Ulllted States Patent [19]
`Woudenberg et al.
`
`US005928907A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,928,907
`*Jul. 27, 1999
`
`[54] SYSTEM FOR REAL TIME DETECTION OF
`glggl?glééécn) AMPLIFICATION
`
`[75] Inventors: Timothy M- woudenberg, Half Moon
`Bay; Kevin 5- Bodner, San Mateo;
`Charles R. Connell, Redwood City, all
`of Calif; Alan M. Ganz, Trumbull,
`Conn; Lincoln J. McBride, Belmont,
`Calif; Paul G_ Saviano, Norwalk,
`Conn; John Shigeura, Fremont, Calif‘;
`David H_ Tracy, NOrWa1k;Eugene R
`Young, Wilton, both of Conn‘; Linda
`G_ Lee, Palo Alto, Calif
`
`FOREIGN PATENT DOCUMENTS
`0108524 5/1984 European Pat. Off .
`0421156 4/1991 European Pat. Off .
`0 421 156 A2 10/1991 European Pat. Off ..... .. G01N 21/64
`(List continued on next page.)
`
`OTHER PUBLICATIONS
`_
`Haugland, pp. 111—112 and 221—229 in Handbook of Fluo
`rescent Probes and Research Chemicals, 5th Edition,
`1992—1994 (Molecular Probes, Eugene, OR, 1992), pp.
`111—112: “Physiological indicators and tracers” pp.
`221—229: “Nucleic acid stains”.
`
`[73] Assignee: The Perkin-Elmer C0rp0rati0n.,
`égphceghlglosystems Dlvlslon’ Foster
`y’
`'
`
`(Llst Con?rmed on next page)
`Primary Examiner—Kenneth R. Horlick
`Attorney, Agent, or Firm—Wilson Sonsini Goodrich &
`
`[*] Notice:
`
`Rosan
`[57]
`
`ABSTRACT
`
`This patent issued on a continued pros-
`ecution application ?led under 37 CFR
`1.53(d), and is subject to the tWenty year
`patent term provisions of 35 USC Asystem is provided for carrying out real time ?uorescence
`154(a)(2)_
`based measurements of nucleic acid ampli?cation products.
`In a preferred embodiment of the invention, an excitation
`beam is focused into a reaction mixture through a surface,
`the reaction mixture containing
`a ?rst ?uorescent indi
`cator capable of generating a ?rst ?uorescent signal Whose
`intensity is proportional to the amount of an ampli?cation
`product in the volume of the reaction mixture illuminated by
`the excitation beam and (ii) a second ?uorescent indicator
`homogeneously distributed throughout the reaction mixture
`capable of generating a second ?uorescent signal propor
`tiehal t0 the Volume of reaetieh IhiXthre illuminated by the
`excitation beam Preferabhh the excitation beam is focused
`into the reaction mixture by a lens through a portion of a Wall
`of a Closed reaction Phamber Containing the reaction miX'
`ture. The same lens is used to collect the ?rst and second
`?uorescent signals generated by the ?rst and second ?uo
`rescent indicators, respectively, in response to the excitation
`beam. The ratio of the ?uorescent intensities of the ?rst and
`second ?uorescent signals provides a stable quantitative
`indicator of the amount of ampli?cation product synthesized
`in the Course of the ampli?cation reaction‘
`
`_
`[21] Appl' NO" 08/752’973
`[22]
`Filed;
`Dec, 2, 1996
`
`Related US. Application Data
`
`[63] Continuation of application No- 08/235,411, Apr- 29, 1994,
`abandoned
`[51] Int. Cl.6 ............................. .. C12Q 1/68; C12P 19/34
`[52] US. Cl. ........................ .. 435/912; 435/6; 422/8205;
`422/8206; 422/8207; 422/8208; 422/8211
`[58] Field of Search ............................ .. 422/8205, 82.06,
`422/8207 8208 8211, 435/6 911 912
`’
`’
`’
`’
`’
`
`[56]
`
`.
