Monitoring amplification of DNA during PCR

Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Methods for quantitating amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.

MICROFICHE APPENDIX
 This application includes a microfiche appendix. 1 microfiche, 54 pages.
 This invention relates generally to observing fluorescence signals
 resulting from hybridization in conjunction with the polymerase chain
 reaction. More specifically, the present invention relates to observing
 hybridization with fluorescence during and/or immediately after PCR and
 using this information for product identification, sequence alteration
 detection, and quantification.
 The polymerase chain reaction (PCR) is fundamental to molecular biology and
 is the first practical molecular technique for the clinical laboratory.
 Despite its usefulness and popularity, current understanding of PCR is not
 highly advanced. Adequate conditions for successful amplifications must be
 found by trial and error and optimization is empirical. Even those skilled
 in the art are required to utilize a powerful technique without a
 comprehensive or predictive theory of the process.
 PCR is achieved by temperature cycling of the sample, causing DNA to
 denature (separate), specific primers to attach (anneal), and replication
 to occur (extend). One cycle of PCR is usually performed in 2 to 8 min
 requiring 1 to 4 hours for a 30-cycle amplification. The sample
 temperature response in most PCR instrumentation is very slow compared to
 the times required for denaturation, annealing, and extension. The
 physical (denaturation and annealing) and enzymatic (extension) reactions
 in PCR occur very quickly. Amplification times for PCR can be reduced from
 hours to less than 15 min. Incorporated herein by reference in their
 entireties are each of the following individual applications, which
 disclose such a rapid cycling system: U.S. application Ser. No.
 08/818,267, filed Mar. 17, 1997, entitled Method for Detecting the Factor
 V Leiden Mutation, which is a continuation-in-part of U.S. patent
 application Ser. No. 08/658,993, filed Jun. 4, 1996, now abandoned,
 entitled System And Method For Monitoring PCR Processes., which is a
 continuation-in-part of U.S. patent application Ser. No. 08/537,612, filed
 Oct. 2, 1995, entitled Method For Rapid Thermal Cycling of Biological
 Samples, which is a continuation-in-part of U.S. patent application Ser.
 No. 08/179,969, filed Jan. 10, 1994, (now U.S. Pat. No. 5,455,175),
 entitled Rapid Thermal Cycling Device, which is a continuation-in-part of
 U.S. patent application Ser. No. 07/815,966 filed Jan. 2, 1992, (now
 abandoned) entitled Rapid Thermal Cycling Device which is a
 continuation-in-part of U.S. patent application Ser. No. 07/534,029 filed
 Jun. 4, 1990, (now abandoned) entitled Automated Polymerase Chain Reaction
 Device. Copending U.S. application filed in the U.S. Patent and Trademark
 Office on Jun. 4, 1997, entitled System and Method for Carrying Out and
 Monitoring Biological Processes as Ser. No. 8/869,275 and naming with Carl
 T. Wittwer, Kirk M. Ririe, Randy P. Rasmussen, and David R. Hillyard as
 applicants is also hereby incorporated by reference in its entirety. Rapid
 cycling techniques are made possible by the rapid temperature response and
 temperature homogeneity possible for samples in high surface
 area-to-volume sample containers such as capillary tubes. For further
 information, see also: C. T. Wittwer, G. B. Reed, and K. M. Ririe, Rapid
 cycle DNA amplification, in K. B. Mullis, F. Ferre, and R. A. Gibbs, The
 polymerase chain reaction, Birkhauser, Boston, 174-181, (1994). Improved
 temperature homogeneity allows the time and temperature requirements of
 PCR to be better defined and understood. Improved temperature homogeneity
 also increases the precision of any analytical technique used to monitor
 PCR during amplification.
 Fluorimetry is a sensitive and versatile technique with many applications
 in molecular biology. Ethidium bromide has been used for many years to
 visualize the size distribution of nucleic acids separated by gel
 electrophoresis. The gel is usually transilluminated with ultraviolet
 light and the red fluorescence of double stranded nucleic acid observed.
 Specifically, ethidium bromide is commonly used to analyze the products of
 PCR after amplification is completed. Furthermore, EPA 0 640 828 A1 to
 Higuchi & Watson, hereby incorporated by reference, discloses using
 ethidium bromide during amplification to monitor the amount of double
 stranded DNA by measuring the fluorescence each cycle. The fluorescence
 intensity was noted to rise and fall inversely with temperature, was
 greatest at the annealing/extension temperature (50.degree. C.), and least
 at the denaturation temperature (94.degree. C.). Maximal fluorescence was
 acquired each cycle as a measure of DNA amount. The Higuchi & Watson
 application does not teach using fluorescence to monitor hybridization
 events, nor does it suggest acquiring fluorescence over different
 temperatures to follow the extent of hybridization. Moreover, Higuch &
 Watson fails to teach or suggest using the temperature dependence of PCR
 product hybridization for identification or quantification of PCR
 products.
 The Higuchi & Watson application, however, does mention using other
 fluorophores, including dual-labeled probe systems that generate
 flourescence when hydrolyzed by the 5'-exonuclease activity of certain DNA
 polymerases, as disclosed in U.S. Pat. No. 5,210,015 to Gelfand et al. The
 fluorescence observed from these probes primarily depends on hydrolysis of
 the probe between its two fluorophores. The amount of PCR product is
 estimated by acquiring fluorescence once each cycle. Although
 hybridization of these probes appears necessary for hydrolysis to occur,
 the fluorescence signal primarily results from hydrolysis of the probes,
 not hybridization, wherein an oligonucleotide probe with fluorescent dyes
 at opposite ends thereof provides a quenched probe system useful for
 detecting PCR product and nucleic acid hybridization, K. J. Livak et al.,
 4 PCR Meth. Appl. 357-362 (1995). There is no suggestion of following the
 temperature dependence of probe hybridization with fluorescence to
 identify sequence alterations in PCR products.
 The specific hybridization of nucleic acid to a complementary strand for
 identification has been exploited in many different formats. For example,
 after restriction enzyme digestion, genomic DNA can be size fractionated
 and hybridized to probes by Southern blotting. As another example, single
 base mutations can be detected by "dot blots" with allele-specific
 oligonucleotides. Usually, hybridization is performed for minutes to hours
 at a single temperature to achieve the necessary discrimination.
 Alternately, the extent of hybridization can be dynamically monitored
 while the temperature is changing by using fluorescence techniques. For
 example, fluorescence melting curves have been used to monitor
 hybridization. L. E. Morrison & L. M. Stols, Sensitive fluorescence-based
 thermodynamic and kinetic measurements of DNA hybridization in solution,
 32 Biochemistry 3095-3104, 1993). The temperature scan rates are usually
 10.degree. C./hour or less, partly because of the high thermal mass of the
 fluorimeter cuvette.
 Current methods for monitoring hybridization require a lot of time. If
 hybridization could be followed in seconds rather than hours,
 hybridization could be monitored during PCR amplification, even during
 rapid cycle PCR. The many uses of monitoring hybridization during PCR, as
 will be fully disclosed herein, include, product identification and
 quantification, sequence alteration detection, and automatic control of
 temperature cycling parameters by fluorescence feedback
 The prior art, as explained above, carries out temperature cycling slowly
 and empirically. When analysis of PCR products by hybridization is needed,
 additional time consuming steps are required. Thus, it would be a great
 advance in the art to provide methods for monitoring hybridization during
 PCR and analyzing the reaction while it is taking place, that is, during
 or immediately after temperature cycling without manipulation of the
 sample. By monitoring hybridization during PCR, the underlying principles
 that allow PCR to work can be followed and used to analyze and optimize
 the reaction during amplification.
 BRIEF SUMMARY OF THE INVENTION
 It is an object of the present invention to provide a
 double-strand-specific DNA dye for monitoring product hybridization during
 PCR.
 It is another object of the invention to provide a system for identifying
 PCR-amplified products by their fluorescence melting curves.
 It is also an object of the invention to provide a method for improving the
 sensitivity of PCR quantification with double-strand-specific DNA dyes.
 It is still another objection of the invention for determining the amount
 of specific product amplified by PCR by melting curves to correct for
 nonspecific amplification detected with the double-strand-specific DNA
 dye.
 It is a further object of the invention to provide a method of relative
 quantification of different PCR products with double-strand-specific dyes.
 It is yet another object of the invention to provide a method of product
 quantification by the reannealing kinetics of the product in the presence
 of a double-strand-specific DNA dye.
 It is a still further object of the invention to provide a novel resonance
 energy transfer pair to monitor primer and/or probe hybridization.
 It is still another object of the invention to provide a method of product
 quantification by the reannealing kinetics of a probe to the product using
 a resonance energy transfer pair.
 It is also an object of the present invention to provide a method to
 determine initial template copy number by following the fluorescence of a
 hybridization probe or probes each cycle during PCR amplification.
 It is another object of the invention to provide a system for homogeneous
 detection of PCR products by resonance energy transfer between two labeled
 probes that hybridize internal to the PCR primers.
 It is still another object of the invention to provide a system for
 homogeneous detection of PCR products by resonance energy transfer between
 one labeled primer and one labeled probe that hybridizes internal to the
 PCR primers.
 It is yet another object of the invention to provide a system for detection
 of sequence alterations internal to PCR primers by resonance energy
 transfer and probe melting curves.
 It is a further object of the invention to provide a system for relative
 quantification of different PCR products by probe melting curves.
 It is yet another object of the invention to provide methods to determine
 the initial template copy number by curve fitting the fluorescence vs
 cycle number plot.
 It is still another object of the invention to provide a system and method
 for performing PCR rapidly and also continuously monitoring the reaction
 and adjusting the reaction parameters while the reaction is ongoing.
 It is another object of the invention to replace the nucleic acid probes by
 synthetic nucleic acid analogs or derivatives, e.g. by peptide nucleic
 acids (PNA), provided that they can also be labeled with fluorescent
 compounds.
 These and other objects and advantages of the invention will become more
 fully apparent from the description and claims which follow, or may be
 learned by the practice of the invention.
 The present invention particularly decreases the total time required for
 PCR amplification and analysis over prior art techniques while at the same
 time allowing the option of significantly increasing the quality of the
 reaction by optimizing amplification conditions.
 The present invention provides methods and applications for continuous
 fluorescence monitoring of DNA amplification. Required instrumentation
 combines optical components with structures to provide rapid temperature
 cycling to continuously monitor DNA amplification by a variety of
 different fluorescence techniques. In one illustrative embodiment,
 fluorescence is acquired continuously from a single sample or alternately
 from multiple samples on a rotating carousel with all of the samples being
 simultaneously subjected to rapid thermal cycling. Further information on
 associated instrumentation can be found in the U.S. patent applications
 referenced above.
 In accordance with one aspect of the present invention, fluorescence during
 DNA amplification was monitored by: 1) the double strand-specific dye SYBR
 Green I, and 2) resonance energy transfer of fluorescein to Cy5.TM. or
 Cy5.5.TM. with hybridization probes. Fluorescence data acquired once per
 cycle allow quantification of initial template copy number.
 Furthermore, in contrast to measuring fluorescence once per cycle,
 embodiments of the present invention are disclosed which monitor
 temperature, time and fluorescence continuously throughout each cycle thus
 producing a 3-dimensional spiral. This 3-dimensional spiral can be reduced
 to temperature vs. time, fluorescence vs. time, and fluorescence vs.
 temperature plots. Fluorescence vs. temperature plots of the fluorescence
 from hybridization probes can be used to detect sequence alterations in
 the product. These sequence alterations may be natural, as in mutations or
 polymorphisms, or artificial, as in an engineered alternative template for
 quantitative PCR.
 In accordance with another aspect of the present invention, fluorescence
 monitoring is used to acquire product melting curves during PCR by
 fluorescence monitoring with double-strand-specific DNA specific dyes.
 Plotting fluorescence as a function of temperature as the thermal cycler
 heats through the dissociation temperature of the product gives a PCR
 product melting curve. The shape and position of this DNA melting curve is
 a function of GC/AT ratio, length, and sequence, and can be used to
 differentiate amplification products separated by less than 2.degree. C.
 in melting temperature. Desired products can be distinguished from
 undesired products, including primer dimers. Analysis of melting curves
 can be used to extend the dynamic range of quantitative PCR and to
 differentiate different products in multiplex amplification. Using double
 strand dyes, product denaturation, reannealing and extension can be
 followed within each cycle. Continuous monitoring of fluorescence allows
 acquisition of melting curves and product annealing curves during
 temperature cycling.
 The present invention provides reagents and methods for rapid cycle PCR
 with combined amplification and analysis by fluorescence monitoring in
 under thirty minutes, more preferably in under fifteen minutes, and most
 preferably in under ten minutes.
 A method for analyzing a target DNA sequence of a biological sample
 comprises
 amplifying the target sequence by polymerase chain reaction in the presence
 of two nucleic acid probes that hybridize to adjacent regions of the
 target sequence, one of the probes being labeled with an acceptor
 fluorophore and the other probe labeled with a donor fluorophore of a
 fluorescence energy transfer pair such that upon hybridization of the two
 probes with the target sequence, the donor and acceptor fluorophores are
 within 25 nucleotides of one another, the polymerase chain reaction
 comprising the steps of adding a thermostable polymerase and primers for
 the targeted nucleic acid sequence to the biological sample and thermally
 cycling the biological sample between at least a denaturation temperature
 and an elongation temperature;
 exciting the biological sample with light at a wavelength absorbed by the
 donor fluorophore and detecting the emission from the fluorescence energy
 transfer pair.
 A method for analyzing a target DNA sequence of a biological sample
 comprises
 amplifying the target sequence by polymerase chain reaction in the presence
 of two nucleic acid probes that hybridize to adjacent regions of the
 target sequence, one of the probes being labeled with an acceptor
 fluorophore and the other probe labeled with a donor fluorophore of a
 fluorescence energy transfer pair such that upon hybridization of the two
 probes with the target sequence, the donor and acceptor fluorophores are
 within 25 nucleotides of one another, the polymerase chain reaction
 comprising the steps of adding a thermostable polymerase and primers for
 the targeted nucleic acid sequence to the biological sample and thermally
 cycling the biological sample between at least a denaturation temperature
 and an elongation temperature;
 exciting the sample with light at a wavelength absorbed by the donor
 fluorophore; and
 monitoring the temperature dependent fluorescence from the fluorescence
 energy transfer pair.
 A method of real time monitoring of a polymerase chain reaction
 amplification of a target nucleic acid sequence in a biological sample
 comprises
 (a) adding to the biological sample an effective amount of two nucleic acid
 primers and a nucleic acid probe, wherein one of the primers and the probe
 are each labeled with one member of a fluorescence energy transfer pair
 comprising an acceptor fluorophore and a donor fluorophore, and wherein
 the labeled probe hybridizes to an amplified copy of the target nucleic
 acid sequence within 15 nucleotides of the labeled primer;
 (b) amplifying the target nucleic acid sequence by polymerase chain
 reaction;
 (c) illuminating the biological sample with light of a selected wavelength
 that is absorbed by said donor fluorophore; and
 (d) detecting the fluorescence emission of the sample.
 An improved method of amplifying a target nucleic acid sequence of a
 biological sample comprises
 (a) adding to the biological sample an effective amount of a
 nucleic-acid-binding fluorescent entity;
 (b) amplifying the target nucleic acid sequence using polymerase chain
 reaction, comprising thermally cycling the biological sample using initial
 predetermined temperature and time parameters, and then
 (i) illuminating the biological sample with a selected wavelength of light
 that is absorbed by the fluorescent entity during the polymerase chain
 reaction;
 (ii) monitoring fluorescence from the sample to determine the optimal
 temperature and time parameters for the polymerase chain reaction; and
 (iii) adjusting the initial temperature and time parameters in accordance
 with the fluorescence.
 In one illustrative embodiment, the fluorescent entity comprises a double
 strand specific nucleic acid binding dye, and in another illustrative
 embodiment the fluorescent entity comprises a fluorescently labeled
 oligonucleotide probe that hybridizes to the targeted nucleic acid
 sequence.
 A method for detecting a target nucleic acid sequence of a biological
 sample comprises
 (a) adding to the biological sample an effective amount of a pair of
 oligonucleotide probes that hybridize to the target nucleic acid sequence,
 one of the probes being labeled with an acceptor fluorophore and the other
 probe labeled with a donor fluorophore of a fluorescence energy transfer
 pair, wherein an emission spectrum of the donor fluorophore and an
 absorption spectrum of the acceptor fluorophore overlap less than 25%, the
 acceptor fluorophore has a peak extinction coefficient greater than
 100,000 M.sup.-1 cm.sup.-1 and upon hybridization of the two probes, the
 donor and acceptor fluorophores are within 25 nucleotides of one another;
 (b) illuminating the biological sample with a selected wavelength of light
 that is absorbed by said donor fluorophore; and
 (c) detecting the emission of the biological sample. An illustrative
 resonance energy transfer pair comprises fluorescein as the donor and Cy5
 or Cy5.5 as the acceptor.
 A method of real time monitoring of a polymerase chain reaction
 amplification of a target nucleic acid sequence in a biological sample
 comprises
 amplifying the target sequence by polymerase chain reaction in the presence
 of two nucleic acid probes that hybridize to adjacent regions of the
 target sequence, one of the probes being labeled with an acceptor
 fluorophore and the other probe labeled with a donor fluorophore of a
 fluorescence energy transfer pair such that upon hybridization of the two
 probes with the target sequence, the donor and acceptor fluorophores are
 within 25 nucleotides of one another, the polymerase chain reaction
 comprising the steps of adding a thermostable polymerase and primers for
 the targeted nucleic acid sequence to the biological sample and thermally
 cycling the biological sample between at least a denaturation temperature
 and an elongation temperature;
 exciting the biological sample with light at a wavelength absorbed by the
 donor fluorophore and detecting the emission from the biological sample;
 and
 monitoring the temperature dependent fluorescence from the fluorescence
 energy transfer pair.
 A method of real time monitoring of a polymerase chain reaction
 amplification of a target nucleic acid sequence in a biological sample
 comprises
 amplifying the target sequence by polymerase chain reaction in the presence
 of SYBR.TM. Green I, the polymerase chain reaction comprising the steps of
 adding a thermostable polymerase and primers for the targeted nucleic acid
 sequence to the biological sample and thermally cycling the biological
 sample between at least a denaturation temperature and an elongation
 temperature;
 exciting the biological sample with light at a wavelength absorbed by the
 SYBRTM Green I and detecting the emission from the biological sample; and
 monitoring the temperature dependent fluorescence from the SYBR.TM. Green
 I. Preferably, the monitoring step comprises determining a melting profile
 of the amplified target sequence.
 A method for analyzing a target DNA sequence of a biological sample
 comprises
 (a) adding to the biological sample an effective amount of two nucleic acid
 primers and a nucleic acid probe, wherein one of the primers and the probe
 are each labeled with one member of a fluorescence energy transfer pair
 comprising an acceptor fluorophore and a donor fluorophore, and wherein
 the labeled probe hybridizes to an amplified copy of the target nucleic
 acid sequence within 15 nucleotides of the labeled primer;
 (b) amplifying the target nucleic acid sequence by polymerase chain
 reaction;
 (c) illuminating the biological sample with light of a selected wavelength
 that is absorbed by said donor fluorophore and detecting the fluorescence
 emission of the sample. In another illustrative embodiment, the method
 further comprises the step of monitoring the temperature dependent
 fluorescence of the sample, preferably by determining a melting profile of
 the amplified target sequence.
