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`
`1.1511116144448131
`
`United States Patent
`Mitoma
`
`[19]
`
`[11] Patent Number:
`
`6,144,443
`
`I45] Date of Patent:
`
`Nov. 7, 2000
`
`[54] FLUORESCENCE DETECTING APPARATUS
`
`[15]
`
`Inventor:
`
`Yasutami Mituma. Knnagawa~kert.
`Japan
`
`[73] Assignce: Tosoh Corporation, Yamagnehi—ken‘
`Japan
`
`|21| Appt. Nut: 091517.666
`
`[22
`
`Filed:
`
`Oct. 50. 1996
`
`1211.994 Eerndl
`5.371..“1fi
`11-1995 BIumenJ'eld elal.
`5,473.43?
`FOREIGN PATENT DOCUMENTS
`
`
`
`35111461.}.
`355.141?
`
`(I 137' 381’!
`U 13'? 418
`0 Ron 881
`(I 15o 274 A2
`W0 STr'tHJ't'lo
`
`European 931. Oil. _
`13-1984
`llfl‘Jl‘H European Pal. Ofl'.
`_
`511088
`European Pat. 011:.
`.
`1211992
`l'zutepean Fat. 011.
`_
`llflUS‘! W]PO.
`
`t'lTl-lER PUBLICATIONS
`
`Related U.S. Application Date
`
`I53] Continuation of applimlion NO. IW‘DST,S5S, Jul. 0:
`abandoned.
`
`I‘J‘JJL
`
`[30]
`
`Foreign Application Priority Date
`
`l992 Research pp. 4134]?
`Biofl‘eehnology vol. 10 Apr.
`lliguchi el al Simultaneous Amplification and Detection of
`Specific DNA Sequences.
`
`Primtnjr' Examiner—F. l.. Evans
`Attorney; Agent. or Firmw—Nixon & Vanderhye
`
`Jul. 1?. 1992
`
`[JP]
`
`Japan .................................... 4‘212233
`
`[57]
`
`ABSTRACT
`
`Int. Cl.7 .................................................. GOIN 21/64
`|51|
`|53] U.S. Cl.
`....................... 3561317; Emil-158.1; 4351808;
`436:] 72
`
`I58]
`
`|5()]
`
`356.1311. 318,
`Field of Search
`356.34] 7. 440; 2501458.]. 459.1. 461.1.
`461.2: 435134, 289—291. 808; 42382 117—8108.
`$2.11: 4361172
`
`References Cited
`U3. PATENT DOCUMENTS
`
`3,992,631
`4.498.780
`ERIIHTB
`5152,3314
`
`1 Isttnra Harte .
`231055 Banno el [11.
`....................... 3561441] X
`
`611903 Turner el al.
`.
`103' 1993 Lin ....................................... 250145 8.1
`
`A fluorescence detecting apparalus which allows highly
`precise measurement of fluorescence even with minute
`sample amounts, which has a strong, responsiveness to
`temperature variations. which nllowe simultaneous measure—
`ment ol‘a pluralityf ol'samples, and wherein the light source
`and the container holder, and the container holder and the
`fluorescence detector. are each optically connected by opti-
`cal fibers; and the optical fibers are connected to the eon-
`lainer holder in such it manner that
`the sample in the
`container is excited for fluorescence from below the sample
`container held by [he container holder, and lhnl [hey may
`receive the fluorescent light which is emitted by the sample
`from below the sample container.
`
`13 Claims, 4 Drawing Sheets
`
`APPARATUS
`
`DETECTING
`
`THERMO FISHER EX. 1048
`
`THERMO FISHER EX. 1048
`
`

`

`US. Patent
`
`Nov. 7, 2000
`
`Sheet 1 0r 4
`
`6,144,448
`
`Fig.
