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I11111111111111I11I11S000IIIIIIIII740701~1111111111IIIII111111111111111111(12) United States Patent ( i o ) Patent No.: US 6,174,670 B1Wittwer et al. (45) Date of Patent: Jan. 16,2001

MONITORING AMPLIFICATION OF DNADURING PCR

Inventors: Carl T. Wittwer, Salt Lake City, UT

(US); Kirk M. Ririe, Idaho Falls, ID(US); Randy P. Rasmussen, Salt LakeCity, UT (US)

Assignee: University of Utah ResearchFoundation, Salt Lake City, UT (US)

Under 35 U.S.C. 154(b), the term of thispatent shall be extended for 0 days.

Notice:

Appl. No.: 081869,276

Filed: Jun. 4, 1997

Related U.S. Application Data

Continuation-in-part of application No. 081818,267, filed onMar. 17,1997, which is a continuation-in-part of applicationNo. 081658,993, filed on Jun. 4, 1996, now abandoned.

Int. Cl? ............................... C12Q 1/68; C12P 19134

U.S. C1. ................................. 43516; 435191.2; 436194

Field of Search .......................... 43516, 91.2; 436194

References Cited

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0 318 2550 404 258

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1211990 (EP) .


OTHER PUBLICATIONS

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Primary E x a m i n e r x e n n e t h R. Horlick (74) Attorney, Agent, or F i r m a a r n e s & Thornburg

(57) ABSTRACT

Methods of monitoring hybridization during polymerasechain reaction are disclosed. These methods are achievedwith rapid thermal cycling and use of double stranded DNAdyes or specific hybridization probes. A fluorescence reso-nance energy transfer pair comprises fluorescein and Cy5 orCy5.5. Methods for quantitating amplified DNA and deter-mining its purity are carried out by analysis of melting and

reannealing curves.

107 Claims, 52 Drawing Sheets

Microfiche Appendix Included(1 Microfiche, 54 Pages)

10

Fluorescence RatioAbove Background(1og) 1

+Labeled Primer with singleHybridizationProbe

0.10 10 20 30 40

Cycle

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US 6,174,670 B1Pane 2

U.S. PATENT DOCUMENTS

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4,684,4654,701,4154,708,782

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5,187,0845,210,0155,234,5865,240,5775,316,9135,333,6755,346,6725,348,8535,364,7905,380,4895,415,8395,425,9215,436,1345,455,1755,563,0375,565,322

5,585,2425,599,5045,871,9085,928,907

FOREIGN PATENT DOCUMENTS

0 459 241 A10 475 760 A2 0 488 769 A2 0 519 623 A2

0 566 7510 580 362 A10 640 828A1

0 636 4130 643 140

0 674 0090 686 6992 122 18760212 986

WO 89 09437WO 95 20778

93116194WO 95 13399WO 95 21382WO 95121266WO 95 30139WO 95 32306WO 96 00901WO 96 06354

*

511991 (EP).911991 (EP).

1111991 (EP) .1211992 (EP) .1011993 (EP) .111994 (EP).811994 (EP).211995 (EP).311995 (EP).

911995 (EP).1211995 (EP) .811972 (FR) .311987 (JP) .

1011989 (WO) .1111992 (WO) .811993 (WO).511995 (WO).811995 (WO).811995 (WO).

1111995 (WO) .1111995 (WO) .111996 (WO).211996 (WO).

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the Stability of Base Pair Mismatches in Long DNA Deter-

mined by Temperature-

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U S . Patent Jan. 16,2001 Sheet 1 of 52

Temperature

Equilibruim

Denaturation

US 6,174,670 B1

IExtension

Annealing

Time

Fig. I A

Kinetic

Temperature

Annealing

Fig. 1B Time

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U S . Patent Jan. 16,2001 Sheet 2 of 52 US 6,174,670 B1

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k?n0 J

u

nU

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95

75

55 - 95

7 2

Sample Temperature

55 - 95

75

55 - 95

75

US 6,174,670 B1

Temperature Time for 30Cycles (hr)

I I I

A 4

B 2

C 0.67

D 0.25

PhiX174 RFHaeIIIDigest

55

0 2 4 6 8

Time (min)

Fig. 4

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\' 0/ /

Fig. 5A01 Fig. 5Am

Polymerase

\

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\X

Fig. 5 BO) Fig. 5&zi

Fig. 5Clii Fig. 5CP)

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0

Cy5 MonoFunctional Dye

Fig. 6

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so3-

-.' \ \

Cy5.5MonoFunctional Dye0

Fig. 7

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2000

1500

FluorescentIntensity

1000

500

0450

1 .

