PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 :
`WO 99/60381
`GOlN 21/25, C12Q 1/68
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(11) International Publication Number:
`
`Al
`
`(43) International Publication Date:
`
`25 November 1999 (25.11.99)
`
`(21) International Application Number:
`
`PCT/US99/l 1088
`
`(22) International Filing Date:
`
`17 May 1999 (17.05.99)
`
`(81) Designated States: AU, CA, CN, JP, US, European patent (AT,
`BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU,
`MC, NL, PT, SE).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(30) Priority Data:
`60/085,765
`60/092,784
`
`16 May 1998 (16.05.98)
`14 July 1998 (14.07.98)
`
`US
`US
`
`(71) Applicant (for all designated States except US):
`THE
`PERKIN-ELMER CORPORATION [US/US]; 761 Main
`Avenue, Norwalk, CT 06859-0199 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): GAMBINI, Michael,
`R. [US/US]; 181 Josiesing Road, Monroe, CT 06468
`(US). ATWOOD, John, G. [US/US]; 149 Limekiln Road,
`Redding, CT 06896 (US). YOUNG, Eugene, F. [US/US];
`802 Balboa Lane, Foster City, CA 94404 (US). LAKATOS,
`Edward, J. [US/US]; 56 Ridgedale Road, Bethel, CT 06801
`(US). CERRONE, Anthony, L. [US/US]; 51 Kneeland Road,
`New Haven, CT (US).
`
`(74) Agent: AKER, David; The Perkin-Elmer Corporation, 850
`Lincoln Centre Drive, Foster City, CA 94404 (US).
`
`(54) Title: INSTRUMENT FOR MONITORING POLYMERASE CHAIN REACTION OF DNA
`
`(57) Abstract
`
`instrument monitors PCR
`An optical
`replication of DNA in a reaction apparatus
`having a temperature cycled block with vials
`of reaction
`ingredients
`including dye
`that
`fluoresces
`in presence of double-stranded
`DNA. A beam splitter passes an excitation
`beam to the vials to fluoresce the dye. An
`emission beam from the dye is passed by the
`beam splitter to a CCD detector from which
`a processor computes DNA concentration.
`A reference strip with a plurality of reference
`emitters emit reference beams of different
`intensity, from which the processor selects
`an optimum emitter for compensating for
`drift. Exposure time is automatically adjusted
`for keeping within optimum dynamic ranges
`of the CCD and processor. A module of
`the beam splitter and associated optical
`filters is associated with selected dye, and is
`replaceable for different dyes.
`
`3
`
`B
`
`THERMO FISHER EX. 1007
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`

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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Ci\te d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`Fl
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`THERMO FISHER EX. 1007
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`WO 99/60381
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`PCT/US99/l 1088
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`INSTRUMENT FOR MONITORING POLYMERASE CHAIN REACTION OF DNA
`
`This invention relates to biochemical analyses, and particularly to quantitative monitoring
`
`5
`
`of DNA during a polymerase chain reaction (PCR) process.
`
`BACKGROUND
`
`Polymerase chain reaction (PCR) is a process for amplifying or multiplying quantities of
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`10
`
`double-stranded deoxyribonucleic acid (DNA). In a PCR apparatus, a thermal cycler
`
`block has one or more wells for holding vials containing a suspension of ingredients for a
`
`reaction to produce more DNA starting with "seed" samples of the DNA The starting
`
`ingredients in an aqueous suspension, in addition to the a seed sample, include selected
`
`DNA primer strands, DNA elements, enzymes and other chemicals. The temperature of
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`15
`
`the block is cycled between a lower temperature extension phase of the PCR reaction at
`
`about 60°C, which is the phase where all of the DNA strands have recombined into double
`
`strands, and a high temperature denaturing phase at about 95°C, during which the DNA is
`
`denatured or split into single strands. Such a temperature program essentially doubles the
`
`DNA in each cycle, thus providing a method for replicating significant amounts of the
`2 o DNA from a small starting quantity. The PCR process is taught, for example, in U.S.