 A method of detecting a difference at a selected locus in a first nucleic
 acid as compared to a second nucleic acid comprises
 (a) providing a pair of oligonucleotide primers configured for amplifying,
 by polymerase chain reaction, a selected segment of the first nucleic acid
 and a corresponding segment of the second nucleic acid, wherein the
 selected segment and corresponding segment each comprises the selected
 locus, to result in amplified products containing a copy of the selected
 locus;
 (b) providing a pair of oligonucleotide probes, one of the probes being
 labeled with an acceptor fluorophore and the other probe being labeled
 with a donor fluorophore of a fluorogenic resonance energy transfer pair
 such that upon hybridization of the two probes with the amplified products
 the donor and acceptor are in resonance energy transfer relationship,
 wherein one of the probes is configured for hybridizing to the amplified
 products such that said one of the probes spans the selected locus and
 exhibits a melting profile when the difference is present in the first
 nucleic acid that is distinguishable from a melting profile of the second
 nucleic acid;
 (c) amplifying the selected segment of first nucleic acid and the
 corresponding segment of the second nucleic acid by polymerase chain
 reaction in the presence of effective amounts of probes to result in an
 amplified selected segment and an amplified corresponding segment, at
 least a portion thereof having both the probes hybridized thereto with the
 fluorogenic resonance energy transfer pair in resonance energy transfer
 relationship;
 (d) illuminating the amplified selected segment and the amplified
 corresponding segment with the probes hybridized thereto with a selected
 wavelength of light to elicit fluorescence by the fluorogenic resonance
 energy transfer pair;
 (e) measuring fluorescence emission as a function of temperature to
 determine in a first melting profile of said one of the probes melting
 from the amplified selected segment of first nucleic acid and a second
 melting profile of said one of the probes melting from the amplified
 corresponding segment of second nucleic acid; and
 (f) comparing the first melting profile to the second melting profile,
 wherein a difference therein indicates the presence of the difference in
 the sample nucleic acid.
 A method of detecting a difference at a selected locus in a first nucleic
 acid as compared to a second nucleic acid comprises
 (a) providing a pair of oligonucleotide primers configured for amplifying,
 by polymerase chain reaction, a selected segment of the first nucleic acid
 and a corresponding segment of the second nucleic acid, wherein the
 selected segment and corresponding segment each comprises the selected
 locus, to result in amplified products containing a copy of the selected
 locus;
 (b) providing an oligonucleotide probe, wherein one of the primers and the
 probe are each labeled with one member of a fluorescence energy transfer
 pair comprising an donor fluorophore and an acceptor fluorophore, and
 wherein the labeled probe and labeled primer hybridize to the amplified
 products such that the donor and acceptor are in resonance energy transfer
 relationship, and wherein the probe is configured for hybridizing to the
 amplified products such that said probe spans the selected locus and
 exhibits a melting profile when the difference is present in the first
 nucleic acid that is distinguishable from a melting profile of the second
 nucleic acid;
 (c) amplifying the selected segment of first nucleic acid and the
 corresponding segment of the second nucleic acid by polymerase chain
 reaction in the presence of effective amounts of primers and probe to
 result in an amplified selected segment and an amplified corresponding
 segment, at least a portion thereof having the labled primer and probe
 hybridized thereto with the fluorogenic resonance energy transfer pair in
 resonance energy transfer relationship;
 (d) illuminating the amplified selected segment and the amplified
 corresponding segment with the labeled primer and probe hybridized thereto
 with a selected wavelength of light to elicit fluorescence by the
 fluorogenic resonance energy transfer pair;
 (e) measuring fluorescence emission as a function of temperature to
 determine in a first melting profile of said probe melting from the
 amplified selected segment of first nucleic acid and a second melting
 profile of said probe melting from the amplified corresponding segment of
 second nucleic acid; and
 (f) comparing the first melting profile to the second melting profile,
 wherein a difference therein indicates the presence of the difference in
 the sample nucleic acid.
 A method of detecting heterozygosity at a selected locus in the genome of
 an individual, wherein the genome comprises a mutant allele and a
 corresponding reference allele, each comprising the selected locus,
 comprises
 (a) obtaining sample genomic DNA from the individual;
 (b) providing a pair of oligonucleotide primers configured for amplifying,
 by polymerase chain reaction, a first selected segment of the mutant
 allele and a second selected segment of the corresponding reference allele
 wherein both the first and second selected segments comprise the selected
 locus;
 (c) providing a pair of oligonucleotide probes, one of the probes being
 labeled with an acceptor fluorophore and the other probe being labeled
 with a donor fluorophore of a fluorogenic resonance energy transfer pair
 such that upon hybridization of the two probes with the amplified first
 and second selected segments one of the probes spans the selected locus
 and exhibits a first melting profile with the amplified first selected
 segment that is distinguishable from a second melting profile with the
 amplified second selected segment;
 (d) amplifying the first and second selected segments of sample genomic DNA
 by polymerase chain reaction in the presence of effective amounts of
 probes to result in amplified first and second selected segments, at least
 a portion thereof having both the probes hybridized thereto with the
 fluorogenic resonance energy transfer pair in resonance energy transfer
 relationship;
 (e) illuminating the amplified first and second selected segments having
 the probes hybridized thereto with a selected wavelength of light to
 elicit fluorescence by the donor and acceptor;
 (f) measuring a fluorescence emission as a function of temperature to
 determine a first melting profile of said one of the probes melting from
 the amplified first selected segment and a second melting profile of said
 one of the probes melting from the amplified second selected segment; and
 (g) comparing the first melting profile to the second melting profile,
 wherein distinguishable melting profiles indicate heterozygosity in the
 sample genomic DNA.
 A method of detecting heterozygosity at a selected locus in the genome of
 an individual, wherein the genome comprises a mutant allele and a
 corresponding reference allele, each comprising the selected locus,
 comprises
 (a) obtaining sample genomic DNA from the individual;
 (b) providing a pair of oligonucleotide primers configured for amplifying,
 by polymerase chain reaction, a first selected segment of the mutant
 allele and a second selected segment of the corresponding reference allele
 wherein both the first and second selected segments comprise the selected
 locus;
 (c) providing an oligonucleotide probe, wherein one of the primers and the
 probe are each labeled with one member of a fluorescence energy transfer
 pair comprising an donor fluorophore and an acceptor fluorophore, and
 wherein the labeled probe and labeled primer hybridize to the amplified
 first and second selected segments such that one of the probes spans the
 selected locus and exhibits a first melting profile with the amplified
 first selected segment that is distinguishable from a second melting
 profile with the amplified second selected segment;
 (d) amplifying the first and second selected segments of sample genomic DNA
 by polymerase chain reaction in the presence of effective amounts of
 primers and probe to result in amplified first and second selected
 segments, at least a portion thereof having both the labeled primer and
 probe hybridized thereto with the fluorogenic resonance energy transfer
 pair in resonance energy transfer relationship;
 (e) illuminating the amplified first and second selected segments having
 the labeled primer and probe hybridized thereto with a selected wavelength
 of light to elicit fluorescence by the donor and acceptor;
 (f) measuring a fluorescence emission as a function of temperature to
 determine a first melting profile of said probe melting from the amplified
 first selected segment and a second melting profile of said probe melting
 from the amplified second selected segment; and
 (g) comparing the first melting profile to the second melting profile,
 wherein distinguishable melting profiles indicate heterozygosity in the
 sample genomic DNA.
 A method of determining completion of a polymerase chain reaction in a
 polymerase chain reaction mixture comprising (1) a nucleic acid wherein
 the nucleic acid or a polymerase-chain-reaction-amplified product thereof
 consists of two distinct complementary strands, (2) two oligonucleotide
 primers configured for amplifying by polymerase chain reaction a selected
 segment of the nucleic acid to result in an amplified product, and (3) a
 DNA polymerase for catalyzing the polymerase chain reaction, comprises
 (a) adding to the mixture (1) an effective amount of an oligonucleotide
 probe labeled with a resonance energy transfer donor or a resonance energy
 transfer acceptor of a fluorogenic resonance energy transfer pair, wherein
 the probe is configured for hybridizing to the amplified product under
 selected conditions of temperature and monovalent ionic strength, and (2)
 an effective amount of a reference oligonucleotide labeled with the donor
 or the acceptor, with the proviso that as between the probe and reference
 oligonucleotide one is labeled with the donor and the other is labeled
 with the acceptor, wherein the reference oligonucleotide is configured for
 hybridizing to the amplified product under the selected conditions of
 temperature and monovalent ionic strength such that the donor and the
 acceptor are in resonance energy transfer relationship when both the probe
 and the reference oligonucleotide hybridize to the amplified product;
 (b) amplifying the selected segment of nucleic acid by polymerase chain
 reaction to result in the amplified product, at least a portion thereof
 having both the probe and the reference oligonucleotide hybridized thereto
 with the fluorogenic resonance energy transfer pair in resonance energy
 transfer relationship; and
 (c) illuminating the amplified product having the probe and reference
 oligonucleotide hybridized thereto with a selected wavelength of light for
 eliciting fluorescence by the fluorogenic resonance energy pair and
 monitoring fluorescence emission and determining a cycle when the
 fluorescence emission reaches a plateau phase, indicating the completion
 of the reaction.
 A method of determining completion of a polymerase chain reaction in a
 polymerase chain reaction mixture comprising (1) a nucleic acid wherein
 the nucleic acid or a polymerase-chain-reaction-amplified product thereof
 consists of two distinct complementary strands, (2) two oligonucleotide
 primers configured for amplifying by polymerase chain reaction a selected
 segment of the nucleic acid to result in an amplified product, and (3) a
 DNA polymerase for catalyzing the polymerase chain reaction, comprises
 (a) adding to the mixture an effective amount of a nucleic-acid-binding
 fluorescent dye;
 (b) amplifying the selected segment of nucleic acid by polymerase chain
 reaction in the mixture to which the nucleic-acid-binding fluorescent dye
 has been added to result in the amplified product with
 nucleic-acid-binding fluorescent dye bound thereto; and
 (c) illuminating amplified product with nucleic-acid-binding fluorescent
 dye bound thereto with a selected wavelength of light for eliciting
 fluorescence therefrom and monitoring fluorescence emission and
 determining a cycle when the fluorescence emission reaches a plateau
 phase, indicating the completion of the reaction. Preferably, the
 nucleic-acid-binding fluorescent dye is a member selected from the group
 consisting of SYBR.TM. GREEN I, ethidium bromide, pico green, acridine
 orange, thiazole orange, YO-PRO-1, and chromomycin A3, and more preferably
 is SYBR.TM. GREEN I.
 A method of controlling temperature cycling parameters of a polymerase
 chain reaction comprising repeated cycles of annealing, extension, and
 denaturation phases of a polymerase chain reaction mixture comprising a
 double-strand-specific fluorescent dye, wherein the parameters comprise
 duration of the annealing phase, duration of the denaturation phase, and
 number of cycles, comprises
 (a) illuminating the reaction with a selected wavelength of light for
 eliciting fluorescence from the fluorescent dye and continuously
 monitoring fluorescence during the repeated annealing, extension, and
 denaturation phases;
 (b) determining at least
 (i) duration for fluorescence to stop increasing during the extension
 phase, or
 (ii) Duration for fluorescence to decrease to a baseline level during the
 denaturation phase, or
 (iii) a number of cycles for fluorescence to reach a preselected level
 during the extension phase; and
 (c) adjusting the length of the extension phase according to the length of
 time for fluorescence to stop increasing during the extension phase, the
 length of the denaturation phase according to the length of time for
 fluorescence to decrease to the baseline level during the denaturation
 phase, or the number of cycles according to the number of cycles for
 fluorescence to reach the preselected level during the extension phase.
 A method of determining a concentration of an amplified product in a
 selected polymerase chain reaction mixture comprises
 (a) determining a second order rate constant for the amplified product at a
 selected temperature and reaction conditions by monitoring rate of
 hybridization of a known concentration of the amplified product;
 (b) determining rate of annealing for an unknown concentration of the
 amplified product; and
 (c) calculating the concentration of the amplified product from the rate of
 annealing and the second order rate constant. Preferably, the rate of
 annealing is determined after multiple cycles of amplification. One
 illustrative method of determining the second oder rate constant comprises
 the steps of
 raising the temperature of a first polymerase chain reaction mixture
 comprising a known concentration of the amplified product and an effective
 amount of a double-strand specific fluorescent dye, above the denaturation
 temperature of the amplified product to result in a denatured amplified
 product;
 rapidly reducing the temperature of the first polymerase chain reaction
 mixture comprising the known amount of denatured amplified product to a
 selected temperature below the denaturation temperature of the amplified
 product while continuously monitoring the fluorescence of the first
 polymerase chain reaction mixture as a function of time;
 plotting fluorescence as a function of time for determining maximum
 fluorescence, minimum fluorescence, the time at minimum fluorescence, and
 a second order rate constant for the known concentration of amplified
 product from the equation
 ##EQU1##
 wherein F is fluorescence, F.sub.max is maximum fluorescence, F.sub.min is
 minimum fluorescence, k is the second order rate constant, t.sub.0 is the
 time at F.sub.min, and [DNA] is the known concentration of the amplified
 product.
 A method of determining a concentration of a selected nucleic acid template
 by competitive quantitative polymerase chain reaction comprises the steps
 of:
 (a) in a reaction mixture comprising:
 (i) effective amounts of each of a pair of oligonucleotide primers
 configured for amplifying, in a polymerase chain reaction, a selected
 segment of the selected template and a corresponding selected segment of a
 competitive template to result in amplified products thereof,
 (ii) an effective amount of an oligonucleotide probe labeled with a
 resonance energy transfer donor or a resonance energy transfer acceptor of
 a fluorogenic resonance energy transfer pair, wherein the probe is
 configured for hybridizing to the amplified products such that the probe
 melts from the amplified product of the selected template at a melting
 temperature that is distinguishable from the melting temperature at which
 the probe melts from the amplified product of the competitive template,
 (iii) an effective amount of a reference oligonucleotide labeled with the
 donor or the acceptor, with the proviso that as between the probe and
 transfer oligonucleotide one is labeled with the donor and the other is
 labeled with the acceptor, wherein the reference oligonucleotide is
 configured for hybridizing to the amplified products such that the donor
 and the acceptor are in resonance energy transfer relationship when both
 the probe and the reference oligonucleotide hybridize to the amplified
 products;
 amplifying, by polymerase chain reaction, an unknown amount of the selected
 template and a known amount of the competitive template to result in the
 amplified products thereof;
 (b) illuminating the reaction mixture with a selected wavelength of light
 for eliciting fluorescence by the fluorogenic resonance energy transfer
 pair and determining a fluorescence emission as a function of temperature
 as the temperature of the reaction mixture is changed to result in a first
 melting curve of the probe melting from the amplified product of the
 selected template and a second melting curve of the probe melting from the
 competitive template;
 (c) converting the first and second melting curves to first and second
 melting melting peaks and determining relative amounts of the selected
 template and the competitive template from such melting peaks; and
 (d) calculating the concentration of the selected template based on the
 known amount of the competitive template and the relative amounts of
 selected template and competitive template.
 A fluorogenic resonance energy transfer pair consists of fluorescein and
 Cy5 or Cy5.5.
 A method of determining a concentration of a selected nucleic acid template
 in a polymerase chain reaction comprises the steps of:
 (a) in a reaction mixture comprising:
 (i) effective amounts of each of a first pair of oligonucleotide primers
 configured for amplifying, in a polymerase chain reaction, a selected
 first segment of the selected template to result in an amplified first
 product thereof,
 (ii) effective amounts of each of a second pair of oligonucleotide primers
 configured for amplifying, in a polymerase chain reaction, a selected
 second segment of a reference template to result in an amplified second
 product thereof,
 (iii) an effective amount of a nucleic-acid-binding fluorescent dye;
 amplifying, by polymerase chain reaction, an unknown amount of the selected
 template to result in the amplified first product and a known amount of
 the reference template to result in the amplified second product thereof;
 (b) illuminating the reaction mixture with a selected wavelength of light
 for eliciting fluorescence by the nucleic-acid-binding fluorescent dye and
 continuously monitoring the fluorescence emitted as a function of
 temperature to result in a melting curve of the amplified products wherein
 the first product and second product melt at different temperatures;
 (c) converting the melting curves to melting melting peaks and determining
 relative amounts of the selected template and the reference template from
 such melting peaks; and
 (d) calculating the concentration of the selected template based on the
 known amount of the reference template and the relative amounts of
 selected template and reference template.
 A method of monitoring amplification of a selected template in a polymerase
 chain reaction that also comprises a positive control template comprises
 the steps of:
 (a) in a reaction mixture comprising:
 (i) effective amounts of each of a first pair of oligonucleotide primers
 configured for amplifying, in a polymerase chain reaction, a selected
 first segment of the selected template to result in an amplified first
 product thereof,
 (ii) effective amounts of each of a second pair of oligonucleotide primers
 configured for amplifying, in a polymerase chain reaction, a selected
 second segment of the positive control template to result in an amplified
 second product thereof,
 (iii) an effective amount of a nucleic-acid-binding fluorescent dye;
 subjecting the selected template and the positive control template to
 conditions for amplifying the selected template and the positive control
 template by polymerase chain reaction; and
 (b) illuminating the reaction mixture with a selected wavelength of light
 for eliciting fluorescence by the nucleic-acid-binding fluorescent dye and
 continuously monitoring the fluorescence emitted as a function of
 temperature during an amplification cycle of the polymerase chain reaction
 to result in a first melting peak of the amplified first product, if the
 selected template is amplified, and a second melting peak of the amplified
 second product, if the positive control template is amplified;
 wherein obtaining of the second melting curve indicates that the polymerase
 chain reaction was operative, obtaining the first melting curve indicates
 that the selected first segment was amplifiable, and absence of the first
 melting curve indicates that the selected first segment was not
 amplifiable.