`
`I
`
`
`
`THERMO FISHER EX. 1048
`
`THERMO FISHER EX. 1048
`
`

`

`US. Patent
`
`Nov. 7, 2000
`
`Sheet 2 0r 4
`
`6,144,448
`
`Fig. 2
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`THERMO FISHER EX. 1048
`
`THERMO FISHER EX. 1048
`
`

`

`US. Patent
`
`Nov. T, 2000
`
`Sheet 3 01'4
`
`6,144,448
`
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`US. Patent
`
`Nov. 7, 2000
`
`Sheet 4 0f 4
`
`6,144,448
`
`Fig. 4
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`

`

`6,] 44,448
`
`1
`FLUORESCENCE DETECTING APPARATUS
`
`This is a continuation of application Ser. No. WEBSISSS,
`tile-d Jul. 9. 1993, now abandoned.
`
`[‘1 ELI) 011’ THE INVEN'J‘ION
`
`The present invention relates to a fluorescence detecting
`apparatus, and more specifically, it relates to a fluorescence
`detecting apparatus for measuring variations in the fluores-
`cent properties of substances or their interacted complexes
`in reactions which occur between at
`least
`two types of
`substances capable of interaction between each other. for
`example, reactions involving nucleic acids and interealatory
`fluorescent pigments. lipid bilaycrs and hydrophobic fluo»
`rescenl probes. proteins and fluorescent pigments, organic
`polymers and fluorescent pigments, etc.
`
`DESCRIPTION Oli 'I'IIE PRIOR ART
`
`In reactions which occur between at least two types of
`substances capable of interaction between each other, for
`example, reactions involving nucleic acids and intercalatory
`fluorescent pigments, lipid bilayers and hydrophobic fluo-
`rescent probes, proteins and fluorescent pigments, organic
`polymers and fluorescent pigments, etc.,
`the fluorescent
`properties of these substances or their interacted complexes
`vary depending upon their state. Thus, if the variation in the
`fluorescent properties thereof is measured, it is possible to
`know the state of inte rreaction of the above substances. the
`amount of complex formed, etc.
`In polymerase chain reactions {l’CRs) which are con-
`ducted in the copresence of an intercalatory fluorescent
`pigment {for example Japanese Patent Application Ilei
`3-3] 36 t 6} etc... it is possible to know the state of nucleic acid
`amplification (success of the PCR) by measuring the flue—
`rescent properties at a desired point
`in each cycle of the
`MR, and usually at a point during repetition of scparation
`ot’ the double-stranded nucleic acid into single strands and
`hybridization between single strands, by variation of the
`temperature. In this procedure, a device is required which
`can vary the temperature according to a preset program and
`measure the change in the fluorescent properties of a sample
`in a sample container. Such a device in current use is a
`spectrophotometer containing a temperature—controlling cell
`holder.
`
`SUM MARY OF THE INVENTION
`
`Measurement of fluorescent property variation according
`to the prior art using a spectrophotometer employs a system
`in which exciting light is directed towards a cell using an
`optical system of lenses, mirrors, etc. and fluorescence is
`directed towards a fluorescence detecting apparatus in the
`same manner, and therefore it has been dilticult to effect
`multichannel measurement (simultaneous measurement of
`multiple samples). Furthermore, the responsiveness of the
`sample temperature to the temperature variation is lessened
`by the amount of liquid in the sample, and it is dilficult to
`measure the rapid interactions between compounds which
`accompany phase transitions.
`Various attempts have been made at improving tempera-
`ture responsiveness. but in methods where the volume of the
`sample container is reduced, etc.
`it becomes necessary to
`select
`the beam of the exciting light,
`thus causing new
`problems such as a reduction in the quantity of light and a
`resulting decrease in the precision ol‘ measurement. An
`attempt at improvement has been made by using an intense
`
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`
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`
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`
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`
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`
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`
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`
`65
`
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`light source. to compensate for the reduction in the. quantity
`of light. but in this case the problem of heat waste from the
`device and the influence of heat on the fluorescence detect-
`ing apparatus are considerable, and thus it becomes (liflicult
`to obtain highly precise and repeatable measurement results.