`patent No. 4,683,202.
`
`Quantitative measurements have been made on the DNA production during the PCR
`
`process, to provide measures of the starting amount and the amount produced.
`
`25 Measurements and computation techniques are taught in U.S. patent No. 5,766,889
`
`(Atwood), as well as in an article "Kinetic PCR Analysis: Real-time Monitoring of DNA
`
`Amplification Reactions" by Russel Higuchi, et al., Bio/Technology vol. 11, pp. 1026-
`
`1030 (September 1993), and an article "Product Differentiation by Analysis ofDNA
`
`Melting Curves during the Polymerase Chain Reaction" by Kirk M. Ririe, et al., Analytical
`
`30
`
`Biochemistry vol. 245, pp. 154-160 (1997).
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`Prior measuring techniques have utilized microvolume fluorometers (spectrofluorometers)
`
`and a simple arrangement of a video camera with illumination lamps. Such apparatus
`
`utilize dyes that fluoresce in the presence of double-stranded DNA These techniques and
`
`instruments are not particularly adapted to PCR apparatus for routine monitoring of the
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`5
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`reaction. There also is a need for greater precision during the monitoring and
`
`measurements. Previous instruments that allow real time acquisition and analysis of PCR
`
`data have been very basic devices without the required dynamic range, do not have built-in
`
`calibration means, do not allow operation with sample well caps, or are very expensive.
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`10
`
`An object of the present invention is to provide a novel optical instrument for quantitative
`
`monitoring of DNA replication in a PCR apparatus. Other objects are to provide such an
`
`instrument with improved dynamic range, automatic selection of exposure time to extend
`
`dynamic range, automatic adjustment for drift, simplified operation, relatively low cost,
`
`and easy changing of optics to accommodate different fluorescent dyes.
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`15
`
`2 o
`
`SUMMARY
`
`The foregoing and other objects are achieved, at least in part, by an optical instrument as
`
`described herein for monitoring polymerase chain reaction replication of DNA The
`
`replication is in a reaction apparatus that includes a thermal cycler block for holding at
`
`least one vial containing a suspension of ingredients for the reaction. The ingredients
`
`include a fluorescent dye that fluoresces proportionately in presence of DNA
`
`The instrument includes a light source, means for directing light beams, a light detector,
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`2 5
`
`and means for processing data signals. The light source emits a source beam having at
`
`least a primary excitation frequency that causes the dye to fluoresce at an emission
`
`frequency. A first means is disposed to be receptive of the source beam to effect an
`
`excitation beam having the excitation frequency. A primary focusing means is disposed to
`
`focus the excitation beam into each suspension such that the primary dye emits an
`
`3 o
`
`emission beam having the emission frequency and an intensity representative of
`
`concentration of DNA in each suspension. The focusing means is receptive of and passes
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`2
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`the emission beam. A second means is disposed to be receptive of the emission beam from
`
`the focusing means so as to further pass the emission beam at the emission frequency to
`
`another focusing means that focuses the emission beam onto a detector. The detector
`
`generates primary data signals representative of the emission beam and thereby a
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`5
`
`corresponding concentration of DNA in each vial. A processor is receptive of the primary
`
`data signals for computing and displaying the concentration of DNA.
`
`In a preferred embodiment, the first means and the second means together comprise a
`
`beam splitter that is receptive of the source beam to effect the excitation beam, and
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`10
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`receptive of the emission beam to pass the emission beam to the detector. The block is
`
`configured to hold a plurality of vials, and the focusing means comprises a corresponding
`
`plurality of vial lenses each disposed over a vial such that the emission beam comprises
`
`individual beams each associated with a vial. The focusing means may further comprise a
`
`field lens such as a Fresnel lens disposed cooperatively with the vial lenses to effect
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`15
`
`focusing of the excitation beam into each suspension, and to pass the individual beams to
`
`the second means (beam splitter). The detector preferably comprises an array of
`
`photoreceptors receptive of the individual beams to generate corresponding data signals
`
`such that the processing means computes concentration of DNA in each vial.
