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BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for measuring the
length of a polynucleotide or biomolecular such as DNA, and is
particularly concerned with measuring the length of a not less than 50,
especially 100 kilobase long polynucleotide.
Such polynucleotide length measurement is one of the gene analysis means
useful in fields like medical chemistry, biochemistry and genetics.
In the past, measuring the base length (or molecular weight) of a long DNA
chain has been based on the use of gel electrophoresis migration which
provides electric fields in a certain direction. Polyacrylamide gel is
effective in separating DNA several bases to 2 kilobases long, and agarose
gel is in use for separating DNA 1 to 20 kilobase long.
In conventional electrophoresis using electric fields in a certain
direction, molecular sizes are separated in polyacrylamide gel or agarose
gel according to the spatial spread of the molecules. To be more specific,
the separation is based on whether spatially spread molecules pass through
gel meshes. Larger molecules do not pass but become elongated. After all,
these elongated molecules are separated according to their shorter
diameters. In this case, however, the difference in the size of these
elongated DNA molecules is no more than the difference in their seeming
lengths. This is why long DNA cannot be separated with molecular sieves.
Another conventional technique, pulsed field electrophoresis, varies
pulsatively the electric field direction for electrophoresis in agarose
gel in order to separate molecules tens of megabases to several megabases
long.
A more detailed description of measuring by separation not less than 100
kilobase long DNA may be found in PCT International Publication WO
84/02001 and Nucleic Acid Research, vol. 16, pp. 7563-7582, by B.W.
Birren, 1988. In this method, a mixture of giant DNA fragments subject to
measurement is injected into wells in an electrolyte-containing gel matrix
made of network polymers like agarose, the DNA is allowed to migrate as
the direction of electric fields is varied pulsatively, and the DNA is
separated according to the size. For instance, when migration is performed
for 95 hours in a 0.7% concentration agarose matrix, the direction of 2
V/cm electric fields being varied an angle of +106 degrees with the
migration direction every 30 minutes, it is possible to separate 3 to 10
megabase long DNA. The length of the DNA is considered to be measurable on
the basis of the bandwidth of the separated DNA with about 10% errors.
This pulsed field electrophoresis employs the dependence on elongated
molecule lengths of the time taken by varying the direction of the
orientation of the molecules by changing the direction of the electric
fields (see pp. 364-370, Jikken Igaku, vol. 5 by Hasegawa and Kikuchi
(1987)).
The above conventional technique makes it possible to obtain information on
the length of very large DNA with pulsed field separation. But the problem
is that the longer DNA, the longer time its separation takes. For example,
it generally takes 3 or 4 days to separate 3 to 10 megabase long DNA. This
conventional technique separates DNA according to the size, namely, the
length difference while DNA termini travel through the gel, popping out
from and popping in between network molecules composing the gel matrix.
Naturally, as the distance of the travel through the gel is shorter, the
separation worsens. Moreover, when migration is provided by high electric
field intensity, heating impairs separation. Thus it is unfeasible that
the conventional technique shortens the analysis time. Since the longer
DNA, the longer migration it requires, the technique is not suitable for
the practical use for separating not less than tens of megabases long DNA.
Besides, the fact that the longer DNA, the longer migration it needs, and
the larger bandwidth the separated DNA has deteriorates the precision of
the length separation.
Furthermore, the above conventional electrophoresis including pulsed field
electrophoresis separates and measures the measurement subject as a group
of different molecule sizes, using the difference between molecule sizes
and between electrophoresis rates. This deteriorates separation as the
molecules are longer, and increases the volume that groups of the same
molecule size occupy for a cause such as diffusion, resulting in
inefficient separation of long molecules like DNA.
Pulsed field electrophoresis, which varies the electric field direction,
uses radio isotope labels or ethidium bromide staining for the purpose of
detection. To ensure sufficient detection sensitivity, many molecules
(copies) of the same length are necessary and need to be prepared. The
resolution for DNA size provided by this technique is not very high
because of influences such as molecular heat diffusion, and the molecule
length measurement precision is low.