`References Clted
`us PATENT DOCUMENTS
`_
`
`............................... .. 250/458
`
`(List continued on next page.)
`
`28 Claims, 6 Drawing Sheets
`
`0.7
`
`0.6
`
`0.5
`
`0.4
`
`0.3
`
`02
`
`0.1
`
`
`
`Relative Intensity
`
`2000
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`4000
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`6000
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`8000
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`Time (s)
`
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`5,928,907
`Page 2
`
`US. PATENT DOCUMENTS
`
`4,747,687
`4,916,060
`4,954,435
`5,037,199
`5,038,852
`5,135,717
`5,239,360
`5,538,848
`5,547,861
`
`5/1988 Hoppe et al. .
`4/1990 Weaver .
`9/1990 Krauth ...................................... .. 435/7
`8/1991 Hlousek .
`8/1991 Johnson et al. .
`8/1992 RenZoni et al. ........................ .. 422/61
`8/1993 Moring et al. .
`7/1996 Livak et al. .............................. .. 435/5
`8/1996 Nadeau et al.
`.. 435/91.2
`
`5,556,751
`
`9/1996 Stefano . . . . . . . . .
`
`. . . . . . .. 435/6
`
`5,593,867
`
`1/1997 Walker et al. ....................... .. 435/91.2
`
`FOREIGN PATENT DOCUMENTS
`
`0488769 6/1992 European Pat. Off. .
`0 512 334 A2 11/1992 European Pat. Off. .
`5-38297 2/1993 Japan ............................. .. C12Q 1/00
`89/04302 5/1989 WIPO .
`92/02638 2/1992 WIPO .
`
`OTHER PUBLICATIONS
`Holland et al, Proc. Natl. Acad. Sci., 88: 7276—7280 (1991)
`Detection of speci?c polymerase chain reaction product by
`utilizing the 5‘—>3‘ eXonuclease activity of Thermus aquati
`cus DNA polymerase.
`Higuchi, et al Biotechnology, 11: 1026—1030 (1993) Kinetic
`PCR analysis: Real—time monitoring of DNA ampli?cation
`reactions.
`Higuchi, et al Biotechnology, 10: 413—417 (1992) Simulta
`neous ampli?cation and detection of speci?c DNA
`sequences.
`Lee et al, Nucleic Acids Research, 21: 3761—3766 (1993),
`Allelic discrimination by nick—translation PCR With ?uoro
`genic probes.
`Berg et al, Abstract from meeting entitled “Novel Ampli?
`cation Technologies” held Apr. 20—22, 1994, in San Fran
`cisco, An instrumentation system for the real time ?uores
`cence detection of Q—beta replicase ampli?cation reactions.
`Biochim. Biophys. Acta 723, 169—175, Krause et al. (1983).
`Analyt. Biochem. 104, 315—320, Gershoni et al., (1980).
`
`THERMO FISHER EX. 1019
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`U.S. Patent
`
`Jul. 27, 1999
`
`Sheet 1 0f 6
`
`5,928,907
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`THERMO FISHER EX. 1019
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`U.S. Patent
`
`Jul. 27, 1999
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`Sheet 2 0f 6
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`5,928,907
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`THERMO FISHER EX. 1019
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`U.S. Patent
`
`Jul. 27, 1999
`
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`U.S. Patent
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`Jul. 27, 1999
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`U.S. Patent
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`Jul. 27, 1999
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`THERMO FISHER EX. 1019
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`

`
`1
`SYSTEM FOR REAL TIME DETECTION OF
`NUCLEIC ACID AMPLIFICATION
`PRODUCTS
`
`This application is a continuation of application Ser. No.
`8/235,411, ?led Apr. 29, 1994, abandoned.
`The invention relates generally to the ?eld of nucleic acid
`ampli?cation, and more particularly to a system for mea
`suring in real time polynucleotide products from nucleic
`acid ampli?cation processes, such as polymerase chain
`reaction (PCR).