`
`2 o
`
`The instrument should also include an excitation filter between the light source and the
`
`beam splitter, and an emission filter between the beam splitter and the detector. The
`
`splitter and filters are associated with a selected primary dye in the suspension. In a
`
`further embodiment, a filter module contains the splitter and filters, and the module is
`
`removable from the housing for replacement with another module associated with another
`
`2 5
`
`selected primary dye.
`
`For a reference, a fluorescent reference member emits reference light in response to the
`
`excitation beam. The reference is disposed to be receptive of a portion of the excitation
`
`beam from the first means. A portion of the reference light is passed by the second means
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`3 o
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`as a reference beam to the detector, so as to generate reference signals for utilization in
`
`the computing of the concentration of DNA. Preferably the reference member comprises
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`a plurality of reference emitters, each emitting a reference beam of different intensity in
`
`response to the excitation beam, to allow selection by the processor of a reference set
`
`having the highest data signals that are less than a predetermined maximum that is less
`
`than the saturation limit.
`
`5
`
`The detector is operatively connected to the processing means for the detector to integrate
`
`emission beam input over a preselected exposure time for generating each set of data
`
`signals, and the processing means or the detector or a combination thereof have a
`
`saturation limit for the data signals. ln a further aspect of the invention, the processing
`1 o means comprises adjustment means for automatically effecting adjustments in exposure
`time to maintain the primary data within a predetermined operating range for maintaining
`
`corresponding data signals less than the saturation limit, and means for correcting the
`
`primary data in proportion to the adjustments in exposure time. Preferably, the processor
`
`computes photoreceptor data from the data signals for each photoreceptor, and the
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`15
`
`adjustment means ascertains highest photoreceptor data, determines whether the highest
`
`photoreceptor data are less than, within or higher than the predetermined operating range
`
`and, based on such determination, the exposure time is increased, retained or reduced so
`
`as to effect a subsequent exposure time for maintaining subsequent photoreceptor data
`
`within the predetermined operating range.
`
`20
`
`25
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 is a schematic of an optical train for an optical instrument according to the
`
`invention, associated with a polymerase chain reaction (PCR) reaction apparatus.
`
`FIG. 2 is a perspective of the instrument of FIG. 1 with a side panel removed.
`
`FIG. 3 is an exploded perspective of a module shown in FIG. 2.
`
`3 o
`
`FIG. 4 is a perspective of a reference member in the optical train of FIG. 1.
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`FIG. 5 is a flow chart for computing DNA concentration from data obtained with the
`
`instrument of FIG. 1.
`
`FIG. 6 is a flow chart for determining exposure time for data acquisition in operation of
`
`5
`
`the instrument of FIG. 1 and for computations in the flow chart of FIG. 5.
`
`FIG. 7 is a graph of extension phase data of fluorescence vs. cycles from operation of the
`
`instrument of FIG. 1 with a PCR apparatus.
`
`10
`
`FIG. 8 is a flow chart for computing secondary data for computations in the flow chart of
`
`FIG. 5.
`
`FIG. 9 is a flow chart for computing ratios between the plurality of reference emitter
`
`segments of the reference member of FIG. 4.
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`15
`
`2 o
`
`DETAILED DESCRIPTION
`
`An optical instrument A of the invention is utilized with or incorporated into a reaction
`
`apparatus B that replicates ("amplifies") selected portions of DNA by polymerase chain
`
`reaction (11PCR11
`
`). The reaction apparatus is conventional and should function without
`
`interference from the instrument which monitors the amount of DNA in real time during
`
`replication. Suitable reaction apparatus are described in U.S. patent Nos. 5,475,610 and
`
`5,656,493.
