(19)
`
`(12)
`
`Europäisches Patentamt
`
`European Patent Office
`
`Office européen des brevets
`
`(11)
`
`EP 0 640 828 B1
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`EUROPEAN PATENT SPECIFICATION
`
`(45) Date of publication and mention
`of the grant of the patent:
`10.05.2000 Bulletin 2000/19
`
`(21) Application number: 94112728.4
`
`(22) Date of filing: 16.08.1994
`
`(51) Int. Cl.7: G01N 21/64, C12Q 1/68
`
`(54) Monitoring multiple reactions simultaneously and analyzing same
`Gleichzeitige Kontrolle mehrfacher Reaktionen und Analyse derselben
`Contrôle simultané de réactions multiples, et analyse de telles réactions
`
`(84) Designated Contracting States:
`AT BE CH DE DK ES FR GB GR IE IT LI LU NL PT
`SE
`
`(56) References cited:
`EP-A- 0 266 881
`WO-A-92/05278
`
`EP-A- 0 512 334
`US-A- 5 038 852
`
`(30) Priority: 27.08.1993 US 113168
`05.07.1994 US 266061
`
`(43) Date of publication of application:
`01.03.1995 Bulletin 1995/09
`
`(73) Proprietor:
`F. Hoffmann-La Roche AG
`4002 Basel (CH)
`
`(72) Inventors:
`• Higuchi, Russell G.
`Alameda, california 94501 (US)
`• Watson, Robert M.
`Berkeley, California 94703 (US)
`
`(74) Representative:
`AMMANN PATENTANWAELTE AG BERN
`Schwarztorstrasse 31
`3001 Bern (CH)
`
`• PATENT ABSTRACTS OF JAPAN vol. 11, no. 49
`(P-547) 14 February 1987 & JP-A-61 215 948
`(FUJIREBIO) 25 September 1986
`• PATENT ABSTRACTS OF JAPAN vol. 11, no. 313
`(P-626) 13 October 1987 & JP-A-62 105 031
`(FUJIREBIO) 15 May 1987
`• PATENT ABSTRACTS OF JAPAN vol. 15, no. 331
`(P-1241) 22 August 1991 & JP-A-03 122 552
`(FUJIREBIO) 31 May 1989
`• PATENT ABSTRACTS OF JAPAN vol. 16, no. 63
`(C-0911) 18 February 1992 & JP-A-03 259 099
`(TEIJIN) 19 November 1991
`• PATENT ABSTRACTS OF JAPAN vol. 16, no. 190
`(C-0937) 8 May 1992 & JP-A-04 027 399
`(SHIONOGI) 30 January 1992
`• PATENT ABSTRACTS OF JAPAN vol. 16, no. 308
`(P-1381) 7 July 1992 & JP-A-04 084 751
`(SHIMADZU) 18 March 1992
`
`Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
`notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
`a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
`99(1) European Patent Convention).
`
`Printed by Xerox (UK) Business Services
`2.16.7 (HRS)/3.6
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`THERMO FISHER EX. 1017
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`Description
`
`[0001]
`The present invention relates to detecting
`nucleic acid (DNA or RNA) amplification, and more par-
`ticularly to real-time monitoring of multiple nucleic acid
`amplification reactions, simultaneously. The invention
`also relates to a method for using the accumulated data
`to quantitate the starting concentration of a target
`nucleic acid sequence provided in one or more of the
`mixtures and monitor the effect of amplification reaction
`conditions on the reaction kinetics.
`[0002]
`Various methods for detecting nucleic acid
`amplification are known. A number of assays are known
`which quantitate the number of starting DNA templates
`in a polymerase chain reaction (PCR), for example.
`Some involve the measurement of PCR product at the
`end of temperature thermal cycling and relate this level
`to the starting DNA concentration. Such an "endpoint"
`analysis, as has been typically done using PCR, reveals
`the presence or absence of target DNA but generally
`does not provide a usable measure of the starting
`number of DNA targets.
`[0003]
`Other assays involve the use of a competitor
`amplification product whose template is added at known
`concentration to the reaction mixture before thermal
`cycling. In competitor-product protocols, an aliquot of
`the amplification is examined by gel-electrophoresis.
`The relative amount of target-specific and competitor
`PCR product is measured; this ratio is used to calculate
`the starting number of target templates. The larger the
`ratio of target-specific product to competitor-specific
`product, the higher the starting DNA concentration.
