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BACKGROUND OF THE INVENTION
1. Field Of The Invention
The present invention relates to a probe array for examining various
targets at a time when DNA's, RNA's or proteins are substances to be
detected. In particular, it relates to a DNA probe array for DNA detection
by hybridization which has recently been the object of attention.
2. Description Of The Related Art
With the advance of the Human Genome Program, there is a strong movement to
diagnose diseases and understand life phenomena by understanding living
bodies on the basis of DNA. Investigation on the profile of expressed
genes is effective in understanding life phenomena and investigating the
actions of genes. As an effective means for investigating the gene
expression profile, a DNA probe array obtained by fixing a large number of
DNA probes for different kinds of the DNA probes separately on a solid
surface, or a DNA chip has begun to be used. For producing the chip, there
is, for example, a process of synthesizing an oligomer with a designed
sequence base by base in each of a large number of enclosed cells by
employing a photochemical reaction and a lithography widely used in the
semiconductor industry (Science 251, 767-773 (1991)), or a process of
spotting DNA probes one by one, respectively, to different cells to make a
probe array.
SUMMARY OF THE INVENTION
The key point of the DNA probe arrays is that they are inexpensive and easy
to make for any type of probe. The prior art is disadvantageous in this
point. The mass production of the DNA probe arrays and the DNA chips
requires much labor and time and therefore they are very expensive.
Particularly when the density of the cells where the probes are fixed,
respectively, in a probe array is large, it is getting difficult to
produce the probe array at a low cost. If the size of each cell for a
probe species is large, such a probe array is easy to produce but is
disadvantageous, for example, in that the volume required for a detection
reaction and hence the amounts of samples becomes large as a whole, and in
that measurement with the probe array requires much time and does not have
high sensitivity.
The present invention was made for removing the above disadvantages, and an
object of the present invention is to provide a process which permits easy
production of a desired DNA probe array (consisting of DNA probes having
desired sequences) with a high density and entails a low production cost.
A DNA probe array has various DNA probes fixed in separate cells,
respectively, and target DNA fragments are detected by hybridization with
the DNA probes. The target DNA fragments are labeled with a tag such as a
fluorophore prior to the hybridization and fluorescence or luminescence
and the like are used for the detection of the DNA fragments. The probe
array has been used although the density of cells having probes therein is
not so high. In the conventional probe array, however, the probes are
fixed on each cell that spatially divides the surface of a membrane or the
like, and the whole area of the probe array is about 10 cm.times.5 cm or
larger.
On the other hand, the size of the newly developed DNA probe array is about
1 cm.times.1 cm or less although the number of cells holding DNA probes is
very large. The high density probe array is constructed on a solid support
such as glass or Si wafer which, together with the high density, is good
to reduce the amount of samples consumed for the hybridization. For
example, the size of one cell is as small as 0.1 mm.times.0.1 mm, which
should be compared to the conventional size of 5 mm.times.5 mm. This high
density DNA probe array is called a DNA chip. The DNA chip has many cells
holding various probes on the surfaces, respectively. It is used for
analyzing multiple components in a sample.
In the analysis procedure, at first, all the components in the sample are
labeled with tags such as fluorophores or enzymes. They are placed on the
DNA chip for hybridization. If the sample has a component being hybridized
with probes, the component is held on the corresponding cell. By detecting
fluorescence, the position of the cell emitting fluorescence can be
determined. From the positional information of the fluorescence emitting
cell, the probe species being hybridized with the sample components can be
determined. Although the detection and identification of the hybridized
position are easy, the production of DNA chips is not so easy because the
probe species required for research or testing are changing case by case.
In addition, the mass production of the chips is labor intensive and
expensive. This is mainly due to the high density production cells in a
chip. If the density of cells is as low as the conventional one, the
production is relatively easy. The present inventors have found that if
the cells can be separately produced and then assembled to make a probe
array, the production becomes easy even if the probe components should be
changed. The change will be carried out by selecting the cells having
probes thereon.
