U.S. patent number RE36,826 [Application Number 08/160,747] was granted by the patent office on 2000-08-22 for electrophoresis pattern reading system of fluorescence type.
This patent grant is currently assigned to Hitachi Software Engineering Co., Ltd.. Invention is credited to Hitoshi Fujimiya, Keigi Koga, Shigeo Nakajima, Hisanori Nasu.
United States Patent |
RE36,826 |
Fujimiya , et al. |
August 22, 2000 |
Electrophoresis pattern reading system of fluorescence type
Abstract
An electrophoresis pattern reading system of fluorescent type is
comprised of a detachable migration unit comprising a gel
functioning as a base for electrophoresis and a gel-supporting body
for supporting the gel; an electrophoresis unit, to which the
migration unit is mounted, for performing electrophoresis by
applying migrating voltage to the gel to which a sample labeled
with a fluorescent substance is added; and a reading unit including
an instrumentation subunit for reading an electrophoresis pattern,
to which the migration unit is mounted after electrophoresis and
which receives fluorescence emitted from the fluorescent substance
of the sample on the gel upon application of light to the gel. For
example, a plurality of the migration units and the electrophoresis
units are provided, and each of the plural migration units are
mounted to one reading unit in order after electrophoresis has been
performed with the respective electrophoresis units for a long
period of time, thereby reading the electrophoresis pattern in a
short period of time.
Inventors: |
Fujimiya; Hitoshi (Yokohama,
JP), Nakajima; Shigeo (Yokohama, JP), Nasu;
Hisanori (Yokohama, JP), Koga; Keigi (Yokohama,
JP) |
Assignee: |
Hitachi Software Engineering Co.,
Ltd. (Yokohama, JP)
|
Family
ID: |
15394729 |
Appl.
No.: |
08/160,747 |
Filed: |
December 3, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
533853 |
Jun 6, 1990 |
05069769 |
Dec 3, 1991 |
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Foreign Application Priority Data
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Jun 7, 1989 [JP] |
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1-145859 |
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Current U.S.
Class: |
204/461; 204/606;
204/612; 204/616; 250/458.1; 250/459.1; 250/461.1; 356/344 |
Current CPC
Class: |
G01N
27/44721 (20130101); G01N 27/44704 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); C25B 007/00 (); C25B
009/00 () |
Field of
Search: |
;204/182.7,182.8,182.9,299R,461,606,612,616 ;250/458.1,459.1,461.1
;356/344 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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Aug 1987 |
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EP |
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0 294 524 |
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Sep 1987 |
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EP |
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0 241 904 |
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Oct 1987 |
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EP |
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0 294 996 |
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Jun 1988 |
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EP |
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0 330 120 |
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Aug 1989 |
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EP |
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2 417 305 |
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Oct 1974 |
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DE |
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38 08 613 |
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Nov 1988 |
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DE |
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61-062843 |
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Mar 1986 |
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JP |
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86/00304 |
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Sep 1986 |
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WO |
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Other References
European Patent Search, Apr. 15, 1996. .
Peck et al. (1989) "Single Molecule fluorescence detection:
Autocorrelation criterion and experimental realization with
phycoerythrin," Proc. Natl. Acad. Sci. USA, 86, 4087-4091. .
Mathies,R.A. and Stryer,L. (1986), Applications of Fluorescence in
the Biomedical Sciences, Eds. Taylor,D.L., Waggonner,A.SA.,
Lanni,F., Murphy, R.F., and Birge,R. (Alan R. Liss, Inc., New York)
pp. 129-140. .
Nguyen,D.C.; Kellery, R.A.; Jett, J.H.; Martin, J.C. (1987),
Detection of Single Molecules of Phycoerythrin in Hydrodynamically
Focused Flows by Laser-Induced Fluorescence, Anal.Chem. 59,
2158-2161. .
Peck, K; Stryer, L; Glazer, A.N.; Mathies, R.A. (1989) Single
molecule fluorescence detection: Autocorrelation criterion and
experimental realization with phycoertthrin Proc. Natl. Acad. Sci.
USA, 86, 4087-4091. .
Mathies,R.A.; Peck,K,; Stryer, L. (1990), Optimization of
High-Sensitivity Fluorescence Detection, Anal.Chem. 62, pp.
1786-1791, .
Glaser, A.N.; Peck, K.; Mathies, R.A. (1990), A stable
double-stranded DNA-ethidium homodimer complex: Application to
picogram fluorescence detection of DNA in agarose gels,
Proc.Natl.Acad.Sci. USA, vol. 87, pp 3851-3855, May 1990
Biochemistry. .
Ansorge, W.; Rosenthal, A.; Sproat, B.; Schwager, C.; Stegemann,
J.; and Voss, H. (1988) Non-radioactive automated sequencing of
oligonucleotides by chemical degradation;Nucleic Acids Research
vol. 16, 2203-2206. .
Smith, L.M.; Sanders, J.Z.; Kaiser, R.J.; Hughes, P.; Dodd, C.;
Connell, C.R. Heiner, C.; Kent, S.B.H. & Hood, L.E. (1986),
Fluorescence detection in automated DNA sequence analysis, Nature,
vol. 321, 674-679. .
Sanger, F.; Nicklen, S.; & Coulson, A.R. (1977), DNA Sequencing
with chain-terminating inhibitors, Proc.Natl.Acad.Sci., vol. 74,
No. 12, pp. 5463-5467..
|
Primary Examiner: Niebling; John
Assistant Examiner: Delacroix-Muirhead; C.
Attorney, Agent or Firm: Fay, Sharpe, Beall, Fagan, Minnich
& McKee
Claims
What is claimed is:
1. An electrophoresis pattern reading system of
fluorescence-detection type, useful for analyzing a gel-based
sample, the sample being labeled with a fluorescent substance that
fluoresces upon application of light thereto, comprising:
a detachable migration unit comprising a gel functioning as a base
for a sample to be analyzed by electrophoresis and a gel-supporting
body for supporting the gel;
an electrophoresis unit, to which the migration unit is detachably
mounted, for performing electrophoresis by applying migrating
voltage to the gel to which the sample labeled with a fluorescent
substance is added; and
a reading unit physically separate from the electrophoresis unit
for reading an electrophoresis pattern, the reading unit including
means for detachably mounting the migration unit detached from and
apart from the electrophoresis unit after electrophoresis, and said
reading unit having means for passing light to the detachably
mounted migration unit and for receiving fluorescence emitted from
the fluorescent substance of the sample on the gel upon application
of the light.
2. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 1, wherein a
plurality of electrophoresis units is provided for each reading
unit.
3. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 1, said means for
passing light comprising a spot light source for generating light
for exciting fluorescence of the fluorescent substance, a scanning
means for scanning light from the spot light source in a direction
substantially parallel to the direction of electrophoresis by
radiation upon the gel, and a reading section having a
light-receiving subsection for receiving fluorescence from the
fluorescent substance.
4. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 3, wherein the
light-receiving subsection comprises a one-dimensional image
sensor, wherein the direction in which the light is received is
substantially parallel to the direction of electrophoresis.
5. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 3, wherein said
means for passing light and said scanning means produces the
scanning light of a width in the direction perpendicular to the
direction of electrophoresis that is extended on the basis of the
reading width of the irradiated area in the direction
.Iadd.substantially .Iaddend.parallel to the direction of
electrophoresis.