I

I

490 530 570 610

Wavelength(nm)

', cy5I, Excitation

I

I

.t

650 690

Fig. 8

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U S . Patent

6

4

Jan. 16,2001 Sheet 9 of 52

-I

--

US 6,174,670 B1

m

u"

0.9

0.6

0.3

0 10 20 30 40

Fig. 9 Cycle Number

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2.6 T

2.5

2.4

Maximum Change inFluorescence Ratio

2.3

2.2

2.1

0.1 1 10

Ratio of Cy5 to Fluorescein Oligo

Fig. IO

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Maximum

Change inFluorescence

Ratio

0 0.2 0.4

Concentration of Fluorescein Oligo ( p M )

at a 2:l Cy5:Fluorescein Ratio

Fig. I 1

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2.0

Maximum

Change in

Ratio

Fluorescence 1.5

1o0 2 4 6

Spacing between Labeled Oligos

Fig. 12

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U S . Patent

Lo0

Jan. 16,2001 Sheet 13 of 52

LD03

' L o:u \o

r? c'!x 0 0

rr!0

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nU

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Taq Polymerase - exoi)Stoffel - - - (exo-)Klentaq - - - - - - (exo-)

0.9

0.6

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0.9

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(cy5

) 0.3

0.9

0.6

0.3

pp 75Y

' 60

0 100 200

Time (sec)

0 100 200

T i e sec)

0 10 20 30 40

Fig. 14Cycle #

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c

0

+

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0

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Fluorescence

2.5

2.0

Ratio

1.5

1o15 25 35 45

Fig. 19A

6

5

4

Fluorescence Ratio

3

2

15 25 35 45

C y c l e

Fig. 1 9 6

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2*o

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1.6..

1.4..

1.2..

1 .07

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t. , , “ W t t t ,

b bP).

b

I-

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c

c

c

I I I I I 1

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Coefficientof Variation

12

10-

8- -

6 -4

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Fig. 19D

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84 -.0

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0

m

0

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Fluorescence

40 - -

30 - 9

20 -

lo = -

F =A*Copies*((l+E)%)

A =Scaling

Factor = 4.59e-8Copies = le4E =Efficiency= 0.53n =cycle numberI.2= 0.997

F =A*Copies*((l+E)%)

A =Scaling

Factor = 4.59e-8Copies = le4E =Efficiency= 0.53n =cycle numberI.2= 0.997

40 - -

30 - 9

20 -

lo = -

0 . .II m

I mWII mm I

20 21 22 23 24 25 26 27

Cycle Number

.II m

I mWII mm I

20 21 22 23 24 25 26 27

Cycle Number

Fig. 27

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60

40

Fluorescence

20

0

F =A*Copies*((l+E)*n)

A = Scaling Factor = 3.11e-8Copies = 16,711E = Efficiency = 0.54n = cycle numberI'2= 0.993

Predicted copy number = 15,000

A and E are Constant, # of copies is fit

21 22 23 24 25 24 27

Cycle Number

Fig. 22

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c90

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0

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0

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100

0 GENOMIC

80

60

Relative Fluorescence

40

%o @I 100,000copies =24.9 cycles

Aso6315,000 copies =26.6cycles

+o @ 10,000copies = 28.2cycles

20

0

d I O 20 30 40

Fig. 27 Cycle Number

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28

26

24

22

5000 10000 100000 500000

Fig. 28

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1.oo

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0.75

0.500

Fig. 29A

I I I I .

Temperature

95

("C )

75

20 40

Time (sec)

60

550 20 40

Time (sec) t-.60

Fig. 29B

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v!m

n0

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U S . Patent

11

9

7

Fluorescence

Ratio 5

3

1

Jan. 16,2001 Sheet 33 of 52 US 6,174,670 B1

55 65 75 85 95

Temperature ("C)

Fig. 32A

2.0

Fluorescence

Ratio1.5

1.o55 65 75 85

Temperature ("C)

95

Fig. 32 B

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o!0

c?0

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U S . Patent Jan. 16,2001

0

Sheet 37 of 52 US 6,174,670 B1

r

a'L

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2.0

Fluorescence

1.0

0.0

89 944

Fige 37Temperature ("C)

FIuorescence

86 88 90 92 4

Fig.38 Temperature ("C)

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U S . Patent

Fl

Jan. 16,2001 Sheet 39 of 52

100

80

Relative 60

uorescence

40

20

US 6,174,670 B1

084 86 88 90

Temperature("C)

92

Fig. 39

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Fig; 40A

Fig. 40B

Temperature ("C)

B

AC Over-DNA Specific amplification Primer- _ .

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FIu orescence

Mixed Product

-I

1 - -- -84 86 88 90 92

Temperature ("C)Figo4 I A

2.0

d (Fluorescence)

d (Temperature)

1.0

0.5

0

84 86 88 90 92

Fig. 41BTemperature ("C)

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Relative

Fluorescence

1 11 21 31 41

Cycle Number

Fig.42A

Fig. 42B

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F1uo rescence

I 1 I I I I 1

70 72 74 76 78 80 82 84 86 88 90 92

Fig.