SUMMARY OF THE INVENTION
The present invention, therefore, has as its principal object the provision
of a method and apparatus for quickly measuring the base length of
biomoleculars having long molecule length like DNA with high measurement
precision.
Another object of the present invention is to provide the above-mentioned
method and apparatus particularly in the case where only a few molecules
(copies) of the same length are available.
A further important object of the present invention is to provide a process
and apparatus for quickly measuring the length of not less than 1 megabase
long DNA with high measurement precision.
To achieve the above objects, one process in accordance with the present
invention labels at least both termini of biomoleculars like DNA with
fluorophore, stretches these polymers with electrophoresis having electric
fields in a certain direction or with the outflow from apertures to shape
them into almost straight lines, transfers them to a buffer solution in
which there are no substances like gel causing polymers to migrate at a
certain rate irrespective of their length and measures their length.
Molecule length is measured by attaching fluorophore labels to both
termini of biomoleculars, exciting them with laser beams and then
detecting the fluorescence emitted by them.
How to label both termini of a biomolecular like DNA with fluorophore is
known. The migration rate of a biomolecular needs only to be less than
that rupturing the DNA chain. The upper limit depends on the length of the
biomolecular, and can be specified with experiments.
In electrophoresis having electric fields in a certain direction, polymers
of large molecule sizes are elongated in the electrophoresis medium, and
these polymers oriented in the form of straight lines are transferred to a
buffer solution to migrate with their orientation kept straight. The
fluorophore labeling both termini of such a biomolecular is irradiated
with a laser beam focused into not more than 1 .mu.m at the photodetecting
portion and emits fluorescence. The interval between the passing of the
fluorescence label at one terminus of the polymer through the portion to
which the laser beam has been directed and the passing of the fluorescence
label at the other terminus through this portion is measured. Since in a
buffer solution where there is no gel a constant electric field intensity
causes biomoleculars to migrate at a constant rate, it is possible to
calculate the distance between the fluorophore at one terminus of a
biomolecular and that at the other terminus from the time the fluorophore
labeling both termini of the biomolecular takes to pass through the
portion to which the laser beam has been directed and also from the
passing rate. This makes it possible to find out the molecule length and
base length of DNA. The molecule length can be measured for even 1
molecule (1 copy) by this process. The absence of diffusion, occurring in
a measurement of molecule length by molecular migration of a group of many
molecules, makes this process capable of measuring molecule length or base
length with high precision.
The above-mentioned biomolecular length is measured with length measuring
apparatus comprising means for orienting into straight lines the
biomolecular subject to this measurement and transferring it, a light
source for exciting the fluorophore labeling both termini of a
biomolecular, means for detecting the fluorescence emitted by the excited
fluorophore and means for measuring the interval between the detection of
fluorescence emitted from one terminus of the biomolecular and that of
fluorescence emitted from the other terminus. The means for orienting
biomoleculars into straight lines and transferring them has means for
causing electrophoresis and/or means for causing buffer liquid flow, the
biomolecular transfer path having an area up to 10 .mu.m, preferably up to
6 .mu.m, most preferably up to 1 .mu.m in diameter or an area equivalent
to these. The provision of an area not more than 10 .mu.m in diameter for
the biomolecular transfer path is for the purpose of measuring the length
of biomoleculars in terms of each molecule, that is, by drawing the DNA
molecules one by one.
To achieve the objects of the present invention, another process binds a
label to one terminus of DNA, fixes the other terminus to a matrix
physically or chemically, stretches the DNA by means of electric fields or
liquid flow and detects the position of the terminus bound to the label to
measure the length of the DNA. In this manner, the length of DNA is
measured for each molecule by stretching the DNA with an external force
and measuring the distance between the two termini of a molecule. Here, it
is not always necessary to stretch DNA fully and measure its absolute
length. Since fluctuations due to thermal vibration occur to longer DNA,
it is unfeasible to stretch it to the full. This difficulty can be
overcome by obtaining a calibration curve from DNA whose lengths are known
and then finding the length of the DNA subject to the measurement. To fix
DNA, only one terminus needs to be used so that the DNA can be stretched
with certainty. Materials like nitrocellurose film or nylon inducer
conventionally in wide use for hybridization cannot be used because they
fix DNA at more than one point.