 A method of detecting the factor V Leiden mutation in an individual,
 wherein the factor V Leiden mutation consists of a single base change at
 the factor V Leiden mutation locus as compared to wild type, comprises the
 steps of:
 (a) obtaining sample genomic DNA from the individual;
 (b) providing wild type genomic DNA as a control;
 (c) providing a pair of oligonucleotide primers configured for amplifying
 by polymerase chain reaction a selected segment of the sample genomic DNA
 and of the wild type genomic DNA wherein the selected segment comprises
 the factor V Leiden mutation locus to result in amplified products
 containing a copy of the factor V Leiden mutation locus;
 (d) providing an oligonucleotide probe labeled with a resonance energy
 transfer donor or a resonance energy transfer acceptor of a fluorogenic
 resonance energy transfer pair, wherein the probe is configured for
 hybridizing to the amplified products such that the probe spans the
 mutation locus and exhibits a melting profile when the factor V Leiden
 mutation is present in the sample genomic DNA that is differentiable from
 a melting profile of the wild type genomic DNA;
 (e) providing a transfer oligonucleotide labeled with the resonance energy
 transfer donor or the resonance energy transfer acceptor, with the proviso
 that as between the probe and transfer oligonucleotide one is labeled with
 the resonance energy transfer donor and the other is labeled with the
 resonance energy transfer acceptor, wherein the transfer oligonucleotide
 is configured for hybridizing to the amplified products such that the
 resonance energy transfer donor and the resonance energy transfer acceptor
 are in resonance energy transfer relationship when both the probe and the
 transfer oligonucleotide hybridize to the amplified products;
 (f) amplifying the selected segment of sample genomic DNA and wild type
 genomic DNA by polymerase chain reaction in the presence of effective
 amounts of oligonucleotide probe and transfer oligonucleotide to result in
 amplified selected segments, at least a portion thereof having both the
 probe and the transfer oligonucleotide hybridized thereto with the
 fluorogenic resonance energy transfer pair in resonance energy transfer
 relationship;
 (g) determining fluorescence as a function of temperature during an
 amplification cycle of the polymerase chain reaction to result in a
 melting profile of the probe melting from the amplified segment of sample
 genomic DNA and a melting profile of the probe melting from the amplified
 segment of wild type genomic DNA; and
 (h) comparing the melting profile for the sample genomic DNA to the melting
 profile for the wild type genomic DNA, wherein a difference therein
 indicates the presence of the factor V Leiden mutation in the sample
 genomic DNA.
 A method of analyzing nucleic acid hybridization comprises the steps of
 (a) providing a mixture comprising a nucleic acid sample to be analyzed and
 a nucleic acid binding fluorescent entity; and
 (b) monitoring fluorescence while changing temperature at a rate of
 .gtoreq.0.1.degree. C./second.
 A method of quantitating an initial copy number of a sample containing an
 unknown amount of nucleic acid comprises the steps of
 (a) amplifying by polymerase chain reaction at least one standard of known
 concentration in a mixture comprising the standard and a nucleic acid
 binding fluorescent entity;
 (b) measuring fluorescence as a function of cycle number to result in a set
 of data points;
 (c) fitting the data points to a given predetermined equation describing
 fluorescence as a function of initial nucleic acid concentration and cycle
 number;
 (d) amplifying the sample containing the unknown amount of nucleic acid in
 a mixture comprising the sample and the nucleic acid binding fluorescent
 entity and monitoring fluorescence thereof; and
 (e) determining initial nucleic acid concentration from the equation
 determined in step (c).
 A fluorescence resonance energy transfer pair is disclosed wherein the pair
 comprises a donor fluorophore having an emission spectrum and an acceptor
 fluorophore having an absorption spectrum and an extinction coefficient
 greater than 100,000 M.sup.-1 cm.sup.-1, wherein the donor fluorophore's
 emission spectrum and the acceptor fluorophore's absorption spectrum
 overlap less than 25%. One illustrative fluorescence resonance energy
 transfer pair described is where the donor fluorophore is fluorescein and
 the acceptor fluorophore is Cy5 or Cy5.5.
 A method for analyzing a target DNA sequence of a biological sample
 comprises
 amplifying the target sequence by polymerase chain reaction in the presence
 of a nucleic acid binding fluorescent entity, said polymerase chain
 reaction comprising the steps of adding a thermostable polymerase and
 primers for the targeted nucleic acid sequence to the biological sample
 and thermally cycling the biological sample between at least a
 denaturation temperature and an elongation temperature;
 exciting the sample with light at a wavelength absorbed by the nucleic acid
 binding fluorescent entity; and
 monitoring the temperature dependent fluorescence from the nucleic acid
 binding fluorescent entity as temperature of the sample is changed.
 Preferably, the nucleic acid binding fluorescent entity comprises a double
 stranded nucleic acid binding fluorescent dye, such as SYBR.TM. Green I.
 The temperature dependent fluorescence can be used to identify the
 amplified products, preferably by melting curve analysis. Relative amounts
 fo two or more amplified products can be determined by analysis of melting
 curves. For example, areas under the melting curves are found by
 non-linear least squares regression of the sum of multiple gaussians.

DETAILED DESCRIPTION
 Before the present methods for monitoring hybridization during PCR are
 disclosed and described, it is to be understood that this invention is not
 limited to the particular configurations, process steps, and materials
 disclosed herein as such configurations, process steps, and materials may
 vary somewhat. It is also to be understood that the terminology employed
 herein is used for the purpose of describing particular embodiments only
 and is not intended to be limiting since the scope of the present
 invention will be limited only by the appended claims and equivalents
 thereof.
 It must be noted that, as used in this specification and the appended
 claims, the singular forms "a," "an," and "the" include plural referents
 unless the context clearly dictates otherwise.
 In describing and claiming the present invention, the following terminology
 will be used in accordance with the definitions set out below.
 As used herein, "nucleic acid," "DNA," and similar terms also include
 nucleic acid analogs, i.e. analogs having other than a phosphodiester
 backbone. For example, the so-called "peptide nucleic acids," which are
 known in the art and have peptide bonds instead of phosphodiester bonds in
 the backbone, are considered within the scope of the present invention.
 As used herein, "continuous monitoring" and similar terms refer to
 monitoring multiple times during a cycle of PCR, preferably during
 temperature transitions, and more preferably obtaining at least one data
 point in each temperature transition.
 As used herein, "cycle-by-cycle" monitoring means monitoring the PCR
 reaction once each cycle, preferably during the annealing phase of PCR.
 As used herein, "fluorescence resonance energy transfer relationship" and
 similar terms refer to adjacent hybridization of an oligonucleotide
 labeled with a donor fluorophore and another oligonucleotide labeled with
 an acceptor fluorophore to a target nucleic acid such that the donor
 fluorophore can transfer resonance energy to the acceptor fluorophore such
 that the acceptor fluorophore produces a measurable fluorescence emission.
 If the donor fluorophore and acceptor fluorophore are spaced apart by too
 great a distance, then the donor fluorophore cannot transfer resonance
 energy to the acceptor fluorophore such that the acceptor fluorophore
 emits measurable fluorescence, and hence the donor fluorophore and
 acceptor fluorophore are not in resonance energy transfer relationship.
 Preferably, when the two labeled oligonucleotides are both probes and
 neither functions as a PCR primer ("probe-probe"), then the donor and
 acceptor fluorophores are within about 0-25 nucleotides, more preferably
 within about 0-5 nucleotides, and most preferably within about 0-2
 nucleotides. A particularly preferred spacing is 1 nucleotide. When one of
 the labeled oligonucleotides also functions as a PCR primer
 ("probe-primer"), then the donor and acceptor fluorophores are preferably
 within about 0-15 nucleotides and more preferably within about 4-6
 nucleotides.
 As used herein, "effective amount" means an amount sufficient to produce a
 selected effect. For example, an effective amount of PCR primers is an
 amount sufficient to amplify a segment of nucleic acid by PCR provided
 that a DNA polymerase, buffer, template, and other conditions, including
 temperature conditions, known in the art to be necessary for practicing
 PCR are also provided.
 PCR requires repetitive template denaturation and primer annealing. These
 hybridization transitions are temperature-dependent. The temperature
 cycles of PCR that drive amplification alternately denature accumulating
 product at a high temperature and anneal primers to the product at a lower
 temperature. The transition temperatures of product denaturation and
 primer annealing depend primarily on GC content and length. If a probe is
 designed to hybridize internally to the PCR product, the melting
 temperature of the probe also depends on GC content, length, and degree of
 complementarity to the target. Fluorescence probes compatible with PCR can
 monitor hybridization during amplification.
 In accordance with the present invention, which is preferably used in
 connection with rapid cycling (fully described in the above-mentioned U.S.
 Ser. No. 08/658,993, filed Jun. 4, 1996, abandoned, entitled System And
 Method For Monitoring PCR Processes, and U.S. Ser. No. 08/537,612, filed
 Oct. 2, 1995, entitled Method For Rapid Thermal Cycling of Biological
 Samples), a kinetic paradigm for PCR is appropriate. Instead of thinking
 about PCR as three reactions (denaturation, annealing, extension)
 occurring at three different temperatures for three time periods (FIG.
 1A), a kinetic paradigm for PCR is more useful (FIG. 1B). With a kinetic
 paradigm, the temperature vs. time curve consists of continuous
 transitions between overlapping reactions. Denaturation and annealing are
 so rapid that no holding time at a particular temperature is necessary.
 Extension occurs over a range of temperatures at varying rates. A complete
 analysis would require knowledge of all relevant rate constants over all
 temperatures. If rate constants of all reactions were known, a
 "physicochemical description of PCR" could be developed. Determining these
 rates would require precise sample temperature control and is greatly
 simplified by reaction monitoring during temperature cycling.
 FIG. 2 illustrates useful temperature v. time segments for fluorescence
 hybridization monitoring. Product melting curves are obtained during a
 slow temperature increase to denaturation. By quickly lowering the
 temperature after denaturation to a constant temperature, product, probe,
 or primer annealing can optionally be followed. Probe melting curves are
 obtained by slowly heating through temperatures around the probe Tm. The
 embodiment represented in FIG. 2 provides all analysis during temperature
 cycling with immediate real time display. Fluorescent probes are included
 as part of the amplification solution for continuous monitoring of primer,
 probe, or product hybridization during temperature cycling.
 The fluorescence hybridization techniques contained herein are based on
 rapid cycling, with its advantages in speed and specificity.
 A sample temperature profile during rapid cycle PCR is shown in FIG. 3.
 Denaturation and annealing appear as temperature "spikes" on these
 figures, as opposed to the broad plateaus of conventional temperature
 cycling for PCR, e.g. FIG. 1A. Rapid temperature cycling is contrasted to
 conventional temperature cycling in FIG. 4, wherein it is shown that 30
 cycles of amplification can be completed in 15 minutes and the resulting
 PCR products contain many fewer side products. Thus, with rapid cycling
 the required times for amplification are reduced approximately 10-fold,
 and specificity is improved.
 EXAMPLE 1
 FIG. 4 shows the results of four different temperature/time profiles (A-D)
 and their resultant amplification products after thirty cycles (A-D). The
 profiles A and B in FIG. 4 were obtained using a prior art heating block
 device using a prior art microfuge tube. As can be seen in FIG. 4, the
 transitions between temperatures are slow and many nonspecific bands are
 present in profiles A and B. Profile B shows improvement in eliminating
 some of the nonspecific bands (in contrast to profile A) by limiting the
 time each sample remains at each temperature, thus indicating that shorter
 times produce more desirable results.
 Profiles C and D were obtained using a rapid temperature cycler. As can be
 seen in FIG. 4, amplification is specific and, even though yield is
 maximal with 60-second elongation times (C), it is still entirely adequate
 with 10-second elongation times (D).
 The optimal times and temperatures for the amplification of a 536 bp
 fragment of beta-globin from human genomic DNA were also determined.
 Amplification yields and product specificity were optimal when
 denaturation (93.degree. C.) and annealing (55.degree. C.) were less than
 1 second. No advantage was found to longer denaturation or annealing
 times. The yield increased with longer elongation times at 77.degree. C.,
 but there was little change with elongation times longer than 10-20
 seconds. These unexpected results indicate that the previously available
 devices used for DNA amplification are not maximizing the conditions
 needed to optimize the physical and enzymatic requirements of the
 reaction.
 Further information can be obtained from: C. T. Wittwer et al., Rapid Cycle
 Allele-Specific Amplification with Cystic Fibrosis delta F(508) Locus, 39
 Clinical Chemistry 804 (1993) and C. T. Wittwer et al., Rapid DNA
 Amplification, THE POLYMERASE CHAIN REACTION 174 (1994), which are both
 now incorporated herein by this reference. The instrumentation used for
 fluorescence acquisition and rapid temperature cycling is fully disclosed
 in Ser. No. 08/537,612, supra.
 As indicated earlier, the polymerase chain reaction can be performed
 rapidly. In addition to facilitating rapid heat transfer, the use of
 optically clear capillary tubes allows for continuous fluorescence
 monitoring of DNA amplification in accordance with the present invention.
 Fluorescent probes can be used to detect and monitor DNA amplification.
 Useful probes include double-stranded-DNA-specific dyes and
 sequence-specific probes. Three different fluorescence techniques for
 following DNA amplification are compared in FIG. 5. In FIG. 5A,
 fluorescence depends on the hybridization of PCR product as detected with
 a double-strand-specific DNA dye. In FIG. 5B, fluorescence depends on the
 hydrolysis of a 5'-exonuclease quenching probe, which is well known in the
 art as discussed above. FIG. 5C diagrams a hybridization scheme based on
 resonance energy transfer between fluorophores on two adjacent probes. The
 method of FIG. 5A is not sequence specific, although product specificity
 can be determined by melting curves, one aspect of the current invention.
 Both FIG. 5B and 5C are sequence specific. However, the hybridization
 method also allows analysis with melting curves, another aspect of the
 current invention.
 In monitoring fluorescence from reactions involving hydrolysis probes as in
 FIG. 5B and from reactions involving hybridization probes as in FIG. 5C,
 it is advantageous to measure fluorescence emitted by both the donor
 fluorophore and the acceptor fluorophore. In practice, the majority of the
 fluorescence emitted by hydrolysis probes is from the donor fluorophore,
 and the majority of the fluorescence emitted by hybridization probes is
 from the acceptor fluorophore.
 Double-strand-specific DNA dye selection. Those skilled in the art will be
 familiar with the use of ethidium bromide in fluorescence techniques. When
 a double strand-specific fluorescent dye is present during amplification,
 fluorescence generally increases as more double stranded product is made,
 see R. Higuchi et al., Simultaneous amplification and detection of
 specific DNA sequences, 10 Bio/Technology 413-417 (1992). A fluorescence
 PCR assay for hepatitis C RNA using the intercalater, YO-PRO-1 is also
 known in the art. See T. Ishiguro et al., Homogeneous quantitative assay
 of hepatitis C virus RNA by polymerase chain reaction in the presence of a
 fluorescent intercalater, 229 Anal. Biochem. 207-213 (1995). It is
 preferred that SYBR.TM. Green I, which is well known in the art and
 available from Molecular Probes of Eugene, Oregon, be used as a
 double-strand-specific dye. The molecular structure of this dye is a trade
 secret, but it is recommended by the manufacturer as a more sensitive
 double-strand-specific dye for DNA detection on gels. SYBR.TM. Green I is
 heat labile, however, and thus is not useful for fluorescence monitoring
 of PCR according to conventional methods where the temperature of the
 reaction mixture is maintained at melting temperatures for extended
 periods of time. Because of this heat lability, it was unexpected to
 discover that SYBR.TM. Green I can be used to monitor PCR reactions when
 melting temperatures are not maintained for extended periods, i.e. when
 PCR is carried out by rapid cycling according to the kinetic paradigm
 described above.
 EXAMPLE2
 Different double-strand-specific DNA dyes were compared by monitoring the
 amplification of a 110 base pair fragment from the PCO3/PCO4 primer pair
 of the human beta-globin gene from 10,000 template copies. Primers were
 synthesized by standard phosphoramidite chemistry as known in the art,
 namely, using Pharmacia Biotech Gene Assembler Plus (Piscataway, N.J.).
 The human beta-globin primers PCO3/PCO4 (110 base pairs) are described in
 C. T. Wittwer et al., Automated polymerase chain reaction in capillary
 tubes with hot air, 17 Nucl. Acids. Res. 4353-4357 (1989), which is now
 incorporated herein by reference. DNA amplification was performed in 50 mM
 Tris, pH 8.5 (25.degree. C.), 3 mM MgCl.sub.2, 500 .mu.g/ml bovine serum
 albumin, 0.5 .mu.M of each primer, 0.2 mM of each deoxynucleoside
 triphosphate and 0.2 U of Taq polymerase per 5 .mu.l sample unless
 otherwise stated in the following examples. Purified amplification product
 was used as DNA template and was obtained by phenol/chloroform extraction
 and ethanol precipitation, see D. M. Wallace, Large- and small-scale
 phenol extractions and precipitation of nucleic acids, 152 Methods in
 Enzymology 33-48 (1987), followed by removal of primers by repeated
 washing through a Centricon 30 microconcentrator (Amicon, Danvers, Mass.).
 Template concentrations were determined by absorbance at 260 nm.
 A(260):A(280) ratios of templates were greater than 1.7.
 SYBR.TM. Green I (Molecular Probes, Eugene, Oreg.) was used at a 1:10,000
 dilution, ethidium bromide was at 5 .mu.g/ml, and acridine orange was at 3
 .mu.g/ml. These concentrations were determined to be optimal
 concentrations for maximizing the fluorescence signal observed during
 amplification for each dye. Excitation was through a 450-490 nm
 interference filter from a xenon arc source, except for ethidium bromide,
 where a 520-550 nm excitation was used. For SYBR.TM. Green I, the emmision
 at 520-550 was monitored. Ethidium bromide fluorescence was observed
 through a 580-620 nm bandpass. The acridine orange signal was taken as the
 ratio of green (520-550 nm) to red (&gt;610 nm) fluorescence. The
 fluorescence of the sample before amplification was compared to the
 fluorescence after 35 cycles (94.degree. C. max, 60.degree. C. for 20 sec)
 at 60.degree. C. The fluorescence increase was 5.3-fold for SYBR.TM. Green
 I, 1.7-fold for ethidium bromide, and 1.2-fold for acridine orange. In
 separate experiments, the fluorescence from SYBR.TM. Green I was stable
 for greater than 30 min at 70.degree. C. It is also conveniently excited
 with visable light and is claimed to be less of a mutagen than ethidium
 bromide. Background fluorescence in all cases arose primarily from the
 primers.
 SYBR.TM. Green I is a preferred double-strand-specific dye for fluorescence
 monitoring of PCR, primarily because of superior sensitivity, arising from
 greater discrimination between double stranded and single stranded nucleic
 acid. SYBR.TM. Green I can be used in any amplification and is
 inexpensive. In addition, product specificity can be obtained by analysis
 of melting curves, as will be described momentarily.
 Resonance energy transfer dye selection for hybridization probes.
 Fluorescence resonance energy transfer can occur between 2 fluorophores if
 they are in physical proximity and the emission spectrum of one
 fluorophore overlaps the excitation spectrum of the other. Introductory
 theory on fluorescence resonance energy transfer can be found in many
 recent review articles. The rate of resonance energy transfer is:
 (8.785E-5)(t.sup.-1)(k.sup.2)(n.sup.-4)(q.sub.D)(R.sup.-6)(J.sub.DA),
 where:
 t=excited state lifetime of the donor in the absence of the acceptor;
 k.sup.2 =is an orientation factor between the donor and acceptor;
 n=refractive index of visible light in the intervening medium;
 q.sub.D =quantum efficiency of the donor in the absence of the acceptor;
 R=distance between the donor and acceptor (in angstroms);
 J.sub.DA =the integral of (F.sub.D) (e.sub.A) (W.sup.4) with respect to W
 at all overlapping wavelengths with:
 F.sub.D =peak normalized fluorescence spectrum of the donor,
 e.sub.A =molar absorption coefficient of the acceptor (M.sup.-1 cm.sup.-1),
 and
 W=wavelength (nm).