In order to achieve the above object, in the present invention, solid
pieces holding probes, respectively, are composed of small particles so as
to be movable, and the small particles are sparsely arrayed and then moved
to produce a probe array having a dense structure. First, various DNA
probes are prepared by synthesis. These DNA probes are fixed on the
surfaces, respectively, of small particles (beads), so that the kinds of
the DNA probes may be different on the different small particles. A large
amount of the DNA probes can be fixed on solid surfaces, respectively, by
utilizing a method utilizing the combination of biotin and avidin, a
method of fixing DNA probes on Au (gold) surfaces through a SH group
(Biophysical Journal 71, 1079-1086 (1996)), a method of fixing DNA probes
on glass surfaces (Analytical Biochemistry 247, 96-101 (1997)), a method
of fixing DNA probes on an element matrix of acrylamide gel applied on
glass surfaces (Proc Natl. Acad. Sci. USA 93, 4913-4918 (1996)), or the
like.
The various small particles holding the DNA probes on their surfaces are
placed in a holder for examination in a predetermined order so as to
indicate the kinds, respectively, of the DNA probes, or the small
particles are arrayed and fixed on a solid surface in a predetermined
order so as to indicate the kinds, respectively, of the DNA probes,
whereby the probe array is produced. The small particles are spherical
such as beads. As to their sizes, their diameters range from several
micrometers to 1 mm, depending on purpose of use. The small particles may
be square, discoidal or the like, depending on purpose of use. For usual
examinations, spherical beads with a diameter of 0.1 mm to 0.2 mm can be
easily used.
The beads holding the probes, respectively (hereinafter referred to also as
"probe beads") are supplied together with a solvent one by one to a groove
for producing probe array. Necessary kinds of probes can easily be arrayed
in the groove, depending on examinations in which they are used. Since the
beads holding the probes, respectively, can be prepared at a low cost, the
probe array itself can be produced at a low cost. These beads having the
probes attached thereto which have been arrayed in the groove are used
after being placed in a capillary for examination or a cell having a
narrow space. The employment of a capillary as a probe array holder is
advantageous in that the amounts of sample DNA's to be examined can be
reduced. It is advantageous also in that the capillary can easily be
connected to a solvent-introducing system.
On the other hand, when solid particles having the probes, respectively,
fixed thereon are made distinguishable from one another, there is such an
advantage that the trouble of arraying the probes by a definite method can
be saved.
As explained above, according to the present invention, an arbitrary probe
array can be produced easily at a low cost. Moreover, a probe array which
reduces the amount of reagents and permits easy injection of the reagents
and easy washing can be provided by its construction in a capillary.
Typical examples of the present invention are summarized below. In the
typical examples, of the present invention, there is used at least one
probe array obtained by arraying particles having various probes,
respectively, fixed thereon (hereinafter referred to also as "probe
particles") in a definite order in a holder. A plurality of capillaries or
grooves packed with various kinds, respectively, of probe particles are
arrayed in parallel, and one of the particles contained in each capillary
or groove is injected into another capillary or groove to produce a probe
array in which the various kinds of probe particles are arrayed in a
constant and definite order. Various fluorophore-labeled DNA's are
measured at the same time by attaching various probes to particles,
respectively, of different sizes. The present invention permits easy
production of a probe array composed of various fixed DNA probes, and
provides a probe array for detecting various DNA's which is composed of
various fixed arbitrary DNA probes.
There are summarized below characteristics of the DNA probe array for
examining many items of the present invention and a process for production
thereof.
(1) A probe array for examining many items which comprises an array of a
plurality of particles having probes, respectively, fixed thereon, said
probes being capable of binding to different target substances to be
examined (e.g. DNA's, proteins or the like), respectively.
