(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
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`21 February 2008 (21.02.2008) (10) International Publication Number
`
`WO 2008/021010 A2
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`(51) International Patent Classification:
`C12M 1/00 (2006.01)
`C12Q 1/68 (2006.01)
`
`(21) International Application Number:
`PCT/US2007/017245
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`(22) International Filing Date:
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`1 August 2007 (01.08.2007)
`
`(25) Filing Language:
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`(26) Publication Language:
`
`Tnglish
`
`English
`
`(30) Priority Data:
`60/836,435
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`7 August 2006 (07.08.2006)
`
`US
`
`(71) Applicant (for all designated States except US): STRATA-
`GENE CALIFORNIA [US/US]; 11011 North Torrey
`Pines Road, La Jolla, CA 92037 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): SORGE, Joseph, A.
`[US/US]; 3545 South Park Drive, Jackson, WY 83002
`(US).
`
`(74) Agents: WILLIAMS,Kathleen et al.; Edwards Angell
`Palmer & Dodge Llp, P.o. Box 55874, Boston, MA 02205
`(US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of nationalprotection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG,
`ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU,ID,IL,
`IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK,
`LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW,
`MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH,PI.,
`PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY,
`TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA,
`7M, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KL, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, ‘TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK,EE, ES, FI,
`FR, GB, GR,ITU,IE, IS, IT, LT, LU, LV, MC, MT, NL,PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,CI, CM,
`GA, GN, GQ, GW, MI, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`uponreceipt of that report
`
`(54) Title: METHODS FOR REAL-TIME QUANTITATIVE PCR
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`(57) Abstract: Disclosed herein is a process for the qualitative and quantitative determination of at least one in vitro amplified
`nucleic acid in a sealed reaction chamber, wherein subsequent to the amplification of the nucleic acid, at least one probe interacts
`with an at least partially denatured amplification product(s) in an optically detectable, differential manner, which varies as a function
`of decreasing temperature applied through a cooling gradient. This process allows the quantity and character of the amplification
`product(s) to be characterized. The entire amplification reaction, may be carried out in a sealed reaction chamber withoutintermittent
`opening, permitting an automated method of analyzing DNA and RNA amplification in qualitative and quantitative fashion on large
`series of samples.
`
`
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`2008/021010A2/INTINNITINAIIIANTIMMIIATMA
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`WO2008/021010
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`PCT/US2007/017245
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`METHODS FOR REAL-TIME QUANTITATIVE PCR
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`This application claims the benefit of US 60/836,435 filed on August 7, 2006, the
`entirety of which is incorporated by reference.
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`BACKGROUND OF THE INVENTION
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`Described herein is a process for characterizing one or more nucleic acid
`complexes, such as a nucleic acid homodimeror a nucleic acid heterodimer, allowing
`for the analysis of one or more one in vitro amplified nucleic acid, either in or outside
`the reaction chamber.
`
`The polymerase chain reaction (PCR) is a useful analytical tool which
`utilizes basic principles of molecular biology, and appliesto all the fields of analysis
`where nucleic acids play a direct or indirect role. In principle, PCR analytic
`techniques [R. K. Saiki et al. Science 239, 487-491 (1988)] and other enzymatic
`amplification techniques [J. C. Guatelli et al., Proc. Natl. Acad. Sci. 87, 1874-1878
`(1990)] allow the detection of low titers of DNA or RNA copies in an aqueous
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`solution.
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`;
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`WO 91/02815 describes the detection of specific DNA and RNA from
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`biological sample material using a DNA/RNAamplification method in combination
`with, e.g., temperature gradient gel electrophoresis [cf., K. Henco & M. Heibey
`Nucleic Acids Res. 19, 6733-6734 (1990); J. Kang et al. Biotech. Forum Europe8,
`590-593, (1991); G. Gilliland et al. Proc. Natl. Acad. Sci. 87, 2725-2729 (1990)].