`[0004]
`In addition
`to
`requiring
`"downstream"
`processing, such as hybridization or gel electrophore-
`sis, these other assays are more limited in dynamic
`range (i.e., sensitivity to a range of target nucleic acid
`concentrations). In competitor assays, the sensitivity to
`template concentration differences is compromised
`when either the target or added competitor DNA is
`greatly in excess of the other. The dynamic range of
`assays that measure the amount of end product can
`also be limited in that at the chosen number of cycles
`some reactions may have reached a "plateau" level of
`product. Differences in starting template levels in these
`reactions are therefore not well reflected. Furthermore,
`small differences in the measured amount of product
`result in widely varying estimates of the starting tem-
`plate concentration, leading to great inaccuracy due to
`variable reaction conditions, variations in sampling, or
`the presence of inhibitors.
`[0005]
`Optimization of PCR conditions typically is
`accomplished by measuring the effect of different condi-
`tions on the final yield and specificity of PCR product. In
`order to obtain information throughout amplification,
`many replicate samples are placed in a thermal cycler
`so that each can be removed from the thermal cycler at
`a different temperature cycle. The removed tubes then
`are analyzed by gel electrophoresis as a function of
`
`cycle number. As is apparent from this description, such
`optimization is complex and time-consuming in that it
`requires numerous sample manipulation steps as well
`as downstream electrophoresis analysis.
`[0006]
`Any assay intended for large-scale (e.g.,
`clinical) use should not only be reliable, but should be
`simplified as much as possible in order to facilitate its
`automation. Thus, there is a need for an apparatus and
`method for collecting data indicative of nucleic acid
`amplification that can be used, for example, to quanti-
`tate sample starting concentrations and optimize reac-
`tion conditions, that provides reliable results and is
`suitable for automation.
`[0007]
`The aim of the present invention is to provide
`a nucleic acid amplification detection method and appa-
`ratus which makes possible to monitor the amplification
`of multiple amplification reaction mixtures, simultane-
`ously in real-time, and which thereby makes possible to
`overcome the problems and disadvantages of the prior
`art.
`[0008]
`is
`According to the invention this aim
`achieved by providing an apparatus that is character-
`ized in that it comprises:
`
`a thermal cycler including a heat conducting mem-
`ber having multiple recesses formed therein; and
`a sensor arranged for detecting light emitted from
`said recesses, simultaneously.
`
`[0009]
`In a preferred embodiment the apparatus
`according to the invention it further comprises a light
`source optically coupled to said thermal cycler and
`arranged to distribute light over a portion of said heat
`conducting member having a plurality of said recesses
`formed therein.
`[0010]
`In another preferred embodiment of the
`apparatus according to the invention said recesses of
`the heat conducting member of the thermal cycler are
`formed through a surface thereof for receiving reaction
`vessels containing a nucleic acid amplification reaction
`mixtures; and said sensor is an imaging device optically
`coupled to said heat conducting member for generating
`an image of said surface and reaction vessels when
`said vessels are disposed in said recesses of the heat
`conducting member.
`[0011]
`In a further preferred embodiment of the
`apparatus according to the invention said heat conduct-
`ing member of the thermal cycler has multiple recesses
`formed therein and adapted for receiving nucleic acid
`amplification reaction mixtures, and the apparatus fur-
`ther comprises
`
`a housing positioned over said heat conducting
`member and coupled to said thermal cycler;
`a light source arranged to emit light in said housing
`and toward said recesses; and
`a dichroic mirror positioned in said housing and
`above said recesses, said dichroic mirror being
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`transmissive to light having a first wavelength and
`reflective to light having a second wavelength that
`differs from said first wavelength; and
`light
`said sensor being arranged
`to receive
`reflected from said second surface of said dichroic
`mirror.
`
`[0012]
`In a further preferred embodiment of the
`apparatus according to the invention said heat conduct-
`ing member of the thermal cycler has multiple recesses
`formed therein and adapted for receiving nucleic acid
`amplification reaction mixtures including a nucleic acid
`sequence and a fluorescent binding agent and the
`apparatus further comprises
`
`a housing positioned over said heat conducting
`member and being coupled to said thermal cycler;
`a light source arranged to emit light in said housing;
`a sensor arranged in said housing; and
`a dichroic mirror positioned in said housing and
`above said recesses, said dichroic mirror being
`transmissive to light having a wavelength corre-
`sponding to the wavelength of light generated by
`the excitation light source and reflective to light hav-
`ing a wavelength corresponding to the wavelength
`of fluorescence emitted from a nucleic acid amplifi-
`cation mixture, including a fluorescent binding
`agent, when that mixture is disposed in one of the
`recesses formed in the heat conducting member
`and exposed to light from the excitation light
`source, said mirror be oriented to form an optical
`path between said recesses and said sensor.