-dF/dT

43ATemperature ( "C )

-0.05 l ' a ' r , . , , , , , , , ,

78 83 aa73

Fig. 436 Temperature ("C)

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CFTR0.25

0.2

0.15

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0.1

0.05

Fig. 44

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U S . Patent

100

80

60

%Area

40

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US 6,174,670 B3

5 6fluorescence as a function of temperature as the thermalcycler heats through the dissociation temperature of theproduct gives a PCR product melting curve. The shape and

position of this DNA melting curve is a function of GCiATratio, length, and sequence, and can be used to differentiateamplification products separated by less than 2" C. inmelting temperature. Desired products can be distinguishedfrom undesired products, including primer dimers. Analysisof melting curves can be used to extend the dynamic rangeof quantitative PCR and to differentiate different products in

multiplex amplification. Using double strand dyes, productdenaturation, reannealing and extension can be followed

within each cycle. Continuous monitoring of fluorescenceallows acquisition of melting curves and product annealingcurves during temperature cycling.

The present invention provides reagents and methods forrapid cycle PCR with combined amplification and analysisby fluorescence monitoring in under thirty minutes, morepreferably in under fifteen minutes, and most preferably inunder ten minutes.

A method for analyzing a target DNA sequence of abiological sample comprises

amplifying the target sequence by polymerase chain reac-

tion in the presence of two nucleic acid probes thathybridize to adjacent regions of the target sequence,one of the probes being labeled with an acceptorfluorophore and the other probe labeled with a donorfluorophore of a fluorescence energy transfer pair suchthat upon hybridization of the two probes with thetarget sequence, the donor and acceptor fluorophores

are within 25 nucleotides of one another, the poly-merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targetednucleic acid sequence to the biological sample andthermally cycling the biological sample between atleast a denaturation temperature and an elongationtemperature;

exciting the biological sample with light at a wavelength

absorbed by the donor fluorophore and detecting theemission from the fluorescence energy transfer pair.

A method for analyzing a target DNA sequence of a


tion in the presence of two nucleic acid probes thathybridize to adjacent regions of the target sequence,one of the probes being labeled with an acceptorfluorophore and the other probe labeled with a donorfluorophore of a fluorescence energy transfer pair such

biological sample comprises

wherein one of the primers and the probe are eachlabeled with one member of a fluorescence energytransfer pair comprising an acceptor fluorophore and a

donor fluorophore, and whereill the labeled probehybridizes to an amplified copy of the target nucleic

acid sequence within 15 nucleotides of the labeledprimer;

(b) amplifying the target nucleic acid sequence by poly-merase chain reaction;

(c) illuminating the biological sample with light of a

selected wavelength that is absorbed by said donorfluorophore; and

s

l o

(d) detecting the fluorescence emission of the sample.An improved method of amplifying a target nucleic acid

(a) adding to the biological sample an effective amount of a nucleic-acid-binding fluorescent entity;

(b) amplifying the target nucleic acid sequence usingpolymerase chain reaction, comprising thermallycycling the biological sample using initial predeter-mined temperature and time parameters, and then

(i) illuminating the biological sample with a selectedwavelength of light that is absorbed by the fluores-

cent entity during the polymerase chain reaction;(ii) monitoring fluorescence from the sample to deter-

mine the optimal temperature and time parameters

for the polymerase chain reaction; and(iii) adjusting the initial temperature and time param-

eters in accordance with the fluorescence.In one illustrative embodiment, the fluorescent entity com-prises a double strand specific nucleic acid binding dye, and

in another illustrative embodiment the fluorescent entitycomprises a fluorescently labeled oligonucleotide probe that

A method for detecting a target nucleic acid sequence of

(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 beinglabeled with an acceptor fluorophore and the otherprobe labeled with a donor fluorophore of a fluores-cence energy transfer pair, wherein an emission spec-

trum of the donor fluorophore and an absorption spec-

trum of the acceptor fluorophore overlap less than 25%,the acceptor fluorophore has a peak extinction coeffi-cient greater than 100,000 M-lcm-l and upon hybrid-ization of the two probes, the donor and acceptorfluorophores are within 25 nucleotides of one another;

sequence of a biological sample comprises

2o

2s

30

3s hybridizes to the targeted nucleic acid sequence.

a biological sample comprises

4o

4s

that upon hybridization of the two probes with the sotarget sequence, the donor and acceptor fluorophores

are within 25 nucleotides of one another, the poly-merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targetednucleic acid sequence to the biological sample and ssthermally cycling the biological sample between at

least a denaturation temperature and an elongationtemperature;

exciting the sample with light at a wavelength absorbed

monitoring the temperature dependent fluorescence from

A method of real time monitoring of a polymerase chainreaction amplification of a target nucleic acid sequence in a

by the donor fluorophore; and 60

the fluorescence energy transfer pair.