`
`SUMMARY OF THE INVENTION
`Disclosed herein is a process for the qualitative and quantitative analysis ofat
`least one in vitro amplified nucleic acid product in a reaction chamber, the chamber
`preferably being a sealed reaction chamber. Subsequent to the amplification reaction,
`
`at least one probe whichis detectable and capable of interacting with at least one
`amplified nucleic acid product placed in contact with amplification products and
`exposedto the action of a decreasing temperature gradient, wherein the initial
`temperature of the gradient is capable of at least partially denaturing nucleic acids,
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`though full denaturation is preferable, with at least one measurable parameter under-
`going variation through the action of the gradient. The entire amplification reaction,
`including qualitative and quantitative analysis using a cooling gradient, may be
`carried out in a reaction chamber/detection compartment), preferably without
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`intermittent opening of the compartment.
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`The probe, the parameter of which is to be detectable spectroscopically,
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`preferably contains at least one labeled residue, for example a fluorescentresidue, and
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`preferably has intercalating properties, and a nucleic acid component. The interaction
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`with the target nucleic acid, such as for example, in vitro amplified nucleic acid, as a
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`function of its denaturation condition, is accompanied by a change in the
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`spectroscopically measured signal. This, for example, may take place byintercalation
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`of the dye into the nucleic acid double helix or by dilution or concentration effects
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`within the measuring compartment.
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`In another embodimentof the process disclosed herein, the nucleic acid
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`renaturation process initiated by the decreasing temperature gradient is detected using
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`wave length variation and/or shift in fluorescent intensity and/or variation in excited
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`state lifetime, or using the principle of the so-called energy transfer (Forster Transfer),
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`or via concentration effects, or using various, preferably hydrophobicinteractive
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`properties of the labeled probe.
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`The methods described herein permit simultaneous or sequential detection of
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`multiple different amplified nucleic acids, or other target nucleic acids to be analyzed.
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`This is effected by using a multiplicity of dyes which may be distinguished from each
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`other spectroscopically, and which permit the analysis of the various amplified
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`nucleic acids and/or other target nucleic acids of interest, through whichat least one
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`independentcalibrating substanceis introduced. This, in particular, is possible where
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`the various nucleic acids to be analyzed interact with differently labeled participants
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`in hybridization.
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`Detection of the measuring signal is conducted, for example, by measuring the
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`fluorescence generated by the dyes which,in particular, may be excited continuously
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`or in pulses byalaser.
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`An embodimentof the process disclosed herein is based on the premise that
`the amplified nucleic acids contain at least one co-amplified nucleic acid standard, the
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`sequence of which is homologousto a sequence of one of the nucleic acids of interest
`to be analyzed and preferably identical to that sequence. In another embodiment, the
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`nucleic acid standard may contain one or more point mutations which,in particular,
`lies in a sequence region of lowest stability. However, care must be taken that any
`point mutation lies outside the primer binding sites if enzymatic amplifications are
`performed. The nucleic acid standard may also be a natural component of the nucleic
`acid to be analyzed.
`According to the process disclosed herein, it is also possibile to observe
`successful amplification of a specific nucleic acid without adding a labeled standard
`fragmentto the reaction batch after amplification has taken place. In this preferred
`procedure ofthe invention, specifically those primers required for amplification are
`employed which then hybridize at the corresponding sites in the sequence of the
`nucleic acids in question. However, the corresponding sequences between the primer
`sites may be so different that when passing the cooling temperature gradient, both
`sequences--the amplified test and standard sequence--renature separately, and
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`preferably, renature in co-operative fashion.
`This allows use of sequences having a sequence deviation to such extent that
`heteroduplex formation is no longer possible. In such a situation,it is no longer
`necessary to add a labeled standard fragment after amplification has taken place.
`Different melting temperatures of both sequences may be influenced, for example, by
`greatly varying the length of the sequenceor by selecting a poly-A/T-sequence. The
`question of whether an amplification reaction has taken place may then be decided by
`employing the process described herein, for example, in a cooling temperature
`gradient with simultancous presence of ethidium bromide, where quantitative
`
`detection is likewise possible.