`
`[0013]
`In one embodiment of the apparatus accord-
`ing to the invention, a CCD-camera detects the accumu-
`lation of double-stranded DNA (dsDNA) in each of
`multiple polymerase chain reactions, simultaneously,
`using the increase in the fluorescence of a detectable
`fluorescent dye initially introduced into each amplifica-
`tion reaction mixture. The fluorescence results from the
`fluorescent dye binding duplex DNA. This embodiment
`advantageously eliminates the need for fiber-optic leads
`and the accompanying problems associated with cou-
`pling the fiber-optics to the individual reaction mixtures.
`[0014]
`According to another aspect of the invention,
`the optical system that moves excitation light from a
`source to the multiple reaction mixtures being amplified
`in a thermal cycler, which forms part of the apparatus, is
`configured to provide excitation light to the reaction mix-
`tures in a uniform manner. That is, the optical system
`permits excitation light to be essentially uniformly dis-
`tributed over the thermal cycler heat exchanger so that
`each amplification reaction mixture receives essentially
`the same amount of excitation light.
`[0015]
`With this apparatus, the fluorescence data
`can be collected and used to quantitate the initial
`amount of target nucleic acid sequence. In a preferred
`method for quantitation, multiple amplification reaction
`
`mixtures are provided. One amplification reaction mix-
`ture has an unknown concentration of a specific nucleic
`acid sequence. The other reaction mixtures include the
`same specific nucleic acid sequence in differing but
`known concentrations. The amplification reaction mix-
`tures of known and unknown nucleic acid concentration
`are thermally cycled in parallel for multiple cycles. The
`fluorescence emitted from the reaction mixtures is mon-
`itored in real time and the number of cycles necessary
`for each reaction mixture to fluoresce at a certain inten-
`sity value determined . The number of cycles necessary
`for the mixture of unknown nucleic acid concentration to
`reach that value is compared to the number of cycles
`necessary for the mixtures of known nucleic acid con-
`centration to reach that value to obtain the initial quan-
`tity of said specific nucleic acid sequence in the mixture
`of unknown concentration.
`[0016]
`It has been found that sensitivity to a range
`of target nucleic acid concentration of at least six orders
`of magnitude is possible because the amplifications are
`monitored in real-time. In addition, sensitivity to as few
`as 100 ssDNA templates in the background of 40,000
`cell-equivalents of complex genomic DNA can be
`obtained.
`[0017]
`The invention further advantageously pro-
`vides a way to process the fluorescence data to reliably
`analyze the effect of different reaction conditions on the
`amplification kinetics. Since multiple amplifications can
`be monitored simultaneously, the effect of many differ-
`ent reaction variables can be assessed rapidly. This is
`useful in optimizing amplification reactions for optimum
`yield and efficiency.
`[0018]
`Real-time monitoring also gives the advan-
`tage of detecting instances of partial inhibition of PCR,
`which can greatly affect the ability to quantitate accu-
`rately. As shown in Fig. 10D, these instances can be
`detected by their reaction profile so that these samples
`can be repurified and tested again. In addition,
`instances of total inhibition, which might otherwise lead
`to the false conclusion that there is no nucleic acid tar-
`get in a sample, can be detected in that there is the
`expectation that given enough cycles, even PCRs with-
`out target DNA will produce fluorescence that is due to
`nonspecific amplification products. If such fluorescence
`is not seen by the expected cycle, the presence of inhib-
`itors can be inferred.
`[0019]
`A further advantage of the present invention
`is that the need for additional time-consuming manipu-
`lations to determine the yield of many reactions at the
`end of the amplification is eliminated. Thus, the need for
`downstream hybridization or gel electrophoresis, for
`example, is avoided.