6. An electrophoresis pattern reading system as claimed in claim 3,
wherein the scanning means includes a concave lens for modulating
the light from the spot light source.
7. An electrophoresis pattern reading system as claimed in claim 6,
wherein the concave lens is arranged to modulate the light from the
spot light source to enlarge reading size with respect to a
direction substantially perpendicular to the direction of
electrophoresis.
8. An electrophoresis pattern reading system as claimed in claim 3,
wherein the scanning means includes a convex lens for modulating
the light from the spot light source.
9. An electrophoresis pattern reading system as claimed in claim 8,
wherein the convex lens is .[.disclosed.]. .Iadd.disposed
.Iaddend.substantially parallel to the direction of
electrophoresis.
10. An electrophoresis pattern reading system as claimed in claim
1, wherein the sample comprises a nucleic acid.
11. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 2, said means for
passing light comprising a spot light source for generating light
for exciting fluorescence of the fluorescent substance, a scanning
means for scanning light from the spot light source in a direction
substantially parallel to the direction of electrophoresis by
radiation upon the gel, and a reading section having a
light-receiving subsection for receiving fluorescence from the
fluorescent substance.
12. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 11, wherein the
light-receiving subsection comprises a one-dimensional image
sensor, wherein the direction in which the light is received is
substantially parallel to the direction of electrophoresis.
13. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 11, wherein said
means for passing light and said scanning means produces the
scanning light of a width in .[.the.]. .Iadd.a .Iaddend.direction
perpendicular to the direction of electrophoresis that is extended
on the basis of .[.the.]. .Iadd.a .Iaddend.reading width of
.[.the.]. .Iadd.an .Iaddend.irradiated area in the direction
.Iadd.substantially .Iaddend.parallel to the direction of
electrophoresis.
14. An electrophoresis pattern reading system as claimed in claim
11, wherein the scanning means includes a concave lens for
modulating the light from the spot light source.
15. An electrophoresis pattern reading system as claimed in claim
14, wherein the concave lens is arranged to modulate the light from
the spot light source to enlarge reading size with respect to a
direction substantially perpendicular to the direction of
electrophoresis.
16. An electrophoresis pattern reading system as claimed in claim
11, wherein the scanning means includes a convex lens for
modulating the light from the spot light source.
17. An electrophoresis pattern reading system as claimed in claim
16, wherein the convex lens is .[.disclosed.]. .Iadd.disposed
.Iaddend.substantially parallel to the direction of
electrophoresis.
18. An electrophoresis pattern reading system as claimed in claim
2, wherein the sample comprises a nucleic acid.
19. A method for reading an electrophoresis pattern of
fluorescence-detection type, comprising:
the step of pouring a sample into a gel in a migration unit, the
step of mounting the migration unit in an electrophoresis unit,
thereafter the step of performing electrophoresis of the sample,
the step of thereafter removing the migration unit with the
electrophoresed sample from the electrophoresis unit, the step of
mounting the removed migration unit with the electrophoresed sample
in a reading unit, and the step of reading the electrophoresis
pattern of the electrophoresed sample in the migration unit by the
reading unit.
20. A method for reading an electrophoresis pattern of
.[.fluorescent.]. .Iadd.fluorescence-detection .Iaddend.type as
claimed in claim 19, further comprising the step of providing a
plurality of the migration units and a plurality of the
electrophoresis units, wherein the step of mounting the migration
unit in the reading unit is carried out serially for each of the
plurality of the migration units in a common reading unit.
21. A method for reading an electrophoresis pattern of
fluorescence-detection type as claimed in claim 19, wherein a
direction substantially parallel to a direction of electrophoresis
is set as a first direction and a direction perpendicular to the
direction of electrophoresis is set as a second direction, and the
step of reading the electrophoresis pattern includes the step of
defining a pixel size having a longer dimension in the first
direction than in the second direction.
22. A method for reading an electrophoresis pattern of
.[.fluorescent.]. .Iadd.fluorescence-detection .Iaddend.type as
claimed in claim 21, wherein the step of reading the
electrophoresis pattern includes the step of obtaining data of a
pattern of distribution of the .[.fluorescent substance.].
.Iadd.electrophoresed sample.Iaddend., and thinning out the data by
at least one pixel in the second direction.
23. A method for reading an electrophoresis pattern .Iadd.of
fluorescence-detection type .Iaddend.as claimed in claim 19,
wherein said .[.step of pouring uses the.]. sample comprises a
nucleic acid.
24. A method for reading an electrophoresis pattern of
fluorescence-detection type, comprising:
(a) pouring a plurality of samples one each into a like plurality
of gels one each .[.into.]. .Iadd.in .Iaddend.a like plurality of
migration units;
(b) mounting each migration unit one each in a like plurality of
electrophoresis units;
(c) thereafter performing electrophoresis of each sample;
(d) thereafter removing one migration unit with its electrophoresed
sample from a first one of the plurality of electrophoresis
units;
(e) mounting the removed migration unit with its electrophoresed
sample in a reading unit;
(f) reading the electrophoresis pattern of the
.[.electrophoresis.]. .Iadd.electrophoresed .Iaddend.sample in the
migration unit by the reading unit; and
(g) repeating the steps (d) through (f) for each remaining
electrophoresed sample, and using the same reading unit for each
reading step. .Iadd.
25. An electrophoresis pattern reading system of
fluorescence-detection type as claimed in claim 1, wherein said
means for receiving fluorescence further comprises means for
scanning the sample with the light as excitation light in one axial
direction which is a direction nearly parallel or nearly
perpendicular to the direction of electrophoresis, and means for
moving the sample in a direction perpendicular to the one axial
direction. .Iaddend..Iadd.26. An electrophoresis pattern reading
system of fluorescence-detection type as claimed in claim 25,
wherein said means for scanning provides said one axial direction
substantially parallel to a direction of electrophoresis of the
sample. .Iaddend..Iadd.27. An electrophoresis pattern reading
system of fluorescence-detection type as claimed in claim 1,
wherein said means for receiving fluorescence has an
optical axial extending in a direction different from an optical
axis of said means for passing light. .Iaddend..Iadd.28. An
electrophoresis pattern reading system of fluorescence-detection
type as claimed in claim 1, wherein said means for passing light
irradiates the fluorescent substance with exciting light in a plane
at a constant optical angle of incidence with respect to a surface
defined by the electrophoresis pattern. .Iaddend..Iadd.29. An
electrophoresis pattern reading system of fluorescence-detection
type as claimed in claim 1, wherein said means for passing light
and said means for receiving light are located on a common side of
and spaced from a surface defined by the electrophoresis pattern.