(2) A probe array for examining many items which comprises a plurality of
particles having probes, respectively, fixed thereon, said probes being
capable of binding to different target substances to be examined,
respectively, wherein said particles are arrayed in a line in a
predetermined order, and said order is such that the arraying positions of
said particles correspond to the kinds, respectively, of said probes fixed
on said particles.
(3) A probe array according to the item (2), wherein the sizes or shapes of
said particles holding said probes correspond to the kinds, respectively,
of said probes fixed on the surfaces of said particles.
(4) A probe array according to the item (2), wherein said particles holding
said probes are labeled with different dyes or fluorophores, respectively,
depending on the kinds of said probes held by the particles.
(5) A probe array according to the item (2), wherein said probes are
arrayed to form a layer on a two-dimensional plane.
(6) A probe array according to the item (2), wherein said particles are
one-dimensionally arrayed, and the order of particles (therefore the
probes), is predetermined.
(7) A probe array according to the item (2), wherein said particles are
held in a container having a transparent window.
(8) A probe array according to the item (2), wherein said particles holding
said probes are held in a capillary.
(9) A probe array according to the item (2), wherein said particles holding
said probes are held in a groove formed on a flat solid surface or a
groove formed between two flat surfaces.
(10) A probe array according to the item (2), wherein said particles
holding said probes are two-dimensionally arrayed at a predetermined
position(s) by arraying a plurality of capillaries holding said particles
holding said probes, or by arraying said particles holding said probes in
grooves formed on a flat surface.
(11) A probe array according to the item (2), wherein said particles
holding said probes are held in a gel-like substance.
(12) A probe array for examining many items which comprises a plurality of
particles having probes, respectively, fixed thereon, said probes being
capable of binding to different target substances to be examined,
respectively, wherein said particles are arrayed so that characteristics
of said particles may correspond to the kinds, respectively, of said
probes.
(13) A probe array according to the item (12), wherein the sizes or shapes
of said particles holding said probes correspond to the kinds,
respectively, of said probes fixed on the surfaces of said particles.
(14) A probe array according to the item (12), wherein said particles
holding said probes are labeled with different dyes or fluorophores,
respectively, depending on the kinds of said probes held by the particles.
(15) A probe array according to the item (12), wherein said probes are
arrayed to form a layer on a two-dimensional plane.
(16) A probe array according to the item (12), wherein said particles are
one-dimensionally arrayed.
(17) A probe array according to the item (12), wherein said particles are
held in a container having a transparent window.
(18) A probe array according to the item (12), wherein said particles
holding said probes are held in a capillary.
(19) A probe array according to the item (12), wherein said particles
holding said probes are held in a groove formed on a flat solid surface or
a groove formed between two flat surfaces.
(20) A probe array according to the item (12), wherein said particles
holding said probes are two-dimensionally arrayed at a predetermined
position(s) by arraying a plurality of capillaries holding said particles
holding said probes, or by arraying said particles holding said probes in
grooves formed on a flat surface.
(21) A probe array according to the item (12), wherein said particles
holding said probes are held in a gel-like substance.
(22) A process for producing a probe array which comprises a step of fixing
probes on the surfaces, respectively, of particles, and a step of arraying
a plurality of said particles having said probes fixed thereon.
(23) A process for producing a probe array according to the item (22),
wherein said particles are arrayed on a straight line in a predetermined
order so as to indicate the kinds, respectively, of said probes fixed on
said particles.
(24) A process for producing a probe array according to the item (22),
wherein the plurality of said particles having said different probes,
respectively, fixed thereon are transferred to a groove or probe array
holder for arraying said particles, by using a plurality of capillaries or
grooves for transferring said particles having said probes fixed thereon,
and said particles are arrayed on a straight line in a predetermined order
so as to indicate the kinds, respectively, of said probes fixed on said
particles.