For example, as shown in the scheme in FIG. 8, one terminus of DNA 101,
which is subject to length measurement and bound to a label 102 at the
other terminus, is bound to a particle 103 whose diameter is about 0.2
.mu.m or 10 .mu.m, preferably 0.2 .mu.m to 6 .mu.m, most preferably 0.2
.mu.m to 1 .mu.m. Then, this DNA bound to the particle 103 is led to a
matrix 104 having apertures 106 passable to DNA 101 but not to the
particle 103. The DNA can pass one of the apertures 106 except its portion
bound to the particle 103, which allows one terminus of the DNA to be
fixed in a specific position. Applying electric fields between electrodes
105a and 105b or using liquid flow causes DNA to migrate, fixes the
particle 103 at the mouth of the apertures and stretches the DNA. The
label is searched for along the apertures 106, and it is detected that the
label 102 is in a position corresponding to the particle 103 at the fixed
terminus.
To bind a particle to the DNA terminus to be fixed, such methods using a
bifunctional reagent as the carbodiimide method (this will be described
later in connection with the first example of the present invention), the
glutaric aldehyde method and the N-succinimidyl 3- (2-pyridylditio)
propionate method (SPDP) can be employed to bind a particle to primer
having an amino group, and then the ligation method can be employed to
bind the particle to the subject DNA. As the particles to be bound to the
fixed terminus of DNA, latex particles or acryl-like particles are used.
The other terminus of the DNA can be bound to a label such as fluorophore
by the ligation method which is known. The matrix to be fixed to DNA at a
terminus is produced by employing semiconductor processing techniques such
as electron beam processing or sputtering to form in quartz glass or a
silicon wafer apertures not less than 0.1 .mu.m in diameter and smaller
than the diameter of the particle to be fixed. The diameter of apertures
are not less than 0.1 .mu.m, since the lower limit of production processes
is 0.1 .mu.m.
According to the present invention, one terminus of DNA is fixed, the DNA
is stretched by applying an external force to it, and then the distance
between the fixed terminus and the other terminus is measured. The
terminus opposite to the fixed one is bound to a label such as fluorophore
or an enzyme. The position of the fixed terminus, in a case such as where
DNA bound to a particle is fixed to an aperture, can be specified in
advance because the positions of the apertures are known. This position
can also be specified by binding a label to the fixed terminus as well as
the other terminus. The other terminus bound to a label can be found out
by detecting fluorescence if the label is fluorophore or by measuring
oxygen activity if the label is oxygen. For instance, 1 megabase long
double helical DNA measures about 0.34 mm when stretched. Since the fixed
terminus and the label measure not more than 1 .mu.m, it is easy to
recognize the fixed terminus of DNA and the other terminus bound to a
label both separated from each other. Thus the length of DNA can be
measured for each molecule.
The intensity of electric fields to be applied for stretching DNA needs to
be not less than 40 V/cm to 50 V/cm such that the DNA cannot be ruptured.
The detection of label fluorophore positions can be performed by changing
the location to which the laser beam is directed.
The use of an enzyme as a label bound at one terminus of DNA for detection
is possible by this method: binding alkaline phosphatase to one terminus
of DNA by the SPDP method, applying this terminus to a chemiluminescent
assay using 3-(2'spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)-phenyl-1,
2-dioxetane (AMPPD) as the substrate and then detecting emission points
with a CCD camera or a camera equipped with an image intensifier.