`The process disclosed herein permits the amplification to be performed in a
`homogenousphaseor on a solid phase, preferably using a primer which is attached to
`a solid phase and which has an extended sequence to which the labeled probe can
`hybridize. Thus, the concentration of the probe can be determined either specifically
`at the solid phase support or within the free solution. Preferably, at least one molecule
`of fluorescent dye is linked to a nucleic acid molecule, the sequence of whichis
`identical or homologousto the nucleic acid to be detected or to the co-amplified
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`nucleic acid standard.
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`Oncethe nucleic acid molecule with the fluorescent dye linked thereto has
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`been added to the reaction mixture after amplification has taken place, hybridization
`with the amplified nucleic acids is effected, preferably by effecting thermal
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`denaturation followed with a subsequent cooling gradient during which renaturation
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`occurs. However, it is also possible to add the nucleic acid molecule having the linked
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`fluorescent dye to the reaction mixture before amplification has taken place. Here, the
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`probeis to be added as a non-amplifiable double-stranded RNA oras a non-
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`amplifiable chemically modified nucleic acid.
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`For nucleic acid amplification, a possible embodiment uses a primerof the
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`primer pair employed for amplification, which primer contains a G:C-rich region at
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`the 5' terminus, for example from 15 to 20 G:C residues.
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`In an embodiment of the standardization and quantification of the process
`described herein by the above embodiments thereof, the fluorescent probes used for
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`standardization and/or quantification are added subsequent to effected amplification.
`This meansthat initially, during the amplification reaction, the fluorescent probes
`used for standardization and/or quantification are stored spatially separated from the
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`amplification process. Further, in some embodiments, the probes used for
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`standardization differ in more than one position from the target nucleic acid being
`analyzed. In order to improvethe signal/noise ratio in the following determination
`using the employed probe,it is desirable not to add toolittle probe to the mixture to
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`be amplified.
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`In one embodiment of the process according to the invention, the probe used
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`for standardization and/or quantification and/or analysis of the target polynucleotide is
`a single-stranded oligo- or polynucleotide which, however, cannot participate in the
`amplification reaction because of chemical modification. Only by suitable
`manipulation following the amplification reaction, is the single-stranded probeis
`exposed which then is capable of hybridizing with the corresponding nucleic acids
`being analyzed. Thus, for example, the probe, if:present in the form of a single-
`stranded nucleic acid, may be inactivated in the form of a "hairpin structure" and may
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`thus be prevented from participating in the amplification reaction.
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`The oligo- or polynucleotides to be used as probesin a particularly preferred
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`fashion have one or morestructural elements with at least two chemical substituents,
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`each being capable of interacting with electromagnetic waves, with cleavage or
`linkage of stable bonds, or by absorption or emission ofradiation. For a substituent
`whichis particularly suitable for interacting with electromagnetic radiation, with
`cleavage and linkage of stable bonds, such as covalent bonds, psoralen or its
`derivatives have proven successful. For a structural element serving as an actual
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`marker of the probe, luminescent dyes such as fluorescent dyes having high quantum
`yield such as dyes from the thiazole orange class have proven beneficial. Preferable
`are dyes having large Stokes shift which, dependent on hybridization condition, alter
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`the luminescent properties.
`It may be advantageous that the spectra of the structural elements at the
`respective sensitive sites which, on the one hand, are to be excited for cleavage and
`linkage of, for instance, covalent bondsor, but on the other hand, are to be regarded as
`absorption or emission maxima,are far enough apart so that each excitation will not
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`interfere with the fiction of the otherstructural element.
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`Likewise,it is possible to combine both chemical structures in a single
`chemical structure if linkage and cleavage of bonds each occur at different wave
`lengths such as is possible with maximum fluorescence ofthis structure. Thus, the
`respective functions, probe fixation within a non-amplified structure on the one hand,
`and spectroscopic identification of said structure on the other hand, cannotinterfere
`with each other. Where two separated structural elements have said separated
`_ functionsit is preferable that they should not fall below a distance ofat least 10
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`nucleotides on the oligo- or polynucleotide strand.