(25) A process for producing a probe array which comprises a step of fixing
probes on the surfaces, respectively, of particles, and a step of arraying
a plurality of said particles having said probes fixed thereon, as a
mixture on a plane, wherein the kinds of said probes fixed on said
particles are distinguished by the shapes or sizes or any other physical
or chemical properties of the particles or fluorophores labeling said
particles, respectively.
(26) A process for producing a probe array according to the item (25),
wherein the plurality of said particles having said different probes,
respectively, fixed thereon are transferred at the same time to a groove
or probe array holder for arraying said particles, by using a plurality of
capillaries or grooves for transferring said particles having said probes
fixed thereon.
(27) A process for producing a probe array according to the item (25),
wherein said particles having said probes fixed thereon are held in
different particle reservoirs for the different kinds of said probes,
supplied one from each reservoir to a groove for arraying said particles,
through a capillary or a groove to be arrayed, and transferred to a probe
array holder while maintaining the array, whereby a probe array is
produced.
(28) A process for producing a probe array according to the item (25),
wherein said particles having said probes fixed thereon are held in
different particle reservoirs for the different kinds of said probes,
supplied one at a time from each reservoir to a groove for arraying said
particles, through a capillary or a groove to be arrayed, and transferred
to a probe array holder by means of an electric force while maintaining
the array, whereby a probe array is produced.
(29) A process for producing a probe array according to the item (25),
wherein said particles having said probes fixed thereon are held in
different particles reservoirs for the different kinds of said probes,
supplied one at a time from each reservoir to a groove for arraying said
particles, through a capillary or a groove to be arrayed, and transferred
to a probe array holder by means of a solution flow while maintaining the
array, whereby a probe array is produced.
(30) A method for detecting target substances to be examined which bind to
probes, respectively,, held on the surfaces, respectively, of particles,
said method comprising a step of labeling said target substances with a
fluorophore or a material emitting phosphorescence or any tag and a step
of irradiating said particles with light (laser beams), followed by
optical detection of the fluorescence or phosphorescence emitted.
(31) A method for detecting target substances to be examined according to
the item (30), wherein a light source (laser beams) is scanned along a
straight line on which said particles are arrayed, and said fluorescence
or phosphorescence emitted from tags is detected with an optical sensor.
(32) A method for detecting target substances to be examined according to
the item (30), wherein said light (laser beams) is cast along a straight
line on which said particles are arrayed, and said fluorescence or
phosphorescence emitted from each of the positions at which said
particles, respectively, are arrayed, is detected.
(33) A method for detecting target substances to be examined according to
the item (30), wherein a pattern of scattering of said light (laser beams)
by said particles is obtained by the irradiation with said light (laser
beams), fluorescence or phosphorescence emitted from said target
substances binding to said probes fixed on said particles is detected, the
shapes of said particles or fluorescences emitted by the fluorophores
labeling said particles, respectively, are detected, whereby the amounts
of said target substances attached to said probes are determined.
(34) A method for detecting target substances to be examined according to
the item (30), wherein said particles are detected while being allowed to
flow.
(35) A method for detecting target substances to be examined according to
the item (30), wherein said target substances attached to said probes
fixed on said particles are measured as fluoroscopic images.
(36) A method for detecting target substances to be examined according to
the item (30), wherein said particles are measured as two-dimensional
images.
(37) A process for producing a probe array which comprises a first step of
fixing probes on the surfaces, respectively, of particles, a second step
of dividing a plurality of said particles having said probes fixed
thereon, into groups and arraying particles in each group in a compartment
on a solid surface, and a third step of reacting said probes fixed on said
particles with target substances to be examined, in said compartments,
wherein the state of distribution of said particles in the first step is
different from the state of distribution of said particles in said
compartments where said third step is carried out.
(38) A probe array which comprises an array of a plurality of small
particles having probes, respectively, thereon, wherein said probes are
arrayed one-dimensionally or two-dimensionally, and wherein an order of
arrangement of small particles having probes is predetermined, or
positions of arrangement of small particles having probes are
predetermined.