BRIEF DESCRIPTION OF THE DRAWINGS
In the accompanying drawings:
FIGS. 1a and 1b are schematic illustrations of the first principle of
measuring the length of biomoleculars such as DNA according to the present
invention;
FIG. 2 is a sectional view of an embodiment of the present invention
illustrating an apparatus for measuring the length of biomoleculars such
as DNA;
FIG. 3 is a graph of fluorescence detection intervals resulting from the
measurement in an embodiment of the present invention;
FIG. 4 is a sectional view of another embodiment of the present invention
illustrating an apparatus using capillary tubes to measure the length of
biomoleculars such as DNA;
FIG. 5 is a sectional view of still another embodiment of the present
invention illustrating an apparatus using fine grooves to measure the
length of biomoleculars such as DNA;
FIG. 6 is a sectional view of a further embodiment of the present invention
illustrating an apparatus using a molecular sieve to measure the length of
biomoleculars such as DNA;
FIG. 7 is a schematic representation of the structure of single-stranded
DNA whose termini both are labeled with fluorescence and which is bonded
through hybridization to fluorescence-labeled DNA probes at other
locations;
FIG. 8 is a schematic illustration of the second principle of measuring the
length of biomoleculars such as DNA according to the present invention;
FIG. 9 is a schematic representation of another embodiment of the present
invention illustrating an apparatus for preparing a DNA fragment; and
FIG. 10 is a schematic plan view of an embodiment of the present invention
illustrating a container for measuring the length of biomoleculars such as
DNA.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring now to FIGS. 1a, 1b and 2, the fundamentals of embodiments of the
present invention in which both termini of DNA are labeled with
fluorescence will be described. FIG. 1a illustrates the digestion of DNA
with an enzyme, FIG. 1b the fluorescence labels at DNA termini. These
FIGS. 1a and 1b show the principle of biomolecular length measurement,
such as DNA molecule length measurement, of the present invention. To take
DNA molecules as an example, the principle of biomolecular length
measurement of the present invention will be sketched as this procedure:
(1) digesting DNA molecules with enzymes (FIG. 1a), (2) binding
fluorescence labels to the termini of digested DNA to produce DNA fragment
molecules whose termini have been labeled with fluorescence (FIG. 1b), (3)
injecting the fluorescence-labeled DNA fragment molecules into an upper
buffer solution 15 (FIG. 2) and applying them to electrophoresis in the
gel to orient the DNA fragment molecules into straight lines, (4)
transferring DNA fragment molecules from the electrophoresis agarose gel
migration path outlet not more than 1 .mu.m in diameter to a buffer
solution with their orientation kept straight. Incidentally, the lower
limit of the diameter of the outlet, that is, the outflow part of the
electrophoresis migration path virtually depends on the limit of
production processes, (5) directing a very thin laser beam to the DNA
fragment molecules not away (not more than 1 mm) from the electrophoresis
migration path outlet, (6) receiving the fluorescence coming from the DNA
fragment molecule termini labeled with fluorescence by using the laser
beam, and detecting it with a photodetector 7 (FIG. 2), using clocking
means to measure the time the fluorescence at both termini of the
fluorescence-labeled DNA fragment molecules takes to pass through the
above-noted portion to which the laser beam has been directed and
obtaining the length of the DNA fragment molecules from the measurement
results and the flow rate of DNA molecules.
EXAMPLE 1
Now, an example of the present invention will be described in detail. (1):
First, in the sample solution 10 of the DNA molecules to be measured for
length and base length, a restriction enzyme (e.g., Not I) is used for
causing a characteristic chemical reaction in each base portion (FIG. 1a)
so as to digest the DNA into a plurality of DNA fragment molecules (FIG.