 For any given donor and acceptor, a distance where 50% resonance energy
 transfer occurs can be calculated and is abbreviated R.sub.0. Because the
 rate of resonance energy transfer depends on the 6th power of the distance
 between donor and acceptor, resonance energy transfer changes rapidly as R
 varies from R.sub.0. At 2R.sub.0, very little resonance energy transfer
 occurs, and at 0.5R.sub.0, the efficiency of transfer is nearly complete,
 unless other forms of de-excitation predominate (i.e., collisional
 quenching). R.sub.0 values for many different donor and acceptor pairs
 have been compiled and vary between 22 and 72 angstroms.
 In double helical DNA, 10 bases are separated by about 34 angstroms. By
 labeling the bases of DNA with donor and acceptor fluorophores, resonance
 energy transfer has be used as a spectroscopic ruler to observe the
 helical geometry of DNA and analyze the structure of a four-way DNA
 junction. Resonance energy transfer can also be used as a monitor of
 hybridization. If a labeled oligonucleotide is hybridized to a labeled
 template strand, R can be brought from much greater than R.sub.0 to well
 below R.sub.0, increasing resonance energy transfer dramatically.
 Alternately, 2 labeled probes can be hybridized to the same template
 strand for a similar change in fluorescence energy transfer.
 The practical use of resonance energy transfer to monitor hybridization
 depends on the sensitivity required and how much time is available. Using
 a competitive hybridization technique with 1 nM labeled probes,
 PCR-amplified DNA was detected after 15 min at 40.degree. C. Faster signal
 generation is desirable. If only seconds were required for hybridization,
 PCR products could conveniently be quantified each cycle of amplification.
 Even further, the extent of probe hybridization could be monitored within
 a temperature cycle.
 Hybridization is a second order process (see B. Young & M. Anderson,
 Quantitative analysis of solution hybridization, In: Nucleic Acid
 Hybridization: A Practical Approach 47-71, (B. Hames, S. Higgins eds.,
 1985). When the concentration of the probe is much greater than the
 concentration of the target, the hybridization rate is inversely
 proportional to concentration of probe. For example, by doubling the probe
 concentration, the hybridization time is cut in half. High probe
 concentrations would be necessary for cycle-by-cycle monitoring during
 PCR, because hybridization must occur before the hybridization site is
 covered by polymerase extension.
 The high probe concentrations required for hybridization monitoring during
 PCR require a resonance energy transfer pair with unique characteristics.
 Consider excitation of a donor (D) and an acceptor (A) pair with light.
 The number of fluorophores of D and A directly excited will be
 proportional to the extinction coefficient (e) of each fluorophore at the
 excitation wavelength, or:
EQU Number of D molecules directly excited=(K)(e.sub.D)
EQU Number of A molecules directly excited=(K)(e.sub.A)
 where K is a proportionality constant. De-excitation of the donor will
 occur by fluorescence, resonance energy transfer, and other processes
 summarized as thermal quenching. If p.sub.F =probability of resonance
 energy transfer, and P.sub.TD =probability of donor thermal quenching,
 then the probability of donor fluorescence is:
EQU 1-p.sub.F -p.sub.TD
 and the number of fluorescing donor molecules is:
 (K)(e.sub.D)(1-p.sub.F -p.sub.TD)
 If the probability of detecting a donor emission in the donor emission
 window (for example, a bandpass filter window) is p.sub.DD, then the
 number of observed donor emissions is:
EQU (p.sub.DD)(K)(e.sub.D)(1-p.sub.F -p.sub.TD)
 Now, the number of excited acceptor fluorophores is the sum of those
 directly excited and those excited through resonance energy transfer:
EQU (K)(e.sub.A)+(K)(e.sub.D)(p.sub.F)
 If p.sub.TA =the probability of thermal quenching of the acceptor, then the
 probability of acceptor fluorescence is:
EQU 1-p.sub.TA
 and the number of fluorescing acceptor molecules is:
EQU [(K)(e.sub.A)+(K)(e.sub.D)(p.sub.F)][1-(p.sub.TA)]
 If the probability of detecting an acceptor emission in the acceptor
 emission window is p.sub.AA, then the number of observed acceptor
 emissions is:
 (p.sub.AA)[(K)(e.sub.A)+(K)(e.sub.D)(p.sub.F)][1-(p.sub.TA)]
 Finally, if the probability of observing a donor emission in the acceptor
 emission window is p.sub.DA, then the number of observed emissions (both D
 and A) in the acceptor emission window is:
EQU (p.sub.AA)[(K)(e.sub.A)+(K)(e.sub.D)(p.sub.F)][1-(p.sub.TA)]+(p.sub.
 DA)(K)(e.sub.D)(1-p.sub.F -.sub.TD)
 Since fluorescence measurements are relative and K is present in all terms,
 if we remove K and rearrange, the observed intensity at the donor window
 is proportional to (donor excitation)-(energy transfer):
EQU (e.sub.D)(p.sub.DD)(1p.sub.TD)-(e.sub.D)(p.sub.DD)(p.sub.F) 1)
 and the observed intensity at the acceptor window is proportional to
 (acceptor excitation)+(energy transfer)+( donor emission in the acceptor
 window):
EQU (e.sub.A)(p.sub.AA)(1-p.sub.TA)+(e.sub.D)(p.sub.DD)(p.sub.F)(1-p.sub.
 TA)+(e.sub.D)(p.sub.DA)(1-p.sub.TD -p.sub.F)
 As resonance energy transfer increases, the donor signal decreases and the
 acceptor signal increases. The percent signal change depends on the
 background light intensity in each window. With high concentrations of
 probes, this background light intensity is high. During PCR, when varying
 target (product) concentrations need to be monitored, it is not possible
 to match the donor concentration to the target concentration. The excess
 donor molecules contribute to the background light intensity in both the
 donor and acceptor windows and partially mask the energy transfer
 phenomena. Background light in the acceptor window comes from not only
 donor emission in the acceptor window, but also from direct excitation of
 the acceptor. This background can be minimized with a low e.sub.A and a
 low p.sub.DA.
 The fluorescein/rhodamine fluorescence energy transfer pair, commonly used
 for nucleic acid detection, has high background fluorescence. Both direct
 acceptor excitation (e.sub.A,, ca. 10% e.sub.MAX) and emission of the
 donor at wavelengths used to detect acceptor emission (p.sub.DA, ca. 20%
 peak emission) are high. This pair can be used to monitor hybridization if
 the probe concentration is near to the target concentration and enough
 time is allowed for complete hybridization. It is not a useful pair of
 fluorophores for continuous monitoring of PCR because high probe
 concentrations are required and the template concentration in PCR is
 continually changing.
 Monitoring product concentration during PCR by hybridization has not been
 possible in the past because an acceptable resonance energy transfer pair
 had not been found. There have been few attempts to use resonance energy
 transfer for direct "noncompetitive" detection of hybridization. For
 example, U.S. Pat. No. 5,565,322 states "the observed energy transfer
 efficiency in terms of re-emission by the acceptor was relatively low." At
 probe concentrations that are high enough for significant hybridization to
 occur in seconds, the background fluorescence is too high.
 Fluorescein is perhaps the most widely used fluorophore. Its extinction
 coefficient and quantum efficiency are high and it is extensively used in
 microscopy, immunoassays, and flow cytometry. It is the donor in a
 commonly used resonance energy transfer pair with rhodamine. Cy5 is a
 popular red-emitting fluorophore with a very high extinction coefficient.
 The structure of the N-hydroxysuccinimide ester of Cy5 is shown in FIG. 6,
 and the structure of the related dye, Cy5.5, is shown in FIG. 7. These
 dyes are indodicarbocyanine dyes that are used commonly in flow cytometry
 and automated fluorescence sequencers and are available from Amersham
 (Pittsburg, Pa.). Both fluorescein and Cy5 are commercially available as
 amidites for direct, automated incorporation into oligonucleotides.
 However, Cy5 has never been reported as a resonance energy transfer pair
 with fluorescein. Intuitively, fluorescein emission and Cy5 absorption do
 not overlap enough for resonance energy transfer to be considered. The
 emission spectrum of fluorescein and absorption spectrum of Cy5 attached
 to oligonucleotides are shown in FIG. 8. When the areas under the curves
 are normalized, the overlap from the technical spectra is 19%. Cy5.5
 excitation is shifted to the red by about 25 nm, further decreasing the
 overlap with fluorescein emission to about 15%. Working in the
 red/infrared region of the spectrum is advantageous when choosing optical
 components for instrumentation. Laser diodes can be used for illumination,
 photodiode detectors have excellent sensitivity, and most materials have
 minimal autofluorescence in the pertinent spectral region.
 Despite low spectral overlap, it has been discovered that fluorescein and
 either Cy5 or Cy5.5 make an excellent resonance energy transfer pair for
 hybridization monitoring during PCR.
 EXAMPLE 3
 A 110 bp beta-globin fragment was amplified from 50 ng human genomic DNA
 according to the procedure of Example 2 with the internal probes
 CAAACAGACA CCATGGTGCA CCTGACTCCT GAGGA-fluorescein (SEQ ID NO:3) and
 Cy5-GAAGTCTGCC GTTACTGCCC TGTGGGGCAA G-p (SEQ ID NO:18) at 0.2 .mu.M each
 and 0.8 U KlenTaql polymerase (a 5'-exonuclease deficient variant of Taq
 polymerase-U.S. Pat. No. 5,436,149) in a 10 .mu.l reaction. The probes
 hybridized internal to the primers on the same strand and were immediately
 adjacent without any intervening bases.
 Probes and primers were synthesized by standard phosphoramidite chemistry
 as known in the art, using a Pharmacia Biotech Gene Assembler Plus
 (Piscataway, N.J.). The 3'-fluorescein-labeled probe was synthesized on a
 fluorescein-labeled CPG cassette (Glen Research, Sterling, Va.) with the
 final trityl-ON to assist with C18 reverse phase HPLC purification. The
 late eluting peak was collected and the trityl group was removed on a
 PolyPack (Glen Research). The fluorescein-labeled oligo was eluted with
 50% acetonitrile and again purified by C18 reverse phase HPLC. The
 5'-Cy5-labeled probe was synthesized with a chemical phosphorylation agent
 on the 3'-end (Glen Research) and adding a Cy5 amidite (Pharmacia) to the
 5'-end during trityl-OFF synthesis. Failure sequences were removed by C18
 reverse phase HPLC. Probe purity was checked with polyacrylamide
 electrophoresis and the absorbance of the dye and the oligo.
 HPLC was perfomed on a 4.times.250 mm Hypersil ODS column (Hewlett Packard)
 with a 0.1 M triethanolamine:acetate mobile phase and an acetonitrile
 gradient at 1 ml/min. The eluate was monitored for both absorbance
 (A.sub.260) and fluorescence (490 nm excitation, 520 nm emission for
 fluorescein and 650 nm excitation, 670 nm emission for Cy5). Tritylated-
 and fluorescein-labeled oligonucleotides were eluted with a 10-20%
 acetonitrile gradient, and Cy5-labeled oligonucleotides eluted over a
 10-40% acetonitrile gradient.
 Temperature cycling was 94.degree. C. for 0 sec with a programmed approach
 rate of 20.degree. C./sec, 60.degree. C. for 20 sec with an approach rate
 of 20.degree. C./sec, and 75.degree. C. for 0 sec with an approach rate of
 1.degree. C./sec in a capillary fluorescence rapid temperature cycler.
 During temperature cycling, fluorescein and Cy5 fluorescence were acquired
 each cycle at the end of the annealing/extension segment. Resonance energy
 transfer was observed as both a decrease in fluorescein fluorescence, and
 an increase in Cy5 fluorescence beginning around cycle 26 of amplification
 (FIG. 9). In general, observing the fluorescence ratio of Cy5 to
 fluorescein fluorescence is perferred.
 The unexpectedly good results with the fluorescein/Cy5 pair can at least
 partly be rationalized. The overlap integral, J.sub.DA depends not only on
 spectral overlap, but also on the extinction coefficient of the acceptor
 (Cy5 has an extinction coefficient of 250,000 M.sup.-1 cm.sup.-1 at 650
 nm), and on the 4th power of the wavelength. Both of these factors will
 favor a high J.sub.DA for Cy5, even given low spectral overlap. Recently,
 phycoerythrin and Cy7 were shown to be an effective tandem probe for
 immunofluorescence, despite low spectral overlap. In a later example, the
 utility of fluorescein and Cy5.5 as labels on hybridization probes is
 demonstrated. Fluorescence resonance energy transfer can be used to
 monitor nucleic acid hybridization even when the interacting dyes have low
 spectral overlap. The use of fluorescein with Cy5, Cy5.5 and other red or
 infrared emitting dyes as resonance energy transfer pairs for monitoring
 hybridization has not been previously recognized. Fluorescein has a long
 emission "tail" that goes out to 600 nm, 700 nm and beyond that can be
 used to excite these far red and infrared dyes. The rate of energy
 transfer is dependent on the overlap integral, but is also effected by the
 6th power of the distance between the fluorophores. If the probes are
 designed so that the resonance energy transfer dyes are in close
 proximity, the transfer rate is high. At least with fluorescein/Cy5,
 fluorescein/Cy5.5 and like pairs, resonance energy transfer appears to
 predominate over collisional quenching and other forms of energy loss when
 the fluorophores are close together, as in the above example where the
 fluorophores are attached to adjacent probes with no intervening bases.
 The potential usefulness of a resonance energy transfer pair for
 hybridization probes can be judged by observing the change in the ratio of
 light intensity in the donor and acceptor windows at minimal and maximal
 resonance energy transfer. One way to obtain minimal and maximal transfer
 is to attach both fluorophores to the same oligonucleotide and measure
 fluorescence ratio before and after digestion with phospodiesterase.
 EXAMPLE 4
 The dual-labeled fluorescein/Cy5 probe Cy5-CTGCCG-F-TACT GCCCTGTGGG GCAAGGp
 (SEQ ID NO:19) was synthesized by standard phosphoramidite chemistry,
 where p is a terminal 3'-phosphate (chemical phosphorylation reagent, Glen
 Research), F is a fluorescein residue introduced as an amidite with a
 2-aminobutyl-1,3-propanediol backbone to maintain the natural 3-carbon
 internucleotide phosphodiester distance (ClonTech, Palo Alto, Calif.), and
 Cy5 is added as the amidite (Pharmacia). The ratio of Cy5 to fluorescein
 fluorescence in 0.1 M Tris, pH 8.0 was obtained before and after exhastive
 hydrolysis with phosphodiesterase (Sigma, St. Louis, Mo.). The change in
 the fluorescence ratio was 220-fold after hydrolysis. A dual-labeled
 fluorescein/rhodamine probe F-ATGCCCT*CCC CCATGCCATC CTGCGTp (SEQ ID
 NO:20) was purchased from Perkin Elmer (Foster City, Calif.), where F is
 fluorescein and * is a rhodamine attached to a modified T residue by an
 amino-linker arm. The change in the fluorescence ratio (rhodamine to
 fluorescein) was 22-fold after hydrolysis with phosphodiesterase.
 The potential signal from the fluorescein/Cy5 pair was 10-fold that of the
 fluorescein/rhodamine pair.
 EXAMPLE 5
 The effect of the ratio, concentration, and spacing of fluorescein and
 Cy5-labeled adjacent hybridization probes during PCR was studied.
 Amplification of the beta globin locus and probe pair of Example 3 was
 used and the maximum change in the fluorescence ratio of Cy5 to
 fluorescein was observed. The maximal signal occurred when the ratio of
 Cy5 to fluorescein-labeled probes was 2:1 (FIG. 10). At this 2:1 ratio,
 the best signal occurred at a fluorescein probe concentration of 0.2 .mu.M
 and a Cy5-labeled probe concentration of 0.4 .mu.M (FIG. 11). The optimal
 number of intervening bases between adjacent hybridization probes during
 PCR was also determined. Several probes of the same length but slightly
 shifted in their hybridization position were synthesized according to
 Example 3 so that when they hybridized to the beta globin target, 0, 1, 2,
 3, 4, or 6 bases remained between the probes. The highest signal during
 PCR occurred with one intervening base (FIG. 12). Although some resonance
 energy transfer was detected at a spacing of 15 and even 25 bases, much
 better transfer occurred at 0-5 bases.
 Heller et al. (U.S. Pat. No. 4,996,143), found that energy transfer
 efficiency decreased as the number of nucleotides between fluorophores
 decreased from 4 to 0 units. In contrast, the best energy transfer with
 the fluorescein/Cy5 pair was seen at 0 to 2 intervening nucleotides.
 Hybridization probe method. If 2 probes are synthesized that hybridize
 adjacently on a target and each is labeled with one fluorophore of a
 resonance energy transfer pair, the resonance energy transfer increases
 when hybridization occurs (FIG. 5C). The fluorescein/rhodamine pair is
 most commonly used for nucleic acid detection.
 One aspect of this invention is to provide a sequence-specific homogeneous
 hybridization method for detection of PCR products. It is not obvious how
 to achieve this. Using hybridization probes during amplification is
 counterintuitive. It does not seem that both probe hybridization and
 polymerase extension can occur. To get sequence specific fluorescence, the
 probes must be hybridized, but the probes cannot be hybridized if the
 polymerase is to complete primer extension and exponentially amplify DNA.
 One solution to this problem is to use a dual-labeled single probe and
 utilize the 5'-exonuclease activity of common heat stable DNA polymerases
 to cleave the probe during extension, thereby separating the 2
 fluorophores. In this case, the fluorescence signal arises from separation
 of the resonance energy transfer pair upon probe hydrolysis (FIG. 5B),
 rather than approximation of the fluorophores by adjacent hybridization
 (FIG. 5C). However, dual-labeled probes are difficult to make, requiring
 manual addition of at least one fluorophore to the oligo and usually
 require extensive purification. The probes are expensive, and two
 dual-labeled probes are necessary for competitive quantification of a
 target or for mutation detection. A further concern is that the observed
 fluorescence depends on the cumulative amount of probe hydrolyzed, not
 directly on the amount of product present at any given cycle. This results
 in a continued increase in fluorescence even after the PCR plateau has
 been reached. Finally and most importantly, probe hydrolysis does not
 always occur during polymerase extension, an effect that is not well
 understood. For example, the dual-labeled fluorescein/Cy5 probe of Example
 4 showed very poor hydrolysis during PCR when it was flanked by primers.
 Indeed, several dual-labeled fluorescein/Cy5 probes, including those with
 terminal labels, were made and all showed poor hydrolysis and signal
 generation during amplification.
 Homogeneous detection of PCR products with adjacent hybridization probes
 would solve many of the problems of the 5'-exonuclease system. Synthesis
 of adjacent hybridization probes is relatively simple because amidites for
 both fluorescein and Cy5 are available for direct incorporation during
 automated synthesis and dual labeling of one probe is not required.
 Because their fluorescence results from hybridization, not hydrolysis, the
 temperature dependence of probe fluorescence could be used for mutation
 detection and quantification. However, use of adjacent hybridization
 probes for homogeneous detection of PCR products has not been reported
 previously. Surprisingly, both hybridization for signal generation and
 amplification by polymerase extension through the area blocked by the
 probes can occur.