.Iaddend..Iadd.30. An electrophoresis pattern reader for reading an
electrophoresis pattern defining a sample plane in a sample that
has been labeled with a fluorescent substance that fluoresces at a
second wavelength upon application of light thereto at a first
wavelength, comprising:
a sample holder for detachably holding the sample apart from any
electrophoresis unit and further defining the sample plane;
a light source emitting the light of the first wavelength to the
sample in a light source direction angularly intersecting the
sample plane;
a scanner for scanning by moving the light emitted by the light
source and the sample holder relative to each other in a first
direction, which is a direction nearly parallel or nearly
perpendicular to the direction of electrophoresis, in the sample
plane and in a second direction in the sample plane at a
perpendicular angle with respect to the first direction, to
irradiate the fluorescent substance, to excite the fluorescent
substance and to produce fluorescence at the second wavelength;
the light source direction being at an angle of incidence with
respect to the sample plane sufficient to transmit the light to the
fluorescent substance;
a collector for collecting the fluorescence at the second
wavelength along an emittance direction at an angle of emittance
relative to the sample plane, and including a spectral filter for
rejecting light of the first wavelength and passing light of the
second wavelength;
the light source forming the light source direction and the
collector forming the emittance direction to intersect each other
at a scanning location on the sample; and
a sensor for sensing the fluorescence at the second wavelength
after the fluorescence is collected and filtered by said collector,
over a period of time for each of a plurality of the scanning
locations, to produce a correlated electrical analog signal having
an analog value proportional to quantity of the fluorescence at the
second wavelength received during the
period of time. .Iaddend..Iadd.31. An electrophoresis pattern
reader according to claim 30, including an analog to digital
converter for converting the analog value of the analog signal to a
digital value; and
means for outputting a scan of the digital values correlated to
the
scanning of the sample. .Iaddend..Iadd.32. An electrophoresis
pattern reader according to claim 30, wherein said scanner provides
the first direction parallel to a direction of electrophoresis in
the electrophoresis pattern and provides the second direction
substantially perpendicular to the first direction.
.Iaddend..Iadd.33. An electrophoresis pattern reader according to
claim 30, further including a mirror for reflecting fluorescence of
the second wavelength to said collector. .Iaddend..Iadd.34. An
electrophoresis pattern reader according to claim 30, wherein said
light source is a laser. .Iaddend..Iadd.35. An electrophoresis
pattern reader according to claim 31, wherein said scanner includes
a vibrating mirror, said collector includes a collector lens and a
condenser;
wherein said sensor is a photomultiplier for photoelectrical
conversion; and
further including an amplifier between said sensor and said analog
to digital converter. .Iaddend..Iadd.36. An electrophoresis pattern
reader according to claim 30, further including means for changing
scanning speed by varying an exciting light spot size at the
scanning location through changing the angle of incidence of the
exciting light to change the ratio of exciting light spot size on
the electrophoresis pattern in said first direction and said second
direction. .Iaddend..Iadd.37. An electrophoresis pattern reader
according to claim 30, wherein said scanner moves the light emitted
by the light source in only one scanning plane as one dimensional
scanning in the first direction and relatively moves the light
source and the sample in the second direction while maintaining
substantially constant the angle of incidence defined by the angle
of intersection between the light source plane and the sample
plane;
said sensor spatially separating light from the surface of the
sample of the fluorescence from scattered light from the light
source and the sample plane;
said sensor producing the analog signal with spatially separate
electrical components separated according to the spatially separate
light components; and
means for extracting only the electrical component of the
fluorescence from other electrical components on the basis of the
spatially separate electrical components for improving a signal to
noise ratio.
.Iaddend..Iadd.38. An electrophoresis pattern reader according to
claim 37, wherein the angle of intersection between the light
source plane and the sample plane is sufficiently smaller than
90.degree. to physically separate scattered/reflected light to be
spaced from an optical axis of the sensor wherein the greatest
intensity of received fluorescence is detected, as viewed at the
sensor. .Iaddend..Iadd.39. An electrophoresis pattern reader
according to claim 38, wherein the scattered/reflected light
produces intensity peaks at said sensor physically spaced from
intensity peaks of the received fluorescence, disposed parallel to
the sample plane. .Iaddend.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to an electrophoresis pattern reading
system of a fluorescence type and, more particularly, to a pattern
reader for electrophoresis of fluorescence type, in which
detachable migration units are mounted to a plurality of
electrophoresis units which are electrophoresed simultaneously with
each other, and an electrophoresis pattern is read by a common
reader unit, thereby efficiently implementing electrophoresis and
reading the electrophoresis pattern.
The pattern reader for electrophoresis of the fluorescence type has
the advantage that it does not require dangerous and expensive
radioisotopes.
Generally speaking, electrophoresis analysis methods using
fluorescence method had been used for analysis of various genetic
structures including DNA sequencing (determination of a sequence of
bases of the gene), mass spectrometry of proteins such as amino
acids and analysis of polymer structures. Such an electrophoresis
analysis method involves implementing electrophoresis using a
sample of fragments labeled with a fluorescent substance and a
distribution pattern developed by electrophoresis is analyzed to
thereby analyze the samples.
Description will be made of a DNA sequencing device as a
representative example of an electrophoresis pattern reader.
In the DNA sequencing using the DNA sequencing device, a sample of
a DNA whose structure is determined is first cut into fragments
with a restriction enzyme by controlling reactivity against a
chemical reaction specific to a site of each a base and labeling
them with a fluorescent substance. The fragments are different in
length from each other and have each a particular base selected
from four kinds of bases labeled at their cut ends, consisting of
adenine (A), cytosine (C), guanine (G) and thymine (T). As the
fragmented DNA sample can be separated by electrophoresis in
accordance with the length of the fragment, each fragment is
separated by means of electrophoresis and radiated with laser light
to excite the fluorescent substance labeled on each of the
fragments. A measurement of the distribution in intensity of the
fluorescence emitted from the fluorescent substance permits the
reading of a sequence of bases, thereby determining the structure
of the DNA.
FIG. 13 is a view showing an example of the distribution of DNA
fragments obtained by electrophoresis. As the distance of migration
varies with lengths of DNA fragments (the difference of their
molecular weights), the fragments having the same molecular weights
gather together as time passes, and in an electrophoresis pattern
70, as shown in FIG. 13, bands 66 are formed so as to correspond to
the molecular weights of the DNA fragments. As a whole, the
electrophoresis pattern is provided such that the bands 66 are
formed in lanes 71, 72, 73 and 74. It is to be noted herein that,
as there is the difference in molecular weight by one base or more
among the bases A, G, C and T of the fragments, the distances of
migration for all fragments are different from the other. Hence, it
can be theoretically concluded that the bands 66 in the lanes 71 to
74 are not disposed transversely in a row with each other. For DNA
.[.sequening.]. .Iadd.sequencing.Iaddend., the pattern of the bands
66 is read for each of the bases, A, G, C and T in the respective
lanes 71 to 74 in order from the bottom of the pattern, thereby
analyzing the DNA sequence.
The above description has been made of the electrophoresis analysis
method using the DNA sequencing for analyzing the sequence of the
bases of each DNA as an example. It is to be noted, however, that
the electrophoresis method can likewise be applied to analysis of
other samples. The electrophoresis of the sample subject to
analysis allows the sample to be separated into different molecular
weights and to form bands corresponding to the separated molecular
weights. Hence, the difference in molecular weight between the
components of the sample can be determined by reading the
distribution of the bands formed. Furthermore, the electrophoresis
may be applied to assumption of a molecular weight of a compound or
determination of the presence or absence of a given molecule by
measuring the distance of migration of the sample and judgment of
the presence or absence of the band in a predetermined
position.
By pouring a sample labeled with a fluorescent substance into a gel
functioning as a base for electrophoresis and electrophoresing the
gel, the gel is provided with a distribution of bands after
electrophoresis in accordance with the molecular weights of
components of the sample so that the distribution of the bands is
measured. A measurement of the band distribution is made by
radiating the electrophoresed gel with light such as laser light
that generates fluorescence upon excitement of the fluorescent
substance, and the distribution pattern of the bands is measured by
detecting the fluorescence emitted upon reaction with a
photoelectrically converting element.