1a). (2): An enzyme (ligase) is used for binding the termini of these
digested DNA fragments to DNA oligomers having fluorescence labels 14a and
14b so as to obtain a labeled DNA sample 11 (FIG. 1b). When a restriction
enzyme is used for binding fluorescence-labeled DNA oligomer in the form
of double strands to the termini of the DNA digested with the restriction
enzyme, one or more than one piece of fluorophore are introduced into each
double strand of DNA. Not less than two fluorophore molecules can be bound
to one terminus of digested DNA. For the fluorophore, FITC (fluoreceine
isothiocyanate: 525 nm maximum emission wavelength) or Texas Red (615 nm
maximum emission wavelength) can be used. (3): When the DNA fragment
molecules 11 whose termini both have been labeled with fluorescence are
injected with a sample injector 12 shown in FIG. 2 into an upper buffer
solution 15 of apparatus shown in FIG. 2 for measuring the length of
polymers like DNA, and they migrate in electrophoresis in the agarose gel
in a capillary tube 20, the DNA fragment molecules 11 whose termini have
been labeled with fluorescence stretch in the direction of the migration
and are oriented in the form of straight lines. The diameter of the
capillary tube electrophoresis portion may be the conventional size, 50
.mu.m to 100 .mu.m or more than that. Since orienting the DNA fragment
molecules is the chief objective here, the length of the area having
agarose gel in the molecular orientation migration portion 1 is 1 cm,
migration length in general being not less than 1 cm. But to separate the
DNA fragment molecules roughly, 5 cm or more is a suitable migration
distance. (4): If the outlet (portion for sample elution) of the
electrophoresis agarose gel migration path is in the form of a capillary
tube whose inside diameter is not more than 6 .mu.m, and there is liquid
flow 2b for sheath flow provided by supplying a buffer solution through a
buffer solution inlet 2a and an electric field provided by an upper
electrode 17 and a lower electrode 18, a flow path 3 for oriented DNA from
the outlet where the labeled DNA sample molecules 11 pass in an area not
more than 1 .mu.m in diameter with their orientation kept straight can be
formed in a sheath flow forming tube 19. Incidentally, one end of the
agarose gel is in contact with an upper buffer solution 15 in an upper
buffer vessel 21, the migration being caused by the upper electrode 17 and
also the lower electrode 18 in a lower buffer vessel 22 filled with a
lower buffer solution 16. (5): A very thin laser beam 4a is directed to
the flow path 3 for oriented DNA not away (not more than 1 mm) from the
above-noted outlet of the electrophoresis migration path so as to excite
the fluorophore at one of the two termini of the DNA fragment molecules 11
whose termini both have been labeled with fluorescence. For the light
source 4 of the laser for exciting the fluorophore, either the Ar ion
laser (488 nm, 10 mW) or He-Ne Laser (594 nm, 3 mW) are suitable, but this
example employs a combination of Texas Red and He--Ne laser used as the
fluorophore and the laser light source respectively. He--Ne laser is
thinned into not more than 1 .mu.m with the laser beam thinning lens 5 and
directed to the flow path 3 for oriented DNA. Then, the fluorescence sent
out by the fluorescence-labeled DNA fragment molecules 11 is detected from
the direction almost at right angles with incident angle of the laser
beam. The presence of DNA fragment molecule termini in the area of 1
(.mu.m).sup.3 means that at least 2 pieces of fluorophore are included in
the volume of 1 (.mu.m) . This corresponds to a concentration of
3.times.10.sup.-9 M which sends out much stronger light than the
background light (usually weaker than 3.times.10.sup.-11 M fluorophore).
(6): The flow rate of DNA on the flow path 3 for oriented DNA depends on
the electric field intensity, but in this example the DNA flow rate for 25
V/cm electric field intensity is about 3 mm per second regardless of DNA
length (Bio/Technology, vol. 6, pp. 816-821 (1988)). The
fluorescence-labeled DNA fragment molecules 11 take about 0.3 ms to pass
through the portion to which the laser beam has been directed. In the
meantime the fluorophore emits about 6.times.10.sup.4 fluorescence
photons. These fluorescence photons pass through a light receiving lens 6
and a filter (not shown in the drawings) and are detected with a
photodetector 7. In this example, the light receiving efficiency of the
light receiving system for the emitted fluorescence is about 5%, and the
photoelectric transducing efficiency of the photodetector 7 is about 5%.