`
`In an embodimentof the process described herein, masked oligo- or
`polynucleotide probes are added to the above-described mixture of substances, the
`amplification reaction is carried out as described, and subsequently, the masked probe
`is released, by radiation for example, and hybridizes with the amplification product
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`under analysis and, and the hybridized complex is detected by a time/temperature
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`cooling gradient in homogenoussolution.
`Using this embodiment, it is possible to perform the analysis in such a way
`that the hybridization labeled probe no longerhas to be located separately within the
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`compartmentof the reaction chamber in order to add.it to the reaction mixture
`immediately prior to the actual analysis. Thus, it is possible to provide the labeled
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`probe with the other reagents in a form in which it cannot participate in the
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`subsequent amplification process. Thereby, it is no longer necessary to separate
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`amplification mixture and probe within the measuring chamber.
`In order to operate the process, sheet-like systems having hollow pockets or
`recesses serving as reaction chambers (compartments) are preferably used. Preferably,
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`the sheet systems are thermally weidable and suited to accommodate ready-for-use
`reagent mixtures in freeze-dried or matrix-bound form. Furthermore,direct optical
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`measurement of the reaction chamber contents is possible. Hence, the sheet material is
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`translucent or transparent at least for specific wave length regions of electromagnetic
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`radiation. Preferably, the reagents needed to perform the process according to the
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`invention are stored in spatially separated matrices, and subsequentto sealing the
`reaction chamber, are introduced into the reaction process. Preferably, the reaction
`chambers are separated from each other at a distance in which the holes are separated
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`in a commercially available microtiter plate. This has the advantage that equipment
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`suitable for processing microtitration plates may be used in the technology described
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`herein. '
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`In one embodiment, in order to analyze a mixture of amplified nucleic acids, a
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`time-controlled cooling temperature gradient is applied after addition of substances
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`needed in the reaction, and the renaturation behavior of the nucleic acids is measured.
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`This is done through the variation of spectroscopic parameters of the substance
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`interacting with the nucleic acid. Variation of the spectroscopic parameteris
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`monitored over time or in equivalent fashion as a function of temperature change.
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`Evaluation of the function of variation in spectroscopic behaviorof the
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`substanceinteracting with the nucleic acid permits the determination of the presence
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`or numberor degree of homology of an examined nucleic acid with the corresponding
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`standard. Preferably, evaluation of these data is done on-line using a data processing
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`system.
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`In addition to eliminating the above-mentioned drawbacksin priorart, the
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`process according to the invention is advantageousin that amplification of nucleic
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`acids and subsequent analytics maybe carried outin a single hermetically sealable
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`reaction compartment. Thereby, disposal of these biological materials without
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`opening the compartments is possible, and a potential source of contamination is
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`eliminated. Furthermore, such procedure also permits storage of test sheets of the
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`above-mentioned type in sealed condition over prolonged periods of time so that
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`archiving of the often valuable substances is made possible. However, storage
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`preferably is done in frozen condition. Likewise, the process accordingto the
`invention advantageously permits the experiments to be repeatable, optionally at a
`later time even after prolonged interim storage, or the amplified mixture to be
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`preparatively processable and analyzable.
`The device for performing the process disclosed herein has a meansfor time-
`dependent regulation of the temperature ofthe reaction chambers to be used in the
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`process. In one embodiment, the time-dependent regulation of the temperatureis
`controlled by a programmable unit. The read-out unit of the device preferably
`consists of an optical unit capable ofregistering photons. Particularly preferred are
`such units which are suitable for registering emitted fluorescent light. Likewise
`contemplated is equipment capable of detecting other spectroscopic properties such as
`nuclear spin or electron spin etc., which can be correlated to conformational changes
`of the nucleic acid double-helix or other structural variables, or the use of
`chromatographic procedures. Using the method of hydrophobic interaction
`chromatography, molecules having hydrophobic ligands, as represented by partially
`denaturing structures ofthe substances to be analyzed, may be separated from the
`duplexes.
