WORLD IN'I'ELLEC'I'UAL_ PROPERTY ORGANIZATION
`International Bureau
`
`
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`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 3
`
`(11) International Publication Number:
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`WO 95/30139
`
`G01N 21/64, C12Q 1/68
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`_
`_
`_
`(43) International Publication Date:
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`9 November 1995 (09.11.95)
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`(21) International Application Number:
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`PCT/US95/04818
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`(22) International Filing Date:
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`19 April 1995 (1904.95)
`
`(74) Agents‘ MACEVICZ Stephen: C- *3‘ 31-? PeTkin'E1m€f COT‘
`poration, Applied Biosystems Division, 850 Lincoln Centre
`Drive’ F0516‘ City: CA 94404 (US)-
`
`(30) Priority Data:
`08/235,411
`
`29 April 1994 (29.04.94)
`
`US
`
`(81) Designated States: AU, CA, JP, European patent (AT, BE,
`CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT,
`SE).
`
`(71) Applicant: PERKIN-ELMER CORPORATION [US/US]; Ap-
`plied Biosystems Division, 850 Lincoln Centre Drive, Foster
`City, CA 94404 (US).
`
`Published
`With international search report.
`
`(72) Inventors: WOUDENBERG, Timothy, M.; 360 Bridgeport
`Drive, Half Moon Bay, CA 94019 (US). BODNER, Kevin,
`S.; Apartment C, 44 East 44th Place, San Mateo, CA 94403
`(US). CONNELL, Charles, R.; 167 King Street, Redwood
`City, CA 94062 (US). GANZ, Alan, M.; 145 Sterling
`Road, Trumbull, CT 06611 (US). MCBRIDE, Lincoln,
`J.; 400 Alameda de las Pulgas, Belmont, CA 94002 (US,.
`SAVIANO, Paul, G.; 1 Hillcrest Place, Norwalk, CT 06850
`(US). SI-IIGEURA, John; 5110 Keystone Drive, Fremont.
`CA 94536 (US). TRACY, David, H.; 581 Beldon Hill Road,
`Norwalk, CT 06850 (US). YOUNG, Eugene, F.; 34 Lambert
`Common, Wilton, CT 06850 (US). LEE, Linda, G.; 3187
`Stelling Drive, Palo Alto, CA 94303 (US).
`
`|
`
`(54) Title: SYSTEM FOR REAL TIME DETECTION OF NUCLEIC ACID AMPLIFICATION PRODUCTS
`
`(57) Abstract
`
`A system is provided for carrying out real time fluorescence-based measurements of
`nucleic acid amplification products. In a preferred embodiment of the invention, an excitation
`beam is focused into a reaction mixture through a surface, the reaction mixture containing:
`(1)
`a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is
`proportional to the amount of an amplification product in the volume of the reaction mixture
`illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously
`distributed throughout the reaction mixture capable of generating a second fluorescent signal
`proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably,
`the excitation beam is focused into the reaction mixture by a lens through a portion of a wall
`of a closed reaction chamber containing the reaction mixture. The same lens is used to collect
`the first and second fluorescent signals generated by the first and second fluorescent indicators,
`respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the
`first and second fluorescent signals provides a stable quantitative indicator of the amount of
`amplification product synthesized in the course of the amplification reaction.
`
`llllll.
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`_
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`FOR THE PURPOSES OF INFORMATION ONLY
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`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE-
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CS
`CZ
`DE
`DK
`ES
`FI
`FR
`GA
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central Africa.n Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Spain
`Finland
`France
`Gabon
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukra.ine
`United States of America
`Uzbekistan
`Viet Nam
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`WO 95/30139
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`PCT/US95/04818
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`SYSTEM FOR REAL THVIE DETECTION OF NUCLEIC ACTD
`
`AMPLIFICATION PRODUCTS
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`The invention relates generally to the field of nucleic acid amplification, and
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`more particularly to a system for measuring in real time polynucleotide products from
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`nucleic acid amplification processes, such as polymerase chain reaction (PCR).