`Very recently, dcviccs which employ optical fibers in an
`optical system (Biotechnology. Vol. 10, p. 413, 1992) have
`become known, but in these devices the sample is excited
`from the top 01‘ the sample container, and the fluorescence
`from the sample is also received from the top, and therefore
`if the amount of the sample is small,
`there is a loss of
`exciting light and fluorescence from the sample in the air
`layer between the sample and the tip of the optical fibers
`The proceSs is thus complicated by the necessity of replac-
`ing the sample container from time to time, depending on the
`volume ol‘ the sample, in order to overcome this problem.
`and therefore improvement thereof is in order.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`is a drawing showing the construction of an
`'I
`FIG.
`apparatus according to the present invention:
`FIG. 2 is a drawing showing the periphery of the container
`holder of the apparatus in FIG. 1;
`FIG. 3 is a graph showing the results of Example 1, with
`the fluorescence intensity during the. PC'R cycle for various
`concentrations of ).DNA plotted on the vertical axis, and the
`number of PC‘R cycles plotted on the horizontal axis. In the
`figure. ‘3" represents a case where the amount of DNA is 3.5
`ng. "b" a case where it is (1.25 ng. and “c” a case where it
`is 0.025 ng.
`FIG. 4 is a graph showing the results of Example 2, with
`the fluorescence intensity during a PCR conducted in the
`presence of an intercalatory fluorescent pigment plotted on
`the vertical axis, indicating temperatures of the sample at the
`time of measurement of the fluorescence intensity on the
`right—hand side. and the time plotted on the horizontal axis.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The present invention relates to a fluorescence detecting
`apparatus which comprises a sample container which holds
`a sample. a container holder which holds the sample eon~
`tainer and is capable of varying the temperature 01‘ the
`sample in the sample container which it holds, a tluores~
`cence detector for measuring the fluorescence from the
`sample, and a light source which emits exciting light
`to
`excite the sample for fluorescence, characterized in that the
`light source and the container holder, and the container
`holder and the fluorescence detector. are each optically
`connected by optical
`litters; and said optical
`fibers are
`connected to the container holder in such a manner that the
`sample in the container is excited for fluorescence from
`below the sample container held by the container holder. and
`that they may receive the fluorescent light which is emitted
`by the sample [rum below the sample container. A more-
`detailed description 01‘ the present
`invention is provided
`below. The sample container for holding the sample may he
`of any shape, but
`in order to excite the sample from the
`bottom of the container and to receive fluorescence of the
`sample from the bottom ofthe container, a container is used
`of which at least the bottom is light-permeable. to reaction
`systems such as the PCR, wherein there exists the danger of
`infection of viral nucleic acids, etc. and the slightest con—
`tamination between two or more samples results in a large
`experimental error. it is preferable to use a scalable reaction
`container and to supply the samples to the detector under
`
`THERMO FISHER EX. 1048
`
`THERMO FISHER EX. 1048
`
`

`

`5,1 44,448
`
`"J‘t
`
`.10
`
`4
`fluorescence from the sample is received from below the
`sample container. For example, if the container holder is in
`the form of a holder shaped to fit the container. then the
`fibers may be connected so as to provide a through-opening
`on the concave section thereof, hut in order to allow for
`highly precise and repeatable measurement of small
`amounts of samples. they are preferably connected at
`the
`deepest area of the concave section.
`The end olithe optical fiber line for directing the exciting
`light and the one for receiving the fluorescent light may each
`be connected separately to the container holder. For
`example. the ends ofthe exciting light radiation fibers or the
`fluorescent light receiving fibers may be arranged so as to
`intersect over an extension wire. or they may be arranged to
`be parallel to each other. For a more wositive and repeatable
`measurement of fluorescence according to the present
`invention. it is preferable to construct the ends of the fibers
`of the two systems in proximity to each other, and it is
`particularly preferable to construct the ends using fibers of
`two coaxial systems. It‘ the ends are constructed using fibers
`of two coaxial system. then it is preferable to position the
`ends of the filters for receiving the fluorescent light from the
`sample on the exterior. and the ends of the optical fibers for
`directing the exciting light in the center. since this allows for
`reception of weaker fluorescence. As mentioned previously.