(b) illuminating the biological sample with a selectedwavelength of light that is absorbed by said donor

fluorophore; and

(c) detecting the emission of the biological sample. Anillustrative resonance energy transfer pair comprises

fluorescein as the donor and Cy5 or Cy5.5 as theacceptor.A method of real time monitoring of a polymerase chain

reaction amplification of a target nucleic acid sequence in a


amplifying the target sequence by polymerase chain reac-tion in the presence of two nucleic acid probes thathybridize to adjacent regions of the target sequence,one of the probes being labeled with an acceptor

fluorophore and the other probe labeled with a donorbiological sample comprises 65 fluorophore of a fluorescence energy transfer pair such

that upon hybridization of the two probes with thetarget sequence, the donor and acceptor fluorophores

(a) adding to the biological sample an effective amount of two nucleic acid primers and a nucleic acid probe,

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US 6,174,670 B3

7are within 25 nucleotides of one another, the poly-merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targetednucleic acid sequence to the biological sample andthermally cycling the biological sample between atleast a denaturation temperature and an elongationtemperature;

exciting the biological sample with light at a wavelengthabsorbed by the donor fluorophore and detecting theemission from the biological sample; and

monitoring the temperature dependent fluorescence fromthe fluorescence energy transfer pair.

A method of real time monitoring of a polymerase chainreaction amplification of a target nucleic acid sequence in abiological sample comprises


tion in the presence of SYBRTMGreen I, the poly-

merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targetednucleic acid sequence to the biological sample andthermally cycling the biological sample between atleast a denaturation temperature and an elongation

temperature;exciting the biological sample with light at a wavelength

absorbed by the SYBRTM Green I and detecting theemission from the biological sample; and

monitoring the temperature dependent fluorescence fromthe S YBRT MGreen I. Preferably, the monitoring stepcomprises determining a melting profile of the ampli-fied target sequence.

A method for analyzing a target DNA sequence of a

(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 eachlabeled with one member of a fluorescence energytransfer pair comprising an acceptor fluorophore and a

donor fluorophore, and wherein the labeled probehybridizes to an amplified copy of the target nucleicacid sequence within 15 nucleotides of the labeledprimer;

(b) amplifying the target nucleic acid sequence by poly-merase chain reaction;

(c) illuminating the biological sample with light of aselected wavelength that is absorbed by said donorfluorophore and detecting the fluorescence emission of the sample. In another illustrative embodiment, themethod further comprises the step of monitoring thetemperature dependent fluorescence of the sample,preferably by determining a melting profile of theamplified target sequence.

A method of detecting a difference at a selected locus in

a first nucleic acid as compared to a second nucleic acidcomprises

(a) providing a pair of oligonucleotide primers configuredfor amplifying, by polymerase chain reaction, aselected segment of the first nucleic acid and a corre -sponding segment of the second nucleic acid, whereinthe selected segment and corresponding segment eachcomprises the selected locus, to result in amplifiedproducts containing a copy of the selected locus;

(b) providing a pair of oligonucleotide probes, one of theprobes being labeled with an acceptor fluorophore andthe other probe being labeled with a donor fluorophoreof a fluorogenic resonance energy transfer pair such


S

10

1s

20

2s

30

3s

aC

40

4s

so

5s

60

65

8that upon hybridization of the two probes with theamplified products the donor and acceptor are in reso -nance energy transfer relationship, wherein one of theprobes is configured for hybridizing to the amplifiedproducts such that said one of the probes spans theselected locus and exhibits a melting profile when thedifference is present in the first nucleic acid that is

distinguishable from a melting profile of the secondnucleic acid;

(c) amplifying the selected segment of first nucleic acid

and the corresponding segment of the second nucleicacid by polymerase chain reaction in the presence of effective amounts of probes to result in an amplifiedselected segment and an amplified correspondingsegment, at least a portion thereof having both theprobes hybridized thereto with the fluorogenic reso-nance energy transfer pair in resonance energy transferrelationship;

(d) illuminating the amplified selected segment and theamplified corresponding segment with the probeshybridized thereto with a selected wavelength of lightto elicit fluorescence by the fluorogenic resonanceenergy 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 amplifiedselected segment of first nucleic acid and a secondmelting profile of said one of the probes melting fromthe amplified corresponding segment of second nucleicacid; and

( f ) comparing the first melting profile to the secondmelting profile, wherein a difference therein indicatesthe presence of the difference in the sample nucleicacid.

A method of detecting a difference at a selected locus infirst nucleic acid as compared to a second nucleic acid

(a) providing a pair of oligonucleotide primers configured

for amplifying, by polymerase chain reaction, aselected segment of the first nucleic acid and a corre -

sponding segment of the second nucleic acid, whereinthe selected segment and corresponding segment eachcomprises the selected locus, to result in amplifiedproducts containing a copy of the selected locus;

(b) providing an oligonucleotide probe, wherein one of the primers and the probe are each labeled with onemember of a fluorescence energy transfer pair com-prising an donor fluorophore and an acceptorfluorophore, and wherein the labeled probe and labeledprimer hybridize to the amplified products such that thedonor and acceptor are in resonance energy transferrelationship, and wherein the probe is configured forhybridizing to the amplified products such that said

probe spans the selected locus and exhibits a meltingprofile when the difference ispresent in the first nucleicacid that isdistinguishable from a melting profile of thesecond nucleic acid;