`
`BACKGROUND
`
`Nucleic acid sequence analysis is becoming increasingly
`important in many research, medical, and industrial ?elds,
`e.g. Caskey, Science 236: 1223—1228 (1987); Landegren et
`al, Science, 242: 229—237 (1988); and Arnheim et al, Ann.
`Rev. Biochem., 61: 131—156 (1992). The development of
`several nucleic acid ampli?cation schemes has played a
`critical role in this trend, e.g. polymerase chain reaction
`(PCR), Innis et al, editors, PCR Protocols (Academic Press,
`NeW York, 1990); McPherson et al, editors, PCR: APractical
`Approach (IRL Press, Oxford, 1991); ligation-based ampli
`?cation techniques, Barany, PCR Methods and Applications
`1:5—16 (1991); and the like.
`PCR in particular has become a research tool of major
`importance With applications in cloning, analysis of genetic
`expression, DNA sequencing, genetic mapping, drug
`discovery, and the like, eg Arnheim et al (cited above);
`Gilliland et al, Proc. Natl. Acad. Sci., 87: 2725—2729 (1990);
`Bevan et al, PCR Methods and Applications, 1: 222—228
`(1992); Green et al, PCR Methods and Applications, 1:
`77—90 (1991); BlackWell et al, Science, 250: 1104—1110
`(1990).
`AWide variety of instrumentation has been developed for
`carrying out nucleic acid ampli?cations, particularly PCR,
`e.g. Johnson et al, US. Pat. No. 5,038,852 (computer
`controlled thermal cycler); WittWer et al, Nucleic Acids
`Research, 17: 4353—4357 (1989)(capillary tube PCR);
`Hallsby, US. Pat. No. 5,187,084 (air-based temperature
`control); Garner et al, Biotechniques, 14: 112—115 (1993)
`(high-throughput PCR in 864-Well plates); Wilding et al,
`International application No. PCT/US93/04039 (PCR in
`micro-machined structures); Schnipelsky et al, European
`patent application No. 903010619 (publ. No. 0381501
`A2)(disposable, single use PCR device), and the like. Impor
`tant design goals fundamental to PCR instrument develop
`ment have included ?ne temperature control, minimiZation
`of sample-to-sample variability in multi-sample thermal
`cycling, automation of pre- and post-PCR processing steps,
`high speed cycling, minimiZation of sample volumes, real
`time measurement of ampli?cation products, minimiZation
`of cross-contamination, or sample carryover, and the like. In
`particular, the design of instruments that permit PCR to be
`carried out in closed reaction chambers and monitored in
`real time is highly desirable. Closed reaction chambers are
`desirable for preventing cross-contamination, e. g. Higuchi et
`al, Biotechnology, 10: 413—417 (1992) and 11: 1026—1030
`(1993); and Holland et al, Proc. Natl. Acad. Sci., 88:
`7276—7280 (1991). Clearly, the successful realiZation of
`such a design goal Would be especially desirable in the
`analysis of diagnostic samples, Where a high frequency of
`false positives and false negatives Would severely reduce the
`value of the PCR-based procedure. Real time monitoring of
`a PCR permits far more accurate quantitation of starting
`target DNA concentrations in multiple-target ampli?cations,
`
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`as the relative values of close concentrations can be resolved
`by taking into account the history of the relative concentra
`tion values during the PCR. Real time monitoring also
`permits the ef?ciency of the PCR to be evaluated, Which can
`indicate Whether PCR inhibitors are present in a sample.
`Holland et al (cited above) and others have proposed
`?uorescence-based approaches to provide real time mea
`surements of ampli?cation products during a PCR. Such
`approaches have either employed intercalating dyes (such as
`ethidium bromide) to indicate the amount of double stranded
`DNA present, or they have employed probes containing
`?uorescer-quencher pairs (the so-called “Tac-Man”
`approach) that are cleaved during ampli?cation to release a
`?uorescent product Whose concentration is proportional to
`the amount of double stranded DNA present.
