line B--B in
FIG. 7.
With this embodiment, an electrophoretic gel 2 is covered with a mirror 62
except at an end face thereof provided with a fluorescence receiving
bundle 44 of optical fibers and at a small area where the gel is scanned
with an incident exciting beam 15.
The mirror 62 covering the gel enables the apparatus to utilize the
fluorescence and exciting beam 15 with a further improved efficiency and
to exhibit increased sensitivity.
The foregoing embodiments include a bundle 44 of optical fibers as means
for receiving fluorescence from an end face of the electrophoretic gel 2.
Since the light receiving means is used merely for guiding the
fluorescence from the gel end face to the sensitive surface of a
photoelectric device, some other means, such as a molded optical product,
is usable insofar as such means performs the above function.
Although a fluorescent substance is used as a labeling pigment for the
above embodiments, a phosphorescent substance is alternatively usable (see
Japanese patent application SHO 62-2230).
With the base sequence determining apparatus of the invention, a slab of
electrophoretic gel having nucleic acid fragments developed therein is
exposed to an exciting beam projected thereto in the direction of
thickness of the gel, and the luminescence emitted by a labeling pigment
on the fragments is received at the end face of the gel. Consequently, the
luminescence can be received from a direction at an angle of 90 degrees
with the exciting beam in which direction Rayleigh scattering of the beam
very objectionable to the measurement of the luminescence is minimum.
Since the position of measurement is specified by the location where the
exciting beam is incident, there is no need to receive the light from a
particular location as distinguished as such. This obviates the need for
an image forming lens, assures a great solid angle, results in a higher
S-N ratio, eliminates the need for an expensive one- or two-dimensional
array type sensor of high sensitivity and ensures the measurement with use
of one photomultiplier tube or like photoelectric device. The light
receiving assembly is therefore available at about 1/100 the conventional
cost.
Unlike the apparatus of FIG. 10, the present apparatus is so adapted that
the fluorescence is propagated through the electrophoretic gel.
Consequently, no problem arises even if the gel is thin.
The fluorescence is slightly greater than the exciting beam in wavelength
and is less susceptible than the beam to Rayleigh scattering due to the
gel.
Since the fluorescence is propagated by total reflection for detection, the
deformation or distortion of the gel poses no problem unlike the apparatus
of FIG. 10.
Thus, base sequences can be determined accurately with high sensitivity
using the apparatus of the invention.
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