(c) amplifying the selected segment of first nucleic acidand the corresponding segment of the second nucleicacid by polymerase chain reaction in the presence of effective amounts of primers and probe to result in anamplified selected segment and an amplified corre-

sponding segment, at least a portion thereof having thelabled primer and probe hybridized thereto with thefluorogenic resonance energy transfer pair in resonanceenergy transfer relationship;

omprises

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9(d) illuminating the amplified selected segment and the

amplified corresponding segment with the labeledprimer and probe hybridized thereto with a selectedwavelength of light to elicit fluorescence by the fluo-rogenic 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 segmentof first nucleic acid and a second melting profile of saidprobe melting from the amplified corresponding seg-

ment of second nucleic acid; and

( f ) comparing the first melting profile to the secondmelting profile, wherein a difference therein indicatesthe presence of the difference in the sample nucleicacid.

A method of detecting heterozygosity at a selected locusin the genome of an individual, wherein the genome com-prises 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 configuredfor amplifying, by polymerase chain reaction, a first

selected segment of the mutant allele and a secondselected segment of the corresponding reference allelewherein both the first and second selected segmentscomprise the selected locus;

(c) providing a pair of oligonucleotide probes, one of theprobes being labeled with an acceptor fluorophore andthe other probe being labeled with a donor fluorophoreof a fluorogenic resonance energy transfer pair suchthat upon hybridization of the two probes with theamplified first and second selected segments one of theprobes spans the selected locus and exhibits a firstmelting profile with the amplified first selected segmentthat is distinguishable from a second melting profilewith the amplified second selected segment;

(d) amplifying the first and second selected segments of

sample genomic DNA by polymerase chain reaction inthe presence of effective amounts of probes to result inamplified first and second selected segments, at least aportion thereof having both the probes hybridizedthereto with the fluorogenic resonance energy transferpair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selectedsegments having the probes hybridized thereto with aselected wavelength of light to elicit fluorescence bythe donor and acceptor;

( f ) measuring a fluorescence emission as a function of temperature to determine a first melting profile of saidone of the probes melting from the amplified firstselected segment and a second melting profile of saidone of the probes melting from the amplified second

selected segment; and(g) comparing the first melting profile to the secondmelting profile, wherein distinguishable melting pro-files indicate heterozygosity in the sample genomicDNA.

A method of detecting heterozygosity at a selected locusin the genome of an individual, wherein the genome com-prises a mutant allele and a corresponding reference allele,each comprising the selected locus, comprises


(b) providing a pair of oligonucleotide primers configuredfor amplifying, by polymerase chain reaction, a firstselected segment of the mutant allele and a second

10selected segment of the corresponding reference allelewherein both the first and second selected segmentscomprise the selected locus;

(c) providing an oligonucleotide probe, wherein one of the primers and the probe are each labeled with onemember of a fluorescence energy transfer pair com-prising an donor fluorophore and an acceptor

fluorophore, and wherein the labeled probe and labeledprimer hybridize to the amplified first and secondselected segments such that one of the probes spans the

selected locus and exhibits a first melting profile withthe amplified first selected segment that is distinguish-

able from a second melting profile with the amplifiedsecond selected segment;

(d) amplifying the first and second selected segments of sample genomic DNA by polymerase chain reaction inthe presence of effective amounts of primers and probeto result in amplified first and second selectedsegments, at least a portion thereof having both thelabeled primer and probe hybridized thereto with thefluorogenic resonance energy transfer pair in resonanceenergy transfer relationship;

(e) illuminating the amplified first and second selectedsegments having the labeled primer and probe hybrid-ized thereto with a selected wavelength of light to elicitfluorescence by the donor and acceptor;

( f ) measuring a fluorescence emission as a function of temperature to determine a first melting profile of saidprobe melting from the amplified first selected segmentand a second melting profile of said probe melting fromthe amplified second selected segment; and

(g) comparing the first melting profile to the secondmelting profile, wherein distinguishable melting pro-

files indicate heterozygosity in the sample genomicDNA.

A method of determining completion of a polymerasechain reaction in a polymerase chain reaction mixture com-prising (1) a nucleic acid wherein the nucleic acid or a

4o polymerase-chain-reaction-amplified product thereof con-sists of two distinct complementary strands, (2) two oligo-

nucleotide primers configured for amplifying by polymerasechain reaction a selected segment of the nucleic acid to resultin an amplified product, and (3) a DNA polymerase for

(a) adding to the mixture (1) an effective amount of anoligonucleotide probe labeled with a resonance energytransfer donor or a resonance energy transfer acceptorof a fluorogenic resonance energy transfer pair, whereinthe probe is configured for hybridizing to the amplifiedproduct under selected conditions of temperature andmonovalent ionic strength, and (2) an effective amountof a reference oligonucleotide labeled with the donor orthe acceptor, with the proviso that as between the probe

and reference oligonucleotide one is labeled with thedonor and the other is labeled with the acceptor,wherein the reference oligonucleotide is configured forhybridizing to the amplified product under the selectedconditions of temperature and monovalent ionicstrength such that the donor and the acceptor are inresonance energy transfer relationship when both theprobe and the reference oligonucleotide hybridize tothe amplified product;