In other words, since about 150 entities of fluorescence are countable
among those emitted during the passing of fluorophore through the
detection portion, the time that the passing takes can be obtained
accurately. The number of photons for measurement can also be increased to
about 1000 by adding to the amount of laser emission, the amount of
fluorophore for labeling or the efficiency of light receiving. This makes
it possible to take a more detailed measurement of the time that the
passing takes. Using clocking means to obtain the time between the passing
of one terminus of DNA through the portion to which the laser beam has
been directed and the passing of the fluorescence label at the other
terminus through this portion can calculate the length of the fluorescence
molecule with the help of the above-noted DNA flow rate. The processing of
required data is performed by a data processor 8, and results of this
processing are issued from output 9. FIG. 3 shows an example of a
measurement result according to the present invention. The x-axis is used
for the interval between two signals, namely, the interval between the
fluorescence emission from the fluorophore at one terminus and that from
the fluorophore at the other terminus of the fluorescence-labeled DNA
fragment molecules 11, and the y-axis for counts of the fluorescence
photons emitted by the DNA fragment molecules 11. In this example, flow
takes 0.3 ms to travel 1 .mu.m, so the period 1 ms corresponds to about 10
kilobase long DNA. A signal 13 resulting from the labeled DNA samples 11,
which is shown in 13, represents labeled 200 kilobase long DNA samples.
When the labeled DNA samples 11 have comparatively many molecules
(copies), the interval is obtained between the fluorescence emission from
the fluorescence label at the rear terminus of the first DNA molecule (the
fluorescence from the front terminus from the first DNA molecule has
already been measured) and the fluorescence emission from the fluorescence
label at the front terminus of the second DNA molecule. This time is
random and has a greater distribution. On the other hand, the interval
between the occurrence of a fluorescence signal resulting from the
fluorescence label at the front terminus and that from the fluorescence
label at the rear terminus of one labeled DNA sample molecule 11 is
invariably dependent upon the length of the fragment, so accumulating the
time data gives a peaked distribution and obtains the lengths of DNA
fragments. Obviously, it is also possible to obtain them from one DNA
fragment molecule (one copy).
EXAMPLE 2
In the above example, electrophoresis agarose gel migration and sheath flow
are used for the formation of the flow path 3 for oriented DNA on which
DNA fragment molecules pass with their orientation kept in the form of
straight lines. But the straight-line orientation can be achieved by
drawing biomolecules out of a capillary tube with electric fields and
buffer solution flow. FIG. 4 illustrates an example of such apparatus.
Fluorescence-labeled DNA samples 11 are injected from an upper buffer
solution inflow opening 15a. Upper buffer solution 15b is supplied to an
upper buffer vessel 21. The path where a laser beam 4a goes and a lower
buffer vessel 22 are filled with a lower buffer solution 16. To cause
samples to migrate, voltage is applied to an upper electrode 17 and a
lower electrode 18. The upper buffer vessel 21 is equipped with sample
spewing capillary tubes 23a whose bottoms are spewing outlets 23b smaller
(not more than 6 .mu.m) in diameter than the tops, and is also equipped
with sample passing capillary tubes 25 whose inside is not more than 6
.mu.m in diameter. The electric field provided by the upper electrode 17
and the lower electrode 18 and the flow of a buffer solution formed with
the spewing capillary tubes 23a make it possible to orient the
fluorescence-labeled DNA samples 11 into straight lines and draw them out
of the spewing outlets (spewing outlet portion) 23b and to forcibly orient
the fluorescence-labeled DNA samples 11 through the sample passing
capillary tubes 25. The laser beam 4a is directed to the proximity of the
spewing outlets 23b (not more than about 1 mm from the outlets), and the
fluorescence emitted from the spewed labeled DNA is detected from the
direction almost perpendicular to the surface of the FIG. 4 page, or from
the bottom of the lower buffer vessel 22. The optical system for
fluorescence excitation and detection is the same as in FIG. 2.