`Unfortunately, successful implementation of these
`approaches has been impeded because the required ?uores
`cent measurements must be made against a very high
`?uorescent background. Thus, even minor sources of instru
`mental noise, such as the formation of condensation in the
`chamber during heating and cooling cycles, formation of
`bubbles in an optical path, particles or debris in solution,
`differences in sample volumes—and hence, differences in
`signal emission and absorbence, and the like, have hampered
`the reliable measurement of the ?uorescent signals.
`In vieW of the above, it Would be advantageous if an
`apparatus Were available Which permitted stable and reliable
`real time measurement of ?uorescent indicators of ampli?
`cation products resulting from any of the available nucleic
`acid ampli?cation schemes.
`
`SUMMARY OF THE INVENTION
`
`The invention relates to a system for carrying out real time
`?uorescence-based measurements of nucleic acid ampli?
`cation products. In a preferred embodiment of the invention,
`an excitation beam is focused into a reaction mixture con
`taining
`a ?rst ?uorescent indicator capable of generating
`a ?rst ?uorescent signal Whose intensity is proportional to
`the amount of an ampli?cation product in the volume of the
`reaction mixture illuminated by the excitation beam and (ii)
`a second ?uorescent indicator homogeneously distributed
`throughout the reaction mixture and capable of generating a
`second ?uorescent signal proportional to the volume of
`reaction mixture illuminated by the excitation beam. It is
`understood that the proportionality of the ?uorescent inten
`sities is for a constant set of parameters such as temperature,
`pH, salt concentration, and the like, that independently
`in?uence the ?uorescent emissions of organic dyes.
`Preferably, the excitation beam is focused into the reac
`tion mixture by a lens through a portion of a Wall of a closed
`reaction chamber containing the reaction mixture. In further
`preference, the same lens collects the ?rst and second
`?uorescent signals generated by the ?rst and second ?uo
`rescent indicators, respectively, in response to the excitation
`beam; thus, variability in the collected signal due to mis
`alignment of excitation and collection optics is avoided. In
`this embodiment, Whenever the lens directs the excitation
`beam through a portion of a Wall of the closed reaction
`chamber Which is not in contact With the reaction mixture,
`that portion of the Wall is heated so that condensation from
`the reaction mixture does not form in the optical pathWay of
`the ?uorescent signals being collected by the lens, thereby
`removing another source of variability in the collected
`signal.
`In the most preferred embodiment, the reaction chamber
`is a tube With a closed end, referred to herein as the bottom
`
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`5,928,907
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`DEFINITIONS
`
`3
`of the tube, and an open end, referred to herein as the top of
`the tube, Which can be closed With a cap such that a
`leak-proof seal is formed. In other Words, once a reaction
`mixture is placed in the tube and the cap is attached a closed
`reaction chamber is formed. In this most preferred
`embodiment, (1) the reaction mixture ?lls a portion of the
`tube, generally at the bottom of the tube, such that a void is
`left betWeen the cap of the tube and a top surface of the
`reaction mixture, and (2) the lens Without contacting the cap
`focuses the excitation beam through the cap into the reaction
`mixture through its top surface and collects the resulting
`?uorescence generated by the ?rst and second ?uorescent
`indicators. As mentioned above, the portion of the tube
`through Which the excitation beam passes—the cap in this
`embodiment—is heated to prevent the formation of conden
`sation Which Would introduce an added source of variability
`in the measurement of the collected ?uorescent signals.
`Potential variability that could arise from sequential analysis
`of the ?rst and second ?uorescent signals is eliminated by
`simultaneously analyZing the signals by spectrally separat
`ing the signal light onto an array of photo detectors, eg by
`diffracting the signal onto a charged-coupled device (CCD)
`array.
`As discussed more fully beloW, an excitation beam gen
`erated by a single light source, eg a laser, is conveniently
`distributed to a plurality of closed reaction chambers by ?ber
`optics. Likewise, the same ?ber optics can collect the
`?uorescent signals from the plurality of reaction chambers
`for analysis by a single detection and analysis system.
`Preferably, the system is employed With the PCR ampli
`?cation of nucleic acids.