`
`Background
`
`Nucleic acid sequence analysis is becoming increasingly important in many
`
`research, medical, and industrial fields, e.g. Caskey, Science 236: 1223-1228 (1987);
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`Landegren et al, Science, 242: 229-237 (1988); and Amheim et al, Ann. Rev. Biochem.,
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`61: 131-156 (1992). The development of several nucleic acid amplification schemes has
`
`played a critical role in this trend, e. g. polymerase chain reaction (PCR), Innis et al,
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`editors, PCR Protocols (Academic Press, New York, 1990); McPherson et al, editors,
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`PCR: A Practical Approach (IRL Press, Oxford, 1991); ligation-based amplification
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`20
`
`techniques, Barany, PCR Methods and Applications 1: 5-16 (1991); and the like.
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`PCR in particular has become a research tool of major importance with
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`applications in cloning, analysis of genetic expression, DNA sequencing, genetic
`
`mapping, drug discovery, and the like, e.g. Arnheim et al (cited above); Gilliland et al,
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`-Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan et al, PCR Methods and
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`25
`
`Applications, 1: 222-228 (1992); Green et al, PCR Methods and Applications, 1: 77-
`
`90 (1991); Blackwell et al, Science, 250: 1104-1110 (1990).
`
`A wide variety of instrumentation has been developed for carrying out nucleic
`
`acid amplifications, particularly PCR, e.g. Johnson et al, U.S. patent 5,038,852
`
`(computer—controlled thermal cycler); Wittwer et al, Nucleic Acids Research, 17:
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`30
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`4353-4357 (1989)(capillary tube PCR); Hallsby, U.S. patent 5,187,084 (air-based
`
`temperature control); Gamer et al, Biotechniques, 14: 112-115 (1993)(high-
`
`throughput PCR in 864-well plates); Wilding et al, International application No.
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`-1-
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`PCT/US93/04039 (PCR in micro-machined structures); Schnipelsky et al, European
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`patent application No. 90301061.9 (publ. No. 0381501 A2)(disposable, single use
`
`PCR device), and the like. Important design goals fundamental to PCR instrument
`
`development have included fine temperature control, minimization of sample-to-
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`sample variability in multi-sample thermal cycling, automation of pre- and post-PCR
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`processing steps, high speed cycling, minimization of sample volumes, real time
`
`measurement of amplification products, minimization of cross-contamination, or
`
`sample carryover, and the like. In particular, the design of instruments that permit
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`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. I-Iiguchi 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
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`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 amplifications, as the relative values of close concentrations can be
`
`resolved by taking into account the history of the relative concentration values during
`
`the PCR. Real time monitoring also permits the efiiciency 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 fluorescence—based
`
`approaches to provide real time measurements of amplification 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 fluorescer-quencher pairs (the so-called "Tac-Man" approach) that are
`
`. cleaved during amplification to release a fluorescent product whose concentration is
`
`proportional to the amount of double stranded DNA present.
`
`Unfortunately, successfiil implementation of these approaches has been
`
`impeded because the required fluorescent measurements must be made against a very
`
`high fluorescent background. Thus, even minor sources of instrumental noise, such as
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`the formation of condensation in the chamber during heating and cooling cycles,
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`formation of bubbles in an optical path, particles or debris in solution, differences in
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`sample vo1umes—-and hence, diiferences in signal emission and absorbence, and the
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`like, have hampered the reliable measurement of the fluorescent signals.
`
`In view of the above, it would be advantageous if an apparatus were available
`
`which permitted stable and reliable real time measurement of fluorescent indicators of
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`amplification products resulting from any of the available nucleic acid amplification
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`schemes.
