United States Patent [19J
`Wittwer et al.
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US006140054A
`6,140,054
`[11] Patent Number:
`[45] Date of Patent:
`Oct. 31, 2000
`
`[54] MULTIPLEX GENOTYPING USING
`FLUORESCENT HYBRIDIZATION PROBES
`
`[75]
`
`Inventors: Carl T. Wittwer; Philip S. Bernard,
`both of Salt Lake City, Utah
`
`[73] Assignee: University of Utah Research
`Foundation, Salt Lake City, Utah
`
`[21]
`
`Appl. No.: 09/164,023
`
`[22]
`
`Filed:
`
`Sep. 30, 1998
`
`[51]
`[52]
`
`[58]
`
`[56]
`
`Int. CI.7 ....................................................... C12Q 1/68
`U.S. Cl . ............................ 435/6; 435/91.2; 536/23.1;
`935/77; 935/78
`Field of Search ..................... 435/6, 91.2; 536/23.1;
`935/77, 78
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,403,707
`5,475,098
`5,591,578
`5,888,739
`
`4/1995 Atwood et al. ............................. 435/5
`12/1995 Hall et al. .............................. 536/23.7
`1/1997 Meade et al. ............................... 435/6
`3/1999 Pitner et al. ................................ 435/6
`
`FOREIGN PAfENT DOCUMENTS
`
`8/1993 WIPO .............................. C12Q 1/68
`WO 93/16194
`WO 97/46707 12/1997 WIPO .
`WO 97/46712 12/1997 WIPO .
`WO 97/46714 12/1997 WIPO .
`
`01HER PUBLICATIONS
`
`Lay et al., "Real-Time Fluorescence Genotyping of Factor
`V Leiden During Rapid-Cycle PCR", Clinical Chemistry
`43, No. 12, pp. 2262-2267, 1997.
`
`Bernard et al., "Integrated Amplification and Detection of
`the C677T Point Mutation in the Methylenetetrahydrofolate
`Reductase Gene by Fluorescence Resonance Energy Trans(cid:173)
`fer and Probe Melting Curves", Analytical Biochemistry
`255, pp. 101-107, 1998.
`Ririe et al., "Product Differentiation by Analysis of DNA
`Melting Curves During the Polymerase Chain Reaction",
`Analytical Biochemistry 245, pp. 154-160, 1997.
`
`Wittwer et al., "Continuous Fluorescence Monitoring of
`Rapid Cycle DNA Amplification", Bio Techniques 22, pp.
`130-138, Jan., 1997.
`
`Wittwer et al., "The LightCyclerTM: A Microvolume Multi(cid:173)
`sample Fluorimeter with Rapid Temperature Control", Bio(cid:173)
`Techniques 22, pp. 176-181, Jan. 1997.
`
`Morrison et al., "Quantification of Low-Copy Transcripts
`by Continuous SYER® Green I Monitoring During Ampli(cid:173)
`fication", BioTechniques, vol. 24, No. 6, pp. 954-962, 1998.
`
`Primary Examiner-Kenneth R. Horlick
`Assistant Examiner-Janell E. Taylor
`Attorne}\ Agent, or Firm-Barnes & Thornburg
`
`[57]
`
`ABSTRACT
`
`The present invention is directed to a mutation detection kit
`and method of analyzing multiple loci of one or more
`nucleic acid sequences for the presence of mutations or
`polymorphisms. More particularly, the present invention
`relates to the use of the polymerase chain reaction (PCR) and
`fiuorescently labeled oligonucleotide hybridization probes
`to identify mutations and polymorphisms based on melting
`curve analysis of the hybridization probes.