`if the two systems are pmvided [or by a single optical fiber
`line, then the end ofthe fiber line may be simply connected
`to the container holder.
`
`3.5
`
`40
`
`3
`sealed conditions. An example of a scalable sample con-
`tainer
`is a commercially available centrifuge tube (for
`example. an Eppenclorf Inicroeentrit‘uge lube, etc). Also,
`there is not particular restriction on the material, provided it
`satisfies the conditions described above.
`
`The container holder used to hold the sample container
`has a function for varying the temperature of the sample. and
`also serves to hold the container. It may be, for example, in
`the form of a container holder shaped to [it the container
`used. or be constructed. for example, in a large box shape
`with said container simply being placed therein.
`If the container holder is constructed in the form of a
`container holder shaped to lit the container, then it may be
`constructor] so that variation in the temperature of samples
`may be achieved by. for example, varying the temperature of
`the container holder itself to vary the temperature of the
`sample container. thus varying the temperature ot'thc sample
`in the sample container. In this case. the container holder and
`sum ple container are [ureter-ably constructed of a highly
`then‘noconductive mate-rial. An example which may be
`mentioned is a heater. etc. installed on the surface of contact
`of the container holder with the container,
`in a position
`which does not interfere with excitement or reception of the
`fluorescent light. If the container holder is constructed in the
`form of a large box shape,
`then the temperature of the
`sample may be varied by installing a heater therein. creating
`a refrigerant circuit. or by air-conditioning or Liquid hath.
`etc. In addition to these, a variety of other methods may be
`employed to vary the temperature of the sample according to
`the present invention. Since there is no need for healing
`when varying the temperature of the sample within a tem-
`perature range of, for example, room temperature or lower,
`in such cases only cooling mearts may he provided. Thus. the
`container holder is preferably constructed with attention to
`the desired range of variation of the sample temperature.
`The fluorescence detector may be any one which is
`capable of measuring fluorescence front a sample. Here,
`“fluorescence" includes fluorescence intensity. fluorescence
`spectrum, etc., and these can be measured using a photo-
`multiplier or a photodiode.
`The light source to be used may he a xenon lamp, :1 DZ
`lamp, a mercury lamp, a halogen lamp, a discharge tube,
`laser light, or the like. According to the present invention,
`optical fibers are used both to direct the exciting light from
`the light source to the sample, and to direct the fluorescent
`light from the sample to the detecting apparatus. However,
`this does not exclude a construction wherein. for example.
`common optical parts such as lenses or mirrors are used near
`the light source in addition to the optical fibers. and the light
`l'ocussed thereby is directed to the optical fibers.
`Two systems of optical fibers are used. one for directing
`the exciting light to the sample, and the other for directing
`fluorescent light from the sample to the fluorescence detec—
`tor. Also, by using, for example a dichroic mirror or the like.
`the above mentioned 2 systems may be provided [or by a
`single optical fiber {one line}. The ends of the optical fibers
`ofeach system are connected directly to the light source and
`the fluorescence detector, respectively, via the above men-
`tioned optical pans or if necessary a light amplifier, etc. If
`the two systems are provided for by a single optical fiber
`line, then an auxiliary optical system is attached thereto to
`direct light branched at the dichroic mirror provided at one
`end to the light source or the detector. The other end of the
`optical fiber line is connected to the container holder so that
`exciting light
`is radiated to the sample from below the
`sample container held by the container holder. and so that
`
`The source of the exciting light is normally selected to
`maximize the light intensity, but in cases where there exists
`the possibility of deterioration of the sample due to the
`radiation of the exciting light, a light-interrupting shutter
`may be provided to prevent the exciting light from radiating
`onto the sample. and the shutter may be opened and closed
`only at the time of measure-merit of the fluorescence to avoid
`constant bombardment of the exciting light onto the sample.