EXAMPLE 3
In the same manner as the drawing of samples out of capillary tubes shown
in FIG. 4, it is possible as shown in FIG. 5 to draw biomoleculars out of
fine grooves and orient them into straight lines by using electric fields
and a buffer solution 15a. To be more specific, DNA molecules can be
oriented by causing them to flow out from a group of fine grooves 29 whose
width and depth are about 1 .mu.m, a little larger than the shorter
diameter of DNA molecules (and which are made of two plates 28 of such
material as silicon crystal or glass). The width and depth of the fine
grooves 29 can be not more than 1 .mu.m. Tapering the fine grooves 29 made
of two plates 28, namely, flow paths for oriented DNA can strengthen the
electric field intensity and stretch DNA more, resulting in the migration
of samples oriented in the form of straight lines. Such flow paths for
oriented DNA can be provided by a semiconductor pattern formation
technique such as grooving the surface of silicon crystal plates or glass
materials or a processing technique such as etching.
EXAMPLE 4
As shown in FIG. 6, it is possible to measure the length of molecules like
DNA by means of electrophoresis using electric fields and a molecular
sieve 30 to spew samples out of the molecular sieve. For the molecular
sieve 30, such products as the OMEGA Cell by Filtron Inc. in the U.S. can
be used.
In the examples shown in FIGS. 5 and 6, as in FIG. 4, fluorescence labels
are excited with the laser beam 4a, and the excited fluorescence is
detected from the direction almost perpendicular to the surface of the
drawing page, or from the bottom of the lower buffer vessel 22.
In the above examples, the combination of Texas Red fluorophore having a
single maximum emission wavelength and He--Ne laser is used But it is also
possible to digest DNA molecules with more than one restriction enzyme and
also to label both termini of DNA fragment molecules with fluorophore
having emission wavelengths different to each other terminus so as to
detect these termini separately according to their wavelengths. In this
case, the fluorescence label primer (in the form of double strands)
capable of junction with both termini of double-stranded DNA is bound to
both termini by the ligation method enzyme reaction.
It is also possible to use as labels minute particles which have adsorbed
and are bound to fluorophore and thereby to label both termini of
biomoleculars with one kind or different kinds of fluorophore. As such
minute particles the latex particles by Ployscience, Inc. can be used
which are 0.1 .mu.m in diameter and contain a coumarin-like coloring
matter and have carboxyl groups on the surface.
Since DNA with a double helix is about 3.4 A long for 1 base, it is about 1
.mu.m long for 3 kilobases. Laser beams, if their convergence conditions
are optimized, can be thinned into about 0.5 .mu.m. Thus the length of DNA
can be measured to a precision of 1 to 2 kilobases.
Reducing the diameter of the directed laser beam to the quantity equal to
its wavelength or a smaller one by such means as fiber can increase the
precision of DNA length measurement.
The measurement of DNA length has been described so far. According to this
process, as shown in FIG. 7, it is also possible to produce a detailed DNA
map by binding both termini of single-stranded DNA 27 to fluorescence
labels 14a and 14b of different kinds or to fluorescence labels 14a and
14a of the same kind, forming more than one DNA probe 26a, 26b and 26c
having fluorescence labels 28a, 28b and 28c at various locations and a
hybrid, orienting the DNA into a straight line, causing it to migrate and
detecting the fluorescence labels 28a, 28b and 28c sequentially with the
apparatus described earlier.
As understood from the above, according to the present invention relating
to labeling both the termini of a biomolecular with fluorescence and then
measuring its length, a measurement is taken with the DNA molecules
stretched, i.e., oriented into straight lines, so the advantage is that
the precision is higher than the conventional method of separating lengths
of DNA according to the difference in the spread of DNA molecules, and
that the length of DNA molecules can be measured by using fewer DNA
molecules. The measurement precision is 1 to 2 kilobases, so it is
possible to measure tens of kilobases to hundreds of kilobases long DNA or
DNA longer than that. This precision is tens of times as high as that by
conventional methods.