`
`20 Claims, 23 Drawing Sheets
`
`Temperature (°C)
`
`0
`
`20
`
`40
`
`60
`
`Time (sec)
`
`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 1of23
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`6,140,054
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`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 2 of 23
`
`6,140,054
`
`95
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`SAMPLE
`TEMP('C)
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`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 3 of 23
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`6,140,054
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`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 4 of 23
`
`6,140,054
`
`Cy5 MonoFunctional Dye
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`Fig. 4
`
`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 5 of 23
`
`6,140,054
`
`Cy5.5 MonoFunctlonal Dye
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`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 6 of 23
`
`6,140,054
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`
`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 7 of 23
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`6,140,054
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`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 8 of 23
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`6,140,054
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`U.S. Patent
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`Oct. 31, 2000
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`Sheet 9 of 23
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`

`
`U.S. Patent
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`Oct. 31, 2000
`
`Sheet 10 of 23
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`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 11 of 23
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`6,140,054
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`
`U.S. Patent
`
`Oct. 31, 2000
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`Sheet 12 of 23
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`
`U.S. Patent
`
`Oct. 31, 2000
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`Sheet 13 of 23
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`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 16 of 23
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`6,140,054
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`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 19 of 23
`
`6,140,054
`
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`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 20 of 23
`
`6,140,054
`
`2.2
`Fluorescence
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`
`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 21 of 23
`
`6,140,054
`
`1.9
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`
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`
`70
`
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`
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`
`Fig. 18
`
`THERMO FISHER EX. 1028
`
`

`
`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 22 of 23
`
`6,140,054
`
`187G
`
`G845
`
`3
`
`-dF/dT
`2
`
`1
`
`/
`J
`!
`I
`I
`I
`I
`I
`I .
`
`50
`
`55
`
`60
`65
`Temperature ( °C)
`
`70
`
`75
`
`Fig. 19
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`THERMO FISHER EX. 1028
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`U.S. Patent
`
`Oct. 31, 2000
`
`Sheet 23 of 23
`
`6,140,054
`
`G845
`
`3
`
`Fluorescence
`(-dF/dT) 2
`
`1
`
`_.: \
`•• ••
`•• ··"
`
`\.
`..
`~.
`0 -1--1:.-..~--,r--~~.....,..·'~·.-...:::;.~.;.;.._~~--____;::.oi~
`75
`50
`60
`55
`65
`70
`
`845A
`
`·. . .
`
`C187
`: · .
`
`187G
`
`,_.,
`
`I
`I
`I
`
`Temperature (°C)
`Fig. 20
`
`THERMO FISHER EX. 1028
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`
`6,140,054
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`s
`
`1
`MULTIPLEX GENOTYPING USING
`FLUORESCENT HYBRIDIZATION PROBES
`
`US GOVERNMENT RIGHTS
`
`This invention was made v.ith United States Government
`support under Grant No. GM5 1647, awarded by the
`National Institute of Health. The United States Government
`has certain rights in the invention.
`
`FIELD OF THE INVENTION
`
`The present invention is directed to a method of analyzing
`multiple loci of one or more nucleic acid sequences for the
`presence of mutations or polymorphisms. More particularly,
`the present invention relates to the use of the polymerase
`chain reaction (PCR) and fluorescently labeled oligonucle(cid:173)
`otide hybridization probes to identify mutations and poly(cid:173)
`morphisms based on melting curve analysis of the hybrid(cid:173)
`ization probes.
`
`BACKGROUND OF THE INVENTION
`
`2
`possible by the use of high surface area-to-volume sample
`containers such as capillary tubes. The use of high surface
`area-to-volume sample containers allows for a rapid tem(cid:173)
`perature response and temperature homogeneity throughout
`the biological sample. Improved temperature homogeneity
`also increases the precision of any analytical technique used
`to monitor PCR during amplification.
`In accordance with the present invention amplification of
`a nucleic acid sequence is conducted by thermal cycling the
`10 nucleic acid sequence in the presence of a thermostable
`DNA polymerase. The method comprises the steps of plac(cid:173)
`ing a biological sample comprising the nucleic acid
`sequence in a capillary vessel, raising the temperature of the
`biological sample from a first temperature to a second
`15 temperature wherein the second temperature is at least 15°
`C. higher than the first temperature, holding the biological
`sample at the second temperature for a predetermined
`amount of time, lowering the temperature of the biological
`sample from the second temperature to at lea"St the first
`20 temperature and holding the biological sample at a tempera(cid:173)
`ture at least as low as the first temperature for a pre(cid:173)
`determined length of time. The temperature of the biological
`sample is then raised back to the second temperature, and the
`biological sample is thermocycled a predetermined number
`25 of times. In one embodiment, the method of amplifying a
`DNA sequence comprises a two temperature cycle wherein
`the samples are cycled through a denaturation temperature
`and an annealing temperature for a predetermined number of
`repetitions. However the PCR reaction can also be con-
`30 ducted using a three temperature cycle wherein the samples
`are cycled through a denaturation temperature, an annealing
`temperature and an elongation temperature for a predeter(cid:173)
`mined number of repetitions.