`The system of the invention permits accurate real time
`monitoring of nucleic ampli?cation reactions by providing
`apparatus and ?uorescent reagents for generating a stable
`?uorescent signal proportional to the amount of ampli?ca
`tion product and independent of variations in the volume of
`reaction mixture. The availability of data shoWing the
`progress of ampli?cation reactions leads to more accurate
`estimates of relative starting concentrations of target nucleic
`acids, to rapid assessment of the ef?ciency of the ampli?
`cation reactions, and opens the possibility of reduced
`reagent usage and feedback reaction control.
`
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1 diagrammatically illustrates a preferred embodi
`ment of the sample interface components of the system of
`the invention.
`FIG. 2 diagrammatically illustrates a preferred embodi
`ment for simultaneously monitoring a plurality of ampli?
`cation reactions by sequentially interrogating reactions via a
`?ber optic multiplexer.
`FIG. 3 shoWs spectrally separated ?uorescent intensity
`data for a tetramethylrhodamine ?uorescent indicator, a
`?uorescein ?uorescent indicator, and instrument background
`registered by a CCD array of the preferred embodiment
`described beloW.
`FIG. 4 shoWs the time dependence of ?uorescent signals
`from a ?uorescein dye proportional to the ampli?cation
`product (?rst ?uorescent indicator) and a tetramethyl
`rhodamine dye employed as a second ?uorescent indicator
`during a typical PCR.
`FIG. 5 shoWs the cycle dependence of the ratio of the
`intensities of the ?uorescein and tetramethylrhodamine dyes
`from the same PCR Whose time dependent data is shoWn in
`FIG. 3.
`FIG. 6 shoWs data relating the amount of ampli?cation
`product to cycle number in separate PCRs having different
`starting concentrations of the same target nucleic acid.
`
`As used herein, the term “stable” in reference to a
`?uorescent signal means that the root means square (RMS)
`deviation in the signal due to noise is less than or equal to
`tWo percent of the average signal magnitude. More
`preferably, stable means that the RMS deviation in the signal
`due to noise is less than or equal to one percent of the
`average signal magnitude.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The invention is a ?uorescence-based system for moni
`toring in real time the progress of a nucleic acid ampli?ca
`tion reaction. The type of ampli?cation scheme used With
`the system is not critical, but generally the system requires
`either the use of a nucleic acid polymerase With exonuclease
`activity or a population of double stranded DNA Which
`increases during the course of the reaction being monitored.
`Exemplary ampli?cation schemes that may be employed
`With the system of the invention include PCR, ligase-based
`ampli?cation schemes, such as ligase chain reaction (LCR),
`Q-beta replicase-based ampli?cation schemes, strand dis
`placement ampli?cation (SDA) schemes, such as described
`by Walker et al, Nucleic Acids Research, 20:1691—1696
`(1992), and the like. Acomprehensive description of nucleic
`acid ampli?cation schemes is provided by Keller and
`Manak, DNA Probes, Second Edition (Stockton Press, NeW
`York, 1993). Fundamental to the system is the measurement
`of ratios of ?uorescent intensities of a ?rst ?uorescent
`indicator and an internal standard, referred to herein as a
`second ?uorescent indicator. The ?rst and second ?uores
`cent indicators must be spectrally resolvable. That is, their
`respective emission spectra must be suf?ciently non
`overlapping so that separate emission peaks are observed in
`the combined spectrum. Clearly, the system may be gener
`aliZed to include a plurality of ?rst ?uorescent indicators,
`eg to monitor the simultaneous ampli?cation of several
`target nucleic acids in a single reaction, so that a plurality of
`?uorescent intensity ratios are monitored. Several spectrally
`resolvable dyes suitable for use in such embodiments are
`disclosed in Fung et al, US. Pat. No. 4,855,225; Menchen
`et al, US. Pat. No. 5,188,934; Bergot et al, International
`Application PCT/JS90/05565; and like references.