The electrophoresis device of fluorescence detecting type is
described, for example, in Japanese Patent Unexamined Publication
(kokai) No. 61-62,843/1986.
Description will now be made of the electrophoresis device of such
fluorescence type.
FIG. 9 is a perspective view showing an outlook of a conventional
electrophoresis device. As shown in FIG. 9, the conventional
electrophoresis device comprises an electrophoresis and
instrumentation unit 51 for carrying out electrophoresis and
instrumenting the distribution of fluorescence, a data processor
unit 52 for processing data instrumented, and a cable 53 connecting
them to each other. The electrophoresis and instrumentation unit 51
has a door 51a and the door 51a is opened to pour a gel functioning
as a base for electrophoresis of DNA fragments and then a given
amount of a sample to be analyzed. Then the door 51a is closed and
a switch is turned on to start up electrophoresis. As
electrophoresis has been started up, an operational state is
displayed and monitored on a display panel 51b of the
electrophoresis and instrumentation unit 51. The data instrumented
is then transferred to the data processor unit 52 and is subjected
to desired data processing in accordance with preset programs. The
data processor unit 52 comprises predominantly a main body of a
computer 54 consisting of a microprocessor, memory and so on, a
keyboard 55 from which instructions are given by the operator, a
display 56 for display processing results and states, and a printer
57 for recording the processed results.
FIG. 10 is a block diagram showing the construction of the
electrophoresis and instrumentation unit. As shown in FIG. 10, the
electrophoresis and instrumentation unit 51 (FIG. 9) comprises an
electrophoresis subunit 63 and a signal processor subunit 64. The
electrophoresis subunit 63 further comprises an electrophoresis
section 5 in which electrophoresis is performed, a first electrode
2a and a second electrode 2b for applying voltage to the
electrophoresis section 5, a support plate 3 for supporting the
electrophoresis section 5 and the first and second electrodes 2a
and 2b, a power supply 4 for electrophoresis for applying voltage
to the electrophoresis section 5, a light source 11 for generating
light to excite a fluorescent substance, an optical fiber 12 for
leading the light from the light source 11, a condenser 14 of an
optic system for condensing and collecting fluorescence 13
generated by the fluorescent substance, an optical filter 15 for
selectively passing the light having a particular wavelength
therethrough, and an optical sensor 16 for converting the condensed
light into electrical signals. The signal processor subunit 64
further comprises an amplifier 17 for amplifying the electrical
signals from the optical sensor 16, an analog-digital converting
circuit 18 for converting analog signals of the electrical signals
into digital data, a signal processing section 19 for implementing
pre-processing of the digital data converted, for example, by
addition average processing or the like, an interface 20 for
implementing the interface processing for feeding the pre-processed
data to an external data processor, and a control circuit 10 for
performing an entire control of the electrophoresis and the signal
processing. The digital signal OUT is generated from the signal
processor subunit 64 and then supplied to the data processor unit
52 (FIG. 9), thereby implementing the data processing such as
analysis processing and so on.
Description will now be made of operation of the electrophoresis
device which is constructed in the manner as described
hereinabove.
Reference is made to FIGS. 9 and 10. After the door 51a of the
electrophoresis and instrumentation unit 51, a gel is poured into
the electrophoresis section 5 disposed within the unit 51 and
thereafter a sample of DNA fragments labeled with a fluorescent
substance is poured thereinto. A switch of the display panel 51b is
turned on to give an instruction for starting-up electrophoresis,
and then voltage is applied from the first and second electrodes 2a
and 2b of the power supply 4 to the electrophoresis section 5,
thereby starting up electrophoresis. The electrophoresis allows the
sample labeled with the fluorescent substance to be migrated in the
lanes 71, 72, 73 and 74, thereby gathering the molecules having the
same molecular weights together and forming the bands 66, for
instance, as shown in FIG. 13. The molecules having smaller
molecular weights are allowed to migrate at a rate faster than
those having greater molecular weights so that the former is
migrated in a distance longer than the latter within the same time
unit. The bands 66 are detected in a manner as shown in FIG. 11a by
leading light from the light source 11 through the optical fiber 12
and radiating the gel in the electrophoresis section 5 on its
optical path, thereby forcing the fluorescent substance labeled on
the bands 66 of the gel to emit fluorescence 13.
Referring to the front view as shown in FIG. 11a and to the
longitudinally sectional view as shown in FIG. 11b, the
electrophoresis section 5 comprises a gel 5a consisting of
polyacrylic amide or the like and support plates 5b and 5c made of
glass for supporting and interposing the gel 5a from the both
sides. For example, a sample of DNA fragments is poured into the
gel 5a of the electrophoresis section 5 from its upper portion and
electrophoresis is carried out by applying voltage to the first
electrode
2a and the second electrode 2b (FIG. 10). Light radiated from the
light source, for example, laser light, passes through the light
path 61 in the gel 5a from the optical fiber 12 and radiated to the
fluorescent substance on the light path 61. This allows the
fluorescent substance present on the light path 61 to be excited to
emit fluorescence 13. The fluorescence 13 emitted is led to a
substage condenser 14 of optics consisting of a combination of
lenses and then selected by the optical filter 15 after being
condensed, thereby converting it into electrical signals by means
of the sensor 16. The electrical signals obtained by the sensor 16
is amplified to a desired level by the amplifier 17 and subjected
to analog-digital conversion by the analog-digital circuit 18
followed by a supply to the signal processing section 19. The
signal processing section 19 processes the signals by means of
addition-average processing or the like in order to improve a
signal-noise ratio. The data of the digital signals which has been
subjected to signal processing is fed to the data processor subunit
52 through the interface 20.
FIGS. 12a and 12b are views describing an embodiment of
fluorescence intensity pattern signals of the DNA fragments to be
transferred from the electrophoresis and instrumentation subunit
51. For instance, as shown in FIG. 12a, as the laser light is
radiated upon the electrophoresis section 5 in which the
electrophoresis is performed, the fluorescent substance of the gel
present on the light path 61 is excited to emit fluorescence 13.
This fluorescence 13 is detected in predetermined detection
positions in each lane in the direction of electrophoresis in the
course of lapse of time. This allows the fluorescence 13 to be
detected when the bands 66 in each lane pass through the positions
on the light path 61, thereby detecting a pattern signal of
fluorescence intensity in each lane, as shown in FIG. 12b.
Therefore, the pattern signal of the magnitude of fluorescence
intensity as shown in FIG. 12b is represented as a pattern signal
of fluorescence intensities of the bands 66 in the electrophoresis
direction 62. The data processor unit 52 performs data processing
for comparing molecular weights and determining the sequence of DNA
from data of the pattern of fluorescence intensity. The sequence of
the bases or the like determined by data processing is symbolized
and then generated, thereby being displayed on a display screen 56
or printed by a printer 57. The data of the result obtained by data
processing may be recorded in magnetic recording media as needed.