 EXAMPLE 6
 A 110 bp beta-globin fragment was amplified from genomic DNA with adjacent
 fluorescein- and Cy5-labeled probes as described in Example 3. Either 0.4
 U (Taq) or 0.8 U (Stoffel fragment, Perkin Elmer, or KlenTaql) of enzyme
 was used in 10 .mu.l reactions. Unless indicated otherwise, temperature
 cycling was 94.degree. C. for 0 sec with a programmed approach rate of
 20.degree. C./sec, 60.degree. C. for 20 sec with an approach rate of
 20.degree. C./sec, and 75.degree. C. for 0 sec with an approach rate of
 1.degree. C./sec. FIG. 13 shows the development of fluorescence by 2
 adjacent hybridization probes immediately after the template was amplified
 for 30 cycles. After a brief denaturation at 94.degree. C., the
 temperature was lowered to 60.degree. C. and fluorescence increased for
 about 20 sec. The magnitude of the signal is greater with an exonuclease
 deficient polymerase (Stoffel fragment) than with native Taq polymerase
 that includes a 5'-exonuclease activity. After about 20 sec., the
 fluorescence drops as the polymerase displaces and/or hydrolyzes the
 probes. The relative decrease in fluorescence is slightly faster when the
 polymerase has 5'-exonuclease activity (Taq DNA polymerase) then when it
 lacks this activity (Stoffel fragment).
 In FIG. 14 (top panel), the temperature is cycled between 94.degree. C. and
 60.degree. C. with a 20 sec hold at 60.degree. C. Fluorescence is acquired
 at the end of the 20 sec when fluorescence is maximal. Good amplification
 occurs with Taq (exo.sup.+), but not with Stoffel fragment (exo.sup.-) as
 verified by both fluorescence development and agarose gels (gels not
 shown). However, if the time at 60.degree. C. is increased from 20 sec to
 120 sec (FIG. 14, middle panel), the exo.sup.- polymerase amplifies well.
 The slower rate of probe displacement with an exo.sup.- polymerase
 apparently requires more time at 60.degree. C. for efficient amplification
 than the exo.sup.+ polymerase. The time required by exo.sup.- polymerases
 can be reduced by slowly increasing the temperature from 60.degree. C. to
 75.degree. C. (FIG. 14, bottom panel). The polymerase stalls when it
 reaches the probe. However, at the probe melting temperatures, the probes
 melt off the template and the polymerase continues unencumbered to
 complete polymerization of the strand. Polymerization is completed as long
 as the temperature is not raised too quickly after probe melting. FIG. 14
 (bottom panel) shows one exo.sup.+ polymerase (Taq) and two exo.sup.-
 polymerases (Stoffel fragment and KlenTaq1).
 When exonuclease activity is present, some of the probe is hydrolyzed each
 cycle as evidenced by an the decrease in fluorescence with extensive
 amplification. This is observed in FIGS. 13 and 14 (middle and bottom
 panels), but is does not occur with exo.sup.- polymerases. Because the
 fluorescence is stable on extensive amplification, exo.sup.- polymerases
 such as KlenTaql are preferred.
 The success of using adjacent hybridization probes to monitor PCR depends
 on several factors. Resonance energy transfer is maximized when there is
 either 0 to 2 intervening bases between adjacent hybridization probes. To
 increase the fraction of strands that hybridize to the probes before the
 primer extends through the area of probe hybridization, the probe melting
 temperatures should be greater than the primer melting temperatures
 (preferably &gt;5.degree. C.).
 Cycle-by-cycle fluorescence. Conventional endpoint analysis of DNA
 amplification by gel electrophoresis identifies product size and estimates
 purity. However, because amplification is at first stochastic, then
 exponential, and finally stagnant, the utility of endpoint analysis is
 limited for quantification. One aspect of the present invention includes
 cycle-by-cycle monitoring for quantification of initial template copy
 number with hybridization probes. As will be appreciated by those skilled
 in the art, once-per-cycle monitoring of multiple samples undergoing DNA
 amplification is a powerful quantitative tool. Cycle-by-cycle monitoring
 is achieved by acquiring fluorescence during the extension or combined
 annealing/extension phase of each cycle and relating the fluorescence to
 product concentration.
 EXAMPLE 7
 Cycle-by-cycle monitoring of PCR was performed by three different
 fluorescence techniques. Fluorescence was monitored by (i) the
 double-strand-specific dye SYBR.TM. Green I, (ii) a decrease in
 fluorescein quenching by rhodamine after exonuclease cleavage of a
 dual-labeled hydrolysis probe and (iii) resonance energy transfer of
 fluorescein to Cy5 by adjacent hybridization probes. Amplification
 reagents and conditions were as described in Example 2. The human
 beta-globin primers RS42/KM29 (536 base pairs) and PC03/PC04 (110 base
 pairs) are described in C. T. Wittwer et al., Automated polymerase chain
 reaction in capillary tubes with hot air, 17 Nucl. Acids. Res. 4353-4357
 (1989), which is now incorporated herein by reference. Temperature cycling
 for beta-globin was 95.degree. C. maximum, 61.degree. C. minimum, 15 sec
 at 72.degree. C. and an average rate between temperatures of 5.2.degree.
 C./sec. The beta-actin primers and fluorescein/rhodamine dual probe were
 obtained from Perkin Elmer (no. N808-0230). Temperature cycling for
 beta-actin was 94.degree. C. maximum, 60.degree. C. for 15 sec with an
 average rate between temperatures of 6.2.degree. C./sec. The single
 labeled probes 5'-CAAACAGACA CCATGGTGCA CCTGACTCCT GAGGA-fluorescein-3'
 (SEQ ID NO:3) and 5'-Cy5-AAGTCTGCCG TTACTGCCCT GTGGGGCAAGp (SEQ ID NO:4)
 were synthesized as in Example 3. These adjacent probes hybridize internal
 to the PC03/PC04 beta-globin primer pair on the same DNA strand and are
 separated by one base pair. Temperature cycling was 94.degree. C. maximum,
 59.degree. C. for 20 sec with an average rate between temperatures of
 7.0.degree. C./sec. Hybridization probes (beta-actin and beta-globin) were
 used at 0.2 .mu.M each.
 When multiple samples are monitored once each cycle with SYBR.TM. Green I,
 a 10.sup.7 -10.sup.8 range of initial template concentration can be
 discerned as represented in FIG. 15. This amplification is of a 536 base
 pair fragment of the beta-globin gene, with SYBR.TM. Green I as the
 double-strand specific dye. When the data were normalized as the percent
 maximal fluorescence of each sample, one hundred initial copies were
 clearly separated from ten copies. However, the difference between one and
 ten copies was marginal, and no difference was observed between zero and
 one average copies per sample.
 In contrast, sequence-specific probes have a similar dynamic range but,
 appear to discriminate even a single initial template copy from negative
 controls. Signal generation with 5'-exonuclease probes (beta-actin
 fragment, FIG. 16) is dependent not only on DNA synthesis, but requires
 hybridization and hydrolysis between the fluorophores of the dual-labeled
 probe. This hydrolysis reduces quenching and the fluorescence ratio of
 fluorescein to rhodamine emission increases. Whereas the fluorescence from
 double strand dyes levels off with excess cycling (FIG. 15), the signal
 from exonuclease probes continues to increase with each cycle (FIG. 16).
 Even though no net product is being synthesized, probe hybridization and
 hydrolysis continue to occur. As the template copy number decreases below
 10.sup.3, signal intensity decreases, but low copy numbers can still be
 quantified because the negative control signal is stable.
 In FIG. 17, amplification is monitored using adjacent hybridization probes
 and is expressed as a ratio of Cy5 to fluorescein fluorescence. The change
 in fluorescence ratio is largely due to an increase in Cy5 fluorescence
 from resonance energy transfer (FIG. 9). In contrast to dual-labeled
 hydrolysis probes, the fluorescence signal of hybridization probes
 decreases at high cycle numbers if the polymerase contains an exonuclease
 activity (see also FIG. 14).
 The present invention's feasibility using two different methods for
 resonance energy transfer detection of hybridization during PCR will now
 be demonstrated. The first method uses two adjacent hybridization probes,
 one labeled 3' with fluorescein and the other labeled 5' with Cy5. As
 product accumulates during PCR, the probes hybridize next to each other
 during the annealing segment of each cycle. The second method uses a
 primer labeled with Cy5 and a single hybridization probe. The labeled
 primer is incorporated into the PCR product during amplification and only
 a single hybridization is necessary.
 EXAMPLE 8
 Cycle-by-cycle monitoring of PCR was performed by resonance energy transfer
 between a Cy5-labeled primer and a fluorescein-labeled hybridization
 probe. This was compared to monitoring with adjacent Cy5/fluorescein
 hybridization probes. The Cy5-labeled primer was CAACTTCATC CACGT*TCACC
 (SEQ ID NO:21) where T* is a modified T base with Cy5 attached and the
 corresponding probe was GTCTGCCGTT ACTGCCCTGT GGGGCAA-fluorescein (SEQ ID
 NO:22). The adjacent hybridization probes were CCTCAAACAG ACACCATGGT
 GCACCTGACT CC-fluorescein (SEQ ID NO:23) and Cy5-GAAGTCTGCC GTTACTGCCC
 TGTGGGGCAAp (SEQ ID NO:24). The hybridizaton probes were synthesized
 according to Example 3 and used at 0.2 .mu.M. The Cy5-labeled primer was
 synthesized in two steps. Automated synthesis was used to incorporate an
 amino-modifier C6dT (Glen Research) at the desired T position. Then, the
 monovalent N-hydroxysuccinimide ester of Cy5 (FIG. 6) was manually
 conjugated to the amino linker according to the manufacturer's
 instructions (Amersham). HPLC purifification was as described in Example
 3.
 The Cy5-labeled primer (0.5 .mu.M) was used instead of PCO4 to amplify the
 110 base pair beta-globin fragent from human genomic DNA as in Example 3,
 except that 0.4 U of Taq polymerase was used per 10 .mu.l. The adjacent
 hybridization probes also monitored amplification of the same beta-globin
 fragment. Temperature cycling was done at 94.degree. C. for 0 sec and
 60.degree. C. for 20 sec. The fluorescence was monitored once each cycle
 at the end of the annealing/extension segment. In both methods,
 fluorescence energy transfer to Cy5 increases with hybridization and is
 plotted as a ratio of Cy5 to fluorescein fluorescence (FIG. 18).
 In additional experiments, the number of bases separating the Cy5-label and
 the fluorescein label were varied. The best fluorescence resonance energy
 transfer was observed with about 4-6 bases between the fluorophores,
 although a signal was detectable up to at least 15 bases.
 In contrast to hydrolysis probes, the fluorescence signal from
 hybridization probes is not cumulative and develops anew during each
 annealing phase. The fluorescence is a direct measure of product
 concentration because the hybridization is a pseudo-first order reaction.
 Because the concentration of probe is much greater than the product, the
 traction of product hybridized to probe is independent of product
 concentration. These characteristics indicate that using a single
 hybridization probe along with a labeled primer will provide a superior
 monitor of product accumulation for quantification. The inherent variance
 of different fluorescence techniques during cycle-by-cycle monitoring is
 also important for quantification.
 EXAMPLE 9
 DNA amplification was perfomed according to Example 2 for each of three
 different fluorescence monitoring methods. SYBR.TM. Green I was used at a
 1:10,000 dilution in the amplification of a 205 base pair human
 beta-globin fragment from primers KM29 and PC04. The hydrolysis probe and
 conditions are those specified in Example 7. The hybridization probe,
 TCTGCCGTTA CTGCCCTGTG GGGCAAG-fluorescein (SEQ ID NO:5) was used with KM29
 and the Cy5-labeled primer CAACTTCATCCACGTT*CACC (SEQ ID NO:6) where T*
 was a Cy5-labeled T base synthesized as in example 8. All amplifications
 were performed in ten replicates with 15,000 template copies (50 ng of
 human genomic DNA/10 .mu.l). The temperature cycles were 31 sec long
 (94.degree. C. maximum, 60.degree. C. for 20 sec, average rate between
 temperatures 6.2.degree. C./sec). Fluorescence was acquired for each
 sample between seconds 15 and 20 of the annealing/extension phase.
 FIG. 19 allows comparison of three fluorescence monitoring techniques for
 PCR. The fluorescence probes are the dsDNA dye SYBR.TM. Green I (FIG.
 19A), a dual-labeled fluorescein/rhodamine hydrolysis probe (FIG. 19B),
 and a fluorescein-labeled hybridization probe with a Cy5-labeled primer
 (FIG. 19C). All probes had nearly the same sensitivity with detectable
 fluorescence occurring around cycle 20. With extended amplification, the
 signal continued to increase with the hydrolysis probe, was level with
 SYBR.TM. Green I, and slightly decreased with the hybridization probe. The
 precision of the three fluorescence monitoring techniques are compared in
 FIG. 19D. The mean +/- standard deviations are plotted for each point. The
 data are plotted as the coefficient of variation (standard deviation/mean)
 of the fluorescence ratio above baseline (taken as the average of cycles
 11-15).
 Although the change in fluorescence ratio from the hydrolysis probe is
 greater than that from the hybridization probe (FIGS. 19B and 19C), the
 coefficient of variation of fluorescence from the hydrolysis probes is
 greater (FIG. 19D). That is, the fluorescence resulting from the
 hybridization probe method is more precise than using a hydrolysis probe,
 even though the absolute signal levels are lower. This is an unexpected
 advantage of hybridization probes over the more usual dual-labeled
 hydrolysis probes.
 Quantification of initial template copy number. Quantitative PCR has become
 an important technique in both biomedical research and in the clinical
 laboratory. The process of quantification often includes running a
 standard curve of samples containing known copy 5 numbers of the target
 sequence. The copy number of an unknown sample is determined by
 extrapolation between the known values. When a complete amplification
 curve is monitored cycle-by-cycle using fluorescence, radioactivity or any
 other method that gives a signal proportional to the amount of DNA
 present, many data points are available for analysis and it is not obvious
 which value to choose to represent a standard or unknown. Prior art is to
 choose a "threshold value" of the signal and then use the cycle number
 when the standard or unknown crosses that threshold as the representative
 value (see Higuchi & Watson, EPA 0 640 828 A1). This approach uses a very
 small amount of the available data in an amplification curve. In addition,
 the assignment of the threshold value is highly subjective and is subject
 to conscious or unconscious bias. More of the available data could be used
 objectively by applying non-linear curve fitting techniques to the data in
 an amplification curve. Preferably, equations could be found that describe
 the shape of the amplification curves by modeling factors of the
 underlying process.
 A number of different equations could be used to fit the data produced
 during amplification. DNA amplifications typically have a log linear
 segment and the data in this segment can be fit to an equation that
 describes an exponential increase like that expected in a DNA
 amplification. The log-linear portion of a DNA amplification can be
 described by the equation:
EQU y=A*[DNA]*(1+E).sup.n
 wherein A is a scaling factor that converts units of signal to units of
 DNA; [DNA] is the starting concentration of DNA in the reaction; E is the
 efficiency of the reaction; and n is the cycle number.
 A quantification process would involve: (1) fitting the known standards to
 this equation allowing the parameters A and E to float, and (2) fitting
 the unknown samples to the equation using the values of A and E from the
 standards and allowing [DNA] to float. This technique uses much more of
 the data and uses the portion of the data, the log-linear portion, that is
 likely to be most informative. FIGS. 20, 21 and 22 show an example of this
 approach. Ten-fold dilutions of a purified PCR product were amplified as a
 standard curve and an "unknown" human genomic DNA standard was used. FIG.
 20 shows that the log-linear portion is easily identified either by the
 user or by software. FIG. 21 shows a fit of the equation
 y=A*[DNA]*(1+E).sup.n to the 10.sup.4 copy standard. FIG. 22 uses average
 values from several standards for A and E and fits [DNA]. The fit value of
 16,700 is very close to the theoretical value for a single copy gene in
 genomic DNA (15,000 copies).
 Using all the data in an amplification curve would include the background
 level and the plateau value. While at high copy number the plateau is
 uninformative, at low copy number it is often proportional to starting
 copy number. The background level could be useful in determining the first
 point that shows a significant increase in signal. At this time all the
 factors involved in the shape of the DNA amplification curve are not
 known, so one approach is to describe the shape of the curve. FIG. 23
 shows amplification curves using fluorescent hybridization probes to
 detect a five order of magnitude range of DNA template concentrations.
 Each curve is fit to the equation:
EQU y=((as*x+ab)-(ds*x+db))/(1+(x/c) b)+(ds*x+db)
 wherein "as" is the background of the slope line, "ab" is the y intercept
 of the background line, "ds" is the slope of the plateau line, "db" is the
 y intercept of the slope line, "c" is cycle number where the reaction is
 half way from background to plateau (A.sub.50), and "b" is the slope of
 the log-linear portion of the amplification.
 This equation gives good fits to this amplification data, and FIG. 24 shows
 that the value of the A.sub.50 correlates well with the log of the
 starting copy number across seven orders of magnitude. FIG. 25 shows the
 same equation fit to data from amplifications that used a hydrolysis probe
 to detect DNA template over a 5 order of magnitude range. This equation
 gives good fits to this amplification data, and FIG. 26 shows that the
 value of the A.sub.50 correlates well with the log of the starting copy
 number. This demonstrates the flexibility of the full curve fit approach
 as the equation has given good fits to both the sharp plateaus of the
 hybridization probe amplification curves and the steadily increasing
 "plateaus" of the hydrolysis probe curves.
 Total curve fits are not limited to this equation. FIG. 27 shows an
 amplification of three concentrations of DNA template fit to the equation:
EQU y=(((as*x+ab)-(dmax*x/dd+x))/(1+(x/c) b))+(dmax*x/dd+x),
 which is similar to the first 6 parameter equations except that the plateau
 is defined by a hyperbolic curve rather than by a line. FIG. 28 shows that
 the A.sub.50 for this equation correlates well to the starting copy
 number.
 While the A.sub.50 has been used in these examples and level between the
 background and the plateau could be chosen if a particular technique is
 more robust lower or higher in the amplification profile. For example a
 series of amplification standard curves are evaluated for the best
 correlation between the starting copy number and the A.sub.50, the
 A.sub.40, the A.sub.30, the A.sub.20, and the A.sub.10. The level of
 amplification that best correlates with the known starting copy number is
 determined. This will be different for different detections systems. FIG.
 19 shows that coefficient of variation for various detection systems. The
 level of amplification that is the best predictor is likely to be the
 level with the lowest coefficient of variation.
 As the DNA amplification reaction itself is better understood, other
 equations that have parameters that reflect physical processes could be
 used. The plateau of the DNA amplification curve has different causes in
 different reactions. It is often due to the inability of the primers to
 compete with product reannealing in the latter cycles. This effect could
 be captured with a parameter that is dependent on the square of the
 concentration of product in the reaction (as reannealing rate is
 proportional to the square of the product concentration). Another cause of
 the plateau can be the depletion of the primers. Primer limited reactions
 have a characteristic shape, they have a very sharp plateau that can be
 recognized. Primer limited reaction fits will include parameters that
 define this sharp top. Enzyme limited reactions have a very rounded
 plateau that can be fit accordingly. Weighting factors can be devised that
 reflect the known coefficients of variation for the given system to more
 heavily weight the more reliable data points. By fitting more of the
 points in an amplification profile, more accurate and robust estimates of
 starting copy number can be obtained. One or more of the parameters of
 these fits can be used to estimate the starting copy number of unknown
 samples.