`The system includes a sample interface—that is, optical
`components operationally associated With a closed reaction
`chamber—Which comprises a lens for focusing an excitation
`beam into the reaction mixture and for collecting the result
`ing ?uorescence and a ?ber optic for transmitting both the
`excitation beam from a light source to the lens and the
`?uorescent signals from the lens to a detection and analysis
`means. Preferably, the reaction mixture is contained in a
`closed reaction chamber to prevent cross-sample
`contamination, or so-called “carryover.” The lens therefore
`focuses the excitation beam and collects ?uorescence
`through a portion of a Wall of the closed reaction chamber.
`As mentioned above, the preferred reaction chamber is a
`tube, e.g. having the geometry and volume of a conventional
`Eppendorf tube. The tube is closed after the reaction mixture
`is added by attaching a cap to the open end of the tube. In
`a preferred embodiment of the sample interface for PCR, the
`lens directs the excitation beam and collects ?uorescence
`through the cap of the tube, as illustrated in FIG. 1. In the
`illustrated con?guration, a ?rst end ?ber optic 2 is held by
`ferrule 4, housing 6, and plate 10 in a co-axial orientation
`With lens 8. A second end of ?ber optic 2 (not shoWn) is
`operationally associated With a light source and detection
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`and analysis means, discussed more fully below. The dis
`tance between the end face of ?ber optic 2 and lens 8 is
`determined by several factors, including the numerical aper
`ture of the ?ber optic, the geometry of tube 18, the focal
`length of lens 8, the diameter of lens 8, and the like.
`Guidance for selecting values for such variables in any
`particular embodiment is readily found in standard texts on
`optical design, e.g. Optics Guide 5 (Melles Griot, Irvine,
`Calif., 1990), or like reference. In the illustrated
`embodiment, lens 8 has a diameter of 8 mm and is composed
`of material BK7, available from Edmund Scienti?c
`(Barrington, N.J Fiber optic 2 has a numerical aperture of
`2. Preferably, the design permits maximal transmission of
`excitation beam 28 to reaction mixture 22. For example, lens
`8, numerical aperture of ?ber optic 2, and the distance
`betWeen the end of ?ber optic 2 and lens 8 are selected so
`that the diameter of lens 8 equals or exceeds the diameter of
`excitation beam 28 Where beam 28 impinges on the lens (as
`illustrated in FIG. 1). Excitation beam 28 is focused through
`cap 16, void 24, and top surface 26 of reaction mixture 22
`to a region approximately 1—3 times the diameter of the ?ber
`optic just beloW, e.g. 1—3 mm, surface 26. This degree of
`focusing is not a critical feature of the embodiment; it is a
`consequence of adapting the sample interface to the geom
`etry and dimensions of a sample holder of a commercially
`available thermal cycler. In other embodiments, the geom
`etry and dimension may permit a sharper focus into the
`reaction mixture.
`The lens of the invention may have a variety of shapes
`depending on particular embodiments. For example, the lens
`may be a sphere, truncated sphere, cylinder, truncated
`cylinder, oblate spheroid, or truncated oblate spheroid, or the
`like, and may be composed of any suitably transparent
`refractive material, such as disclosed by Hlousek, US. Pat.
`No. 5,037,199; Hoppe et al, US. Pat. No. 4,747,87; Moring
`et al, US. Pat. No. 5,239,360; Hirsch?eld, US. Pat. No.
`4,577,109; or like references.
`Fluorescent light generated by excitation beam 28 is
`collected by lens 8 along approximately the same optical
`pathWay as that de?ned by excitation beam 28 and focused
`onto the end of ?ber optic 2 for transmission to optical
`separation and analysis components of the system.
`In further preference, the sample interface also includes
`means for heating the portion of the Wall of the reaction
`chamber used for optical transmission in order to reduce
`variability due to scatter and/or absorption of the excitation
`beam and signal from condensation of reaction mixture
`components. In the embodiment of FIG. 1, the portion of the
`reaction chamber (tube 18) Wall used for optical transmis
`sion is cap 16. Accordingly, heating element 12 and heat
`conductive platen 14 are employed to heat cap 16.