(b) amplifying the selected segment of nucleic acid bypolymerase chain reaction to result in the amplifiedproduct, at least a portion thereof having both the probeand the reference oligonucleotide hybridized thereto

5

10

’O

’’

3o

35

45 catalyzing the polymerase chain reaction, comprises

50

5s

60

65

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11with the fluorogenic resonance energy transfer pair inresonance energy transfer relationship; and

(c) illuminating the amplified product having the probeand reference oligonucleotide hybridized thereto with aselected wavelength of light for eliciting fluorescenceby the fluorogenic resonance energy pair and monitor-ing fluorescence emission and determining a cycle

when the fluorescence emission reaches a plateauphase, indicating the completion of the reaction.A method of determining completion of a polymerase

chain reaction in a polymerase chain reaction mixture com-prising (1) a nucleic acid wherein the nucleic acid or apolymerase-chain-reaction-amplified product thereof con-sists of two distinct complementary strands, (2) two oligo-nucleotide primers configured for amplifying by polymerasechain reaction a selected segment of the nucleic acid to resultin an amplified product, and (3) a DNA polymerase forcatalyzing 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 bypolymerase chain reaction in the mixture to which thenucleic-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 selectedwavelength of light for eliciting fluorescence therefromand monitoring fluorescence emission and determininga cycle when the fluorescence emission reaches aplateau phase, indicating the completion of the reac-

tion. Preferably, the nucleic-acid-binding fluorescentdye is a member selected from the group consisting of S YBRT MGREEN I, ethidium bromide, pic0 green,acridine orange, thiazole orange, YO-PRO-1, and chro-

momycinA3, and more preferably is SYBRTMGREENI.

A method of controlling temperature cycling parameters

of a polymerase chain reaction comprising repeated cyclesof annealing, extension, and denaturation phases of a poly-merase chain reaction mixture comprising a double-strand-specific fluorescent dye, wherein the parameters compriseduration of the annealing phase, duration of the denaturationphase, and number of cycles, comprises

(a) illuminating the reaction with a selected wavelength of light for eliciting fluorescence from the fluorescent dyeand continuously monitoring fluorescence during therepeated annealing, extension, and denaturation phases;

(i) duration for fluorescence to stop increasing duringthe extension phase, or

(ii) Duration for fluorescence to decrease to a baselinelevel during the denaturation phase, or

(iii) a number of cycles for fluorescence to reach apreselected level during the extension phase; and

(c) adjusting the length of the extension phase accordingto the length of time for fluorescence to stop increasingduring the extension phase, the length of the denatur-ation phase according to the length of time for fluo-rescence to decrease to the baseline level during thedenaturation phase, or the number of cycles accordingto the number of cycles for fluorescence to reach thepreselected level during the extension phase.

A method of determining a concentration of an amplifiedproduct in a selected polymerase chain reaction mixturecomprises

(b) determining at least

12(a) determining a second order rate constant for the

amplified product at a selected temperature and reac-tion conditions by monitoring rate of hybridization of aknown concentration of the amplified product;

(b) determining rate of annealing for an unknown con-

centration of the amplified product; and

(c) calculating the concentration of the amplified product

from the rate of annealing and the second order rateconstant. Preferably, the rate of annealing isdeterminedafter multiple cycles of amplification. One illustrativemethod of determining the second oder rate constantcomprises the steps of raising the temperature of a first polymerase chain

reaction mixture comprising a known concentrationof the amplified product and an effective amount of a double-strand specific fluorescent dye, above thedenaturation temperature of the amplified product toresult in a denatured amplified product;

rapidly reducing the temperature of the first polymerasechain reaction mixture comprising the knownamount of denatured amplified product to a selectedtemperature below the denaturation temperature of the amplified product while continuously monitoring

the fluorescence of the first polymerase chain reac-tion mixture as a function of time;

plotting fluorescence as a function of time for deter-mining maximum fluorescence, minimumfluorescence, the time at minimum fluorescence, anda second order rate constant for the known concen -

tration of amplified product from the equation

5

10

1s

20

2s

30

Fmax- FmmF=Fm , -

k ( r - o ) [DNA]+ 1

3s

wherein F is fluorescence, F,,, is maximumfluorescence, F,, is minimum fluorescence, k is thesecond order rate constant, to is the time at Fmi,, and[DNA] is the known concentration of the amplified

A method of determining a concentration of a selectednucleic acid template by competitive quantitative poly-

merase chain reaction comprises the steps of

40 product.