`
`The device for operating the process disclosed herein is capable of
`accommodating a means for operating the process which is assembled of a system of
`reaction compartments, preferably a sheet system with ready-to-use reagents in
`freeze-dried form. Preferably, the reaction compartments are arranged in
`microtitration form. Preferably, the reagents of the means for operating the process
`are fixated and/or stored in at least one water-soluble matrix. Preferably, the matrix
`contains stabilizers such as sugars, particularly trehalose or saccharose. Preferably,
`the means for operating the process of the invention comprises reaction compartments
`and/or other reagent reservoirs, amplification primers, buffer components, andat least
`one polymerase and usual co-factors for performing the amplification reaction. In
`another preferred embodiment of the meansfor operating the process of the invention,
`the reaction chamberor reaction compartmentis provided with an additional separate
`reagent reservoir in a matrix located within the sheet sealing the compartment. Here,
`preferably, the labeled probe with the buffer substances required for hybridization are
`stored.
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`Such a device may include for example Stratagene’s Mx4000® Multiplex
`Quantitative PCR System whichis further adapted so that after runningaPCR
`reaction, the system is able to run a cooling curve in which the starting temperature
`can be as high as 100°C. As the temperature decreases the single strand product
`renatures to form a double stranded productby either a step-wise or continuous
`decrease in temperature, with fluorescence data being collected at each step. The
`magnitude of the increase in fluorescenceintensity of the SYBR Green dye dueto its
`intercalation into dsDNA provides an indicator of the amount ofrenatured dsDNA at
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`each point in the cooling curve. In thermal renaturation profiles for complex nucleic
`acid mixtures such as those generated during PCRreactions , two or more semi-
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`discrete populations with different transition temperatures can typically be identified.
`Populations with a Tm of 80°C or higher correspond to larger PCR products and can
`usually be assigned to the specific DNA product. DNA products displaying meiting
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`temperatures of <75°C correspond to non-specific DNA products. Exemplary
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`fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5,
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`Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa Fluor 488,
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`Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor
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`647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green,
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`BODIPY,fluorescein isothiocyanate (FITC), carboxy-fluorescein (FAM),
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`phycoerythrin, rhodamine, dichlororhodamine (dRhodaminew), carboxy
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`tetramethylrhodamine (TAMRAw), carboxy-X rhodamine (ROXm), LIZ, VIC, NED,
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`PET, SYBR, PicoGreen, RiboGreen, and the like. Descriptions of fluorophores and
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`their use can be found in, amongother places, R. Haugland, Handbook of Fluorescent
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`Probes and Research Products, (2002), Molecular Probes, Eugene, OR; M. Schena,
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`Microarray Analysis (2003), John Wiley & Sons, Hoboken, NJ; Synthetic Medicinal
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`Chemistry 2003/2004 Catalog, Berry and Associates, Ann Arbor, M1; G. Hermanson,
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`Bioconjugate Techniques, Academic Press (1996); and Glen Research 2002 Catalog,
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`Sterling, VA. Near-infrared dyes are expressly within the intended meaning of the
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`terms fluorophore and fluorescent reporter group.
`In one embodiment, the means for operating the process of the invention is
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`arranged in kit systems comprising reaction vessels such as sheet systems with
`storable and directly usable reagent mixtures, where it
`is merely necessary to charge
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`the reaction vessels with the sample to be analyzed which, in a hermetically sealed
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`condition, is then subjected to an amplification procedure and subsequentanalysis.