`Preferably, heating element 12 comprises resistance heating
`elements and temperature sensors that permit programmed
`controlled of the temperature of cap 16. Cap 16 is main
`tained at a temperature above the condensation points of the
`components of the reaction mixture. Generally, cap 16 may
`be maintained at a temperature in the range of 94—110° C.
`Preferably, cap 16 is maintained at a temperature in the range
`of about 102° C. to about 105° C. since the principal solvent
`in the reaction mixture is usually Water. More preferably, cap
`16 is maintained at 103° C. Preferably, in embodiments
`employing thermal cycling, the cap-heating components
`described above are thermally isolated from heating
`conducting component 20 employed to cyclically control the
`temperature of reaction mixture 22.
`Selection of appropriate materials for the components
`described above is Well Within the skill of an ordinary
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`mechanical engineer. Exemplary criterion for material selec
`tion include
`degree of thermal expansion, especially for
`ampli?cation schemes employing thermal cycling, and its
`affect on the alignment of the optical components, (ii)
`optical transmission properties in the excitation Wavelengths
`and ?uorophore emission Wavelengths employed, (iii)
`chemical inertness of the reaction chamber relative to com
`ponents of the reaction mixture, (iv) degree to Which critical
`reaction components, eg polymerases, target nucleic acids,
`Would tend to adsorb onto chamber Walls, (v) minimiZation
`of ?uorescent materials in the optical pathWay, and the like.
`Typically, tubes containing ampli?cation reaction mixtures
`are made of polypropylene or like materials.
`The sample interface shoWn in FIG. 1 may be employed
`individually or it may be employed as one of a plurality of
`identical interfaces in a single instrument, as shoWn dia
`grammatically in FIG. 2. In the illustrated embodiment,
`individual sample interfaces 31, arrayed in holder 30 (Which
`may, for example, be a heating block associated With thermal
`cycler 32, such as described in Mossa et al, European patent
`application No. 913110904, publ. No. 0488769 A2) are
`connected by ?ber optics 34 to ?ber optic multiplexer 36,
`Which selectively permits transmission betWeen individual
`?ber optics and port 35, eg under user control via a
`programmed microprocessor. In a preferred con?guration,
`excitation beam 41, generated by light source 52 and con
`troller 54, passes through beam splitter 40 and is focused
`onto port 35 by lens 38, Where it is sequentially directed by
`?ber optic multiplexer 36 to each of a predetermined set, or
`subset, of ?ber optics 34. Conversely, a ?uorescent signal
`generated in a reaction chambers is collected by lens 8 and
`focused onto a ?ber optic Which, in turn, transmits the signal
`to a detection and analysis means, possibly via a ?ber optic
`multiplexer. Returning to FIG. 2, a ?uorescent signal col
`lected by a sample interface is directed to ?ber optic
`multiplexer 36 Where it emerges through port 35 and is
`collected and collimated by lens 38. Lens 38 directs the
`?uorescent signal to beam splitter 40 Which, in turn, selec
`tively directs the signal through cut-off ?lter 42, Which
`prevents light from the excitation beam from reaching the
`signal detection components. Beam splitter 40 may be a
`conventional dichroic mirror, a fully re?ective mirror With
`an aperture to pass the excitation beam (eg as disclosed in
`US. Pat. No. 4,577,109), or like component. After passing
`through cut-off ?lter 42, the ?uorescent signal is directed by
`lens 44 to a spectral analyZer Which spectrally separates the
`?uorescent signal and measures the intensities of a plurality
`of the spectral components of the signal. Typically, a spectral
`analyZer comprises means for separating the ?uorescent
`signal into its spectral components, such as a prism, diffrac
`tion grating, or the like, and an array of photo-detectors,
`such as a diode array, a charge-coupled device (CCD)
`system, an array of bandpass ?lters and photomultiplier
`tubes, or the like. In the preferred embodiment of FIG. 2, the
`spectral analyZer comprises diffraction grating 46 (e.g.,
`model CP-140, Jobin-Yvon, NJ) and CCD array 48 (e.g.,
`model S2135 Princeton Instruments, NJ), Which is linked to
`CCD controller 50.