`[0020]
`Although the non-fiber-optic embodiment of
`the present invention illustrated in Figs. 2 and 3 has
`numerous advantages as described above, the sensor,
`which preferably is a video camera, is positioned a sig-
`nificant distance from the reaction mixtures in the heat-
`conducting member to minimize parallax. Accordingly,
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`the light source also is significantly spaced from the
`reaction mixtures in the heat-conducting member in
`order to provide uniform illumination and avoid interfer-
`ence with the sensor's vision. This arrangement gener-
`ally requires a significant amount of space which, in
`turn, may require an entire darkroom to be allocated to
`the apparatus. However, many clinical laboratories do
`not have darkrooms or additional space to allocate to
`constructing a darkroom. Although X-ray rooms, which
`can readily be found in clinical laboratories, can provide
`a suitable environment for the optical paths of the exci-
`tation light and fluorescence emissions, the space in
`these rooms is essentially completely allocated to X-ray
`equipment.
`[0021]
`According to a further embodiment of the
`present invention, an excitation light and fluorescence
`sensing configuration is incorporated into the apparatus
`so that the optical paths of the excitation light and fluo-
`rescence emissions can be folded to make the appara-
`tus more compact and to simplify enclosing the optical
`paths in a light-tight environment. In this manner, the
`apparatus can be readily used on the bench without the
`need of a darkroom.
`[0022]
`According to this embodiment, the appara-
`tus for monitoring multiple nucleic acid amplifications
`simultaneously includes a thermal cycler having a heat-
`conducting member that includes multiple recesses
`formed therein for receiving nucleic acid amplification
`mixtures. A housing is positioned over the heat-con-
`ducting member so that a light-tight chamber is formed.
`A light source is arranged to emit light in the housing
`chamber to excite amplification mixtures disposed
`therein. A dichroic mirror is positioned in the housing
`and above the recesses. The dichroic mirror is transmis-
`sive to light having a first wavelength and reflective to
`light having a second wavelength that differs from the
`first wavelength. In the preferred embodiment, the mir-
`ror is a low pass dichroic mirror. The mirror is con-
`structed such that it is transparent or transmissive to
`light having a wavelength corresponding to the wave-
`length of light generated by the light source and reflec-
`tive to light having a wavelength corresponding to the
`wavelength of fluorescence emitted by the amplification
`mixtures when exposed to the excitation light. Alterna-
`tively, a high pass dichroic mirror can be used. In that
`case, the mirror is reflective to light corresponding to the
`wavelength of light generated by the light source and
`transparent or transmissive to light having a wavelength
`corresponding to the wavelength of fluorescence emit-
`ted by the amplification mixtures when exposed to the
`excitation light. A sensor also is arranged in the housing
`for sensing fluorescence emitted from the amplification
`mixtures. In the low pass dichroic mirror arrangement,
`the sensor is arranged to receive fluorescence reflected
`from the mirror. However, in the high pass arrangement,
`the sensor and light source positions are reversed.
`[0023]
`With this arrangement, the excitation light
`source and sensor both can be positioned relatively
`
`close to the heat-conducting member, e.g., from about 6
`to 12 inches therefrom, thereby permitting the optical
`paths of the excitation light source and the fluorescence
`emissions to take up a relatively small amount of space.
`The compactness of this arrangement facilitates enclos-
`ing the optical paths in the housing, which is coupled to
`the thermal cycler to effectively form a light-tight cham-
`ber or darkroom for the excitation and amplification mix-
`ture emission light. In this manner, the apparatus can be
`used most anywhere, without the need for dedicating a
`darkroom to the apparatus. The housing further advan-
`tageously prevents extraneous light, such as light emit-
`ted from monitors used with the apparatus and LED's
`associated with the thermal cycler and computer equip-
`ment, from reaching the sensor.
`[0024]
`A further advantage of having the sensor
`positioned very close to the heat conducting member in
`which the nucleic acid amplification mixtures are to be
`disposed is that sensitivity requirements of the sensor
`can be reduced. Since the sensor is moved closer to the
`heat-conducting member, the intensity of the light that is
`emitted from the reaction mixtures and reaches the sen-
`sor increases. Accordingly, when using a CCD camera
`type sensor, as in the preferred embodiment, a less
`expensive CCD camera with acceptable low light
`response and low thermal noise can be substituted for a
`relatively expensive, cooled CCD camera which is gen-
`erally required when sensitivity requirements are
`greater.
`[0025]
`The close proximity of the light source to the
`reaction mixture recesses also provides advantages. By
`moving the light source closer to the reaction mixture
`recesses, the intensity of the excitation light, which
`reaches the reaction mixtures that are placed in the
`recesses, increases, thereby increasing the intensity of
`the fluorescence emissions. In this manner, smaller
`concentrations of the target sequence can be detected
`at an earlier stage of thermal cycling. In addition, some
`of the increased fluorescence can be traded for better
`wavelength resolution by using a filter with a more nar-
`row band pass. In this manner, a broader range of
`wavelengths can be detected and labels with very close
`wavelengths can be distinguished.