`The Light—interrupting shutter is provided in the optical path
`running from the light source to the container holder, but the
`simplest construction is one in which a movable shutter is
`constructed between the ends of the optical libers and the
`light source, and the shutter is mechanically or electrically
`moved to open and close in synchronization with the timing
`of measurement of the fluorescence. This timing may be, for
`example, the time at which the temperature of the container
`- holder falls within a desired range (i.e., the time at which the
`sample in the container is thought to be in the desired range).
`Such a procedure is particularly effective in cases where
`continuous (intermittent) detection of. fluorescence from two
`identical samples is made, such as in the fluorescence
`measurement
`in PCRs. as disclosed in Japanese Patent
`Application Hei 3-313616.
`The variation of the temperature of the container holder
`for varying the sample temperature may be elfectcd within
`a desired range, and this maybe stored in a program. and a
`controller for controlling the temperature of the container
`holder may be attached to the apparatus according to the
`present invention. The controller may include a sensor for
`sensing the temperature of the container holder and means
`for storing the above mentioned progra m. and may compare
`the signal from the above mentioned sensor and the contents
`of the program, outputting an indication for heating or
`cooling of the container holder, and this purpose may be
`achieved by using a microcomputer or the like.
`If the
`controller functions to control the detector. light source and
`shutter, etc. of the present invention. then it
`is possible to
`achieve an automatic fluorescence detecting apparatus
`which varies the temperature of a sample when the sample
`
`5t]
`
`55
`
`6t]
`
`65
`
`THERMO FISHER EX. 1048
`
`THERMO FISHER EX. 1048
`
`

`

`6,144,448
`
`5
`container is placed in the container holder. and which can
`measure fluorescence by opening and closing the shutter in
`accordance with the timing.
`The apparatus according to the present invention is suit"
`able for use in PCRSJH this case. it is particularly preferable
`that the above mentioned sample container be sealed. The
`PCR is a reaction which amplifies the nucleic acid in a
`sample, and for the purpose of preventing secondary eon-
`taminalion a lluorexcnce detecting apparatus according to
`the present invention which employs a sealed sample con-
`tainer is most ell'ectivc. This is because. by using a sealed
`container, it is possible to keep to a minimum the contami-
`nation of nucleic acids by other sam ples. which is a cause of
`false positivity in PCRs.
`In most PCRs. completion is not after a single procedure.
`This is because the separation of double-stranded nucleic
`acids into single-stranded nucleic acids by varying the
`temperature of the sample, annealing ol‘ a primer to each of
`the single-stranded nucleic acids. and the extension reaction
`of nucleic acids originating from the primer (which results
`in the appearance of double—stranded nucleic acids) are
`conducted as one cycle. and the cycle is usually repeated.
`Thus, an apparatus according to the present invention which
`is suitable for PCRs. is preferably provided with the above
`mentioned shutter.
`
`Because the above mentioned cycle is repeated, the tem-
`perature variation of the container holder is actually the
`variation between the temperature at which the nucleic acids
`can exist in double-stranded form (if the object nucleic acid
`is double-stranded. then this temperature range is applied at
`the start of the above mentioned cycle, at
`the time of
`extension of the primer. and appearance of the double-
`stranded nucleic acid resulting from the above mentioned
`extension} and the temperature at which they can only exist
`in single-stranded form (this temperature range is applied for
`division of the double-stranded nucleic acid into single
`strands).
`With reactions involving nucleic acids and intercalatory
`fluorescent pigments as a case in point. here the fluorescent
`properties ol‘ the fluorescent pigment themselves vary in a
`temperature-dependent manner, and thus the timing ol'varia-
`tion of the temperature of a sample in a PCR and opening
`and shutting of the above mentioned shutter is preferably
`such that the measurement is made at a stage wherein a fixed
`temperature has been reached after completion of a series of
`temperature variations, rather than during variation of the
`temperature. Furthermore.