According to the present invention, in addition to DNA length measurement,
it is also possible to produce a detailed DNA map by forming fluorescence
label DNA probes and a hybrid at various locations other than the termini
in single-stranded DNA, orienting the DNA into a straight line, causing it
to migrate and then detecting labels.
Now, an example will be described in which one terminus of DNA 101 is bound
to a label 102 such as fluorophore, the other terminus is physically or
chemically fixed to a matrix 104, the DNA is stretched by means of
electric fields or liquid flow, the position of the terminus bound to the
label is detected, and then the length of the DNA is measured.
First, DNA is stretched with an external force, the distance between the
termini is measured, and the length of the DNA is measured for each
molecule. Here, it is not always necessary to stretch DNA fully and
measure its absolute length. Since fluctuations due to thermal vibration
occur to longer DNA, it is unfeasible to stretch it to the full. This
difficulty can be overcome by obtaining a calibration curve from DNA whose
lengths are known and then finding the length of the DNA subject to the
measurement. Incidentally, it is known that in general when DNA is fully
stretched, it is 0.21 nm long for each base. To fix DNA, only one terminus
needs to be used so that the DNA can be stretched with certainty.
Materials like nitrocellurose film or nylon inducer conventionally in wide
use for hybridization cannot be used because they fix DNA at more than one
point.
As shown in FIG. 8, one terminus of DNA 101 subject to measurement and
having a label 102 at the other terminus is bound to a particle 103 whose
diameter is about 0.2 .mu.m to 10 .mu.m. In fact since this particle needs
to be sufficiently smaller than the DNA length, its size is up to 10
.mu.m, more preferably up to 6 .mu.m, most preferably up to 1 .mu.m. The
apertures for capturing the particle are at least about 0.1 .mu.m in size.
Thus the particle size may be at least 0.2 .mu.m. The DNA bound to the
particle 103 is passed through the matrix 104 having apertures through
which the particle 103 cannot pass. The DNA can pass one of the apertures
106 except its portion bound to the particle 103, which allows one
terminus of the DNA to be fixed in a specific position. It is possible
then to fix the DNA to the matrix 104 and stretch it with the help of the
particle 103 by applying electric fields between electrodes 105a and 105b
or using liquid flow. In this process, the length of DNA is found by
detecting that the label 102 is in a position corresponding to the fixed
terminus. Using a container having grooves or apertures not passable to
the particle 103 fixes the particle 103 to the mouths of the grooves or
apertures, and stretches the DNA along the grooves or apertures provided
in a certain direction. A search for the label 102 therefore needs to be
made only along the grooves or apertures. Naturally, gel matrices can be
used for fixing the particle 103 and stretching the DNA. In this case, the
particles 103 are captured at the mouths of the gel matrices, and the DNA
main portion and the label 102 stretch inside the gel matrices. In
addition, the distance between one DNA terminus fixed with the fixing
particle 103 and the label 102 at the other terminus can be measured by
using the fixing particle 103 as the label specifying an origin. The DNA
subject to measurement can be fixed also by means of association reaction.
One terminus of the target DNA is bound to primer DNA whose series is
known, and this terminus is allowed to fuse through hybridization with a
matrix bound to DNA primer complementary to the primer whose series is
known, resulting in fixing the DNA bound to a label. In this case also,
the length of DNA can be measured, the DNA being stretched by such means
as electric fields. Instead of complementary DNA chains, biotin-avidin
association reaction or immunoreaction can be used. Now, Examples 5 and 6
of the present invention will be described in detail.
EXAMPLE 5
The measurement subject is DNA prepared from human leucocytes. A DNA
fragment was digested | | |