`[0026]
`According to another aspect of this embodi-
`ment, a shutter is coupled to the light source to intermit-
`tently expose the reaction mixtures to the excitation
`light. Preferably, the shutter is timed to expose the mix-
`tures to the excitation light during the annealing/exten-
`sion phase where maximum fluorescence generally can
`be exhibited. This configuration reduces the mixture or
`sample exposure to the excitation light, which can be
`very intense due to its close proximity to the heat-con-
`ducting member. By minimizing sample exposure to the
`excitation light, which typically is UV light, but can be of
`other wavelengths, the shutter improves the system per-
`formance by increasing sample stability. Otherwise the
`intense excitation light can cause photo-deactivation of
`the sample which is commonly known as "bleaching".
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`[0027]
`According to a further aspect of this embod-
`iment, a field lens is positioned between the dichroic
`mirror and the sensor to minimize parallax across the
`field therebetween.
`[0028]
`A further advantageous feature of the inven-
`tion involves a heated window which is provided imme-
`diately above the heat-conducting member and can
`actually be placed on the tubes containing the amplifica-
`tion mixtures. The heated window prevents heat loss
`from the reaction tubes, helps maintain the desired ther-
`mal cycling temperature profiles of the mixtures and
`minimizes reflux, while permitting the transmission of
`light therethrough. According to the preferred embodi-
`ment, the heated window is maintained at the denatura-
`tion temperature which is generally about 95-105°C.
`[0029]
`A filter or wheel of filters preferably is cou-
`pled to the sensor so that the sensor can selectively
`detect light of different wavelengths. The filter(s) also
`can prevent excitation light from reaching the sensor. By
`using a plurality of filters, which can be arranged on a fil-
`ter wheel, the filter can be readily changed during a par-
`ticular annealing/extension phase, for example, so that
`emissions of different wavelengths, for example, from
`different homogeneous nuclease probes (described
`below), can be monitored. Different emission wave-
`lengths and, thus, nucleic acid sequences in a given
`sample can be monitored and the presence of the target
`sequence determined.
`[0030]
`In addition, the high sensitivity of the system
`permits very accurate detection of fluorescence that is
`produced using a homogenous assay system such as
`that described in U.S. Patent No. 5,210,015 to Gelfand
`et al. This assay system uses the 5' to 3' nuclease activ-
`ity of a nucleic acid polymerase to cleave annealed,
`labeled oligonucleotides from hybridized probe target
`duplexes and release labeled oligonucleotide fragments
`for detection. This assay is particularly useful for detect-
`ing multiple nucleic acid targets in the same amplifica-
`tion reaction mixture. Such probes also are useful in
`confirming target nucleic acid presence when there are
`nonspeciflc amplification products.
`[0031]
`The homogenous assay uses such a probe
`whose fluorescence is normally quenched by fluores-
`cence energy transfer, or FET, to a second label. The 5'
`nuclease activity of the DNA polymerase separates the
`fluorophore from the probe and quencher, disrupting the
`FET and restoring the fluorescence. No processing is
`required to detect this change, which may be seen
`using the monitoring apparatus of the present invention
`that incorporates the dichroic mirror arrangement.
`These probes, if labeled with different fluorophores, can
`be used to detect multiple nucleic acid targets.
`[0032]
`The following terms are used in this specifi-
`cation. The accompanying definitions are provided to
`aid disclosure, rather than limit the invention.
`[0033]
`The term "target nucleic acid sequence"
`refers to a purified or partially purified nucleic acid to be
`amplified.
`
`[0034]
`The term "amplification reaction mixture"
`refers to an aqueous solution comprising the various
`reagents used to amplify a target nucleic acid. These
`include enzymes, aqueous buffers, salts, amplification
`primers, target nucleic acid, and nucleoside triphos-
`phates. Depending upon the context, the mixture can be
`either a complete or an incomplete amplification reac-
`tion mixture.
`[0035]
`The term "primer" refers to an oligonucle-
`otide capable of acting as a point of initiation of DNA
`synthesis when annealed to a nucleic acid template
`under conditions in which synthesis of a primer exten-
`sion product is initiated, i.e., in the presence of four dif-
`ferent nucleotide triphosphates and a DNA polymerase
`in an appropriate buffer (pH, ionic strength, cofactors,
`etc.) and at a suitable temperature.