`
`25
`
`The reaction apparatus (FIG. 1) is conventional and has two main components, namely a
`
`thermal cycler block 1 with wells la for holding at least one vial lb containing a
`
`suspension of ingredients for the reaction, and a thermal cycle controller le for cycling the
`
`temperature of the block through a specified temperature program. The starting
`
`ingredients of the aqueous suspension of sample materials include a "seed" sample of
`3 o DNA, selected DNA primer strands, DNA elements, enzymes and other chemicals. The
`block, typically aluminum, is heated and cooled in a prescribed cycle by electrical means,
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`liquid or air coolant, or a combination of these, or other means to achieve the cycling.
`
`The suspensions in the vials are thereby cycled between two temperature phases so as to
`
`effect the polymerase chain reaction. These phases are a lower temperature extension
`
`phase of the PCR reaction at about 60°C, which is the phase where all of the DNA strands
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`5
`
`have recombined into double strands, and a high temperature denaturing phase at about
`
`95°C, during which the DNA is denatured or split into single strands.
`
`For the present purpose the sample also contains a fluorescent dye that fluoresces
`
`proportionately and more strongly inthe presence of double stranded DNA to which the
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`10
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`dye binds, for example SYBR Green dye (available from Molecular Probes, Inc., Eugene,
`
`Oregon) that fluoresces in the presence of double stranded DNA. Another type of
`
`fluorescent dye labeled "probes", which are DNA-like structures with complimentary
`
`sequences to selected DNA strand portions, may also be used. Other dyes that have
`
`similar characteristics may be utilized. As used herein and in the claims, the term "marker
`
`15
`
`dye" refers to the type that binds to double stranded DNA, or to the probe type, or to any
`
`other type of dye that attaches to DNA so as to fluoresce in proportion to the quantity of
`
`DNA. Samples may also contain an additional, passive dye (independent of the DNA) to
`
`serve as a reference as described below. Under incidence oflight having a correct
`
`excitation frequency, generally a dye fluoresces to emit light at an emission frequency that
`
`2 o
`
`is lower than that of the excitation light.
`
`The vials typically are formed conically in a plastic unitary tray containing a plurality of
`
`vials, for example 96 in an array of 12 by 8. The tray preferably is removable from the
`
`block for preparations. A plastic unitary cover with caps ld for the vials may rest or
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`2 5
`
`attach over the vials to prevent contamination and evaporation loss. Other means may be
`
`used for this function, such as oil on the sample surface, in which case caps are not
`
`needed. If used, the caps are transparent to light utilized in the instrument, and may be
`
`convex facing upwardly.
`
`3 o
`
`The monitoring instrument is mounted over the block containing the vials. The instrument
`
`is removable or swings away for access to the vials. In the bottom of the instrument, a
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`platen 2 rests over the vial caps or, if none, directly over the vials. The platen,
`
`advantageously aluminum, has an array of holes 2a therethrough aligned with the vials,
`
`each hole having a diameter about the same as the vial top diameter. If there are caps, the
`
`platen should have its temperature maintained by a film heater or other means for heating
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`5
`
`the platen sufficiently to prevent condensation under the caps without interfering with
`
`DNA replication in the vials, for example holding the platen at slightly higher temperature
`
`than the highest sample temperature that the thermal cycler reaches.
`
`Above each of the vials is a lens 2b positioned for its focal point to be approximately
`
`1 o
`
`centered in the suspension in the vial. Above these lenses is a field lens 3 to provide a
`
`telecentric optical system. Advantageously the field lens is an aspherically corrected
`
`Fresnel lens for minimal distortion. A neutral density pattern (not shown) to correct
`
`nonuniformities in illumination and imaging may be mounted on or in proximity to the field
`
`lens, for example to attenuate light in the center of the image field. A folding optical
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`15
`
`mirror is optionally mounted at 45° for convenient packaging. This may be omitted, or
`
`other such folding optics may be used. Also the field lens, and/or the vial lenses, each may
`
`be comprised of two or more lenses that effect the required focusing, the word "lens"
`
`herein including such multiplicities.