 Continuous fluorescence monitoring of PCR. The present invention's feature
 of continuous monitoring, that is, monitoring many times within each PCR
 cycle, will now be discussed. While fluorescence monitoring during PCR can
 be done once each cycle at a constant temperature, the present invention
 provides the important advantage of providing continuous monitoring
 throughout the PCR cycle. Temperature is important because fluorescence
 changes as the temperature changes. FIGS. 29A&B demonstrate the inverse
 relationship between temperature and fluorescence for SYB.TM. Green I.
 This is a confounding effect during temperature cycling that is usually
 eliminated by considering fluorescence once per cycle at a constant
 extension temperature. However, in accordance with the present invention,
 monitoring fluorescence during temperature changes is very informative.
 Prior to the present invention, continuous fluorescence monitoring within
 each cycle, as opposed to once each cycle, has not been carried out. In
 accordance with the present invention, time, temperature and fluorescence
 is acquired every sec, every 200 msec, every 100 msec or even at a greater
 frequency. Such data can reveal fine details of product denaturation,
 reannealing and extension, and probe annealing and melting during rapid
 cycling not available in previously available methods.
 EXAMPLE 10
 A 180-base-pair fragment of the hepatitis B surface antigen gene was
 amplified from 10.sup.6 copies of purified PCR product using primers
 5'-CGTGGTGGAC TTCTCTCAAT-3' (SEQ ID NO:1), and 5'-AGAAGATGAG GCATAGCAGC-3'
 (SEQ ID NO:2)(Genbank sequence HVHEPB). The amplification conditions of
 Example 2 were followed except that the reaction contained a 1:20,000
 dilution of SYBR.TM. Green I and 2 mM MgCl.sub.2. Each temperature cycle
 was 27 sec long (92.degree. C. maximum, 59.degree. C. minimum, 5 sec at
 70.degree. C., average rate between temperatures 3.0.degree. C./sec).
 Time, temperature, and 2 channels of fluorescence were acquired every 200
 msec and continuously displayed as fluorescence v. cycle number and
 fluorescence v. temperature plots. FIG. 30 shows a 3D trace of
 temperature, time and fluorescence for cycles 10 through 34. This 3D curve
 is also projected in FIG. 30 as 2D plots of temperature v. time,
 fluorescence v. time, and fluorescence v. temperature. The temperature v.
 time projection of FIG. 30 repeats each cycle and provides essentially the
 same information as set forth in FIG. 3. Because fluorescence varies
 inversely with temperature, the fluorescence v. time projection shown in
 FIG. 30 at early cycles is a scaled mirror image of the temperature v.
 time plot (see FIG. 29). As product accumulates, the fluorescence
 increases at all temperatures with double stranded product. However at
 denaturation temperatures, fluorescence returns to baseline since only
 single stranded DNA is present. The fluorescence v. temperature projection
 of double stranded dyes shown in FIG. 30 eliminates the time axis and
 shows the temperature dependence of strand status during DNA
 amplification.
 EXAMPLE 11
 A 536 base pair fragment of the human beta-globin gene was amplified from
 25 ng of genomic DNA and a 1:10,000 dilution of SYBR.TM. Green I in a
 volume of 5 .mu.l. Each temperature cycle was 28 sec long (95.degree. C.
 maximum, 61.degree. C. minimum, 15 sec at 72.degree. C. with an average
 rate between temperatures of 5.2.degree. C./sec). Other conditions are the
 same as those described in FIG. 30. Cycles 15-40 are displayed.
 The temperature dependence of product strand status during PCR is revealed
 by fluorescence v. temperature plots using as shown in FIG. 31. Early
 cycles represented appear identical, with a nonlinear increase in
 fluorescence at lower temperatures. As amplification proceeds, temperature
 cycles appear as rising loops between annealing and denaturation
 temperatures. As the sample is heated, fluorescence is high until
 denaturation occurs. As the sample cools, fluorescence increases,
 reflecting product reannealing. When the temperature is constant during
 extension, increasing fluorescence correlates with additional DNA
 synthesis.
 As will be appreciated by an understanding of this disclosure, continuous
 monitoring within a cycle can provide insight into DNA amplification
 mechanics not previously available in the art. Using the present
 invention, many aspects of DNA amplification that have heretofore been
 little understood are discernable. For example, rapid cycle amplification
 claims to denature the product in less than one second, while the prior
 art uses ten seconds to one minute of denaturation. Observing product
 melting by real time fluorescence monitoring with double strand dyes in
 accordance with the present invention (FIGS. 30 and 31) shows that use of
 the shorter denaturation times is effective. As another example, many
 causes of the known "plateau effect" have been proposed, but few data are
 available to distinguish between alternatives. As shown in FIG. 31,
 product reannealing is very rapid. In fact, during later cycles of
 amplification, a majority of product is reannealed each cycle during
 cooling before the primer annealing temperature is reached. This occurs
 with cooling rates of 5-10.degree. C./sec in rapid cycle instrumentation.
 The product reannealing with slower, prior art temperature cyclers will be
 more extensive and this undesirable effect will be greater. Product
 reannealing appears to be a major, and perhaps the sole, cause of the
 "plateau effect."
 Now consider continuous monitoring of sequence specific probes. As will be
 appreciated by an understanding of this disclosure, continuous monitoring
 within a cycle can identify the nature of probe fluorescence.
 EXAMPLE 12
 Continuous monitoring of amplification every 200 msec was performed with a
 dual-labeled hydrolysis probe (beta-actin) and adjacent hybridization
 probes (beta-globin) as in Example 7. In FIG. 32A, cycles 20-45 of a
 reaction monitored with the hydrolysis probe is shown. Hydrolysis probes
 show a linear change in fluorescence ratio with temperature and a parallel
 increase in fluorescence as more probe is hydrolyzed. In contrast, the
 fluorescence ratio from hybridization probes varies radically with
 temperature (FIG. 32B, cycles 20-40). During the annealing/extension
 phase, the probes hybridize to single stranded product and the
 fluorescence ratio (Cy5/fluorescein) increases. During heating to product
 denaturation temperatures, the probes dissociate around 70.degree. C.,
 returning the fluorescence ratio to background levels.
 EXAMPLE 13
 A 110 base pair beta-globin fragment was amplified from 50 ng of genomic
 DNA in a volume of 10 .mu.l. The amplification conditions and adjacent
 hybridization probes of Example 3 were followed with either 0.4 U of Taq
 polymerase or 0.8 U of KlenTaql. Fluorescence was monitored each 100 msec.
 Fluorescence v. temperature plots using KlenTaql (FIG. 33) and Taq (FIG.
 34) demonstrate melting of the probes at about 70.degree. C. The maximal
 signal with KlenTaql is greater than that with Taq, because of the
 exonuclease activity of the latter. At later cycles with Taq, the
 fluorescence each cycle begins to decrease as the concentration of intact
 probe decreases. Three dimensional plot of temperature, time, and
 fluorescence are shown in FIG. 35 (KlenTaq1) and FIG. 36 (Taq).
 The present invention's combination of (1) continuous fluorescence
 monitoring within each temperature cycle and (2) analysis of the
 temperature and time dependence of hybridization provides advantages not
 otherwise obtainable. FIG. 2 shows that information that was previously
 unobtainable can be extracted by continuous monitoring throughout the
 cycle. Continuous fluorescence monitoring during the product melting phase
 of the cycle provides useful information on the purity, identity, and
 quantity of DNA present during that cycle.
 As a PCR reaction is heated from the extension temperature to the
 denaturation temperature, any DNA in the sample is melted to single
 strands. This denaturation can be observed as a drop in the fluorescence
 of SYBR.TM. Green I. For small PCR products, the melting transition occurs
 over a narrow temperature range and the midpoint of that melting range is
 referred to as the Tm. Similar to sizing by gel electrophoresis, melting
 peak analysis measures a fundamental characteristic of DNA and can be used
 to identify amplified products. Unlike gel electrophoresis, melting curve
 analysis can distinguish products of the same length but different GC/AT
 ratio. In addition, two products with the same length and GC content, but
 differing in their GC distribution (for example, equally distributed vs. a
 GC clamp on one end) would have very different melting curves.
 The temperature at which PCR products melt varies over a large range. Using
 empirical formulas known in the art, the effect of GC content on the
 melting temperature (Tm) of DNA predicts that a 0% GC duplex would melt
 41.degree. C. lower than a 100% GC duplex. Given the same GC content, a 40
 base pair primer dimer would melt 12.degree. C. below a 1000 bp product.
 Hence, the range of Tm for potential PCR products spans at least
 50.degree. C. This wide range allows most PCR products to be
 differentiated by melting curves. Thus, the combination of fluorescence
 monitoring of PCR with melting curve analysis provides simultaneous
 amplification, detection, and differentiation of PCR products.
 EXAMPLE 14
 DNA melting curves for three different PCR products were acquired on a
 microvolume fluorimeter integrated with a 24-sample thermal cycler with
 optics for SYBR.TM. Green I fluorescence (LightCycler LC24, Idaho
 Technology, Idaho Falls, Id.). The primers for the 180 base pair hepatitis
 B surface antigen gene amplification were 5'-CGTGGTGGAC TTCTCTCAAT-3' (SEQ
 ID NO:1) and 5'-AGAAGATGAG GCATAGCAGC-3'(SEQ ID NO:2). The primers for the
 292 base pair prostate specific antigen (PSA) gene amplification were
 5'-CTGTCCGTGA CGTGGATT-3' (SEQ ID NO:7) and 5'-AAGTCCTCCG AGTATAGC-3' (SEQ
 ID NO:8). The 536 base pair human beta-globin gene amplification was done
 as in Example 7. PCR was performed as described in Example 2.
 Amplification products were purified by phenol/chloroform extraction and
 ethanol precipitation, followed by repeated washing through a Centricon 30
 microconcentrator (available from Amicon of Danvers, Mass.). Template
 concentrations were determined by absorbency at 260 nm and had
 A(260)/A(280) ratios greater than 1.7.
 Fifty ng of purified DNA in 50 mM Tris, pH 8.5, 2 mM MgCl.sub.2, and 250
 .mu.g/ml bovine serum albumin and a 5 .mu.l volume were pipetted into the
 open plastic reservoir of composite glass/plastic reaction tubes,
 centrifuged at low speed to place the sample at the tip of the glass
 capillary, and sealed inside with a plastic plug. Fluorescence data for
 melting curves was acquired by integrating the signal over 0.25-2.0
 seconds during a linear temperature transition to 95.degree. C. at
 0.1-10.0.degree. C./second. The fluorescence was continuously acquired and
 displayed at fluorescence v. temperature plots in the LabView programming
 environment (National Instrument, Austin, Tex.). FIG. 37 shows the melting
 curves of the three purified PCR products.
 The Tm's of three products in FIG. 37 span only 6 degrees and two of the
 curves are separated by only 2 degrees. This small separation is ample to
 allow easy differentiation of the products. The importance of GC
 percentage over length on Tm is illustrated by the 292 bp PSA product
 melting at a higher temperature than the longer 536 bp beta-globin
 fragment. Melting curves are often obtained at rates of 0.5.degree.
 C./minute to ensure equilibrium. Moreover, as the heating rate decreases,
 the melting curve shifts to lower temperatures and becomes sharper (FIG.
 38, hepatitis B fragment). Note however, that the melting curves of FIG.
 37 were obtained during a heating rate of 0.2.degree. C./sec (12.degree.
 C./minute) and can differentiate products differing in Tm by 2.degree. C.
 or less.
 The apparent Tm of PCR products is also dependent on double-strand-specific
 DNA dye concentration (FIG. 39, hepatitis B fragment). Higher
 concentrations of dye increase the stability of the DNA duplex and the
 observed Tm.
 For monitoring of melting curves with SYBR.TM. Green I, the preferred
 conditions are 1:7,000-1:30,000 fold dilution of SYBR Green I with heating
 rates of 0.1-0.5.degree. C./second. These conditions allow easy
 differentiation of products that differ in Tm by 20.degree. C.
 More precise temperature control and software for melting peak analysis
 will reduce the detectable difference in Tm to a fraction of a degree.
 This will allow the differentiation of most PCR products. Not all products
 can be differentiated by Tm however, just as it is possible to misread
 electrophoresis results because of comigration of two or more products, it
 is possible that some of the product melting in the expected range may not
 be the intended product. However, if no DNA melts in the range of the
 expected product, it can conclusively be said that none of the expected
 product is present.
 Another form of product differentiation available with melting curve
 analysis is the distinctive patterns of domain melting seen in longer PCR
 products. While short products (&lt;300 bp) usually melt in one transition,
 longer products can have internal melting domains that give melting curves
 of a complex, distinctive shape. These complex melting curves can be used
 as a fingerprint for product identification.
 Melting curve analysis can be used to differentiate intended product from
 nonspecific products such as primer dimers. Primer dimers melt over a wide
 range of low temperatures; very different from the sharp melting curves of
 specific PCR amplification products. Larger heterogeneous products which
 resulted from running many cycles at low annealing stringency have lower
 and broader melting curves when compared with pure PCR product.
 EXAMPLE 15
 Amplification of the 536 beta-globin gene fragment was performed as in
 Example 7 with a 1:30,000 dilution of SYBR.TM. Green I except that the
 conditions were varied. In reaction A (FIG. 40), no template was added and
 the reaction was cycled at 94.degree. C. for 0 sec, 60.degree. C. for 0
 sec, and 72.degree. C. for 10 sec for 30 cycles to produce small
 nonspecific amplification products. In B, amplification of 10.sup.6
 initial copies of purified template at low stringency (94.degree. C. for 0
 sec, 50.degree. C. for 0 sec, and 72.degree. C. for 10 sec) for 55 cycles
 showed a broad size range of amplification products on gel electrophoresis
 and melts across a wide temperature range. In C, 10.sup.6 initial copies
 of purified template were cycled at 94.degree. C. for 0 sec, 60.degree. C.
 for 0 sec, and 72.degree. C. for 10 sec for 30 times and shows a single
 bright band and melts in a sharp transition. The temperature transition
 rate was 0.2.degree. C./sec. A Hind III digest of .lambda. phage DNA (M)
 is used as a marker.
 FIG. 40 shows how melting curves accurately reflect the specificity of a
 PCR reaction. The sharp, high temperature melting curve C corresponds to a
 single band on a gel. The low temperature, broad melting, curve A comes
 from analysis of a no template control that shows only primer dimers.
 Over-amplification of the product in C gives the intermediate melting
 curve B, still clearly differentiable from the specific product.
 The melting curves seen, for example, in FIG. 37, can be better quantified
 by first taking the derivative of fluorescence (F) with respect to
 temperature (T). This derivative is plotted as -dF/dT v. T and converts
 the melting curves to melting peaks.
 EXAMPLE 16
 The purified hepatitis B and beta-globin gene fragments of Example 14 were
 melted individually and together with a temperature transition rate of
 0.2.degree. C./sec and other conditions as specified in Example 14 (FIG.
 41). The somewhat subjective determination of Tm from the melting curves
 (top) is easily called by eye from the melting peaks (bottom). The area
 under the melting peaks can also be quantified by integration of the area
 under the curves. The fluorescence baseline was first subtracted from the
 -dF/dT v. T plot assuming that the magnitude of the baseline varies as the
 area under the curve. Then the peaks were fit by nonlinear least squares
 regression to gaussians with the mean, standard deviation, and height of
 the peak as the fit parameters. The area under each gaussian was taken as
 the peak area. All calculations were performed in the LabView programming
 environment (National Instruments, Austin, Tex.). FIG. 41 shows an example
 of this conversion of melting curves to melting peaks. The code for these
 calculations is included as appendix A.
 The ability to distinguish specific product from primer dimer and other
 reaction artifacts improves the use of double-strand-specific DNA dyes in
 the quantification of low initial copy numbers. Relatively large initial
 template copy numbers have been quantified using ethidium bromide (Higuchi
 & Watson, supra). However, at low initial copy numbers, the background
 amplification of primer dimers and other amplification artifacts
 interferes with the specific amplification signal. With the present
 invention's ability to differentiate specific products from non-specific
 artifacts, double-strand-specific DNA dyes can be used to quantify low
 initial template copy numbers. This is advantageous because of the
 simplicity of using these dyes. The double-strand-specific DNA dyes can be
 used in any amplification and custom fluorescently-labeled
 oligonucleotides are not necessary. Quantification of very low copy
 numbers with double-strand-specific DNA dyes requires very good
 amplification specificity or, as provided by the present invention, a
 means to differentiate the desired product from nonspecific amplification.
 EXAMPLE 17
 The present invention's approach to product purity determination was used
 to improve quantitative PCR based on once-per-cycle monitoring of
 double-strand-specific DNA dye fluorescence. Fluorescence was acquired
 once each cycle after polymerase extension of the product for a series of
 reactions varying in the initial concentration of purified beta-globin
 template (see FIG. 42A). The beta globin template and amplification
 conditions were as given in Example 7. The log-linear increase above
 background fluorescence began at a cycle number dependent on initial
 template concentration. The plots of the five reactions ranging from
 10.sup.6 to 10.sup.2 copies per reaction were separated by about four
 cycles. The sample with an average 10.sup.2 copies per reaction showed a
 decrease in reaction efficiency, and reactions with initial copy number
 below 100 gave fluorescence profiles that were less useful. The
 fluorescence profiles for the reactions containing 10 and 1 (average)
 copies rise in reverse order, and the negative control showed
 amplification after about 30 cycles. This is due to amplification of
 primer dimers and other nonspecific amplification products that cannot be
 distinguished from the intended product by once-per-cycle fluorescence
 monitoring of double-strand-specific DNA specific dyes.
 Melting peaks were acquired for each sample (FIG. 42B) and these were found
 to correlate well with electrophoresis results (FIG. 42C). The reaction
 containing zero and one average initial template copies produced no
 discernible electrophoresis band at the expected 536 base pair location.
 The reactions containing 10 and 100 initial copies of template showed weak
 electrophoresis bands. This agreed well with the melting peak analysis,
 which showed no DNA melting in the range of the intended product
 (90-92.degree. C.) for the reactions containing zero and one initial
 copies and small peaks in this temperature range for 10 and 100 copies.
 Strong electrophoresis bands for the reactions containing 10.sup.3
 -10.sup.6 initial copies correlate well with large melting peaks in the
 expected 90-92.degree. C. range.
 The ratio of intended product to total product, determined by melting peak
 integration, ranged from 0.28 for 10.sup.5 copies to 0.0002 for zero
 initial template copies. Each fluorescence value in FIG. 41A was
 multiplied by the appropriate ratio to give the corrected plot (designated
 "corrected fluorescence" in FIG. 42D). This procedure extended the
 effective dynamic range of quantitation to between 10 and 1 initial
 template copies.
 Melting peaks can distinguish specific products from non-specific products
 (FIG. 40) and they can distinguish two purified PCR products mixed
 together (FIG. 41) so they should also be useful for distinguishing two
 specific products amplified together in a single reaction tube. Melting
 curves obtained by continuous monitoring of PCR reactions according to the
 present invention are useful in multiplex PCR.