`[0036]
`The term "template" refers to a portion of the
`target nucleic sequence to which the primer anneals.
`[0037]
`The above is a brief description of some defi-
`ciencies in the prior art and advantages of the present
`invention.
`[0038]
`Other features, advantages and embodi-
`ments of the invention will be apparent to those skilled
`in the art from the following description, accompanying
`drawings and appended claims.
`
`Fig. 1 graphically shows continuous, real-time mon-
`itoring of a PCR;
`Fig. 1A is an enlarged view of the area within line
`1A in Fig. 1;
`Fig. 2 is a perspective view of the amplification
`apparatus in accordance with the principles of the
`present invention;
`Fig. 3 is an enlarged section of the thermal cycler of
`Fig. 1 to show the thermal cycler heat exchanger
`block and cover therefor;
`Fig. 4 is a block diagram of the apparatus shown in
`Fig. 2;
`Fig. 5 shows examples of digitized images of multi-
`ple reaction mixtures;
`Fig. 6 illustrates a selected pixel array for averaging
`to obtain a single fluorescence value;
`Fig. 7
`represents cycle-to-cycle fluorescence
`measurement drift;
`Fig. 8A shows multiple fluorescence profiles;
`Fig. 8B shows the fluorescence values of Fig. 8A
`after normalization;
`Fig. 8C shows gel electrophoresis of the amplifica-
`tion products represented by the profiles in Figs. 8A
`and B;
`Fig. 9 shows the linear relationship between the log
`of starting template copies and the number of
`cycles required to reach a selected fluorescence
`value;
`Figs. 10A-D represent the effect of changes in reac-
`tion conditions;
`Figs. 11-13 are simplified flow charts of the steps
`for obtaining the fluorescence values;
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`Fig. 14 is a perspective view of the amplification
`apparatus of the present invention illustrating a fur-
`ther embodiment of the excitation light and emis-
`sions/fluorescence detector arrangement in partial
`section; and
`Fig. 15 is an enlarged view of the excitation light
`and emissions/fluorescence detector arrangement
`of Fig. 14 in partial section;
`Fig. 16 shows a transmissivity v. wavelength curve
`that is representative of a dichroic mirror con-
`structed in accordance with principles of the
`present invention; and
`Fig. 17 is a top view of a heating window for the
`upper portion of the reaction mixture containing
`vessels according to the present invention.
`
`[0039]
`The present invention involves nucleic acid
`amplification and the detection, monitoring and quanti-
`tation of amplification products. In order to facilitate
`understanding of the amplification data collection and
`processing system of the present invention, a summary
`of nucleic acid amplification processes especially suited
`for use in conjunction with the invention will first be dis-
`cussed.
`[0040]
`Those of skill will recognize that the present
`invention requires amplification of the duplex form of
`nucleic acid. There exist well-known methods for ampli-
`fying nucleic acids. The means for amplification are not
`critical and this invention will work with any method
`where nucleic duplexes are generated. The various
`methods are reviewed in Bio/Technology 8:290-293,
`April 1990. They include, but are not limited to PCR,
`LCR, Qβ and 3SR. Although 3SR and Qβ do not involve
`thermal cycling, the result of their amplifications can be
`monitored by the fluorescence detecting arrangement
`discussed below and analyzed in accordance with the
`principles of the present invention. Each method is
`briefly described below.
`[0041]
`PCR amplification of DNA involves repeated
`cycles of heat denaturing the DNA, annealing two oligo-
`nucleotide primers to sequences that flank the DNA
`segment to be amplified, and extending the annealed
`primers with DNA polymerase. The primers hybridize to
`opposite strands of the target sequence and are ori-
`ented so that DNA synthesis by the polymerase pro-
`ceeds across the regions between the primers, each
`successive cycle essentially doubling the amount of
`DNA synthesized in the previous cycle. This results in
`the exponential accumulation of the specific target frag-
`ment, at a rate of approximately 2n per cycle, where n is
`the number of cycles. A complete review of this technol-
`ogy can be found in PCR Technology: Principles and
`Applications, Ed. Erlich H.A., Stockton Press, New York
`1989.
`[0042]
`Taq DNA polymerase is preferred when PCR
`is used in conjunction with the present invention
`although this is not an essential aspect of the invention.