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`The process according to the invention is particularly suitable for analyzing
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`mixtures of substances, preferably nucleic acids where at least one componentin the
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`temperature region of the time/temperature cooling gradient is subject to thermal
`conversion. By adding and co-amplifying a standard having precisely known number
`of copies the processof the invention can be standardized and permits quantitative
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`statements about the amplified nucleic acids of the sample to be examined. Using the
`process disclosed herein it is possible, for example, to detect mutations, point
`mutations, deletions, insertions, and rearrangements within the DNA/RNA nucleic
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`acid. Using the quantitative analysis it is also possible to determine the concentrations
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`of such changesin the nucleic acid. The samples may be derived from most various
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`materials such as live, dead, fossil tissue, as well as tissue which no longer displays
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`active metabolism in vivo, or from bodyfluids, from in vitro cell cultures, or from
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`environmental samples. The process disclosed herein allows for qualitative and
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`quantitative detection of cellular genes and genes of infectious pathogens directly or
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`via their RNA gene products as a wild type sequenceor as variants.
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`Moreover, the process disclosed herein may also be employed for the
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`examination and determination of potentially toxic substances or potential
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`pharmaceutical agents or chemical or biological pesticides by examining their effect
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`on nucleic acids or their amplifications in cellular or non-cellular systems.
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`In one embodiment of the process described herein, the gradient can be a
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`continuous gradient with respect to the rate of decrease in temperature over time. In
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`one embodiment the continuous gradient is linear, though non linear gradients are also
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`encompassed by the gradients described herein. The phrase “continuous temperature
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`gradient” is defined as a gradient in which the temperature is changing (i.e.,
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`increasing or decreasing) continuously with respect to a specified time interval,
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`(preferably decreasing with respect to the specified time interval). The phrase
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`“stepwise gradient” is a gradient in which the temperature is changing(i.e., increasing
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`or decreasing) over a given time interval, however the change is fragmented into a
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`series of steps, each step comprising two time periods:a first time period in which the
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`temperature changes (preferably decreases), and a second time period in which the
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`temperature is maintained.
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`In a preferred embodiment, the steps are of equal length.
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`In another embodiment, the steps are of unequal lengths. In one aspect the time
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`periods in a given step are equal, in another aspect, the first time period is shorter than
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`the second time period of a given step, and in a further aspect the first time period is
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`longer than the second time period of a given step.
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`In one embodiment, the total course of the entire temperature gradientis
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`composed of one or more continuous gradients and/or one or more stepwise
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`gradients. If the total course of the entire temperature gradient contains multiple
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`continuousgradients, one or more orall or none of the multiple continuous gradients
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`are identical.
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`Ifthe total course of the entire temperature gradient contains multiple
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`stepwise gradients, one or moreor all or none of the multiple continuous gradients are
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`identical. In another aspect, each of the multiple gradients are arranged in any order
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`in the entire temperature gradient.
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`In an another embodiment of the process described herein, the gradient can be
`a stepwise gradient with respect to the rate of decrease in temperature over time. The
`time intervals for each degree of temperature decrease over time in a stepwise
`cooling gradient can range from about 1 second, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
`14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 55, 60, 90,
`120, 150, 180, 210, 240, 270, 300, 315, 330, 345, 360, 390, 410, 440, 475, 500, 600,
`700, 800, 900, 1000, to about 1500 seconds, including any fraction of the intervals
`listed thereof. Alternatively, the gradient can have both a stepwise component and a
`linear component.
`
`As used herein, the term "polynucleotide(s)” or "Nucleic acid" generally
`refers to any polyribonucleotide or poly-deoxyribonucleotide, which may be
`unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide(s)" include,
`without limitation, single and double-stranded nucleic acids. As used herein, the term
`"polynucleotide(s)"also includes DNAs or RNAsas described above that contain one
`
`or more modified bases. Thus, DNAs or RNAswith backbones modified for stability
`or for other reasons are "polynucleotide(s)". The term "polynucleotide(s)” as it is
`employed herein embraces such chemically, enzymatically or metabolically modified
`forms of polynucleotides, as well as the chemical forms of DNA and RNA
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`characteristic of viruses and cells, including, for example, simple and complex cells..