`
`2 o A light source 11 for a source beam 20 of light is provided, for example a 100 watt
`halogen lamp. Preferably this is mounted at a focal distance of an ellipsoid reflector lla
`
`which produces a relatively uniform pattern over the desired area. Also, advantageously,
`
`the reflector should be dichroic, i.e. substantially reflecting visible light and transmitting
`
`infrared light, to restrict infrared from the other optical components and from overheating
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`2 5
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`the instrument. This is further aided by a heat reflecting mirror 13 in the optical path. A
`
`mechanical or electronic shutter 12 allows blockage of the light source for obtaining dark
`
`data. The type of light source is not critical, and other types may be used such as a
`
`projection lamp or a laser, with appropriate optical elements.
`
`3 o A beam splitter 6 is disposed to receive the source beam 20. In the present embodiment
`this is a dichroic reflector such that, positioned at 45°, it reflects light having an excitation
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`frequency that causes the marker dye to fluoresce at an emission frequency, and passes
`
`light having the emission frequency. Such a conventional optical device typically utilizes
`
`optical interference layers to provide the specific frequency response.
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`5
`
`The beam splitter is positioned to reflect the source beam to the folding mirror. The
`
`source beam is reflected from the splitter as a excitation beam 22 having substantially the
`
`excitation frequency. The excitation beam is focused by the field lens 3 and then as
`
`separated beams 24 by the vial (well) lenses 2b into the center of the vials. The marker
`
`dye is thereby caused to emit light at the emission frequency. This light is passed
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`10
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`upwardly as an emission beam in the form ofindividual beams 26 that are reflected from
`
`the folding mirror 5 to the beam splitter 6 which passes the emission beam through to a
`
`detector 10.
`
`Together the vial lenses 2b and the field lens 3 constitute a primary focusing means for
`
`15
`
`focusing both the excitation beam and the emission beam. In an alternative aspect, the
`
`field lens may be omitted so that the focusing means consists only of the vial lenses 2b.
`
`Alternatively, the vial lenses may be omitted so that the focusing means consists only of an
`
`objective lens in the field lens position to focus the individual emission beams on the
`
`detector.
`
`20
`
`Also, alternatively, the beam splitter 6 may pass the source beam as an excitation beam
`
`and reflect the emission beam, with appropriate rearrangement of the lamp and the
`
`detector. Moreover, other angles than 45° could be used if more suitable for the beam
`
`splitter, such as a more perpendicular reflection and pass through. More broadly, the
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`2 5
`
`beam splitter splits the optical paths for the excitation beam and the emission beam, and
`
`other variations that achieve this may be suitable. It is desirable to minimize source light
`
`reaching the detector, which the dichroic device helps achieve. A non-dichroic beam
`
`splitter may be used but would be less efficient as significant source light may reach the
`
`detector, or may be reflected or transmitted in the wrong direction and lost.
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`To further filter the source light, an excitation filter 7 is disposed between the light source
`
`11 and the beam splitter 6. This passes light having the excitation frequency and
`
`substantially blocks light having the emission frequency. Similarly, an emission filter 8 is
`
`disposed between the beam splitter and the detector, in this case between the splitter and a
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`5
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`detector lens 9 in front of the detector. This filter passes light having the emission
`
`frequency and substantially blocks light having the excitation frequency. Although a
`
`detector lens is preferred, a focusing reflector may be substituted for the detector lens.
`
`Such an emission focusing means (detector lens or reflector) may be located after (as
`
`shown) or before the beam splitter and on either side of the emission filter, and
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`10
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`alternatively may be integrated into the primary focusing means. For example, the field
`
`lens may be an objective lens that focuses the emission beam onto the detector.