(39) A probe array according to the item (38), wherein marker particles are
placed between the small particles having different kinds of probes. The
marker particles are labeled with fluorophores different from the
fluorophores labeling the small particles, and the positions of the marker
particles on the probe array are reference positions for discriminating
the species of the probes on the small particles. (40) A probe array
according to the item (38), wherein species of the probes on each of the
small particles are different from each other.
(41) A probe array according to the item (38), wherein the small particles
include particles having the same species as the probes.
(42) A probe array according to the item (38), wherein each of the probes
is capable of binding to DNA, RNA or a protein.
(43) A probe array according to the item (38), wherein the small particles
are spherical beads with an outer diameter of 1 .mu.m to 10 .mu.m.
(44) A probe array according to the item (38), wherein the small particles
are spherical beads with an outer diameter of 10 .mu.m to 100 .mu.m.
(45) A probe array according to the item (38), wherein the shape of the
small particles is a cubic shape.
(46) A probe array according to the item (38), wherein the shape of the
small particles is a cylindrical shape.
(47) A probe array according to the item (38), wherein the small particles
are made of glass or plastics
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram showing a production process of a DNA probe array of a
first example of the present invention and an examination apparatus using
the DNA probe array.
FIG. 2A is a plan view of a plate having fine grooves which is a part of a
jig for producing a probe array using small particles as a probe-fixing
medium, in the first example of the present invention, and
FIG. 2B is a cross-sectional view of the jig for producing the probe array.
FIG. 3 is a diagram showing an example of a probe array holder in the first
example of the present invention.
FIG. 4 is a schematic diagram showing an example of an apparatus using a
DNA probe array, in which one of the linear probe array and a light source
is scanned in relation to the other, in the first example of the present
invention.
FIG. 5 is a schematic diagram showing an example of an apparatus using a
DNA probe array, in which laser beams are cast along a linear array of
fine particles, in the first example of the present invention.
FIG. 6 is a schematic diagram showing an example of an apparatus using a
DNA probe array, in which light is cast on the whole of a region where the
probe array is present, in the first example of the present invention.
FIG. 7 is a diagram showing an example of results which are obtained by
means of the structure shown in FIG. 6 using a small particle type probe
array, and which are outputted in a monitor, in the first example of the
present invention.
FIG. 8A is a plan view of a jig for introducing small particle holding
probes, respectively, into a two-dimensional probe array holder (a holder
having probes arrayed in a two-dimensional area) 34, in a second example
of the present invention, and
FIG. 8B is a cross-sectional view of the jig.
FIG. 9 is a schematic illustration of an example of a method for
introducing small particles holding probes, respectively, into a groove
for array from small particle reservoirs, in a third example of the
present invention.
FIG. 10 is a schematic diagram showing the structure of an examination
apparatus in which samples are detected by detection of many colors by the
use of a two-dimensional probe array, in a fourth example of the present
invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The examples of the present invention are explained below in detail with
reference to the drawings.
FIRST EXAMPLE
FIG. 1 is a diagram showing a production process of a DNA probe array of
the first example of the present invention and an examination apparatus
using the DNA probe array. Spherical plastic particles (beads) 1
(diameter: 0.2 mm) holding avidin 70 on their surfaces are prepared. The
precision of diameter of the small particles 1 is 5%. A DNA probe obtained
by PCR amplification by using biotin-attached primers is separated into
individual strands, and the resulting biotin 71-attached DNA probe 2 is
combined with the small particle holding avidin. Thus, each kind of DNA
probe is captured by the small particles, respectively, whereby a
plurality of groups of small particles attached with probe 3 are formed.
Needless to say, synthetic DNA strands may be used as DNA probes. In this
case, the DNA probes can be directly attached to solid (small particle)
surfaces, respectively, without the combination of biotin and avidin.