`in this reaction, since a fluores-
`cent pigment
`is taken up in the nucleic acid extension
`reaction which originates with a primer. and the fluorescent
`properties thereof vary. the reaction may be monitored to
`determine whether or not the PC]! is successful (Japanese
`Patent Application llei 3-31 3616); however, here as well the
`temperature range may he one in which the nucleic acids can
`exist in double-stranded form, and a timing which maintains
`a fixed temperature in the interior of the sample container is
`1311: lerablc.
`ln concrete terms, this is at
`the moment of
`completion of the above mentioned cycle.
`An explanation of the present invention will now be given
`with reference to the drawings. FIG. 1 is a drawing showing
`the entire body of an apparatus according to the present
`invention. 1 represents a container holder for holding a
`sample container. provided with a plurality of concave
`sections [or holding containers to allow measurement of a
`plurality of samples, and optical fibers 5 on each concave
`section. 2 represents a fluorescence detector,3 a light source,
`4 a shutter. 5 optical fibers. and 6 a controller. FIG. 1 shows
`
`f."
`
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`
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`a cast: wherein the end surfaces of the optical fibers for
`irradiating exciting light and the end surfaces of the optical
`fibers for receiving fluorescent light are arranged coaxially.
`Also.
`in this embodiment
`the controller used is a
`microcomputer. and it controls the container holder.
`the
`fluorescence detector. the light source and the shutter.
`
`The temperature of the container holder is varied accord-
`ing to the-
`temperature variation program stored in the
`controller. in order to vary the temperature of the sample in
`the sample container {not shown). The light source is on
`during this time, or is controlled to be on in synehronizaLion
`with the timing of measurement of the fluorescence. The
`controller opens and closes the shutter according to a
`prescribed timing, following the signal from a temperature
`sensor (not shown), thus exciting the sample and allowing
`the fluorescence to be measured by the fluorescence detector.
`In this apparatus. the results of measurement by the flue-
`rescence detector may be read by the controller and later
`displayed.
`
`FIG. 2 is a drawing showing the periphery of the container
`holder of the apparatus explained in FIG. 1. 7 represents a
`sealed sample container, 8 a sample in the container, 9 an
`aluminum container holder which holds the sample con~
`tainer {only one of the plurality of concave sections in
`shown). 10 an D-ring and 11 a screw which connects the
`optical fibers to the container holder. In this embodiment, 48
`fibers are bunched together and arranged around the
`perimeter, with 12 for irradiation of the exciting light and the
`remaining 3|“; for receiving the fluorescent light.
`
`EXAMPLES
`
`Examples will now be provided to illustrate the apparatus
`according to the present
`invention and measurement of
`fluorescence using it. without limiting the present invention
`thereto.
`
`Example 1
`
`Using a commercially available DNA synthesis hit
`(GeneAmp, trade name. product of Takara Brewing Co.) and
`the apparatus shown in FIGS. 1 and 2. amplification of
`nucleic acids was elfected using the PCR (1 cycle-
`denaturalion at 94” C. for I minute. annealing at 55° C. for
`1 minute. extension at 72° C. for 1 minute {using Taq
`polymerase). and 30 cycles were re peated in the presence ol‘
`an intercalatory fluorescent pigment {Pigment 33358. prod-
`uct of Hoechst AG). The reaction solution [or the PCR was
`prepared using a calibrating ).-DNA and 1 set of primer
`included in the kit. and following the directions for the kit.
`The fluorescent pigment was added to a concentration of l
`rig/ml. The composition of the reaction solution is listed
`below.
`
`tit-DNA (1 pg, 0.] pg or 0.0} itgtntlt
`ID a reaction butler solution
`(Sodium chloride
`t'J'ris-HC‘I butter solution (pH 3.0]
`(Magnesium chloride
`(Gelatin (weighth'oldmc)
`tnLercalatorv fluorescent pigment
`(final concentmlionl
`[EU fiMt
`Primer 'l and Printer 2
`(Primer l:
`LUNA. corresponding to lljl-HSS.