`An exemplary CCD array suitable for analyZing ?uores
`cent signal from ?uorescein and tetramethylrhodamine is
`partitioned into 21 collection bins Which span the 500 nm to
`650 nm region of the spectrum. Each bin collects light over
`a 8.5 nm WindoW. Clearly, many alternative con?gurations
`may also be employed. An exemplary application of a CCD
`array for spectral analysis is described in Karger et al,
`Nucleic Acids Research, 19: 4955—4962 (1991).
`AnalyZing the ?uorescent signal based on data collected
`by a spectral analyZer is desirable since components of the
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`THERMO FISHER EX. 1019
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`signal due to one or more ?rst ?uorescent indicators and a
`second ?uorescent indicator (from Which intensity ratios are
`calculated) can be analyzed simultaneously and Without the
`introduction of Wavelength-speci?c system variability that
`might arise, eg by misalignment, in a system based on
`multiple beam splitters, ?lters, and photomultiplier tubes.
`Also, a spectral analyZer permits the use of “virtual ?lters”
`or the programmed manipulation of data generated from the
`array of photo-detectors, Wherein a plurality of discrete
`Wavelength ranges are sampled—in analogy With physical
`bandpass ?lters—under programmable control via an asso
`ciated microprocessor. This capability permits a high degree
`of ?exibility in the selection of dyes as ?rst and second
`?uorescent indicators.
`Generally, the detection and analysis means may be any
`detection apparatus to provides a readout that re?ect the
`ratio of intensities of the signals generated by the ?rst and
`second ?uorescent indicators. Such apparatus is Well knoW
`in the art, as exempli?ed by US. Pat. Nos. 4,577,109 and
`4,786,886 and references such as The Photonics Design &
`20
`Applications Handbook, 39th Edition (Laurin Publishing
`Co., Pitts?eld, Mass., 1993).
`Preferably, the system of the invention is employed to
`monitor PCRs, although it may also be employed With a
`variety of other ampli?cation schemes, such as LCR.
`Descriptions of and guidance for conducting PCRs is pro
`vided in an extensive literature on the subject, eg including
`Innis et al (cited above) and McPherson et al (cited above).
`Brie?y, in a PCR tWo oligonucleotides are used as primers
`for a series of synthetic reactions that are catalyZed by a
`DNA polymerase. These oligonucleotides typically have
`different sequences and are complementary to sequences
`that
`lie on opposite strands of the template, or target,
`DNA and (ii) ?ank the segment of DNA that is to be
`ampli?ed. The target DNA is ?rst denatured by heating in
`the presence of a large molar excess of each of the tWo
`oligonucleotides and the four deoxynucleoside triphosphates
`(dNTPs). The reaction mixture is then cooled to a tempera
`ture that alloWs the oligonucleotide primers to anneal to their
`target sequences, after Which the annealed primers are
`extended With DNA polymerase. The cycle of denaturation,
`annealing, and extension is then repeated many times,
`typically 25—35 times. Because the products of one round of
`ampli?cation serve as target nucleic acids for the next, each
`successive cycle essentially doubles the amount of target
`DNA, or ampli?cation product.
`As mentioned above an important aspect of the invention
`is the ?uorescent dyes used as the ?rst and second ?uores
`cent indicators. By examining the ratio of the ?uorescent
`intensities of the indicators, the effects of most sources of
`systematic variability, Which Would be apparent in the
`intensities alone, are eliminated. Generally, in accordance
`With the invention, the ?rst ?uorescent indicator may be a
`complex-forming dye or a dye covalently attached to an
`oligonucleotide probe Which is degraded during polymer
`iZation steps to generate a signal. This later embodiment
`relates to the so-called “Tacman” approach, described by
`Holland et al, Proc. Natl. Acad. Sci., 88: 7276—7280 (1991).
`As used herein, the term “complex-forming” in reference to
`a dye means that a dye is capable of forming a stable