`
`Suitable filters are conventional optical bandpass filters utilizing optical interference films,
`
`each having a bandpass at a frequency optimum either for excitation of the fluorescent dye
`
`15
`
`or its emission. Each filter should have very high attenuation for the other (non-bandpass)
`
`frequency, in order to prevent "ghost" images from reflected and stray light. For SYBR
`
`Green dye, for example, the excitation filter bandpass should center around 485 nm
`
`wavelength, and the emission filter bandpass should center around 555 nm. The beam
`
`splitter should transition from reflection to transmission between these two, e.g. about 510
`
`2 O
`
`nm, so that light less than this wavelength is reflected and higher wavelength light is
`
`passed through.
`
`More broadly, the excitation filter and the beam splitter together constitute a first means
`
`disposed to be receptive of the source beam to effect an excitation beam having the
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`2 5
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`excitation frequency, and the emission filter and the beam splitter together constitute a
`
`second means disposed to be receptive of the emission beam from the focusing means so
`
`as to pass the emission beam at the emission frequency to the detector. Also, as
`
`mentioned above, the beam splitter alternatively may pass the source beam as an excitation
`
`beam and reflect the emission beam to the detector. In another aspect, the filters may be
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`3 O
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`omitted, and the first means is represented by the beam splitter effecting the exitation
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`beam from the source beam, and the second means is represented by the beam splitter
`
`passing the emission beam to the detector.
`
`In another arrangement, the beam splitter may be omitted, and the first means may
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`5
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`constitute an excitation filter for the excitation frequency, the second means may
`
`constitute an emission filter for the emission frequency, with the light source and the
`
`detector being side by side so that the excitation and emission beams are on slightly
`
`different optical paths angularly. The source and detector need not actually be side by side
`
`with one or more folding mirrors. Thus any such arrangement for achieving the effects
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`10
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`described herein should be deemed equivalent. However, use of the beam splitter is
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`preferred so that the excitation and emission beams through the field lens will have the
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`same optical path.
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`Advantageously the beam splitter 6, the excitation filter 7 and the emission filter 8 are
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`15
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`affixed in a module 30 (FIG. 2) that is associated with a selected primary dye for the
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`suspension. The module is removable from the housing 32 of the instrument A for
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`replacement with another module containing different beam splitter and filters associated
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`with another selected primary dye. The instrument includes a lamp subhousing 33 and a
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`camera subhousing 35.
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`20
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`In an example (FIG. 3), each module includes a mounting block 34 with a flange 36 that is
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`affixable to the housing with a single screw 38. The beam splitter 6 is held at 45° in the
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`block with a frame 40 and screws 42. The emission filter 8 mounts (e.g. with glue) into
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`the block. The excitation filter 7 mounts similarly into a mounting member 44 that is held
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`2 5
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`by screws 46 to the block. With the module in place, the instrument is closed up with a
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`side plate 47 that is screwed on. Positioning pins (not shown) ensure repeatable
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`alignment. The replacement module may have the same mounting block and associated
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`components, with the beam splitter and filters replaced.
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`The detector lens 9 (FIG. 1) is cooperative with the vial lenses 2b and the field lens 3 to
`
`focus the individual beams on the detector 10. The lens should be large aperture, low
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`distortion and minimum vignetting.
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`5
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`The detector preferably is an array detector, for example a charge injection device (CID)
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`or, preferably, a charge coupled device (CCD). A conventional video camera containing a
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`CCD detector, the detector lens and associated electronics for the detector should be
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`suitable, such as an Electrim model 1 OOOL which has 7 51 active pixels horizontal and 24 2
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`(non-interlaced) active pixels vertical. This camera includes a circuit board that directly
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`10
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`interfaces to a computer ISA bus. No framegrabber circuitry is required with this camera.
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`Essentially any other digital imaging device or subsystem may be used or adapted that is
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`capable of taking still or freeze-frame images for post processing in a computer.