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`Summary of the Invention
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`The invention relates to a system for carrying out real time fluorescence-based
`
`measurements of nucleic acid amplification products. In a preferred embodiment of
`
`the invention, an excitation beam is focused into a reaction mixture containing (i) a
`
`first fluorescent indicator capable of generating a first fluorescent signal whose
`
`intensity is proportional to the amount of an amplification product in the volume of the
`
`reaction mixture illuminated by the excitation beam and (ii) a second fluorescent
`
`indicator homogeneously distributed throughout the reaction mixture and capable of
`
`generating a second fluorescent signal proportional to the volume of reaction mixture
`
`illuminated by the excitation beam. It is understood that the proportionality of the
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`fluorescent intensities is for a constant set of parameters such as temperature, pH, salt
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`concentration, and the like, that independently influence the fluorescent emissions of
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`organic dyes.
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`Preferably, the excitation beam is focused into the reaction mixture by a lens
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`through a portion of a wall of a closed reaction chamber containing the reaction
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`mixture. In fiirther preference, the same lens collects the first and second fluorescent
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`signals generated by the first and second fluorescent indicators, respectively, in
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`response to the excitation beam; thus, variability in the collected signal due to
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`misalignment of excitation and collection optics is avoided. In this embodiment,
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`whenever the lens directs the excitation beam through a portion of a wall of the closed
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`reaction chamber which is not in contact with the reaction mixture, that portion of the
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`wall is heated so that condensation from the reaction mixture does not fonn in the
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`optical pathway of the fluorescent signals being collected by the lens, thereby removing
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`25
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`another source of variability in the collected signal.
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`In the most preferred embodiment, the reaction chamber is a tube with a closed
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`end, referred to herein as the bottom of the tube, and an open end, referred to herein as
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`the top of the tube, which can be closed with a cap such that a leak-proof seal is
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`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)
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`30
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`the reaction mixture fills 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
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`mixture, (2) the walls of the tube are frosted; that is, they are made of a material that
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`transmits and scatters light, and (3) the lens without contacting the cap focuses the
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`35
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`excitation beam through the cap into the reaction mixture through its top surface and
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`collects the resulting fluorescence generated by the first and second fluorescent
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`indicators. As mentioned above, the portion of the tube through which the excitation
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`beam passes--the cap in this embodiment—-is heated to prevent the formation of
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`condensation which would introduce an added source of variability in the measurement
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`of the collected fluorescent signals. Potential variability that could arise fiom
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`sequential analysis of the first and second fluorescent signals is eliminated by
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`simultaneously analyzing the signals by spectrally separating the signal light onto an
`
`array of photo detectors, e. g. by difiracting the signal onto a charged-coupled device
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`(CCD) array.
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`As discussed more fully below, an excitation beam generated by a single light
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`source, e.g. a laser, is conveniently distributed to a plurality of closed reaction
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`chambers by fiber optics. Likewise, the same fiber optics can collect the fluorescent
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`signals from the plurality of reaction chambers for analysis by a single detection and
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`analysis system.
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`Preferably, the system is employed with the PCR amplification of nucleic acids.
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`The system of the invention permits accurate real time monitoring of nucleic
`
`amplification reactions by providing apparatus and fluorescent reagents for generating
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`a stable fluorescent signal proportional to the amount of amplification product and
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`independent of variations in the volume of reaction mixture. The availability of data
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`showing the progress of amplification reactions leads to more accurate estimates of
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`relative starting concentrations of target nucleic acids, to rapid assessment of the
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`efficiency of the amplification reactions, and opens the possibility of reduced reagent
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`usage and feedback reaction control.
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`Brief Description of the Figures
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`Figure 1 diagrammatically illustrates a preferred embodiment of the sample
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`25
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`interface components of the system of the invention.
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`Figure 2 diagrammatically illustrates a preferred embodiment for
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`simultaneously monitoring a plurality of amplification reactions by sequentially
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`interrogating reactions via a fiber optic multiplexer.
`
`Figure 3 shows spectrally separated fluorescent intensity data for a
`tetrarnethylrhodamine fluorescent indicator, a fluorescein fluorescent indicator, and
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`30
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`instrument background registered by a CCD array of the preferred embodiment
`
`described below.
`
`Figure 4 shows the time dependence of fluorescent signals from a fluorescein
`
`dye proportional to the amplification product (first fluorescent indicator) and a
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`35
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`tetramethylrhodarnine dye employed as a second fluorescent indicator during a typical
`PCR.