 EXAMPLE 18
 In this example, two gene fragments were simultaneously amplified from
 genomic DNA and monitored with SYBR.TM. Green I fluorescence. During each
 amplification cycle, different amplification products denature at melting
 temperatures dependent on the length of the product, GC ratio, and other
 factors well known in the art. The temperature at which each product melts
 can be monitored with the double-strand-specific dye, SYBR.TM. Green I. At
 81 base pair fragment from the cystic fibrosis gene was amplified using
 the primers described herein as SEQ ID NO:14 and SEQ ID NO:15 along with a
 98 base pair fragment of the c-erbB-2 (HER2/neu) oncogene using the
 primers described herein as SEQ ID NO:16 and SEQ ID NO:17.
 Amplification reactions were comprised of 50 mM Tris-HCl, pH 8.3, 3 mM
 MgCl.sub.2, 500 .mu.g/ml of bovine serum albumin, 200 .mu.M of each dNTP,
 and 0.5 .mu.M of the cystic fibrosis primers, 0.3 .mu.M of the HER2/neu
 primers, a 1:30,000 dilution of SYBR.TM. Green I, 1 U AmpliTaq Gold DNA
 polymerase (Perkin Elmer, Foster City, Calif.), and 50 ng of human genomic
 DNA in 10 .mu.l.
 After activation of the polymerase at 95.degree. C. for 30 minutes, the
 samples were cycled at 94.degree. C. for 0 seconds (slope=20), 55.degree.
 C. for 0 seconds (slope=20), and 70.degree. C. for 10 seconds (slope=20)
 for 35 cycles. The samples were cooled to 70.degree. C., and the
 fluorescence was continuously acquired during a 0.2.degree. C./sec ramp to
 94.degree. C. Melting curves (FIG. 43) clearly showed two distinct
 products melting at 78.degree. C. (CFTR) and 88.degree. C. (neu). The two
 products differ in Tm by approximately 10.degree. C. and are easily
 distinguishable.
 Multiplex amplification is useful in cases where an internal control is
 needed during amplification. For example, many translocations are
 detectable by PCR by placing primers on each side of the breakpoint. If no
 amplification occurs, the translocation is not present as long as the DNA
 is intact and no inhibitor is present. These possibilities can be ruled
 out by amplifying a positive control locus in the same reaction mixture.
 Such control amplifications are best done as internal controls with
 simultaneous amplification and detection.
 EXAMPLE 19
 In this example, the procedure of Example 18 was followed except that after
 activation of the polymerase at 95.degree. C. for 30 minutes, the samples
 were cycled at 94.degree. C. for 0 seconds (slope=20), 55.degree. C. for 0
 seconds (slope=20), and 70.degree. C. for 10 seconds (slope=20) for 20
 cycles, followed by 94.degree. C. for 0 seconds (slope=1) , 55.degree. C.
 for 0 seconds (slope=20), and 70.degree. C. for 20 seconds (slope=20) for
 15 cycles. For cycles 26-31, fluorescence was continuously acquired during
 each 1.degree. C./sec transition from 70.degree. C. to 94.degree. C. The
 melting curves were converted to melting peaks and displayed (FIG. 44).
 Note that the amplification efficiency of the CFTR fragment appears
 greater than the neu fragment. The amplification efficiency can be
 rigorously determined by integrating the melting peak data as in Example
 16.
 This kind of quantitative data referenced to a control has many
 applications. For instance, certain oncogenes, such as HER2/neu, are
 amplified in vivo in many tumors. That is, the genes are replicated in
 genomic DNA, sometimes many fold. Often, the clinical behavior of the
 tumor depends on the degree of oncogene replication. Amplification of the
 oncogene and a control template allows quantitative assessment of the
 relative copy number. As a further example, quantification of viral load
 in patients infected with HIV or hepatitis C is important in prognosis and
 therapy. Using a control template and monitoring the efficiency of
 amplification of both control and natural templates during amplification,
 accurate quantification of initial template copy number is achieved.
 The present invention's feature of using melting curves for relative
 quantification will now be explained. In accordance with the present
 invention, an additional use for melting curves is quantitative PCR. FIG.
 42 showed there was a correlation between the area under the melting peak
 and the amount of specific product. Relative quantification of two PCR
 products would be possible if the two products were amplified with similar
 efficiency (or if the differing efficiencies were known and compensated
 for). Relative quantification of two products by integrating melting peak
 areas (see Example 16) is an aspect of the current invention.
 EXAMPLE 20
 The cystic fibrosis and HER-2-neu gene fragments of Example 18 were
 amplified, purified as in Example 2. and adjusted to 175 .mu.g/ml. The
 samples were mixed in various ratios (total 8 .mu.l) and added to buffer
 (1 .mu.l) and SYBR.TM. Green I (1 .mu.l). Final concentrations were 50 mM
 Tris, pH 8.3, 3 mM MgCl.sub.2, 250 .mu.g/ml bovine serum albumin, and a
 1:30,000 dilution of SYBR.TM. Green I. Melting curves were acquired at
 0.2.degree. C./sec, background fluorescence subtracted and the peaks
 integrated as described in Example 16. The results are displayed in FIG.
 45. Excellent correlation was found between the relative areas under
 melting peaks and the relative amounts of the two products.
 Relative quantification of two PCR products is important in many
 quantitative PCR applications. Multiplex amplification of two or more
 products followed by integration of the areas under the melting peaks will
 be extremely useful in these areas. mRNA is often quantified relative to
 the amount of mRNA of a housekeeping gene.
 Another important use of relative quantification is in competitive
 quantitative PCR. Typically a competitor is synthesized that has the same
 priming sites, but differs in length from the original target sequence.
 Known amounts of the competitor are spiked into an unknown sample and
 relative quantitation is performed. Competitors can be made that differ
 from the target sequence in Tm rather than length. The relative amounts of
 the products can be quantified by comparing the areas under their melting
 peaks. As the amount of one of the products is known, the quantity of the
 original target can be obtained. Using the melting peak method is
 significantly easier than the currently used methods which involve running
 multiple tubes for each unknown sample and often pulling tubes at various
 cycle numbers during the reaction to find the log-linear portion of the
 reaction. The relative amounts of the two products must then be
 determined. Usually this is done by labeling one of the dNTPs with a
 radioisotope and then quantifying the amount of label incorporated into
 each band after agarose gel electrophoresis. In comparison, the current
 invention allows the reaction to be monitored continuously so the
 log-linear portion of the amplification can be easily identified. Relative
 quantification can be done quickly by integration of melting peaks. An all
 day process is reduced to less than an hour.
 From the foregoing discussion, it will be appreciated that fluorescence
 monitoring during DNA amplification is an extraordinarily powerful
 analytical technique. When sequence-specific detection and quantification
 are desired, resonance energy transfer probes can be used instead of
 double-strand-specific DNA dyes. The Tm of hybridization probes shifts
 about 4-8.degree. C. if a single base mismatch is present. If a
 hybridization probe is placed at a mutation site, single base mutations
 are detectable as a shift in the probe melting temperature.
 EXAMPLE 21
 The factor V Leiden mutation is a single base change (G to A) that
 substitutes a glutamine residue for an arginine residue at amino acid
 residue 506 (R506Q). For further information, see R. M. Bertina et al.,
 Mutation in Blood Coagulation Factor V Associated with Resistance to
 Activated Protein C, 369 Nature 64-67 (1994) and J. Voorberg et al.,
 Association of Idiopathic Venous Thromboembolism with a Single
 Point-Mutation at Arg.sup.506 of Factor V, 343 Lancet 1535-36 (1994), both
 of which are hereby incorporated by reference. As used herein, "factor V
 Leiden mutation locus" means the nucleotide position in the factor V gene
 at which a guanine base in the wild type is replaced by an adenine base in
 the factor V Leiden mutant. SEQ ID NO:9 shows a portion of the wild type
 factor V gene, and SEQ ID NO:10 shows the corresponding portion of the
 factor V Leiden gene, with the relevant nucleotide at position 31 in each
 case. The complete nucleotide sequence of the factor V gene is described
 at R. J. Jenny et al., Complete cDNA and Derived Amino Acid Sequence of
 Human Factor V, 84 Proc. Nat'l Acad. Sci. USA 4846-50 (1987), hereby
 incorporated by reference, and sequences can also be obtained at Genbank
 locus HUMF10. The amino acid change in the mutant factor V protein makes
 this clotting factor resistant to degradation and increases the tendency
 to clotting and thrombosis. As the most common cause of inherited
 thrombophilia, this mutation is the target of a common laboratory test
 done in clinical molecular genetics laboratories.
 The standard method of analysis for the factor V Leiden mutation is to
 amplify the gene segment by PCR, digest the resulting amplified products
 with a restriction endonuclease that cuts the wild type sequence but not
 the mutant, and distinguish digested wild type and undigested mutant
 products by gel electrophoresis. R. M. Bertina et al., supra. This is a
 method well known in the art for analysis for defined mutations. Such a
 test usually requires about 4 hours, including PCR amplification (2
 hours), enzyme digestion (1 hour), and electrophoresis (1 hour).
 Post-amplification steps include opening the sample tube, adding the
 enzyme, and transferring the digested sample to the electrophoresis
 apparatus. Post-amplification processing increases the risk of end product
 contamination, and manual handling requires care to prevent mislabeling of
 samples. A method that simultaneously amplifies and analyzes for point
 mutations would eliminate these concerns.
 A method for complete amplification and analysis of the factor V Leiden
 mutation within 30 min in the same instrument comprises asymmetrically
 amplifying a portion of a human genomic DNA sample containing the mutation
 locus, followed by obtaining and analyzing a melting curve for the
 amplified DNA. Genomic DNA is prepared according to methods well known in
 the art, e.g. J. Sambrook et al., Molecular Cloning: A Laboratory Manual
 (2d ed., 1989), hereby incorporated by reference. Preferably, the melting
 curve is obtained by the resonance energy transfer methodology described
 above with a fluorogenic hybridization probe. Such an assay easily
 discriminates between homozygous wild type, homozygous mutant, and
 heterozygous genotypes. In a preferred embodiment, the oligonucleotide
 probe is 3'-labeled with fluorescein and designed to hybridize on the
 amplified DNA near to a Cy5-labeled primer for resonance energy transfer.
 This method can be applied to any defined mutation.
 The probe oligonucleotide is preferably about 15-40 nucleotide residues in
 length. The probe could conceivably contain as few as about 10 nucleotide
 residues, however, possible disadvantages of such short oligonucleotides
 include low specificity, low melting temperature, and increased
 background. Oligonucleotides larger than 40 residues could also be used,
 but would be unnecessarily expensive. Thus, the limits on the size of the
 probe oligonucleotide are only those imposed by functionality. The probe
 oligonucleotide should span the mutation, but the mutation preferably does
 not correspond to either the 5'- or 3'-terminal nucleotide residue of the
 probe. Since the present invention is based on melting curves, and lack of
 base pairing at the termini is known to have less of an effect on melting
 properties than at internal sites, the probe should be designed such that
 the mutation occurs at an internal position.
 The oligonucleotide primers for amplification of the selected mutation
 locus are preferably about 15 to 30 residues in length. Primers shorter
 than the preferred range could be used but may not be as specific as would
 be desired. Similarly, primers longer than the preferred range could be
 used, but would be unnecessarily expensive. Thus, the limits on the sizes
 of the PCR primers are only those imposed by functionality.
 The distance between the resonance energy transfer pair is also important
 for the proper functioning of the invention. The optimum distance between
 the resonance energy transfer pair is about 5 nucleotides. A distance of
 about 2 to 8 nucleotides is preferred, although a distance of up to about
 10-15 nucleotides is functional. Having the resonance energy transfer pair
 on adjacent nucleotides is not necessarily beneficial because the distance
 between the resonance energy transfer pair is effected by the position on
 the DNA helix.
 In this example, PCR amplification was carried out in 10 .mu.l reaction
 mixtures comprising 50 mM Tris, pH 8.3, 3 mM MgCl.sub.2, 500 .mu.g/ml
 bovine serum albumin, 200 .mu.M each dNTP, 0.5 .mu.M Cy5-labeled primer
 (SEQ ID NO:11), 0.2 .mu.M unlabeled opposing primer (SEQ ID NO: 12), 0.1
 .mu.M fluorescein-labeled probe (SEQ ID NO:13), 0.4 U Taq polymerase, and
 fifty ng human genomic DNA. Four different samples of DNA were tested:
 human genomic DNA from an individual homozygous for the factor V Leiden
 mutation; human genomic DNA from a heterozygous individual; human genomic
 DNA from an individual homozygous for the wild type factor V allele; and a
 negative control without DNA. The orientation of the Cy5-labeled primer,
 the fluorescein-labeled probe, and the mutation site (identified by
 asterisk) are shown below:
 ##STR1##
 The sequence of the unlabeled opposing primer was TGTTATCACACTGGTGCTAA (SEQ
 ID NO:12) and the amplified product was 186 base pairs in length. The
 Cy5-labeled primer was obtained as in Example 8. Cycling conditions were
 94.degree. C. for 0 sec (slope=20), 50.degree. C. for 10 sec (slope=20) ,
 and 72.degree. C. for 0 sec (slope=1) for 50 cycles, followed by cooling
 to 45.degree. C. and continuous fluorescence monitoring at a slope of
 0.2.degree. C./sec to 94.degree. C. for the melting curve. The highest
 quality melting curves were obtained at the end of amplification with a
 slow temperature transition rate (0.2.degree. C./sec-FIG. 46), although
 monitoring during each cycle at 1.degree. C./sec between 50.degree. C. and
 94.degree. C. also provided clear genotype identification (FIG. 47). The
 melting curves are easiest to visualize by plotting the negative
 derivative of fluorescence with respect to temperature vs temperature
 (-dF/dT vs T). Such a plot allows facile visual identification of all
 possible genotypes from the raw fluorescence data.
 The closer the Cy5 label is to the primer's 3'-end, the greater the
 resonance energy transfer signal. However, the 3'-end must have a free
 3'-hydroxyl for polymerase extension, and placing the Cy5 too close to the
 3'-end (either on the 3' or penultimate base) may inhibit polymerase
 attachment and extension. The 3'-fluorescein probe should hybridize as
 close to the primer as possible (minor overlap of 1-3 bases can be
 tolerated) and the mutation site should be near the middle of the probe. A
 5-base separation between the hybridized fluorophores and a mutation at
 base 8 of a 23-mer probe gave a melting curve shift of 8.degree. C.
 between mutant and wild type sequences (FIG. 46).
 Mutation detection by probe melting can also be performed with 2 labeled
 probes instead of one labeled probe and one labeled primer. In this
 embodiment, one probe is labeled 5' with Cy5 and the other probe is
 labeled 3' with fluorescein. Since both these fluorescent probes can be
 synthesized directly from the amidites, a manual synthesis step is not
 required as it is in the primer/probe system. The fluorescein-labeled
 probe should be designed such that the mutation locus is near the center
 of the fluorescein-labeled probe. The length of the Cy5-labeled probe
 should be designed such that it melts at a higher temperature (&gt;5.degree.
 C.) than the fluorescein-labeled probe which spans the mutation locus.
 Because background from fluorescein is more troublesome than that from
 Cy5, the concentration of the Cy5-labeled probe should preferably be 2-5
 fold that of the fluorescein-labeled probe. The two probes should
 hybridize to the same strand of genomic DNA, and the resonance energy
 transfer pair should be separated by about 0 to 5 nucleotide residues.
 Alternately, the probe that spans the mutation site can be labeled with
 Cy5 and the other probe labeled with fluorescein.
 It will be appreciated that the particular probes and primers disclosed
 herein for detection of the factor V Leiden mutation are merely
 illustrative, and that a person of ordinary skill in the art will be able
 to design other probes and primers for detection of mutations without
 undue experimentation by following the principles and guidelines set forth
 herein. It should also be recognized that although the invention is
 described with respect to detection of a single base mutation in genomic
 DNA, the same principles can be applied to detection of a mutation in
 cDNA. Preparation of the cDNA requires extra process steps and time, as is
 well known in the art, thus it is preferred to use genomic DNA because of
 the advantages of speed and lower cost. Further, the same technique can be
 used to detect insertions and deletions by designing the hybridization
 probe so that it melting temperature changes when the mutation or
 polymorphism is present. The invention can be used to detect any known
 mutation where a probe can be designed to differ in melting temperature
 when hybridized to mutant vs wild type.
 Although fluorescein and Cy5 were used as resonance energy transfer labels
 in the example above, other acceptors, such as Cy5.5, can also be used
 with fluorescein.
 EXAMPLE 22
 The factor V locus of Example 21 was amplified as before except that the
 primer was labeled with Cy5.5 instead of Cy5. Cy5.5 emission was observed
 through a 683 nm long pass dichroic and a 683-703 nm bandpass interference
 filter. The Cy5.5 to fluorescein ratio increased above background at about
 cycle 30 and the ratio approximately doubled by 50 cycles of asymmetric
 amplification. When amplified with wild type DNA, the probe Tm was
 65-66.degree. C. as judged by melting peaks.
 Another example for detecting single base mutations will now be given.
 EXAMPLE 23
 There is a common point mutation in the methylenetetrahydrofolate reductase
 (MTHFR) gene (C.sub.677 T) that converts an alanine to a valine residue
 and results in a thermolabile enzyme. This mutation can reduce MTHFR
 activity and lead to elevated homocysteine plasma levels which has been
 implicated as an independent risk factor for early vascular disease and
 thrombosis as is well known in the art. One of the primers was labeled
 with Cy5 (TGAAGGAGAAGGTGTCT*GCGGGA) (SEQ ID NO:25) where T* represents a
 modified T residue linked to Cy5 (see Example 9 for synthesis and
 purification). The probe sequence was
 fluorescein-CCTCGGCTAAATAGTAGTGCGTCGA (SEQ ID NO:26) and the other primer
 was AGGACGGTGCGGTGAGAGTG (SEQ ID NO:27). A 198 base pair fragment of the
 MTHFR gene was amplified from 50 ng of human genomic DNA in 50 mM Tris, pH
 8.3, 2 MM MgCl.sub.2, 500 .mu.g/ml bovine serum albumin, 0.2 mM of each
 dNTP, 0.5 .mu.M of the Cy5-labeled primer, 0.1 .mu.M of the opposing
 primer, 0.1 .mu.M of the fluorescein-labeled probe, and 0.4 U Taq DNA
 polymerase per 10 .mu.l. Each cycle was 30 sec long and consisted of
 denaturation at 94.degree. C. followed by a 20 sec combined
 annealing/extension step at 60.degree. C. The temperature transition rate
 between steps was 20.degree. C./sec. After 60 cycles, a melting curve was
 acquired as follows: heating from 50-65.degree. C. at 0.5.degree. C./sec,
 65-75.degree. C. at 0.1.degree. C./sec, and 75-94.degree. C. at
 0.5.degree. C./sec. After baseline subtraction and conversion to melting
 peaks, all possible genotypes were easily distinguished (FIG. 48).
 The discriminatory power of hybridization probes is also used to great
 advantage in multiplex or competitive PCR. For example, an artificial
 template is designed with a single internal base change and a
 hybridization probe designed to cover the base change as in Examples 21
 and 23. Relative amplification of the competitor and natural template are
 determined by acquiring and integrating melting peaks as in Example 16.