It is to be noted that the time period required for electrophoresis
by the pattern reader for electrophoresis having the construction
as described hereinabove ranges usually from 5 to 8 hours in the
case of electrophoresis of DNA fragments and the time period for
reading the distribution of the fluorescent substances in the
electrophoresed gel. Hence, it is the current situation in which
the analyzer for the electrophoresis method such as the DNA
sequencer is occupied for most of its treating time by
electrophoresis once the processing for analyzing the substance has
been started up. The data processor unit for data processing of the
results read from the electrophoresis pattern, which is to be used
together with the analyzer for the electrophoresis method of this
kind, is constructed as a separate unit so as to allow a
general-purpose data processor to be utilized therefor. The
electrophoresis and instrumentation unit in which electrophoresis
is performed and the electrophoresis pattern is read is an integral
combination of an electrophoresis subunit which performs
electrophoresis and a signal processor subunit which implements
data processing by reading the band pattern as a result of
electrophoresis. Hence, once electrophoresis for one sample has
started up, the electrophoresis and instrumentation unit is
occupied for a long period of time for a series of analyzing
processes in which the pattern is read. More specifically, as
described hereinabove, for the conventional electrophoresis pattern
reading system, the electrophoresis and instrumentation unit has
been occupied for the total period of time of about 5-8 hours for
electrophoresis and 30 minutes for reading the distribution of the
fluorescent substance developed in the gel, so that it is the
problem that the expensive system cannot effectively be used.
SUMMARY OF THE INVENTION
The present invention has the object to provide an electrophoresis
pattern reading system of fluorescent type in which the
electrophoresis pattern is efficiently read by the fluorescence
method and the system can effectively be utilized.
The present invention has another object to provide a method for
reading an electrophoresis pattern, in which electrophoresis can be
performed by using the electrophoresis pattern reading system of
fluorescent type and a pattern of distribution of the fluorescent
substance electrophoresed can effectively read.
The present invention has a still further object to provide an
electrophoresis pattern reading system of fluorescent type, which
is constructed such that an electrophoresis unit for performing
electrophoresis of fragments of a sample labeled with a fluorescent
substance is disposed separated from a reading unit for reading the
distribution of the fluorescent substance as a result of
electrophoresis and in which the gel as a result of electrophoresis
obtained by using a plurality of .[.plural.]. electrophoresis units
is read with the same reading unit, thereby reading the
electrophoresis pattern efficiently.
In order to achieve the objects, the present invention consisting
of the electrophoresis pattern reading system of fluorescent type
is characterized by a detachable migration unit consisting of a gel
functioning as a base for electrophoresis and a gel-supporting body
for supporting the gel; an electrophoresis unit to which the
migration unit is mounted and in which electrophoresis is performed
by applying the migrating voltage to the gel to which a sample
labeled with a fluorescent substance is added; and a reading unit
to which the migration unit is mounted after electrophoresis and in
which the electrophoresis pattern is read by irradiating the gel
with light and receiving fluorescence generated from the
fluorescent substance of the sample on the gel. By providing the
detachable migration unit comprised of the gel functioning as the
base for electrophoresis and the gel-supporting body for supporting
the gel, the electrophoresis pattern reading system of fluorescent
type is of a separate structure in which the electrophoresis unit
having the migration unit for electrophoresis is disposed
separately from the electrophoresis pattern reading unit with the
migration unit mounted after electrophoresis. Hence, the
electrophoresis unit in which electrophoresis is performed for a
long period of time is of such a structure as being separate from
the electrophoresis pattern reading unit which performs reading the
electrophoresis pattern in a relatively short period of time,
thereby enabling the electrophoresis unit to be separated from the
operation of the reading unit by using the detachable migration
unit composed of the gel functioning as the base for
electrophoresis and the gel-supporting body for supporting the gel.
This can avoid an expensive electrophoresis pattern reading system
of fluorescent type as a whole from being occupied for a long
period of time. The expensive reading unit having a complicated and
highly sophisticated processing mechanism can now be shared with a
plurality of the electrophoresis units prepared at cheaper costs.
This arrangement can also allow electrophoresis to be performed
simultaneously with a plurality of the electrophoresis units and
enables the common reading unit to read the results of
electrophoresis in order, thereby resulting in an efficient
analysis of samples by electrophoresis. As described hereinabove,
by using the detachable migration unit composed of the gel
functioning as the base for electrophoresis and the gel-supporting
body for supporting the gel, which is mounted to the
electrophoresis unit, electrophoresis can be performed by adding
the sample labeled with the fluorescent substance to the gel. After
electrophoresis has been finished, the migration unit is detached
from the electrophoresis unit and the migration unit is then
mounted to the reading unit after electrophoresis after the gel is
removed from the electrophoresed migration unit or while the gel is
held while being supported by the gel-supporting body and, if
necessary, the gel is colored with a colorant or the gel is dried.
In the reading unit, the fluorescent substance on the gel in the
migration unit is irradiated with light and the distribution of the
fluorescent substance on the gel is optically read and recorded as
image data by a recording medium such as paper, film, magnetic
recording medium or the like. Parallel and simultaneous
electrophoresis of different samples under different conditions
using a plurality of migration units which are mounted to a
plurality of electrophoresis units permits the migration units one
after another subsequent to completion of electrophoresis to be
mounted to the reading unit and the distribution data of the
fluorescent substances in the gel to be read in order, thereby
resulting in an efficient reading of electrophoresis patterns.
Therefore, the electrophoresis pattern reading system of
fluorescence type can be arranged, for example, such that a
plurality of researchers share the reading unit and each of them
owns its own electrophoresis unit. This system enables an efficient
electrophoresis analysis by allowing each of the researchers to
perform electrophoresis with its own electrophoresis unit, to mount
the resulting migration unit to the common reading unit, and to
read the results (gels) of electrophoresis by means of the common
reading unit. This system can result in an economical construction
of, for example, an automatic DNA analyzing system. It is further
to be noted that the gel in the migration unit can manually be
analyzed, without the use of a digital analyzer, directly from
information on transcription which may be obtained by transcribing
the original data on distribution of the fluorescent substance,
obtainable by radiation of the gel with light, to the recording
medium as its intact image. This permits the efficient reading
processing of the electrophoresis pattern.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic representation of an overall construction
of the electrophoresis pattern reading system of fluorescent type
according to an embodiment of the present invention.
FIG. 2 is a block diagram showing the construction of an essential
portion of an instrumentation unit.
FIG. 3 is a view showing the position in which the migration unit
to be mounted to the instrumentation unit is mounted.
FIGS. 4a, 4b, 4c, 4d and 4e are views showing the scanning for
reading the migration unit to be mounted to the instrumentation
unit.
FIG. 5a is a view showing an example of pixel data to be obtainable
when no reflecting light is used for scanning with reading
light.
FIG. 5b is a view showing an example of pixel data to be obtainable
when reflecting light is used for scanning with reading light.
FIGS. 6a and 6b are views showing another example of the reading
method with enlarged resolving power by enlarging the size of
pixels for reading a fluorescent substance in a given
direction.
FIG. 7 is a diagrammatic representation showing the construction of
an essential portion in which a one-dimensional sensor of an image
sensor of semiconductor device is used as a light receiving section
for detecting fluorescence from the migration unit.
FIG. 8 is a view showing the reading method for reading the
electrophoresis pattern by using the electrophoresis pattern
reading system of fluorescent type according to an example of the
present invention.
FIG. 9 is a perspective view showing an appearance of the
electrophoresis unit.
FIG. 10 is a block diagram showing the construction of the
instrumentation unit.
FIGS. 11a and 11b are views showing the operational principle of
detecting the electrophoresis pattern by the fluorescence
method.