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`Figure 5 shows the cycle dependence of the ratio of the intensities of the
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`- fluorescein and tetramethylrhodarnine dyes from the same PCR whose time dependent
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`data is shown in Figure 3.
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`Figure 6 shows data relating the amount of amplification product to cycle
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`number in separate PCRs having different starting concentrations of the same target
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`nucleic acid.
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`Definitions
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`As used herein, the term "stable" in reference to a fluorescent signal means that
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`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
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`the RMS deviation in the signal due to noise is less than or equal to one percent of the
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`average signal magnitude.
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`Detailed Description of the Invention
`
`The invention is a fluorescence-based system for monitoring in real time the
`
`progress of a nucleic acid amplification reaction. The type of amplification scheme
`
`used with the system is not critical, but generally the system requires either the use of a
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`nucleic acid polymerase with exonuclease activity or a population of double stranded
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`DNA which increases during the course of the reaction being monitored. Exemplary
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`amplification schemes that may be employed with the system of the invention include
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`PCR, ligase-based amplification schemes, such as ligase chain reaction (LCR), Q-beta
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`replicase-based amplification schemes, strand displacement amplification (SDA)
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`schemes, such as described by Walker et al, Nucleic Acids Research, 20: 1691-1696
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`(1992), and the like. A comprehensive description of nucleic acid amplification
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`schemes is provided by Keller and Manak, DNA Probes, Second Edition (Stockton
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`Press, New York, 1993). Fundamental to the system is the measurement of ratios of
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`fluorescent intensities of a first fluorescent indicator and an internal standard, referred
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`to herein as a second fluorescent indicator. The first and second fluorescent indicators
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`30
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`‘must be spectrally resolvable. That is, their respective emission spectra must be
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`sufiiciently non-overlapping so that separate emission peaks are observed in the
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`combined spectrum. Clearly, the system may be generalized to include a plurality of
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`first fluorescent indicators, e. g. to monitor the simultaneous amplification of several
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`target nucleic acids in a single reaction, so that a plurality of fluorescent intensity ratios
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`35
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`are monitored. Several spectrally resolvable dyes suitable for use in such embodiments
`
`are disclosed in Fung et al, U.S. patent 4,855,225; Menchen et al, U.S. patent
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`5,188,934; Bergot et al, International Application PCT/US90/05565; and like
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`references.
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`The system includes a sample interface--that is, optical components
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`operationally associatedwith a closed reaction chamber--which comprises a lens for
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`focusing an excitation beam into the reaction mixture and for collecting the resulting
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`fluorescence and a fiber optic for transmitting both the excitation beam from a light
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`source to the lens and the fluorescent signals from the lens to a detection and analysis
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`means. Preferably, the reaction mixture is contained in a closed reaction chamber to
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`prevent cross-sample contamination, or so-called "carryover." The lens therefore
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`focuses the excitation beam and collects fluorescence through a portion of a wall of
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`the closed reaction chamber. As mentioned above, the preferred reaction chamber is a
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`tube, e.g. having the geometry and volume of a conventional Eppendorf tube. The
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`tube is closed after the reaction mixture is added by attaching a cap to the open end of
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`the tube. In a preferred embodiment of the sample interface for PCR, the lens directs
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`the excitation beam and collects fluorescence through the cap of the tube, as illustrated
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`in Figure 1. In the illustrated configuration,
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`a. first end fiber optic 2 is held by ferrule
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`4, housing 6, and plate 10in a co-axial orientation with lens 8. A second end of fiber
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`optic 2 (not shown) is operationally associated with a light source and detection and
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`analysis means, discussed more fiilly below. The distance between the end face of
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`fiber optic 2 and lens 8 is determined by several factors, including the numerical
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`aperture of the fiber optic, the geometry of tube 18, the focal length of lens 8, the
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`diameter of lens 8, and the like. Guidance for selecting values for such variables in any
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`particular embodiment is readily found in standard texts on optical design, e.g. Optics
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`Guide 5 (Melles Griot, Irvine, CA, 1990), or like reference. In the illustrated
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`embodiment, lens 8 has a diameter of 8 mm and is composed of material BK7,
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`available from Edmund Scientific (Banington, NJ). Fiber optic 2 has a numerical
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`aperture of .2. Preferably, the design permits maximal transmission of excitation beam
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`28 to reaction mixture 22. For example, lens 8, numerical aperture of fiber optic 2,
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`and the distance between the end of fiber optic 2 and lens 8 are selected so that the
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`diameter of lens 8 equals or exceeds the diameter of excitation beam 28 where beam
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`28 impinges on the lens (as illustrated in Figure 1). Excitation beam 28 is focused
`‘through cap 16, void 24, and top surface 26 of reaction mixture 22 to a region
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`30
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`approximately 1-3 times the diameter of the fiber optic just below, e. g. 1-3 mm,
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`surface 26. This degree of focusing is not a critical feature of the embodiment; it is a
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`consequence of adapting the sample interface to the geometry and dimensions of a
`sample holder of a commercially available thermal cycler. In other embodiments, the
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`geometry and dimension may permit a sharper focus into the reaction mixture.