(a) in a reaction mixture comprising:45 (i) effective amounts of each of a pair of oligonucle-

otide primers configured for amplifying, in a poly-merase chain reaction, a selected segment of theselected template and a corresponding selected seg-ment of a competitive template to result in amplified

(ii) an effective amount of an oligonucleotide probelabeled with a resonance energy transfer donor or aresonance energy transfer acceptor of a fluorogenicresonance energy transfer pair, wherein the probe is

configured for hybridizing to the amplified productssuch that the probe melts from the amplified productof the selected template at a melting temperature thatis distinguishable from the melting temperature atwhich the probe melts from the amplified product of

(iii) an effective amount of a reference oligonucleotidelabeled with the donor or the acceptor, with theproviso that as between the probe and transfer oli-

gonucleotide one is labeled with the donor and theother is labeled with the acceptor, wherein the ref-erence oligonucleotide is configured for hybridizingto the amplified products such that the donor and the

so products thereof,

5s

60 the competitive template,

65

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13 14acceptor are in resonance energy transfer relation-ship when both the probe and the reference oligo-nucleotide hybridize to the amplified products;

amplifying, by polymerase chain reaction, an unknown

amount of the selected template and a known amount of the 5

competitive template to result in the amplified productsthereof;

(b) illuminating the reaction mixture with a selected

wavelength of light for eliciting fluorescence by thefluorogenic resonance energy transfer pair and deter-mining a fluorescence emission as a function of tern-

perature as the temperature of the reaction mixture ischanged to result in a first melting curve of the probemelting from the amplified product of the selectedtemplate and a second melting curve of the probemelting from the competitive template;

(c) converting the first and second melting curves to firstand second melting melting peaks and determiningrelative amounts of the selected template and the com-

petitive template from such melting peaks; and

(d) calculating the concentration of the selected template 2o

based on the known amount of the competitive tem-

plate and the relative amounts of selected template andcompetitive template. amplified;

A fluorogenic resonance energy transfer pair consists of fluorescein and Cy5 or Cy5.5.

A method of determining a concentration of a selectednucleic acid template in a polymerase chain reaction com-prises the steps of

polymerase chain reaction, a selected first segmentof the selected template to result in an amplified firstproduct thereof,

(ii) effective amounts of each of a second pair of oligonucleotide primers configured for amplifying,in a polymerase chain reaction, a selected secondsegment of the positive control template to result inan amplified second product thereof,

(iii) an effective amount of a nucleic-acid-bindingfluorescent dye;

subjecting the selected template and the positive controltemplate to conditions for amplifying the selected templateand the positive control template by polymerase chainreaction; and

(b) illuminating the reaction mixture with a selectedwavelength of light for eliciting fluorescence by thenucleic-acid-binding fluorescent dye and continuouslymonitoring the fluorescence emitted as a function of temperature during an amplification cycle of the poly-

merase 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

wherein obtaining of the second melting curve indi-cates that the polymerase chain reaction was

operative, obtaining the first melting curve indicatesthat the selected first segment was amplifiable, andabsence of the first melting curve indicates that theselected first segment was not amplifiable.

A method of detecting the factor V Leiden mutation in anindividual, wherein the factor V Leiden mutation consists of a single base change at the factor V Leiden mutation locusas compared to wild type, comprises the steps of

2s

(a) in a reaction mixture comprising:(i) effective amounts of each of a first pair of oligo- 30

nucleotide primersconfigured for amplifying, in a

polymerase chain reaction, a selected first segmentof the selected template to result in an amplified first

3sproduct thereof,

(ii) effective amounts of each of a second pair of oligonucleotide primers configured for amplifying,in a polymerase chain reaction, a selected secondsegment 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 unknownamount of the selected template to result in the amplifiedfirst product and a known amount of the reference template 4s

to result in the amplified second product thereof;

(b) illuminating the reaction mixture with a selectedwavelength of light for eliciting fluorescence by thenucleic-acid-binding fluorescent dye and continuouslymonitoring the fluorescence emitted as a function of sotemperature to result in a melting curve of the amplifiedproducts wherein the first product and second productmelt at different temperatures;

(c) converting the melting curves to melting melting

peaks and determining relative amounts of the selected 5stemplate and the reference template from such melting

peaks; and

(d) calculating the concentration of the selected templatebased on the known amount of the reference templateand the relative amounts of selected template and 60

reference template.

40

A method of monitoring amplification of a selected tem-plate in a polymerase chain reaction that also comprises apositive control template comprises the steps of

(a) in a reaction mixture comprising: 65

(i) effective amounts of each of a first pair of oligo-

nucleotide primers configured for amplifying, in a


(b) providing wild type genomic DNA as a control;

(c) providing a pair of oligonucleotide primers configuredfor amplifying by polymerase chain reaction a selectedsegment of the sample genomic DNA and of the wild

type genomic DNA wherein the selected segment com-prises the factor V Leiden mutation locus to result inamplified products containing a copy of the factor VLeiden mutation locus;