`In one embodiment each temperature cycle of the PCR
`35 reaction is completed in approximately 60 seconds or less.
`Rapid cycling times can be achieved using the device and
`techniques described in U.S. Pat. No. 5,455,175, the disclo(cid:173)
`sure of which is expressly incorporated herein.
`In accordance with the present invention PCR amplifica-
`tion of one or more targeted regions of a DNA sample is
`conducted in the presence of a fluorescently labeled hybrid(cid:173)
`ization probes, wherein the probes are synthesized to hybrid(cid:173)
`ize to a specific locus present in a target amplified region of
`the DNA. In a preferred embodiment the hybridization
`probe comprises two oligonucleotide probes that hybridize
`to adjacent regions of a DNA sequence wherein each oli(cid:173)
`gonucleotide probe is labeled with a respective member of
`a fluorescent energy transfer pair. In this embodiment the
`presence of the target nucleic acid sequence in a biological
`sample i"> detected by measuring fluorescent energy transfer
`between the two labeled oligonucleotides.
`Fluorescence resonance energy transfer (FRET) occurs
`between two fluorophores when they are in physical prox-
`55 imity to one another and the emission spectrum of one
`fluorophore overlaps the excitation spectrum of the other.
`The rate of resonance energy transfer is
`
`60
`
`where:
`t=excited state lifetime of the donor in the absence of the
`acceptor;
`k'=an orientation factor between the donor and acceptor;
`n=refractive index of the visible light in the intervening
`medium;
`cw=quantum efficiency of the donor in the absence of the
`acceptor;
`
`As databases for polymorphic markers and disease caus(cid:173)
`ing mutations continue to grow, there is an increasing need
`for procedures that can screen nucleic acid sequences for the
`presence of known polymorphisms and mutations.
`Optimally, the procedure should be capable of analyzing
`multiple DNA sites simultaneously (including nucleic acid
`loci that are physically separated by great distances) for the
`presence of mutations or polymorphisms.
`Current methods for determining the genetic constitution
`of individuals (genotyping) include oligonucleotide ligation,
`allele-specific oligonucleotide hybridization, and PCR(cid:173)
`restriction fragment length analysis. All these methods
`require time consuming multiple manual steps. One alter(cid:173)
`native method of genotyping uses the melting temperature of
`fluorescent hybridization probes that hybridize to a PCR
`amplified targeted region of genomeinucleic acid sequence
`to identify mutations and polymorphisms.
`The polymerase chain reaction (PCR) is a technique of
`synthesizing large quantities of a preselected DNA segment.
`The technique is fundamental to molecular biology and is
`the first practical molecular technique for the clinical labo(cid:173)
`ratory. PCR is achieved by separating the DNA into its two
`complementary strands, binding a primer to each single
`strand, at the end of the given DNA segment where synthesis
`will start, and adding a DNA polymerase to synthesize the
`complementary strand on each single strand having a primer
`bound thereto. The process is repeated until a sufficient
`number of copies of the selected DNA segment have been
`synthesized. During a typical PCR reaction, double stranded
`DNA is separated into its single strands by raising the
`temperature of the DNA containing sample to a denaturing
`temperature where the two DNA strands separate (i.e. the
`''melting temperature of the DNA") and then the sample is
`cooled to a lower temperature that allows the specific
`primers to attach (anneal), and replication to occur (extend).
`In preferred embodiments a thermostable polymerase is
`utilized in the polymerase chain reaction. A preferred ther(cid:173)
`mostable DNA polymerase for use in the PCR reaction is the
`Taq DNA Polymerase and derivatives thereof, including the
`Stoffel fragment of Taq DNA polymerase and KienTaqI
`polymerase (a 5'-exonuclease deficient variant of Taq
`polymerase-see U.S. Pat. No. 5,436,149).
`Thermocycling may be carried out using standard tech- 65
`niques known to those skilled in the art, including the use of
`rapid cycling PCR. Rapid cycling techniques are made
`
`40
`
`45
`
`50
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`6,140,054
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`4
`the hybridization probes to their target complementary
`sequence directly impacts the temperature at which the
`hybridization probe will separate (melt) from the comple(cid:173)
`mentary strand. The greater the difference between the probe
`5 and the target complementary sequence the lower the tem(cid:173)
`perature needed to separate the hybridizing strands.