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`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,
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`truncated cylinder, oblate spheroid, or truncated oblate spheroid, or the like, and may
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`be composed of any suitably transparent refractive material, such as disclosed by
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`Hlousek, U.S. patent 5,037,199;’Hoppe et al, U.S. patent 4,747,87; Moring et al, U.S.
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`patent 5,239,360; Hirschfield, U.S. patent 4,577,109; or like references.
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`Fluorescent light generated by excitation beam 28 is collected by lens 8 along
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`approximately the same optical pathway as that defined by excitation beam 28 and
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`focused onto the end of fiber optic 2 for transmission to optical separation and analysis
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`components of the system.
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`In firrther preference, the sample interface also includes means for heating the
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`portion of the wall of the reaction chamber used for optical transmission in order to
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`reduce variability due to scatter and/or absorption of the excitation beam and signal
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`from condensation of reaction mixture components. In the embodiment of Figure 1,
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`the portion of the reaction chamber (tube 18) wall used for optical transmission is cap
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`16. Accordingly, heating element 12 and heat-conductive platen 14 are employed to
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`heat cap 16. Preferably, heating element 12 comprises resistance heating elements and
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`temperature sensors that permit programmed controlled of the temperature of cap 16.
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`Cap 16 is maintained at a temperature above the condensation points of the
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`components of the reaction mixture. Generally, cap 16 may be maintained at a
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`temperature in the range of 94-1 10°C. Preferably, cap 16 is maintained at a
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`temperature in the range of about 102°C to about 105°C since the principal solvent in
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`the reaction mixture is usually water. More preferably, cap 16 is maintained at 103°C.
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`Preferably, in embodiments employing thermal cycling, the cap-heating components
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`described above are thermally isolated from heating-conducting component 20
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`employed to cyclically control the temperature of reaction mixture 22.
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`As mentioned above, walls of tube 18 are preferably frosted so that any
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`spurious reflections from the walls of heat—conducting component 20 are diflirsed or
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`. scattered to reduce the contributions such reflections may make to the collected signal.
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`Walls of ordinarily translucent or transparent tubes are conveniently frosted by etching
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`or roughening.
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`Selection of appropriate materials for the components described above is well
`‘within the skill of an ordinary mechanical engineer. Exemplary criterion for material
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`selection include (i) degree of thermal expansion, especially for amplification schemes
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`employing thermal cycling, and its affect on the alignment of the optical components,
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`(ii) optical transmission properties in the excitation wavelengths and fluorophore
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`emission wavelengths employed, (iii) chemical inertness of the reaction chamber
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`relative to components of the reaction mixture, (iv) degree to which critical reaction
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`components, e.g. polymerases, target nucleic acids, would tend to adsorb onto
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`chamber walls, (v) minimization of fluorescent materials in the optical pathway, and
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`the like. Typically, tubes containing amplification reaction mixtures are made of
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`‘ polypropylene or like materials.