`anti sense strand
`GATGAGTI‘CGTG'J‘CC’G'i‘ACAAC‘l‘GG,1
`
`t0 rrl
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`THERMO FISHER EX. 1048
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`THERMO FISHER EX. 1048
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`[Printer 2;
`
`EINTI’ mixture
`WuLer
`an poiyrncnnu:
`
`A—DNA, L‘orrcsjttonding to taut—tear).
`sense strand
`(iU'I'IKt‘Co MEI'CAGC'CACACICUCCI
`(LS HEM eat
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`[S unitary”
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`8 ill
`5'15 rnt
`llS Int
`
`In the apparatus shown in FIGS. 1 and 2, optical fibers
`(Superesca, product of Mitsubishi Rayon), a controller
`(DCl’Et'lD, product of Yamatake Honeywell} and a
`light
`source (50 W xenon lamp;
`light power source: Model
`(7-3576, both products of I-Iamamatsu t’hotonics) Were used,
`and the shutter and fluorescence detector were manufactured
`in-house.
`
`For the three different samples prepared in the above
`manner having diflcrent initial l-DNA tItnccntrations. 35 #1
`of each reaction solution was sampled in a microeentrit‘uge
`tube (product of Biobic), mineral oil was. superpositioned on
`the surface thereof to prevent evaporation of the sample.
`which was carried on an aluminum block. and the PCR was
`carried out while raising and lowering the temperature.
`Exciting light [tom the xenon lamp light source and passing
`through a U350 color glass filter was locussed with a lens,
`directed towards the optical fibers. and irradiated from the
`bottom of the sample container to the sample. The fluores-
`cence from the sample was received by the optical fibers at
`the bottom of the reaction container, and the light which
`passed through a 40n-45ti nrn interference filter was mea-
`sured with the in-house manufactured fluorescence detector
`which contained a photodiode. The measurement of the
`fluorescence was made during the final second of the exten-
`sion reaction of the above mentioned reaction cycle.
`The resulting measurement of the variation in fluores-
`cence during the PCR was as shown in 1910.3. ln Flt]. 3, the
`fluorescence intensity during the PCR cycle for each con—
`centration of lt-DNA is plotted on the vertical axis. and the
`number of cycles is plotted on the horizontal axis, and it is
`clear from this graph that the number of cycles required for
`an increase in the fluorescence intensity depended on the
`concenLration ol' the sample; that is, 3 curves were obtained
`with diflcrcnt
`increase points depending on the initial
`amount of DNA in the sample.
`
`Example 2
`
`Using the amplified sample from Example 1. continuous
`measurement was made of the fluorescence intensity at
`temperatures (94, 72, 55° C.) predetermined for the double-
`stranded nucleic acid fragments (approximately 500 base
`pairs) and during a state of continuous variation of the
`temperature to each one, in the presence of an intercalatory
`fluorescent pigment.
`'l'he resulting measurement of the variation in fluores-
`cence was as shown in FIG. 4. In FIG. 4, the time is plotted
`on the horizontal axis. and the fluorescence intensity on the
`vertical axis.
`
`Example 3
`
`t ,ttg
`0f LDNA which had been digested with EcoTI41,
`was suspended in 25 pl of a butler solution [15(th of NaCL
`l ,ugiml ot'interealatory fluorescent pigment (33253. product
`of lloecbst AU), 10 mM of Tris-IICl, pH 8.5). the suspen-
`sion was placed in a 25 ul microcentrifiige tube (product of
`Biohic], and the fluorescence intensity was measured using
`an apparatus similar to the one in Example 3, except that
`
`6, 1 44,448
`
`7
`
`continued
`
`8
`coaxial libers (EElZ-CC‘ZDU, product of Umron) were used.