Methods for attaching DNA probes to solid surfaces, respectively, are
described, for example, in the above-mentioned references (Biophysical
Journal 71, 1079-1086 (1996) and Analytical Biochemistry 247, 96-101
(1997)). The following method may also be employed: a specific sequence of
nucleotides all of which are the same, for example, TTTTT . . . TT is
fixed on each solid (small particle) surface, and each DNA oligomer having
a poly A strand is hybridized with, the nucleotide sequence to be bonded
thereto by binding between complementary strands, whereby the DNA oligomer
is introduced onto the solid (small particle) surface.
The thus prepared small particles holding the probes, respectively, are
arrayed one by one in a transparent capillary tube (a probe array holder
7) to obtain a probe array 4. The kind of the probe held on the surface of
each small particle can be known from the place of this small particle in
the order of the small particles arrayed in the capillary tube (the probe
array holder 7). Therefore, after hybridization between the DNA probes 2
and sample DNA's having a fluorophore tag 8 attached thereto (numeral 9
shows a sample DNA fragment captured by the small particle 1 by binding
between complementary strands), followed by irradiation with light, the
kinds of DNA's in a specimen can be known from the fluorescence emitted.
Sample DNA's having a fluorophore tag 8 attached thereto are injected into
the capillary tube (the probe array holder 7) containing the probe array 4
composed of probes 1, 2, . . . , n, through the sample inlet 5 of the tube
in the sample inflow direction 6 to hybridize the DNA probes 2 with the
sample DNA's having the fluorophore tag 8 attached thereto. Then, the
probe array holder 7 is set on the movable table (not shown) of the
apparatus and laser beams from a laser source 11 are focused by a lens 12
and cast on the moving probe array holder 7. Numeral 5" shows a sample
outlet. The color (wavelength) of fluorescence emitted from sample DNA's
having the fluorophore tag 8 attached thereto which have been captured by
the small particles 1 at positions irradiated with the laser beams is
selectively detected by a filter 12 and the photodetector of a CCD (charge
coupled device) camera 13 which detects the fluorescence from a direction
substantially perpendicular to the direction of the laser beam
irradiation. The fluorescence signals thus detected are displayed in real
time on a monitor 17. By a data processing unit 15, they are processed to
obtain the fluorescence intensity emitted from the particles. The results
are displayed on a display unit 16. The axis of abscissa of an output
pattern displayed in the monitor 17 refers to the positions of the small
particles 1 arrayed in the capillary tube (the probe array holder 7) and
hence the kinds of the probes, and the axis of ordinate refers to
fluorescence intensity which indicates the presence of the sample DNA
fragment bonded to any of the probes by binding between complementary
strands. A controller 14 controls the movement of the abovementioned
movable table, the incorporation of signals from the CCD camera 13 and the
transmission of signals to the data processing unit 15 and the monitor 17.
Whether an objective base sequence is present in any of the sample DNA's
(DNA fragments) or not can be judged from the output in the monitor 17 or
the display unit 16.
It is also possible to carry out the detection by allowing beads to flow
together with a solvent instead of moving the capillary tube holding the
beads.
Although the fluorophore tag is attached to the sample DNA's (DNA
fragments) in the above explanation, it is also possible to attach
different fluorophore tags to the probes 1, 2, . . . , n, respectively,
instead of attaching the fluorophore tag to the sample DNA's (DNA
fragments). In this case, the filter 12 is composed of a many-color filter
capable of selecting a plurality of wavelength regions, or wavelengths of
fluorescence are separated by using optical prism, a diffraction grating
or the like.
Next, a process for producing a probe array using small particles as a
probe-supporting medium in the First Example is explained below.