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`The sample interface shown in Figure 1 may be employed individually or it may
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`be employed as one of a plurality of identical interfaces in a single instrument, as
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`shown diagrammatically in Figure 2. In the illustrated embodiment, individual sample
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`interfaces 31, arrayed in holder 30 (which may, for example, be a heating block
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`associated with thermal cycler 32, such as described in Mossa et al, European patent
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`application No. 91311090.4, publ. No. 0488769 A2) are connected by fiber optics 34
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`to fiber optic multiplexer 36, which selectively permits transmission between individual
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`fiber optics and port 35, e.g under user control via a programmed microprocessor. In
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`a preferred configuration, excitation beam 41, generated by light source 52 and
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`controller 54, passes through beam splitter 40 and is focused onto port 35 by lens 38,
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`where it is sequentially directed by fiber optic multiplexer 36 to each of a
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`predetermined set, or subset, of fiber optics 34. Conversely, a fluorescent signal
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`generated in a reaction chambers is collected by lens 8 and focused onto a fiber optic
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`which, in turn, transmits the signal to a detection and analysis means, possibly via a
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`fiber optic multiplexer. Returning to Figure 2, a fluorescent signal collected by a
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`sample interface is directed to fiber optic multiplexer 36 where it emerges through port
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`35 and is collected and collimated by lens 38. Lens 38 directs the fluorescent signal to
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`20
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`beam splitter 40 which, in turn, selectively directs the signal through cut-off filter 42,
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`which prevents light from the excitation beam from reaching the signal detection
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`components. Beam splitter 40 may be a conventional dichroic mirror, a fully reflective
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`mirror with an aperture to pass the excitation beam (e. g. as disclosed in U.S. patent
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`4,577,109), or like component. Afier passing through cut-off filter 42, the fluorescent
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`signal is directed by lens 44 to a spectral analyzer which spectrally separates the
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`fluorescent signal and measures the intensities of a plurality of the spectral components
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`of the signal. Typically, a spectral analyzer comprises means for separating the
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`fluorescent signal into its spectral components, such as a prism, diffraction grating, or
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`the like, and an array of photo-detectors, such as a diode array, a charge-coupled
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`30
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`device (CCD) system, an array of bandpass filters and photomultiplier tubes, or the
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`like. In the preferred embodiment of Figure 2, the spectral analyzer comprises
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`diffraction grating 46 (e.g., model CP-140, Jobin-Yvon, NI) and CCD array 48 (e.g.,
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`model S2135 Princeton Instruments, NJ), which is linked to CCD controller 50.
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`An exemplary CCD array suitable for analyzing fluorescent signal fiom
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`35
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`fluorescein and tetramethylrhodamine is partitioned into 21 collection bins which span
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`the 500 nm to 650 nm region of the spectrum. Each bin collects light over a 8.5 nm
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`window. Clearly, many alternative configurations may also be employed. An
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`exemplary application of a CCD array for spectral analysis is described in Karger et al,
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`Nucleic Acids Research, 19: 4955-4962 (1991).
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`Analyzing the fluorescent signal based on data collected by a spectral analyzer
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`is desirable since components of the signal due to one or more first fluorescent
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`indicators and a second fluorescent indicator (from which intensity ratios are
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`calculated) can be analyzed simultaneously and without the introduction of
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`wavelength-specific system variability that might arise, e. g. by misalignment, in a
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`system based on multiple beam splitters, filters, and photomultiplier tubes. Also, a
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`spectral analyzer permits the use of "virtual filters" or the programmed manipulation of
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`data generated from the array of photo-detectors, wherein a plurality of discrete
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`wavelength ranges are sampled--in analogy with physical bandpass filters--under
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`programmable control via an associated microprocessor. This capability permits a high
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`degree of flexibility in the selection of dyes as first and second fluorescent indicators.