FIGS. 12a and 12b are views showing examples of pattern signals for
fluorescence intensity of DNA fragments to be fed from the
instrumentation unit 41.
FIG. 13 is a view showing an example of the distribution of
electrophoresed DNA fragments.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
FIG. 1 diagrammatically represents the overall construction of the
electrophoresis pattern reading system of fluorescent type
according to an example of the present invention. As shown in FIG.
1, the electrophoresis pattern reading system of fluorescent type
is overall constructed such that an electrophoresis unit 1 is
disposed separately from a reading unit 6. The electrophoresis unit
1 comprises a migration subunit 5 composed of a gel functioning as
a base for electrophoresis and a gel-supporting body for supporting
the gel, a first electrode 2a and a second electrode 2b for
applying electrophoresis voltage to the migration subunit 5
(hereinafter referred to as migration section) mounted, a
supporting plate 3 for supporting the first electrode 2a and the
second electrode 2b as well as the migration section 5, and a power
supply 4 for electrophoresis for supplying the electrophoresis
voltage. The migration section 5 comprises the gel such as
polyacrylamide or the like on which a sample for electrophoresis is
developed and the gel-supporting body such as glass plates disposed
so as to interpose the gel .[.from the.]. .Iadd.on .Iaddend.both
sides, as .[.describe.]. .Iadd.described .Iaddend.hereinabove (as
shown in FIGS. 11a and 11b). In the electrophoresis unit 1, the
migration section 5 is mounted and a sample of fragments subject to
electrophoresis is supplied from an upper portion of the gel in the
migration section 5. Thereafter, the migration voltage is applied
to the first electrode 2a and the second electrode 2b from the
power supply 4 for electrophoresis, thereby starting up
electrophoresis After electrophoresis, the migration section 5 is
removed from the electrophoresis unit 1 and then mounted to the
reading unit 6.
The reading unit 6 is to perform data processing by mounting the
migration section 5 after electrophoresis as it is (or in such a
state of the gel that is removed from the migration section 5). As
shown in FIG. 1, the reading unit 6 is composed of an
instrumentation subunit 7 as a major component and a data processor
unit 8 and an image printer 9 are added thereto. The data processor
unit 8, the image printer 9 and so on are arranged so as to
generate the electrophoresis pattern data read by the
instrumentation subunit 7 after data processing, image processing
and judgment processing. To the instrumentation subunit 7 is the
migration unit 5 (the migration unit composed of the gel and the
gel-supporting body) after electrophoresis has been performed in
the electrophoresis unit 1, and a reading base is disposed
immediately underneath a lid 7a at an upper portion of the
instrumentation subunit. The migration section 5 removed from the
electrophoresis unit 1 is mounted to the reading base of the
instrumentation subunit 7 electrophoresis unit 1 by opening the lid
7a. After the migration section 5 with the gel as the object for
reading has been mounted, the lid 7a is closed and a switch on an
operational display panel 7b for starting up the reading operation
is then pressed, whereby the instrumentation subunit 7 starts up
the reading of the electrophoresis pattern of the gel of the
migration section 5. As the reading of the electrophoresis pattern
starts up, the light from the light source equipped in the
instrumentation subunit 7 is scanned and the gel of the migration
section 5 mounted is irradiated with exciting light to excite the
fluorescent substance and the distribution of the fluorescent
substance is instrumented by receiving the fluorescence. The data
processor unit 8 is to process data on the basis of the read data
that has been instrumented by the instrumentation subunit 7 and to
control the instrumentation subunit 7. The data processed is
visualized by the image printer 9 or the like.
FIG. 2 is a block diagram showing the construction of the essential
portion of the instrumentation subunit, and FIG. 3 is a view
showing the position in which the migration section is mounted to
the instrumentation subunit.
Description will be made with reference to FIGS. .Iadd.1,
.Iaddend.2 and 3.
In performing electrophoresis analysis of a sample by using the
electrophoresis pattern reading system, the sample labeled with the
fluorescent pigment is first subjected to electrophoresis using
electrophoresis unit 1. After electrophoresis for a given period,
the migration section 5 is removed from the electrophoresis unit 1.
Thereafter, the gel of the migration section 5 removed is placed on
an upper portion of the reading base 7c in the instrumentation
subunit 7 after opening the lid 7a of the reading unit 6 at the
upper portion of the instrumentation subunit 7 in such a state that
the migration section 5 is as it is after removal or that glasses
of the gel-supporting body are removed, as shown in FIG. 3. Then
the lid 7a is closed. This concludes the setting to the
instrumentation subunit 7. At this time, if the gel after
electrophoresis is not yet labeled with the fluorescent pigment,
the processing for labeling the sample with the pigment is
executed. Further, the processing of drying the gel is also
executed.
The operation of giving an instruction of the start-up of reading
the electrophoresis pattern will now be performed. The operation of
starting the reading is executed by giving a start-up instruction
by means of pressing the switch of the operation display panel 7b
for starting the reading or from the data processor unit 8. In
starting the operation of reading by the data processor unit 8, the
state in which the migration unit is mounted to the instrumentation
subunit 7 is fed to the data processor unit 8 through a control
signal line, and the operation of the instrumentation subunit 7 is
controlled by the data processor unit 8 in accordance with the
state. In this case, the operation of starting up the reading is
automatically executed, thereby reducing the burden of operating
the switch on the side of the operator.
The distribution data of the fluorescent pigment read is
transferred to the data processor unit 8. The data processor unit 8
executes desired processings such as processing of detecting peaks
of fluorescent intensity, processing of determining the migration
distance, etc., in accordance with preset programs. The result data
obtained by data processing is printed out, as needed, by the image
printer 9 as image of the fluorescent intensity with light and
shade or as image in which the fluorescent intensity is grouped by
contour lines or by colors or the strength of color. The image
printed out as the light-and-shade picture in accordance with the
fluorescent intensity is the same image as radioactive X-ray film
image which has conventionally been used. The result data which has
been data-processed is stored, as needed, as digital data by a
magnetic or optical recorder.
In the block diagram of FIG. 2 showing the construction of the
instrumentation subunit, laser beams generated from a light source
21 are scanned by a vibration mirror 22 in the direction of the
front and rear sides of this drawing, collected by a lens 23, and
led to the gel of the migration section 5 as the object of reading.
The laser beams scanned by the vibration mirror 22 and collected by
the lens 23 are focused on the gel in the migration section 5. This
allows the fluorescent substance on the light path of the laser
beams scanned to be excited, thereby emitting fluorescence 13. The
fluorescence 13 is collected by a condenser 24, together with
exciting light scattered and so on, and converted into electrical
signals by an optical sensor 26 through an optical filter 25. As
the condenser 26 are a lens, a conical cylinder, an optical fiber,
etc, and they serve as enhancing sensitivity of detecting the
fluorescence received with respect to external light. The optical
filter 25 is to selectively transmit the wavelength component of
the fluorescence, thereby excluding the influence of the scattered
exciting light upon the fluorescence received. As described
hereinabove, the condenser 24 and the optical filter 25 enhances
the sensitivity of receiving the fluorescence to be detected, and
the fluorescence received is converted into electrical signals by
the optical sensor 26. In order to further enhance the sensitivity
of detecting the fluorescence, a photomultiplier having high
efficiency of photo-electrical conversion is used as the optical
sensor 26. The electrical signals obtained by the optical sensor 26
are amplified by an amplifier 27, and the amplified electrical
signals are provided to an analog-digital converting circuit 28 and
converted into digital data. The fluorescence-detecting signal
converted into the digital data is stored by a memory 29, and the
data stored in the memory 29 is supplied to the data processor unit
8 through an interface circuit 30. Overall control of such a series
of signal processings is performed by a control circuit 31.