`Accordingly, an oligonucleotide probe identical in sequence
`to the complementary wild type sequence will dissociate
`from the locus containing a mutation at a lower temperature
`10 than it \vill from the wild type locus. The use of fiuorescently
`labeled hybridization probes enables dynamic monitoring of
`fluorescence as the temperature of the sample is raised and
`the melting curve for the hybridization probe is determined.
`The generated melting curve is then compared to the known
`15 melting curve for the normal, mutant or polymorphic
`sequence to determine the sequence of the target nucleic acid
`locus.
`
`3
`R=distance between the donor and acceptor measured in
`Angstroms;
`J DA. =the integral of (F n) ( e A.) (W4
`) with respect to W at all
`overlapping wavelengths with:
`F n=peak normalized fluorescence spectrum of the
`donor;
`e A. =molar absorption coefficient of the acceptor
`(M- 1cm- 1
`);
`W 4 =wavelength (nm).
`For any given donor and acceptor, a distance where 50%
`resonance energy transfer occurs can be calculated and is
`abbreviated R0 . Because the rate of resonance energy trans-
`fer depend'S on the 6th power of the distance between donor
`and acceptor, resonance energy transfer changes rapidly as
`R varies from R0 . At 2 R0 , very little resonance energy
`transfer occurs, and at 0.5 R0 , the efficiency of transfer is
`nearly complete, unless other forms of de-excitation pre(cid:173)
`dominate.
`Fluorescence resonance energy transfer can be used as a
`labeling system for detecting specific sequences of DNA. In 20
`combination with standard melting curve analysis, single
`point mutations in a gene can be distinguished from the
`normal gene. In accordance with one embodiment such a
`detection system comprises two oligonucleotides that
`hybridize to adjacent loci on DNA. The oligonucleotides are 25
`each labeled, respectively, with one of the fiuorophores of a
`fluorescent resonance energy transfer pair, so that upon
`hybridization of the two labeled oligonucleotides to their
`complementary sequences on the targeted DNA, resonant
`energy is transferred from the donor fiuorophore to the 30
`acceptor fiuorophore. Such an energy transfer event is
`detectable and is indicative of the presence of the target
`nucleic acid sequence.
`The fluorescently labeled oligonucleotides are designed to
`hybridize to the same strand of a DNA sequence resulting in 35
`the donor and acceptor fiuorophores being separated by a
`distance ranging from about 0 to about 25 nucleotides, more
`preferably about 0-5 nucleotides, and most preferably about
`0-2 nucleotides. A particularly preferred spacing bet\veen
`the donor and acceptor fluorophores is about 1 nucleotide. 40
`When one of the labeled oligonucleotides also functions
`as a PCR primer ("probe-primer"), then the two fiuores(cid:173)
`cently labeled oligonucleotides hybridize to opposite strands
`of a DNA sequence. In this embodiment, the donor and
`acceptor fluorophores are preferably within about 0-15 45
`nucleotides and more preferably within about 4-6 nucle(cid:173)
`otides.
`When both of the fiuorescently labeled oligonucleotides
`are not hybridized to their complementary sequence on the
`targeted DNA, then the distance between the donor fluoro- 50
`phore and the acceptor fluorophore is too great for resonance
`energy transfer to occur. Thus the acceptor fluorophore and
`the donor fluorophore are not in resonance energy transfer
`relationship and excitation of the donor fiuorophore will not
`produce a detectable increased fluorescence by the acceptor 55
`fluorophore.
`Acceptable fluorophore pairs for use as fluorescent reso(cid:173)
`nance energy transfer pairs are well know to those skilled in
`the art and include, but are not limited to, fiuorescein/
`rhodamine, phycoerythrin/Cy7, fluorescein/Cy5 or 60
`fiuorescein/Cy5 .5.
`The thermal stability of a DNA duplex relies on duplex
`length, GC content, and Watson-Crick base pairing. Changes
`from Watson-Crick pairing destabilize a duplex by varying
`degrees depending on the length of the mismatched duplex, 65
`the particular mismatch, the position of the mismatch, and
`neighboring base pairs. Accordingly, the percent identity of
`
`SUMMARY OF TIIE INVENTION
`The present invention is directed to the simultaneous
`detection of sequence alterations at two or more loci of an
`amplified DNA sequence as determined by melting-point
`analysis of the DNA-probe complexes formed at the respec(cid:173)
`tive regions of the PCR amplified DNA. More particularly,
`PCR primers are selected to amplify a preselected DNA
`segment containing nucleic acid loci that have been identi(cid:173)
`fied as harboring a particular mutation or polymorphism.