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`Generally, the detection and analysis means may be any detection apparatus to
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`provides a readout that reflect the ratio of intensities of the signals generated by the
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`first and second fluorescent indicators. Such apparatus is well know in the art, as
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`exemplified by U.S. patents 4,577,109 and 4,786,886 and references such as The
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`Photonics Design & Applications Handbook, 39th Edition (Laurin Publishing Co.,
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`Pittsfield, MA, 1993).
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`Preferably, the system of the invention is employed to monitor PCRs, although
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`it may also be employed with a variety of other amplification schemes, such as LCR.
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`Descriptions of and guidance for conducting PCRs is provided in an extensive
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`literature on the subject, e.g. including Innis et al (cited above) and McPherson et al
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`(cited above). Briefly, in a PCR two oligonucleotides are used as primers for a series
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`of synthetic reactions that are catalyzed by a DNA polymerase. These
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`oligonucleotides typically have difierent sequences and are complementary to
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`15
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`20
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`25
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`sequences that (i) lie on opposite strands of the template, or target, DNA and (ii) flank
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`the segment of DNA that is to be amplified. The target DNA is first denatured by
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`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
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`30
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`to a temperature that allows the oligonucleotide primers to armeal to their target
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`sequences, after which the annealed primers are extended with DNA polymerase. The
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`cycle of denaturation, armealing, and extension is then repeated many times, typically
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`25-35 times. Because the products of one round of amplification serve as target
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`35
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`nucleic acids for the next, each successive cycle essentially doubles the amount of
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`target DNA, or amplification product.
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`As mentioned above an important aspect of the invention is the fluorescent
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`dyes used as the first and second fluorescent indicators. By examining the ratio of the
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`fluorescent intensities of the indicators, the eifects of most sources of systematic
`
`variability, which would be apparent in the intensities alone, are eliminated. Generally,
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`in accordance with the invention, the first fluorescent indicator may be a complex-
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`forming dye or a dye covalently attached to an oligonucleotide probe which is
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`degraded during polymerization steps to generate a signal. This later embodiment
`
`relates to the so-called "Tacman" approach, described by Holland et al, Proc. Natl.
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`Acad. Sci., 88: 7276-7280 (1991). As used herein, the term "complex-forrning" in
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`reference to a dye means that a dye is capable of forming a stable non-covalent
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`complex with either double stranded or triple stranded nucleic acid structures, usually
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`DNA, and that the dye's fluorescent characteristics are substantially different in the
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`complexed state as compared to a non-complexed, i.e. usually fi'ee-solution, state.
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`Preferably, the quantum efficiency of fluorescence of an complex-forming dye is
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`enhanced in the complexed state as compared to the free-solution state, thereby
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`resulting in enhanced fluorescent upon complex formation. Exemplary complex-
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`forrning dyes include ethidium bromide, propidium iodide, thiazole orange, acridine
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`orange, daunomycin, mepacrine, 4',6'-diarninidino-2-phenylindole (DAPI), oxazole
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`orange, bisbenzirnidaxole dyes, such as Hoechst 33258 and Hoechst 33342, and
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`heterodimers of various intercalating dyes, such as ethidium, acridine, thiazolium, and
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`oxazolium dyes (known by their acronyms POPRO, BOPRO, YOPRO, and TOPRO),
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`and like dyes, which are described in the following references: Haugland, pgs. 221-
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`229 in Handbook of Fluorescent Probes and Research Chemicals, 5th Edition
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`(Molecular Probes, Inc., Eugene, 1992); Glazer et al, Proc. Natl. Acad. Sci., 87: 3851-
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`3855 (1990); Srinivasan et al, Applied and Theoretical Electrophoresis, 3: 235-239
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`(1993); Kapuscinski et al, Anal. Biochem., 83: 252-257 (1977); Hill, Anal. Biochem.,
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`70: 635-638 (1976); Setaro et al, Anal. Biochem., 71: 313-317 (1976); and Latt et al,
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`J. Histochem. Cytochem., 24:24-33 (1976); and Rye et al, Nucleic Acids Research, 20:
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`2803-2812 (1992). Preferably, when complex-forming dyes are employed as