FIGS. 4a, 4b, 4c, 4d and 4e are diagrammatic representations
showing the scanning for reading the migration unit mounted to the
instrumentation subunit.
Description will be made of the scanning for reading the migration
section 5 (migration unit) as an object of reading, in conjunction
with FIGS. 4a-4e. As shown in FIG. 3, the migration unit (migration
section 5) as the reading object is mounted to the instrumentation
subunit 7 and the migration section 5 mounted is transferred to the
left and right on the reading base 7c once the operation of
starting the reading has started up, thereby reading the
electrophoresis pattern of the gel. The migration section 5 is
first set in the position in such a state as shown in FIG. 4a. As
shown in FIG. 4a, arrow 40 stands for the direction of scanning the
laser beams from the light source, namely, the first scanning
direction (main scanning direction). During the reading operation,
the migration section 5 is transferred to the left end of the
reading base 7c as shown in FIG. 4b, thereby starting up scanning
the laser beams in the main scanning direction. The migration
section 5 is read while being transferred to the right from the
left end at a given speed. The reading finishes as the migration
section 5 reaches the right end as shown in FIG. 4c after the
migration section 5 has started up being read and transferred to
the right at the given speed. In the reading base 7c, the direction
in which the arrow 41 is moved at the stage of transferring the
migration section 5 is a second scanning direction (sub-scan
direction). As described hereinabove, as a result, the distribution
of the fluorescent substance on the gel in the migration section 5
is read as a two-dimensionally distributed image.
In the two-dimensional reading operation by means of the relative
transfer of the migration section 5 in the manner as described
hereinabove, the scanning of the laser beams by the reading light
is equivalent of reading by scanning the surface of the migration
section 5 in both the main scan direction (the scanning direction
indicated by the arrow 40) and the sub-scan direction (the scanning
indicated by the arrow 41), as shown in FIG. 4d. In this case, the
reading is performed in such a manner that the main scan direction
is set to a direction that is the same as the direction of
electrophoresis. In scanning for reading in the manner as described
hereinabove, it is to be noted that the longitudinal direction is
read in the same size as the transverse direction when the speed
for scanning the first scanning direction (main scan direction) is
set to the same as that for scanning the second scanning direction
(sub-scan direction). It is noted, however, that a pattern of
electrophoresis bands of the gel in the migration section as the
object for reading is such that bands expand in a band-like form in
the direction normal to the electrophoresis direction, as shown in
FIG. 13, so that, when the sequence of bases is given, the
resolving ability for reading in the direction (the first scanning
direction) parallel to the direction of electrophoresis is highly
required. On the contrary, the resolving ability for reading in the
second scanning direction is sufficient even if it would be lower
than that for reading in the first scanning direction. Hence, in
this reading operation, the main scan direction is set to the same
direction as the electrophoresis direction, as shown in FIG. 4d,
and the exciting light is applied to the fluorescent substance in
the gel 35 by leading light .[.3.]. .Iadd.32 .Iaddend.from the
light source to the reading surface of the migration section 5 at a
given incidence angle .theta., as shown in FIG. 4e. A mirror 33 is
disposed on the side opposite to the incidence side of the light
32, and light is additionally provided to the fluorescent substance
of the gel 35 as light 34 reflected from the mirror 33. The light
32 entering glass 36 of the migration section 5 is transmitted
through glass 37 disposed on the side opposite to the gel 35 and
reflected on the mirror 33 and then transmitted again through the
glass 37, the gel 35 and the glass 36, thereafter resulting in
reflected light 34 toward the outside. This arrangement can improve
the reading speed in the second scanning direction by enlarging the
area by giving the exciting light to the fluorescent substance as a
result in the second scanning direction.
FIG. 5a is a view showing an example of pixel data to be obtained
in an instance where no reflected light is used for scanning the
reading light, and FIG. 5b is a view showing an example of pixel
data to be obtainable in an instance where reflected light is used
for scanning the reading light. By lowering the resolving power for
detecting the fluorescent substance of the gel in the second
scanning direction by using the reflected light for scanning the
reading light, the pixel data to be obtained is a pixel data to be
read with respect to a pixel size in the region as large as two
times the pixel size in the second scanning direction (in the
direction crossing at a right angle to the direction of
electrophoresis), as shown in FIG. 5b. The method for reading in
the manner as described hereinabove enables the reading speed to
become high while maintaining the resolving power of reading in the
first scanning direction, which is required for determination of
the sequence of bases.
FIGS. 6a and 6b represent other examples of the method for reading
to enhance the reading speed by enlarging the pixel sizes of the
fluorescent substance in the given direction.
As shown in FIG. 6a, for example, the other first method is a
method for applying the exciting light 42 from the light source to
the migration section 5 by enlarging the reading pixel size by
means of a cylindrical lens 43 having a concave lens form in
section in the sub-scan direction (second scanning direction) so as
to become longer with respect to the direction perpendicular to the
sub-scan direction in order to enlarge the reading pixel size with
respect to the sub-scan direction (second scanning direction).
In the instance as described hereinabove, description has been made
of the case where the main scan direction (first scanning
direction) is the same direction as the direction of
electrophoresis. In instances, however, where the main scan
direction is perpendicular to the direction of electrophoresis, the
reading speed can be made higher by enlarging the reading pixel
size of the fluorescent substance in a given direction. In this
case, as shown in FIG. 6b, there is used the cylindrical lens 44 in
a convex form in section .Iadd.and a mirror 45.Iaddend.. In other
words, in instances where the main scan direction is perpendicular
to the direction of electrophoresis, the cylindrical lens 44 in the
convex form in section is disposed in the direction nearly parallel
to the direction of electrophoresis. Likewise, as shown in FIG. 4e,
it is possible to change the reading speed by varying the pixel
size for reading with a ratio of the longitudinal light spot size
to the transverse light spot size by changing the angle of
incidence of the exciting light 42, the location of the cylindrical
lens 44, and so on. Further, where it is acceptable if the
resolving power in the second scanning direction would be lower
than that in the first scanning direction, the data processing for
the read pixel data is performed by thinning out the pixel data in
the transverse direction which is the second scanning direction,
namely, by thinning out the scanning position by one or more pixel
data. This permits the processing of the pixel data at a high
speed, which follows.
Description will be made of variants of examples.
In the above description on the examples, the scanning method is
adopted in which laser beams are scanned for reading using the
vibration mirror 22 in the instrumentation subunit 7. There may
also be used the method using a polygonal mirror or the scanning
method in which the direction of the axis of light is changed by
using the optical characteristics such as refraction, interference
and so on. The use of an image sensor or an array sensor as the
optical sensor for detecting the fluorescence emitted for scanning
by the exciting light applied to the migration segment permits part
of the reading scanning to be performed electronically, thereby
simplifying a scanning mechanism by scanning laser beams for
reading by scanning.