 Alternately, if multiple detection probes are used that sequentially melt
 off different targets at different temperatures, relative quantification
 is achieved by the same analysis. In general, any quantitative technique
 described previously for double-strand-specific DNA dyes can be made
 sequence specific with hybridization probes.
 Absolute Product Concentration by Product Reannealing Kinetics. Product
 concentration determinations are also advantageously carried out using the
 present invention. Continuous monitoring of double stranded DNA formation
 allows DNA quantification at any cycle of amplification by reannealing
 kinetics. The sample temperature is quickly dropped from the denaturation
 temperature and held constant at a lower temperature that is still high
 enough to prevent primer annealing (FIG. 2). The rate of product
 reannealing follows second order kinetics (see B. Young & M. Anderson,
 Quantitative analysis of solution hybridization, In: Nucleic Acid
 Hybridization: A Practical Approach 47-71 (B. Hames & S. Higgins, eds.,
 (1985), which is now incorporated herein by reference). For any given PCR
 product and temperature, a second order rate constant can be measured.
 Once the rate constant is known, any unknown DNA concentration can be
 determined from experimental reannealing data. Cooling is never
 instantaneous, and some reannealing occurs before a constant temperature
 is reached. Rapid cooling will maximize the amount of data available for
 rate constant and DNA concentration determination. The technique requires
 pure PCR product, but such can be assured by melting curves also obtained
 during temperature cycling using the present invention. This method of
 quantification by the present invention is advantageously independent of
 any signal intensity variations between samples.
 EXAMPLE 24
 A 536 base pair fragment of the beta-globin gene was amplified from human
 genomic DNA (Example 7) and purified (see Example 2). Different amounts of
 the purified DNA were mixed with a 1:30,000 dilution of SYBR.TM. Green I
 in 5 .mu.l of 50 mM Tris, pH 8.3 and 3 mM MgCl.sub.2. The samples were
 denatured at 94.degree. C. and then rapidly cooled to 85.degree. C. The
 fluorescence at 520-550 nm was monitored at 85.degree. C. over time. When
 different concentrations of DNA were tested, the shape of the reannealing
 curve was characteristic of the DNA concentration (See FIG. 49). For any
 given PCR product and temperature, a second order rate constant can be
 determined. FIG. 50 shows the determination of a second order reannealing
 rate constant for 100 ng of the 536 base pair fragment in 5 .mu.l at
 85.degree. C. The curve was fit by non-linear least squares regression
 with F.sub.max, F.sub.min, t.sub.0 and k as the floating parameters using
 the second order rate equation shown in FIG. 50. Analysis programs for
 this kind of curve fitting are well known in the art (for example, the
 user defined curve fit of Delta Graph, DeltaPoint, Inc, Monteray, Calif.).
 Once the rate constant is known, an unknown DNA concentration can be
 determined from experimental reannealing data.
 With the rate constant (k) defined, DNA concentrations are determined on
 unknown samples. The fluorescence vs time curves of unknown samples are
 fit by non-linear least squares regression, preferably during temperature
 cycling in real time (for example, using the nonlinear Levenberg-Marquardt
 method described in the LabView programming environment, National
 Instruments, Austin, Tex.). For this fit, F.sub.max, F.sub.min, t.sub.0,
 and [DNA] are the floating parameters and k is constant.
 Since some fluorescent dyes affect reannealing in a concentration dependent
 manner, the assumption of second order kinetics for product reannealing is
 checked by determining the rate constant at different standard DNA
 concentrations. The relationship is defined and alternate formula for
 fitting incorporated as necessary.
 Also within the scope of the present invention is to use probe annealing
 rates to determine product concentrations. The rate of fluorescence
 resonance energy transfer is followed over time after a quick drop to a
 probe annealing temperature that is greater than the primer annealing
 temperature (FIG. 2). For the case of amplification with a labeled primer
 and one labeled probe, the rate of annealing (and fluorescence generation)
 is second order. When using two labeled probes, the rate of fluorescence
 development is third order. These two arrangements are shown in FIG. 18.
 When the concentration of the probe(s) is much greater than the product
 concentration, pseudo-first order and pseudo-second order equations are
 adequate to describe the possibilities. The appropriate rate equations for
 these different conditions are well known in the art (see Young, B. and
 Anderson, M., supra). For the purposes of this invention, it is adequate
 that the prior art suggests appropriate rate equations that are tested
 experimentally and corrected if necessary.
 When probe annealing rates are used to determine product concentrations,
 possible interfering effects include product reannealing (with probe
 displacement by branch migration) and primer annealing and extension
 through the probe. The later is minimized when the probe Tm's are higher
 than the primer Tm's and a probe annealing temperature is chosen to
 minimize primer annealing. FIG. 13 shows that even if extension occurs,
 the fluorescence increases with time for about 20 sec. During this period,
 the fluorescence increase depends on product concentration.
 Probe annealing rates are used to determine product concentration similar
 to the method described above for determining product concentration by
 product reannealing. The steps are summarized as follows: (1) choosing the
 appropriate rate equation for the system, (2) running known DNA standards
 to determine the rate constant, (3) checking the validity of the rate
 equation by comparing different rate constants derived from different
 concentrations, and (4) using the rates constants to determine the DNA
 concentration of unknowns from their probe annealing data.
 Fluorescence Feedback for Control of Temperature Cycling. In contrast to
 endpoint and cycle-by-cycle analysis, the present invention can also
 monitor fluorescence throughout each temperature cycle. Continuous
 fluorescence monitoring can be used to control temperature cycling
 parameters. The present invention uses fluorescence feedback for real time
 control and optimization of amplification. Continuous fluorescence
 monitoring of PCR samples containing a double-strand-specific DNA dye or
 fluorescently labeled oligonucleotide probes can be used to monitor
 hybridization and melting during individual amplification cycles. This
 information can be used by the temperature control algorithms within the
 temperature cycling apparatus to improve and customize thermal cycling
 conditions. Conventional PCR is performed by programming all cycling
 parameters before amplification. With continuous monitoring, determination
 of temperature cycling requirements can occur during amplification, based
 on continuous observation of annealing, extension, and denaturation. The
 potential benefits of using hybridization information to control
 temperature cycling include:
 1. Ensuring complete denaturation of the PCR product each cycle while:
 a. Minimizing exposure to excessively high denaturation temperatures thus
 avoiding heat-induced damage to the amplification products and polymerase.
 Limiting the time product is exposed to denaturation temperatures is
 especially useful for amplification of long products.
 b. Increasing reaction specificity by minimizing the denaturation
 temperature. This selects against products with a Tm higher than the
 intended amplification product.
 2. Maximizing the amplification efficiency by ensuring adequate time for
 primer annealing each cycle while:
 a. Minimizing the amount of time required for amplification by allowing no
 longer than is needed to reach a certain efficiency of primer annealing.
 b. Enhancing reaction specificity by minimizing the time at the annealing
 temperature.
 3. Maximizing the amplification efficiency by ensuring adequate time for
 product extension each cycle while:
 a. Minimizing the amount of time required for amplification by allowing no
 longer than needed to complete product extension.
 b. Enhancing reaction specificity by selecting against products longer than
 the intended amplification product. These would require longer than the
 allotted time to complete product extension.
 4. Initiating thermal cycling changes dependent on the level of
 fluorescence obtained or the current efficiency of amplification. For
 example, over-amplification and nonspecific reaction products can be
 minimized by terminating thermal cycling when the efficiency drops to a
 certain level. As another example, temperature cycling can be modified to
 initiate slower temperature ramps for melting curve acquisition when the
 fluorescence becomes detectable. This saves time because the slower ramps
 need not be used on earlier cycles. Other desirable changes may become
 evident on continued practice of the invention.
 Control is based on an estimate of reaction parameters from the
 fluorescence data. The original fluorescence data is either acquired as a
 change in fluorescence over time (temperature specific rates of
 denaturation, annealing, and extension), a change in fluorescence over
 temperature (product or probe Tm), or a change in extent of amplification
 (amplification yield and efficiency). These rates, Tm's and their first
 and second derivatives are used to determine optimal reaction parameters
 that include denaturation temperature and time, primer annealing
 temperature and time, probe annealing temperature and time, enzyme
 extension temperature and time, and number of cycles.
 Double-strand-specific DNA dyes are used for the control of denaturation,
 control of extension, and to initiate thermal cycling changes at a certain
 amplification level or efficiency. Resonance energy transfer dyes are used
 for the control of annealing as will be described after the following
 example.
 EXAMPLE 25
 A commercial fluorescence monitoring thermal cycler (LC24 LightCycler,
 Idaho Technology Inc., Idaho Falls, Id.) was modified so that the software
 is no longer programmed with temperature/time setpoints, but is programmed
 to acquire fluorescence values, then to use these values for thermal
 cycler control.
 As depicted in the Functional Block Diagram (FIG. 51), the Run-Time Program
 communicates through serial and DAQ-board interfaces with the LightCycler.
 This allows high level access to either temperature or fluorescence data
 and either can be used by the Board-level Software for temperature
 control. However, in the current embodiment of the instrument, only the
 temperature data is converted into digital form at the Controller Hardware
 level. The fluorescence data is sent in analog form through the Digital
 acquisition board interface, is analyzed by the Run-time Program, and is
 sent back to the Board-level software via the serial interface. Product
 melting control:
 A melting peak was acquired for the intended PCR product and a baseline
 fluorescence was acquired for the sample containing the reaction cocktail
 at the temperature at which the product was completely melted.
 Each cycle of the reaction then used this fluorescence value as a target.
 The approach to product denaturation was made in two stages to overcome
 the time-lag due to the requirement of sending the fluorescence value to a
 remote computer for analysis, then returning the instruction that the
 value had been reached. With each product melting step, the temperature
 was increased until the fluorescence reached an intermediate value, then
 the heating power was reduced so that a temperature ramp rate of roughly
 3.degree./sec gave the computer time to analyze the fluorescence and
 signal the thermal cycler that product denaturation had occurred.
 The resulting temperature/time plot (FIG. 52) shows a characteristic
 increase in the melting temperature after cycle 20 as the concentration of
 amplification product increases. Product Tm is a function of product
 concentration.
 Product Annealing/Extension:
 During an extended hold at a combined annealing/extension temperature, the
 fluorescence was monitored and this information was used to ensure that
 adequate, but not excessive time had been allowed for product extension.
 The fluorescence was monitored at 10 second intervals, if the fluorescence
 increased more than a settable ratio (typically 1.00-1.05), then the
 annealing/extension step was continued. Otherwise, the next product
 melting step was initiated. The interval of 10 seconds was chosen to give
 a minimum of 20 seconds at the combined annealing/extension temperature.
 The resulting fluorescence/time plot (FIG. 52) shows a characteristic
 increase in the dwell time at the combined annealing/extension temperature
 as the concentration of amplification product grows. As the primer
 concentration and polymerase become limiting, more time is needed to
 complete product extension with each cycle.
 Amplification Plateau:
 At the end of each annealing/extension step, the fluorescence value was
 acquired and stored. When this value had increased to 1.2 times the lowest
 end-cycle fluorescence value and had subsequently stopped increasing below
 a user settable ratio (typically 1.00-1.02), the thermal cycling was
 terminated. Alternately, a melting-curve acquisition step was initiated by
 entering a slow 0.1-0.2.degree. C./second temperature ramp through the
 product Tm and monitoring the fluorescence of the sample continuously.
 The resulting fluorescence/time plot (FIG. 52) shows that after twenty-five
 cycles of amplification the ratio of cycle-by-cycle fluorescence growth
 fell below 1.00 and the reaction was terminated.
 In one embodiment of the present invention, detection of the amplification
 plateau is used to acquire a high-resolution melting curves for each
 sample in a multiple sample run at the optimal temperature cycle for each
 sample. As a sample reaches its amplification plateau, a melting-curve is
 acquired for that sample, then regular temperature cycling is resumed
 until another reaction reaches its amplification plateau.
 Real time monitoring and control of annealing distinct from extension is
 also provided by the present invention. If one of the primers is
 3'-labeled with Cy5, no extension can occur. However, if labeled primer
 (1-10%) is mixed with unlabeled primer (90-99%), amplification efficiency
 will be slightly decreased, but annealing is observable as fluorescence
 energy transfer from a double-strand-specific dye to Cy5. The primer with
 the lowest Tm (as determined by nearest neighbor thermodynamics as known
 in the art) is labeled with Cy5 and SYBR.TM. Green I is included as a
 double-strand-specific dye. Alternately, primer annealing can be monitored
 indirectly with equivalent complementary oligonucleotides. An
 oligonucleotide of the same length and Tm as the lowest melting primer is
 designed with no complementarity to the amplified sequence. This
 oligonucleotide is 5'-labeled with Cy5 and its complement is 3'-labeled
 with fluorescein or some other resonance energy transfer pair.
 Hybridization of these oligonucleotides is followed by resonance energy
 transfer. The concentration of one probe is made the same as the
 concentration of the lowest Tm primer and the concentration of the other
 probe is made much less than this in order to obtain pseudo-first-order
 kinetics that approximates the pseudo-first-order kinetics of primer
 annealing to product. The efficiency of annealing is monitored and used to
 control annealing temperature and times by one of these methods.
 It is also within the scope of the present invention to entirely replace
 temperature and time setpoints with fluorescence feedback control. For
 example, three samples are placed in a fluorescence temperature cycler
 with feedback capacity. The samples are:
 1. A non-reacting sample containing amplified product and SYBR.TM. Green I.
 2. A non-reacting sample containing complementary fluorescently labeled
 primers with a Tm equal to the lowest Tm primer and concentrations as
 noted above.
 3. The sample to be amplified and SYBR.TM. Green I.
 With each cycle of amplification, product denaturation is ensured by
 monitoring sample 1 as the temperature is increased. A melting curve is
 determined in real-time and when the sample has denatured, the transition
 to the annealing step is begun. Primer annealing is monitored indirectly
 through the hybridization of two complementary primers in sample 2. One of
 the primers is 3' labeled with fluorescein and the other is 5' labeled
 with Cy5 or similar dye. The temperature is decreased until sample 2 shows
 primer hybridization as indicated by an increase in the ratio of
 fluorescence at 670 nm/540 nm. This ratio increases due to resonance
 energy transfer between the fluorophores when they are approximated by
 hybridization. Product extension is followed by monitoring the
 fluorescence of one or more of the actual samples as demonstrated in
 Example 25.
 Summary. From the foregoing discussion, it will be appreciated that
 continuous fluorescence monitoring during DNA amplification to monitor
 hybridization is an extraordinarily powerful analytical technique. Using
 the methods described herein and depending on the number of initial
 template copies present, product identification and quantification can be
 achieved in five to twenty minutes after temperature cycling has begun.
 The present invention achieves several advantages not heretofore available
 in the art. For example, the present invention provides single-color
 fluorescence methods to monitor product purity, relative quantitation by
 multiplex PCR or competitive PCR, absolute product quantification by
 reannealing kinetics, and an improved method for initial template
 quantification by fluorescence vs cycle number plots. The present
 invention also provides dual-color, sequence-specific methods for sequence
 variation detection, relative quantitation by multiplex PCR or competitive
 PCR, product quantification by probe annealing kinetics, and initial
 template quantification by fluorescence vs cycle number plots.
 The following table compares double-strand-specific DNA dyes, hydrolysis
 probes, and hybridization probes useful in continuous monitoring of PCR.
 The fluorescence of double-strand-specific DNA dyes depends on the strand
 status of the DNA. The dual-labeled hydrolysis probes are quenched while
 intact and donor fluorescence increases when the probe is hydrolyzed.
 Hybridization probes depend on increased resonance energy transfer when
 hybridization brings 2 fluorophores closer together.

Summary of Fluorescent Probes
 for Continuous Monitoring of PCR
 Fluorescent Probe
 dsDNA dye Hydrolysis Hybridization
 Mechanism Strand status Quenching Transfer
 Probe Synthesis Unnecessary Difficult Simple
 Specificity Product Tm Sequence Sequence
 Melting Analysis Yes No Yes
 Multicolor Analysis No Yes Yes
 In accordance with the present invention, time, temperature and
 fluorescence are acquired 1-10 times every sec and fine details of product
 and/or probe hybridization are observed during temperature cycling. With
 double-strand-specific DNA dyes, the hybridization of product with respect
 to temperature is used to identify products by melting curves. In
 addition, relative product quantification is achieved by multiplex
 amplification of two or more different products that differ in Tm.
 Further, competitive PCR is performed by altering the sequence internal to
 the common primers so that two or more products have different Tm's.
 Absolute product quantification is obtained by rapidly cooling the
 denatured product and observing reannealing kinetics. The sensitivity of
 initial template quantification with fluorescence vs cycle number plots is
 increased by analysis of product melting curves to control for nonspecific
 amplification and curve fitting algorithms. Finally, immediate
 fluorescence feedback for control of denaturation conditions, elongation
 times and product yield are obtained by monitoring product strand status
 with double-strand-specific DNA dyes.
 The ability to monitor probe hybridization with fluorescence during
 temperature cycling is a powerful tool. The present invention provides
 dual-color fluorescence methods that depend on probe hybridization (not
 hydrolysis) for sequence-specific detection and quantification during PCR.
 The annealing kinetics and melting of hybridization probes provides
 information not available with probes that rely on exonuclease hydrolysis
 between fluorophores. Continuous monitoring of sequence-specific probe
 hybridization can be followed over temperature changes by resonance energy
 transfer. Probe melting occurs at a characteristic temperature determined
 by its sequence and complementarity to the product. Two schemes have been
 detailed by the present invention, (1) two adjacent hybridization probes,
 and (2) one labeled probe that hybridizes to a single stranded PCR product
 that incorporates a labeled primer. The melting temperature of
 sequence-specific probes identifies and discriminates products during PCR.
 DNA polymorphisms or mutations, including single base mutations, are
 detected by probe Tm shifts. In addition, relative product quantification
 is achieved by multiplex amplification of at least two different products
 with one or more probes that melt from their respective products at
 different temperatures. Further, competitive PCR is performed by altering
 the sequence internal to the primers so that one or more probes hybridize
 to the competitor and the natural template at different Tmls. Alternately,
 relative or competitive PCR are performed by multicolor analysis with
 probes labeled with different fluorophores, such as Cy5 and Cy5.5.
 Absolute product concentration is determined by analysis of probe
 annealing kinetics. Initial template copy number is determined from
 fluorescence vs cycle number plots by curve fitting algorithms.
 When multiplex analysis in one PCR reaction is desired, it is common
 practice to use different fluorescent labels with distinguishable emission
 spectra to identify the multiple products. The analysis is complicated by
 the limited number of fluorophores available and the overlapping emission
 spectra of those fluorophores that are available (see HM Shapiro, supra).
 Analysis of product or probe hybridization with melting curves is another
 method to distinguish multiple PCR products. By following hybridization
 during temperature cycling, the number of probes and/or spectral colors
 needed to distinguish multiple products can be minimized.
 The present invention may be embodied in other specific forms without
 departing from its spirit or essential characteristics. The described
 embodiments are to be considered in all respects only as illustrative and
 not restrictive. The scope of the invention is, therefore, indicated by
 the appended claims rather than by the foregoing description. All changes
 which come within the meaning and range of equivalency of the claims are
 to be embraced within their scope.
 Programming code for carrying out melting curve and other analyses is found
 in the Programming Code Appendix (Microfiche).