`The hybridization probes are designed to hybridize to the
`amplified region and span the site containing the mutation or
`polymorphism. In preferred embodiments the hybridization
`probes are labeled with a fluorescent label. Preferably, each
`FRET oligonucleotide pair comprises a pair of oligonucle(cid:173)
`otide probes including a donor oligonucleotide probe,
`labeled with a resonance energy transfer donor, and an
`acceptor oligonucleotide probe, labeled with a resonance
`energy transfer acceptor. The donor oligonucleotide probe
`and the acceptor oligonucleotide probe are designed so the
`two probes hybridize to adjacent regions of the same single
`strand of DNA and resonance energy is transferred from the
`donor fluorophore to the acceptor fiuorophore when both
`probes are bound to their respective complement.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is a temperature v. time chart exemplary of rapid
`temperature cycling for PCR.
`FIG. 2 shows the results of four different temperature/time
`profiles (A-D) and their resultant amplification products
`after thirty cycles (inset).
`FIGS. 3Aand 3B illustrate the mechanism of fluorescence
`generation for fluorescence monitoring of PCR through the
`use of a donor and acceptor oligonucleotide probes. FIG. 3C
`illustrate the mechanism of fluorescence generation for a
`three oligonucleotide probe system.
`FIG. 4 shows the chemical structure of the monovalent
`N-hydroxysuccinimide ester of Cy5T".
`FIG. 5 shows the chemical structure of the monovalent
`N-hydroxysuccinimide ester of Cy5.STM.
`FIG. 6 shows the emission spectrum of fiuorescein (solid
`line) and the excitation spectrum of Cy5 (broken line).
`FIG. 7 is a schematic representation of one embodiment
`of a rapid temperature cycler device with fluorescence
`detection at the tip of the sample containers.
`FIG. 8 is a perspective view of the exterior of the rapid
`temperature cycler device of FIG. 7.
`FIGS. 9A and 9B are perspective and cross sectional
`views, respectively, of a sample handling system for use in
`a rapid temperature cycler device.
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`6,140,054
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`FIG. 19 shows the derivative melting curves of 3 het(cid:173)
`erozygous genotypes. The 3'-fluorescein labeled probe span(cid:173)
`ning the region amplified within exon 2 (see FIG. 16) reveals
`the heterozygous C187G genotype (- - -) at the H63D
`s site and the heterozygousA193T genotype(-) at the S65C
`site. The probe spanning the C282Y site identifies the
`heterozygous G845A genotype (- -- -).
`FIG. 20 shows homogenous multiplex genotyping by
`derivative melting curves for 4 alleles. Shown are 4 samples
`with different C282Y/H63D genotypes: homozygous C187
`(- -
`-), homozygous G845/homozygous 187G
`(- -- -), homozygous 845Nhomozygous C187 (-- -- --)
`and heterozygous G845Nheterozygous C187G (-).
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`20
`
`5
`F1G. 10 is a schematic representation of another embodi(cid:173)
`ment of rapid temperature cycler device which accommo(cid:173)
`dates multiple sample handling trays.
`F1G. 11 shows resonance energy transfer occurring
`between fluorescein- and Cy5-labeled adjacent hybridiza(cid:173)
`tion probes at each cycle during PCR.
`F1G. 12 is a fluorescence ratio v. cycle number plot
`distinguishing two hybridization probe designs monitored
`by resonance energy transfer: ( 0) two hybridization probes
`labeled respectively with fluorescein and Cy5; and ( +) a 10
`primer labeled with Cy5 and a probe labeled with fluores-
`cem.
`F1GS. 13A and 13B show (A) melting curves and (B)
`melting peaks for PCR products of a person heterozygous
`for the factor V Leiden mutation (solid line), homozygous 15
`for the factor V Leiden mutation (dotted line), homozygous
`wild type (broken line), and no DNA control (alternating dot
`and dash).
`FIG. 14 shows a fluorescence ratio v. temperature plot of
`continuous monitoring during cycle 40 of PCR products of
`a sample homozygous for the factor V Leiden mutation
`(solid line), heterozygous for the factor V Leiden mutation
`(dotted line), and homozygous \Vild type (alternating dot and
`dash).
`FIG. 15 shows melting peaks of a homozygous mutant of
`the methylenetatrahydrofolate gene (solid line), homozy(cid:173)
`gous wild type (broken line), heterozygous mutant (dotted
`line), and no DNA control (alternating dot and dash).