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`A detector with a multiplicity of photoreceptors (pixels) 78 is preferable ifthere are a
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`15
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`plurality of vials, to provide separate monitoring of each. Alternatively a scanning device
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`may be used with a single ph,otodetector, for example by scanning the folding mirror and
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`using a small aperture to the detector. Also, a simple device such as a photomultipier may
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`be used ifthere is only one vial. A CCD receives light for a selected integration period
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`and, after analog/digital conversion, reads out digital signal data at a level accumulated in
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`2 o
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`this period. The integration is effectively controlled by an electronic shutter, and a frame
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`transfer circuit is desirable. Signal data are generated for each pixel, including those
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`receiving the individual beams of emitted light from the vials.
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`The instrument preferably includes a fluorescent reference member 4 that emits reference
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`2 5
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`light in response to the excitation beam. Advantageously the reference member is formed
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`of a plurality of reference emitters, e.g. 6, each emitting a reference beam of different
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`intensity in response to the excitation beam. The range of these intensities should
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`approximate the range of intensities expected from the marker dye in the vials; for
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`example each segment may be separated in brightness by about a factor of2.5. The
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`3 o
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`reference member is disposed to receive a portion of the excitation beam from the beam
`
`splitter. A good location is adjacent to the field lens, so that the optical paths associated
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`with the member approximate those of the vials. Most of the reference light passes back
`
`through the beam splitter as a reference beam to the detector. The detector pixels receive
`
`the emission beam to generate reference signals for utilization along with the data signals
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`in the computing of the concentration ofDNA.
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`5
`
`Advantageously the reference member 4 (FIG. 4) comprises a plastic fluorescent strip 4a
`
`and a neutral density filter 4b mounted over the fluorescent strip, optionally with an air
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`space 4h between, such that a portion of the excitation beam and the reference beam are
`
`attenuated by the neutral density filter. The neutral density filter has a series of densities
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`10
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`4c to effect the plurality of reference emitters (segments) each emitting a reference beam
`
`of different intensity. A heating strip 4d and an aluminum strip 4g to smooth the heating
`
`are mounted in a trough 4e on the bottom thereof, and the fluorescent strip is mounted on
`
`the aluminum strip over the heating strip. To prevent heat loss, this assembly preferably is
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`covered by a transparent plexiglass window (not shown, so as to display the varying
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`15
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`density filter). To help maintain constant fluorescence, the heating strip is controlled to
`
`maintain the fluorescent strip at a constant temperature against the thermal cycles of the
`
`cycler block and other effects. This is done because most fluorescent materials change in
`
`fluorescence inversely with temperature.
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`2 o
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`The computer processor 14 (FIG. 1) may be a conventional PC. The computer
`
`programming is conventional such as with "C". Adaptations of the programming for the
`
`present invention will be readily recognized and achieved by those skilled in the art. The
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`processor selectively processes signals from pixels receiving light from the vials and the
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`reference emitters, ignoring surrounding light. The programming therefore
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`25
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`advantageously includes masking to define the pixel regions ofinterest (ROI), e.g. as
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`disclosed in copending provisional patent application serial No. 60/092,785 filed 07/14/98
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`of the present assignee. Mechanical alignment of the optics may be necessary to
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`cooperatively focus the beams into the programmed regions of interest. The analog data
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`signals are fed to the processor through an analog/digital (AID) device 15 which, for the
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`3 o
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`present purpose, is considered to be part of the processor. A saturation level is proscribed
`
`by either the detector or the AID or, preferably, the CCD dynamic range is matched to the
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`ND dynamic range. A suitable range is 8 bits of precision (256 levels), and the CCD
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`amplifier offset is set so that the dark signal output of the CCD (with the shutter 12
`closed) is within the ND range. The processor instructs the detector with selected
`
`exposure time to maintain the output within the dynamic range.
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`5
`
`In a typical operation, fluorescence data are taken from the plurality of vials (e.g. 96
`
`regions of interest) and from the reference emitter segments, for each cycle in a DNA
`
`reaction replication sequence of thermal cycles, typically 40 to 50. Two data sets are
`
`taken (FIG. 5) for each thermal cycle during the extension phase of the PCR reaction at
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`10