FIG. 7 is a diagrammatic representation of an outline of the
construction showing the essential portion in which a
one-dimensional sensor of the image sensor of a semiconductor
device as the optical sensor for the light-receiving section for
detecting fluorescence from the migration segment. As shown in FIG.
7, fluorescence 13 emitted upon receipt of light for excitement
from the light source 21 is condensed by the condenser 46 and led
to an image sensor 48 of a static induction transistor type after
transmitting through the optical filter 47, thereby converting the
fluorescence into electrical signals. The image sensor 48 of the
static induction transistor type is suitable for receiving a slight
degree of fluorescence caused by a noise by a dark current as low
as several orders. Further, a one-dimensional image sensor of a CCD
sensor cooled may provide a light-receiving section of a likewise
high sensitivity.
As described hereinabove, by using the one-dimensional image sensor
as the optical sensor of the light-receiving section to be used for
reading the fluorescence, the scanning in the main scanning
direction can be executed electronically, thereby simplifying the
scanning mechanism for reading. The use of the one-dimensional
sensor can allow the scattered light 13a of the exciting light
emitted on the glass surface of the migration section 5 to separate
the component transmitted through the optical filter 47 from the
component of the fluorescence 13 from the fluorescent substance in
a physical position, thereby extracting only the component of the
fluorescence 13 effectively and improving a ratio of the signal for
detecting the signal for the fluorescence intensity pattern to
noise. This specifically can improve the limit of detecting the
fluorescent signal by one order or more.
FIG. 8 is a diagrammatical representation for describing the
reading method for reading the electrophoresis pattern by using the
electrophoresis pattern reading system of fluorescent type
according to an embodiment of the present invention. As shown in
FIG. 8, this embodiment uses the electrophoresis pattern reading
system of fluorescent type composed of plural electrophoresis units
1a, 1b, . . . , 1n and one reading unit 6 (instrumentation subunit
7). The migration sections 5 are mounted to the plural
electrophoresis units 1a, 1b, . . . , 1n, and each of the migration
sections 5 is subjected to electrophoresis for an analyzing sample.
After electrophoresis, the migration sections 5 are removed from
the electrophoresis units 1a, 1b, . . . , 1n and they are mounted
in order to the instrumentation subunit 7 of the reading unit 6 for
reading the electrophoresis pattern. Although not shown in FIG. 8,
the data processor unit (8; FIG. 1) is connected to the
instrumentation subunit 7 of the reading unit 7, so that the data
processor unit processes data in order for analysis of the sample
from the electrophoresis pattern read. In this case, a conventional
electrophoresis device can be used as an electrophoresis unit and
the electrophoresed gel is mounted to the reading unit 6 and the
pattern of bands on the gel can be read.
As described hereinabove, electrophoresis starts up after preparing
for a sample to be analyzed and setting it to each of the migration
sections 5 which in turn are mounted to the electrophoresis units
1a, 1b, . . . , 1n. Electrophoresis requires about 5 to 8 hours.
After electrophoresis has been finished, the migration section 5 is
removed from the electrophoresis unit and mounted to the
instrumentation subunit 7 of the reading unit 6 to measure the
distribution of the fluorescent substance. The migration section 5
is placed on the reading base 7c, the lid 7a is closed, and the
switch of the display-operating panel 7b is pressed to generate
laser output from the window 7d for reading and to scan the
migration section 5 set. The reading time required is about 0.5
hours for reading a 300 mm.times.400 mm region.
Therefore, each researcher can implement electrophoresis by
exclusively using the electrophoresis unit and read the
electrophoresis pattern using the common reading unit. This
arrangement can effectively use each of the units without occupying
the expensive electrophoresis system of
fluorescent type as a whole for a long period of time.
The embodiments according to the present invention as described
hereinabove including the variants and applications can be
summarized as follows:
(1) The electrophoresis pattern reading system of fluorescent type
is comprised of a separate combination of the electrophoresis unit
and the reading unit. Electrophoresis is performed by using the
migration unit comprised of the gel functioning as a base for
electrophoresis and the gel-supporting body for supporting the gel
and mounting the migration unit to the electrophoresis unit. After
electrophoresis has been finished, the migration unit is removed
from the electrophoresis unit and mounted to the reading unit. In
the reading unit, the gel in the migration unit is irradiated with
light for exciting the fluorescent substance and the fluorescence
emitted from the fluorescent substance of the sample on the gel is
received to read the electrophoresis pattern. The electrophoresis
unit is provided with the power supply for applying the migrating
voltage for electrophoresis to the gel into which the sample
labeled with the fluorescent substance is poured.
(2) One reading unit can be provided with a plurality of the
electrophoresis units. In other words, the electrophoresis units
can be provided in the number more than the reading units.
(3) In reading the electrophoresis pattern by using the
electrophoresis pattern reading system of fluorescent type, the
migration unit comprised of the gel-supporting body for supporting
the gel is mounted to the electrophoresis unit and, after
electrophoresis has been finished, the migration unit is removed
from the electrophoresis unit and mounted to the reading unit,
thereby reading the electrophoresis pattern.
(4) The plural migration units electrophoresed by plural different
electrophoresis units are mounted in order to the common reading
unit and each of the electrophoresis patterns of the gel in the
migration unit is read.
(5) The reading unit is provided with a spot light source for
generating light for exciting fluorescence of the fluorescent
substance, a scanning section (a vibration mirror, a lens and so
on) for scanning the light from the spot light source in the
direction nearly parallel to the direction electrophoresis by
radiation upon the gel, and a reading section having a
light-receiving subsection for receiving the fluorescence from the
fluorescent substance.
(6) The light-receiving subsection is composed of a light-receiving
unit of a one-dimensional image sensor. The direction in which the
light is received is nearly parallel to the direction of
electrophoresis.
(7) When the direction nearly parallel to the direction of
electrophoresis is set as a first direction and the direction
perpendicular to the direction of electrophoresis is set as a
second direction, the electrophoresis pattern is read by making the
pixel size for reading the gel after electrophoresis longer in the
first direction than in the second direction.
(8) When the direction nearly parallel to the direction of
electrophoresis is set as a first direction and the direction
perpendicular to the direction of electrophoresis is set as a
second direction, data of a pattern of distribution of the
fluorescent substance is obtained by reading the electrophoresis
pattern while thinning out the distance equivalent of one pixel or
more in the second direction.
(9) In irradiating the gel with light from the spot light source,
light is used for the reading unit, whose width in the direction
perpendicular to the direction of electrophoresis is extended by
equal times to ten and several times on the basis of the reading
width of the irradiated area in the direction parallel to the
direction of electrophoresis.
(10) The light-receiving subsection is composed of a
light-receiving unit of the image sensor of a static induction
transistor type, and the direction in which the light is received
is nearly parallel to the direction of electrophoresis.
(12) The data from the reading unit may be processed after being
transmitted to the data processor unit provided in a location away
through a data communication path.
Although the present invention has been specifically described by
way of examples, it should be noted that the present invention is
understood to be not restricted to the examples and to include
variations and modifications within the scope of the invention
without departing therefrom.
As have been described hereinabove, the present invention permits
an efficient provision of image of distribution of the fluorescent
substance without occupying the expensive electrophoresis system of
fluorescent type as a whole in analyzing the structure of DNAs by
the fluorescence method, for example, by allowing each researcher
to use the electrophoresis unit for exclusive purposes and to share
the common reading unit with other researchers.
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