`Taq polymerase, a thermostable polymerase, is active
`
`at high temperatures. Methods for the preparation of
`Taq are disclosed in U.S. Patent No. 4,889,818 and
`incorporated by reference. However, other thermostable
`DNA polymerases isolated from other Thermus species
`or non-Thermus species (e.g., Thermus thermophilus
`or Thermotoga maritima), as well as non-thermostable
`DNA polymerase such as T4 DNA polymerase, T7 DNA
`polymerase, E. coli DNA polymerase I, or the Klenow
`fragment of E. coli, can also be used in PCR. Methods
`for providing thermostable DNA polymerases are pro-
`vided in International Patent Applications with publica-
`tion Nos.WO-A- 91/09950 and
`WO-A- 92/03556
`[0043]
`The ligase chain reaction is described in
`International Patent Application with publication No. WO
`89/09835, The process involves the use of ligase to join
`oligonucleotide segments that anneal to the target
`nucleic acid. Ligase chain reaction (LCR) results in
`amplification of an original target molecule and can pro-
`vide millions of copies of product DNA. Consequently,
`the LCR results in a net increase in double-stranded
`DNA. The present detection methods are applicable to
`LCR, as well as PCR. LCR typically requires some
`means for detecting the product DNA such as an oligo-
`nucleotide probe. When used in conjunction with the
`disclosed methods for detecting amplification products,
`such means are unnecessary, and the LCR result is
`immediately detectable.
`[0044]
`Another amplification scheme, Q-beta repli-
`case, exploits the use of the replicase from the RNA
`bacteriophage Qβ. In this amplification scheme, a mod-
`ified
`recombinant bacteriophage genome with a
`sequence specific for the targeted sequence is initially
`ligated to the nucleic acid to be tested. Following enrich-
`ment of the duplexes formed between the bacteri-
`ophage probe and the nucleic acid in a sample, Qβ
`replicase
`is added, which, upon recognizing
`the
`retained recombinant genome, begins making a large
`number of copies.
`The Qβ system does not require primer
`[0045]
`sequences and there is no heat denaturation step as
`with the PCR and LCR amplification systems. The reac-
`tion occurs at one temperature, typically 37°C. The pre-
`ferred template is a substrate for the Qβ replicase,
`midvariant-1 RNA. A very large increase in the tem-
`plates is achieved through the use of this system. A
`review of this amplification system can be found in the
`International Patent Application Pub. No. WO 87/06270
`and in Lizardi et al., 1988, Bio/Technology 6:1197-1202.
`[0046]
`The 3SR system is a variation of an in vitro
`transcription-based amplification system. A transcrip-
`tion-based amplification system (TAS) involves the use
`of primers that encode a promoter sequence as well as
`a complementary sequence to the target strand to gen-
`erate DNA copies of a target strand and the production
`of RNA copies from the DNA copies with an RNA
`polymerase. See, e.g., Example 9B of U.S. Patent No.
`4,683,202 and European Patent Application with publi-
`
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`10
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`15
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`20
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`25
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`THERMO FISHER EX. 1017
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`11
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`EP 0 640 828 B1
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`12
`
`cation No.
`EP-A-0 310,229. The 3SR System is a system which
`uses three enzymes to carry out an isothermal replica-
`tion of target nucleic acids.
`[0047]
`The system begins with a target of single-
`stranded RNA to which a T7 RNA DNA primer is bound.
`By extension of the primer with reverse transcriptase, a
`cDNA is formed, and RNAseH treatment frees the
`cDNA from the heteroduplex. A second primer is bound
`to the cDNA and a double-stranded cDNA is formed by
`reverse transcriptase treatment. One (or both) of the
`primers encodes a promoter, e.g., the promoter for T7
`RNA polymerase, so that the double-stranded cDNA is
`a transcription template for RNA polymerase.
`[0048]
`Transcription competent cDNAs yield anti-
`sense RNA copies of the original target. The transcripts
`are then converted by the reverse transcriptase to dou-
`ble-stranded cDNA containing double-stranded promot-
`ers, optionally on both ends in an inverted repeat
`orientation. These DNAs can yield RNAs, which can
`reenter the cycle. A more complete description of the
`3SR system can be found in Guatelli et al., 1990, Proc.
`Natl. Acad. Sci. USA 87:1874-1878, and European Pat-
`ent Application with publication No. EP-A-0 329 822.
`The TAS system is also described in Gingeras