FIG. 2A is a plane view of a plate having fine grooves which is a part of a
jig for producing a probe array using small particles as a
probe-supporting medium, and FIG. 2B is a cross-sectional view of the jig
for producing the probe array. Small particles 1 having probes,
respectively, attached thereto can be arrayed by means of a device for
arraying fine particles by the use of fine grooves. The plate 18 having
fine grooves for arraying small particles which is shown in the plan view
in FIG. 2A is used after being equipped with the transparent cover 23
which is shown in the cross-sectional view in FIG. 2B. The plate 18 has
the following grooves formed thereon: a plurality of grooves 19 for
arraying small particles holding different kinds, respectively, of probes
in different grooves, respectively; a groove for arraying various
probe-holding small particles (a fine groove for producing a probe array)
20 which intersects (is perpendicular to) the grooves 19; and grooves for
outlet of solution 21 which discharge a solution. The maximum widths and
maximum depths of the grooves 19 and the groove 20 are adjusted to values
at which two small particles cannot enter the same groove (namely, the
maximum widths and maximum depths satisfy the condition 1 that they should
be less than 2R when the diameter of the small particle is taken as R).
The maximum width and maximum depth of the grooves 21 are adjusted to
values at which the small particles cannot pass the grooves 21 (namely,
the maximum width and maximum depth satisfy the condition 2 that they
should be less than R when the diameter of the small particle is taken as
R). That is, the small particles 1 can pass through capillaries formed by
the transparent cover 23 and the fine grooves 19 and 20 formed on the
plate 18. As the forms of section of the grooves 19, 20 and 21, any forms
may be employed so long as they satisfy the above conditions 1 and 2.
In each of the fine grooves 19, small particles holding the same kind of
probes, respectively, are arrayed at random distance intervals. Thus,
small particles holding different kinds of probes are divided into groups
which are held in the fine grooves 19, respectively. For example, small
particles each holding a probe 1 are arrayed in the first groove 19-1
among the grooves 19, small particles each holding a probe 2 in the second
groove 19-2, . . . , small particles each holding a probe n in the n-th
groove 19-n. Various probe-holding small particles are arrayed in the
groove 20 perpendicular to the plurality of the grooves 19. The small
particles 1 having probes, respectively, attached thereto are introduced
into the groove 20 by a solution flow or an electric field. Since two
small particles cannot enter the groove 19 sideways because of their size,
each small particle holding various probes, respectively (probe particles)
is arrayed at an intersection of the array of plurality of the fine
grooves 19 and the groove for arraying probes 20. The grooves 21 after the
intersection are so thin that the particles cannot go forward. At this
point of time, the distances between two particles are random. The
particles arrayed in the groove 20 are introduced into a probe array
holding capillary (a probe array holder 7) (inside diameter: 0.3 mm) by a
solution flow or an electric field in a direction perpendicular to the
grooves 19, namely, along the groove 20 for making an array, to be closely
arrayed. Numeral 22 shows a probe array holder connector (a guide device
for connecting the probe array and solution inlet as well as outlet) which
connects a device for arraying small particles (23 and 20) and the probe
array holder 7. Numeral 5' shows a stopper tube. Particles having DNA
probes, respectively, fixed thereon which are to be used are supplied to
particle reservoirs (see 38 in FIG. 9) communicating with the grooves 19,
respectively. The arraying order of the particle reservoirs holding the
particles having the probes fixed thereon corresponds to the arraying
order of the probes in the groove 20 and the probe array holding
capillary. It is also possible to insert a marker between the particles
having the probes fixed thereon, for making it easy to know the order.
Next, there is explained an example of the probe array holder 7 which holds
probes in a capillary tube, in the First Example.
FIG. 3 is a diagram showing an example of the probe array holder in First
Example. Small particles having probes, respectively, attached thereto are
held in a probe array holder 7 (a capillary) having a sample inlet and a
sample outlet. A terminal adaptor 24 (a terminal adaptor for the capillary
holder and the solution outlet) is attached to each end of the probe array
holder 7 through a stopper tube 5' and a probe array holder connector (a
guide device for connecting the probe array and solution inlet as well as
outlet) 22 in order to prevent the outflow of the small particles 1.