`For comparison. the above mentioned optical fibers were
`affixed to the upper opening of the mierocentrifuge tube For
`measurement in the same manner. For this purpose. the end
`surface-s oftlte optical fibers positioned at the liquid surface
`of the sample in the centrifuge tube and at
`the above
`mentioned opening were separated by a gap of 33 mm.
`The above mentioned optical fibers consisted of one 1 mm
`diameter liber in the center, and 16 lines of 0.25 mm libers
`around the periphery, with the center liber used for irradi-
`ating exciting light, and the peripheral
`fibers used for
`receiving fluorescent
`light.
`In this embodiment, a 50 W
`xenon lamp is used as the light source. and measurement
`was made of the exciting light which was transmitted
`through a U—350 filter and of the fluorescent light which was
`transmitted through an interference filter (400—450 nrn).
`As a result, a fluorescence intensity of 265 rrtV was
`obtained with the apparatus according to the present
`invention. whereas the fluorescence intensity from the same
`sample was measured to be 20 rnV. or only about Via in
`comparison, when the optical fibers were attached to the
`upper opening of the centrifuge tube.
`EFFECT OI" THE INVENTION
`
`According to the present invention, since the light source.
`the container holder (i.e., the sample) and the fluorescence
`detector are all connected by the optical fibers. it is possible
`to arrange all of these separately so that the heal-emitting
`light source and the container holder do not
`aflTec-t
`the
`lluoresee nee detector. Furthermore. multichannel
`measurement, or direction of exciting light from a single
`light wurce to a plurality of samples. and direction of
`fluorescence from a plurality of samples to a detector. may
`be easily realized. If highly photoconductivc fibers are used.
`then the loss of the exciting light and fluorescent light may
`be prevented and the light beam may be selected. and
`therefore it is possible to make high precision measurements
`even in the case of, for example, minute sample amounts.
`etc.
`
`it!
`
`15
`
`EU
`
`30
`
`3.5
`
`40
`
`is
`invention. exciting light
`According to the present
`irradiated onto the bottom of a sample container, and fluo-
`rescent light is received at the bottom of the sample. and
`therefore the air layer between samples may be kept
`to a
`- minimum. As a result, highly precise measurements are
`possible particularly in cases where fluorescence is mea—
`sured from minute sample amounts.
`The significance of using minute sample antouan is that
`the responsiveness of samples to temperature variation may
`be improved. and therefore that in reactions involving sub-
`stances which undergo temperature-dependent phase transi-
`tions and have been diflicult to measure according to the
`prior art _
`_
`. for example, reactions involving intercalatory
`fluorescent pigments and nucleic acids, reactions involving
`lipid bilayers and hydrophobic fluorescent probes, reactions
`involving proteins and fluorescent pigments. and reactions
`involving organic polymers and lluorescenl pigments .
`.
`. the
`measurement of rapid interactions between compounds
`which result from phase interactions due to temperature
`variations may be made by measuring the variation in the
`fluorescent properties thereof.
`Particularly.
`if an apparatus according to the present
`invention is used in a PCR in which an intercalatory pigment
`is added, and the fluorescence intensity is measured accord—
`ing to a desired tinting during each cycle, or the variation in
`the. fluorescence is measured throughout each cycle, then it
`is possible to know the progress of nucleic acid
`
`5t]
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`55
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`Eli]
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`THERMO FISHER EX. 1048
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`THERMO FISHER EX. 1048
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`6,] 44,448
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`9
`from these
`the success of the PCR.
`i.e.,
`amplification,
`measurements. Therefore. according to the present invention
`it is also easily possible to know the success of PCRs, which
`has conventionally been determined by electmphoresis, etc.
`This means that waste may be reduced in cases where the
`initial concentration of the object nucleic acid is high and the
`PCR continues unnoticed even after saturation ofthc ampli—
`fication reaction,
`resulting in the amplification of non-
`specific nucleic acids, and further means that the subsequent
`process of identilication of the nucleic acids mayr be facili-
`tated. If the fluorescent property at an appropriate wave-
`length for the PCR is measured and a