`Polynucleotides can be single stranded or double stranded, and can contain one or
`more portions which are single stranded and/or one or more portions which are double
`Stranded. In typical embodiments, the length of a polynucleotide ranges from as small
`as about 50 bases up to 100 bases, to 500 bases, to 1000 bases to 5 kb to 10 kb or
`higher, including the length of a plasmid, vector, or episome. "Polynucleotide(s)"
`also embraces short polynucleotides often referred to as oligonucleotide(s).
`The terms “oligonucleotide” or “oligo”, as used herein in referring to the
`probe ofthe present invention, is defined as a molecule comprisedof about 15 or
`more nucleotides, preferably more than about 24 and more preferably about 36
`nucleotides. Its exact size will depend upon manyfactors which, in turn, depend upon
`the ultimate function and use of the oligonucleotide. Preferably, an oligonucleotide
`which functions as an extension primerwill be sufficiently long to prime the synthesis
`of extension products in the presence ofa catalyst, e.g., DNA polymerase, and
`deoxynucleotide triphosphates. The exact lengths of the primers will depend on many
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`factors, including temperature, source of primer and use of the method. In diagnostic
`applications, for example, the oligonucleotide primer typically contains 15-25 or more
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`nucleotides, depending on the complexity of the target sequence. For non-extension
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`productapplications, the oligonucleotide generally contains between 10-25
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`nucleotides. Shorter oligonucleotide generally require cooler temperatures to form
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`sufficiently stable hybrid complexes with template.
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`The term “probe” as used herein, in one embodiment encompasses an
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`oligonucleotide or polynucleotide which hybridizes to a target nucleic acid, such as a
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`target ina sample. The term “probe”as used herein, in another embodiment
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`encompasses a dyethat intercalates into double stranded polynucleotides. In another
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`embodiment, the term probe encompasses an oligonucleotide which has attached to it
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`one or more intercalating dyes. In yet a further embodiment, either the probe or the
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`target can be labeled with one, or more than one label. Such labels are well knownto
`one of skill in the art and include intercalating dyes, radioactive labels, fluorescent
`labels, and any kind of detectable label.
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`"Complementary" as used herein refers to the ability of a nucleic acid single
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`strand (or portion thereof) to hybridize to an anti-parallel nucleic acid single strand (or
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`portion thereof) by contiguous base-pairing between the nucleotides (that is not
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`interrupted by any unpaired nucleotides) of the anti-parallel nucleic acid single
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`strands, thereby forming a double-stranded nucleic acid between the complementary
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`strands.
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`A “target” nucleic acid or sample as used herein refers to the nucleic acid used
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`for analysis and to which a polynucleotide for its detection or for an intemal
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`amplification control and/or a pair of PCR primersis hybridized in order to ascertain
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`the presence or absence ofthe nucleic acid.
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`As used herein, "forward amplification primer" refers to a polynucleotide used
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`for PCR amplification that is complementary to the sense strand of the target nucleic
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`acid. "Reverse amplification primer" refers to a polynucleotide used for PCR
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`amplification that is complementary to the antisense strand of the target nucleic acid.
`For a given target, a forward and reverse amplification primer are used to amplify the
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`DNAin PCR.
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`As used herein, "oligonucleotide primers" refer to single-stranded DNA or
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`RNA molecules that are capable of hybridizing to a nucleic acid template and are
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`capable of priming(or initiating) enzymatic synthesis of a second nucleic acid strand.
`In a preferred embodiment, the oligonucleotide primers contain a spacer-
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`linked fluorescent dye capable ofintercalating if the primer is located in a double-
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`helical region. Upon thermal denaturation of the double-helix the fluorescent
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`properties of the dye are modified. as described in Thuong, N. T. & Chassignol, M.
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`Tetrahedron Letters 28, 4157-4160 (1987); Thuong, N. T-. et al., Proc. Natl. Acad. Sci.
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`U.S.A. 84, 5129-5133 (1987); Helene, C., in DNA-Ligand Interactions, Plenum
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`Publishing Corporation, 127-140 (1987); W. Gushelbauer & W. Saenger, Ed.
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`As used herein, "amplifying" refers to producing additional copies of a nucleic
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`acid seque