Needless to say, the adaptor 24 is attached I after introducing the small
particles into the capillary (the probe array holder 7).
DNA samples to be examined are labeled with a fluorophore (in this case,
Cy-5 (maximum emission wavelength: 650 nm) is used) and introduced
together with a solvent into the capillary holding the probe array (the
probe array holder 7) to cause hybridization between the DNA samples and
the probes. After target DNA's are captured on some of the probes
respectively, by the hybridization, the excess DNA samples are washed
away, followed by detection of fluorescence. The linear probe array is
advantageous in that the probe array holder 7 holding the probe array is
easy to scan mechanically, resulting in low consumption of the samples.
The fluorophore tag includes Texas Red (maximum emission wavelength: 615
nm), fluorescein isocyanate (maximum emission wavelength: 520 nm), etc. In
addition to these fluorophore tags, tags capable of emitting
phosphorescence may be used. After the unreacted DNA is washed away, the
residue is introduced into a measuring apparatus. The measuring apparatus
is composed of a laser for excitation and a fluorescence detector. A large
number of the small particles are irradiated at the same time by scanning
laser beams along the capillary tube (the probe array holder) or casting
laser beams on the interior of the capillary tube along the tube, and the
resulting fluorescence images are detected. It is also possible to
introduce the small particles one after another into an irradiation
portion by moving the capillary tube. In addition, the measurement may be
carried out while jetting the solvent and the beads (small particles)
through a nozzle, as in a cell sorter.
Next, an example of an apparatus using a DNA probe array is explained
below.
FIG. 4 is a schematic diagram showing an example of an apparatus using a
DNA probe array, in which one of the linear probe array and a light source
are scanned in relation to the other. In FIG. 4, the same structure as
shown in FIG. 1 is employed, and laser irradiation is carried out as
follows: a laser irradiation position and a detector 13 are fixed and a
probe array holder 7 holding probes is moved in relation to them; or the
probe array holder 7 is fixed, and the laser irradiation position and the
detector 13 are moved in relation to the probe array holder 7. As the
detector 13, a photomultiplier or a lens-equipped cooled CCD camera is
used. Fluorescence is detected from a direction substantially
perpendicular to the laser irradiation direction.
FIG. 5 is a schematic diagram showing an example of an examination
apparatus using a DNA probe array, in which laser beams are cast along a
linear array of fine particles. In FIG. 5, laser beams from a laser source
11 are cast along the axis of a probe array holder 7 in the direction of
said axis. Fluorescence emitted by a fluorophore tag is condensed in a
microlens-array (Cell Fock Lens (a trade name of Nippon Sheet Glass Co.,
Ltd.)) 25 and projected on a line sensor (a CCD line sensor) 27 through a
filter 26. The other constituents shown in FIG. 5 are the same as those in
the structure shown in FIG. 1. Although the structure shown in FIG. 5 is
effective, it can be employed only when the small particles are
transparent.
FIG. 6 is a schematic diagram showing an example of an examination
apparatus using a DNA probe array, in which light is cast on the whole of
a region 30 where the probe array is present (a region where small
particles having probes are held and irradiated with laser). Although the
probe array is one-dimensional in the structure shown in FIG. 6, a cooled
CCD area sensor or the like is used for fluorescence detection so that the
examination apparatus can be used also when the probe array is
two-dimensional. Laser beams from a laser source 11 change their course at
a mirror 28 and the region of irradiation with the laser beams is
one-dimensionally expanded by a beam expander 29, whereby the laser beam
is cast on the whole of the region 30 where small particles having probes
are held and irradiated with the laser, in a probe array holder 7. When a
two-dimensional photodetector such as a CCD area sensor is used,
two-dimensional fluorescence images are obtained and the kinds of probes
are known from the emission positions. The other constituents shown in
FIG. 6 | | |