`F1G. 16 is a schematic representation showing primer and
`probe placement for multiplex amplification and genotyping 30
`of HFE. Upstream (U) and downstream (D) primers are
`illustrated with respect to exon boundaries. Regions of exon
`2 and exon 4 were amplified for analysis of the H63D
`(C187G) and C282Y (G845A) mutations, respectively. The
`fluorescein (F) labeled probes are in fluorescence resonance 35
`energy transfer with the more thermally stable Cy5 (Y)
`labeled probes. The fluorescein labeled probes form a single
`mismatch when hybridizing to the singe-stranded wild type
`allele. The probe hybridizing within exon 2 forms two
`mismatches when hybridizing to a wild type (C187) allele 40
`containing the S65C (193T) polymorphism.
`F1G. 17 shows a real-time amplification and genotyping
`of the C282Y site. The inset shows amplification (Cy5
`fluorescence vs cycle number) of the genotypes: homozy(cid:173)
`gous wild type (- -- -), heterozygous C282Y (-), 45
`homozygous C282Y (- -
`-). A no template control
`(-- -- --) is also included. Data for amplification and melting
`curve analysis were normalized to baseline for each sample
`by dividing each fluorescence value by the minimum fluo(cid:173)
`rescence signal for that sample. Melting curve plots (top) of 50
`Cy5 fluorescence (F) versus temperature (T) are transformed
`into melting peaks (bottom) by plotting -dF/dT vs tempera(cid:173)
`ture. Both amplification and genotyping analysis are com(cid:173)
`pleted within 45 minutes.
`F1G. 18 shows a real-time amplification and genotyping
`of the H63D site. The inset shows amplification (Cy5
`fluorescence vs cycle number) of the 3 genotypes: homozy(cid:173)
`gous \vild type (- -- -), heterozygous H63D (-), and
`-). A no template control
`homozygous H63D (- -
`(-- -- --) is also included. Data for amplification and melting 60
`curve analysis were normalized to baseline for each sample
`by dividing each fluorescence value by the minimum fluo(cid:173)
`rescence signal for that sample. Melting curve plots (top) of
`Cy5 fluorescence (F) versus temperature (T) are transformed
`into melting peaks (bottom) by plotting -dF/dT vs tempera- 65
`ture. Both amplification and genotyping analysis are com(cid:173)
`pleted within 45 minutes.
`
`55
`
`In describing and claiming the invention, the following
`terminology will be used in accordance with the definitions
`set forth 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
`25 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 tempera-
`ture 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
`pair'' refers to a pair of fluorophores comprising a donor
`fluorophore and acceptor fluorophore, wherein the donor
`fluorophore is capable of transferring resonance energy to
`the acceptor fluorophore. In other words the emission spec(cid:173)
`trum of the donor fluorophore overlaps the absorption spec-
`trum of the acceptor fluorophore. In preferred fluorescence
`resonance energy transfer pairs, the absorption spectrum of
`the donor fluorophore does not substantially overlap the
`absorption spectrum of the acceptor fluorophore.
`As used herein, "fluorescence resonance energy transfer
`relationship" and similar terms refer to a donor fiuorophore
`and acceptor fluorophore positioned in sufficient proximity
`and proper orientation to one another to allow the transfer of
`resonance energy from the donor fluorophore to the acceptor
`fluorophore.
`As used herein, "a donor oligonucleotide probe" refers to
`an oligonucleotide that is labeled with a donor fiuorophore
`of a fluorescent resonance energy transfer pair.
`As used herein, "an acceptor oligonucleotide probe"
`refers to an oligonucleotide that is labeled with an acceptor
`fluorophore of a fluorescent resonance energy transfer pair.
`A5 used herein, "FRET oligonucleotide pair'' refers to the
`donor oligonucleotide probe and the acceptor oligonucle(cid:173)
`otide probe pair that form a fluorescence resonance energy
`transfer relationship when the donor oligonucleotide probe
`and the acceptor oligonucleotide probe are both hybridized
`to their complementary target nucleic acid sequences.
`A5 used herein, "melting temperature of the FRET oli(cid:173)
`gonucleotide pair" and "melting temperature of the set of
`donor oligonucleotide probe and acceptor oligonucleotide
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`6,140,054
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`probe", defines the lowest temperature that will disrupt the
`hybridization of at least one of the pair of oli