(d) providing an oligonucleotide probe labeled with aresonance energy transfer donor or a resonance energytransfer acceptor of a fluorogenic resonance energytransfer pair, wherein the probe is configured forhybridizing to the amplified products such that theprobe spans the mutation locus and exhibits a meltingprofile when the factor V Leiden mutation ispresent inthe sample genomic DNA that is differentiable from amelting profile of the wild type genomic DNA,

(e) providing a transfer oligonucleotide labeled with theresonance energy transfer donor or the resonance

energy transfer acceptor, with the proviso that asbetween the probe and transfer oligonucleotide one is

labeled with the resonance energy transfer donor andthe other is labeled with the resonance energy transferacceptor, wherein the transfer oligonucleotide is con-figured for hybridizing to the amplified products suchthat the resonance energy transfer donor and the reso-

nance energy transfer acceptor are in resonance energytransfer relationship when both the probe and thetransfer oligonucleotide hybridize to the amplifiedproducts;

( f ) amplifying the selected segment of sample genomicD N A a n d wild type genomic D N A b y polymerase chain

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15reaction in the presence of effective amounts of oligo-nucleotide probe and transfer oligonucleotide to resultin amplified selected segments, at least a portionthereof having both the probe and the transfer oligo-nucleotide hybridized thereto with the fluorogenic reso-

nance energy transfer pair in resonance energy transferrelationship;

(g) determining fluorescence as a function of temperatureduring an amplification cycle of the polymerase chainreaction to result in a melting profile of the probemelting from the amplified segment of sample genomicDNA and a melting profile of the probe melting fromthe amplified segment of wild type genomic DNA, and

(h) comparing the melting profile for the sample genomicDNA to the melting profile for the wild type genomicDNA, wherein a difference therein indicates the pres-

ence of the factor V Leiden mutation in the samplegenomic DNA.

A method of analyzing nucleic acid hybridization com -

(a) providing a mixture comprising a nucleic acid sampleto be analyzed and a nucleic acid binding fluorescent

entity; and(b) monitoring fluorescence while changing temperature

at a rate of 20.1" C./second.A method of quantitating an initial copy number of a

sample containing an unknown amount of nucleic acidcomprises the steps of

(a) amplifying by polymerase chain reaction at least onestandard of known concentration in a mixture compris-

ing the standard and a nucleic acid binding fluorescententity;

(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 equa-tion describing fluorescence as a function of initialnucleic acid concentration and cycle number;

(d) amplifying the sample containing the unknownamount of nucleic acid in a mixture comprising thesample and the nucleic acid binding fluorescent entityand monitoring fluorescence thereof; and

(e) determining initial nucleic acid concentration from theequation determined in step (c).

A fluorescence resonance energy transfer pair isdisclosedwherein the pair comprises a donor fluorophore having anemission spectrum and an acceptor fluorophore having anabsorption spectrum and an extinction coefficient greaterthan 100,000 M-lcm-l, wherein the donor fluorophore'semission spectrum and the acceptor fluorophore's absorp-

tion spectrum overlap less than 25%. One illustrative fluo-rescence resonance energy transfer pair described is wherethe donor fluorophore is fluorescein and the acceptor fluo-

rophore is Cy5 or Cy5.5.A method for analyzing a target DNA sequence of abiological sample comprises


tion in the presence of a nucleic acid binding fluores-cent entity, said polymerase chain reaction comprisingthe steps of adding a thermostable polymerase andprimers for the targeted nucleic acid sequence to thebiological sample and thermally cycling the biologicalsample between at least a denaturation temperature andan elongation temperature;

exciting the sample with light at a wavelength absorbedby the nucleic acid binding fluorescent entity; and

prises the steps of

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40

4s

so

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16monitoring the temperature dependent fluorescence from

the nucleic acid binding fluorescent entity as tempera-ture of the sample is changed. Preferably, the nucleicacid binding fluorescent entity comprises a doublestranded nucleic acid binding fluorescent dye, such asS YBRT MGreen I. The temperature dependent fluores-cence can be used to identify the amplified products,preferably by melting curve analysis. Relative amountsfo two or more amplified products can be determinedby analysis of melting curves. For example, areas underthe melting curves are found by non-linear least squaresregression of the sum of multiple gaussians.

BRIEF DESCRIPTION OF THE SEVERALVIEWS OF THE DRAWINGS

FIGS. 1 A & B are graphs representing an equilibrium PCRparadigm (A) and a kinetic PCR paradigm (B).

FIG. 2 illustrates useful temperature v. time segments forfluorescence hybridization monitoring.

FIG. 3 is a temperature v. time chart exemplary of rapidtemperature cycling for PCR.

FIG. 4 shows the results of four different temperaturehime

profiles (A-

D) and their resultant amplification productsafter thirty cycles (inset).

FIGS. 5A(1), 5A(2), 5B(1), 5B(2), 5C(1), and 5C(2)illustrate the mechanism of fluorescence generation for threedifferent methods of fluorescence monitoring of PCR: (A)(l)

and (A)(2) double-stranded DNA dye, (B)(l) and (B)(2)hydrolysis probe, and (C)(l) and (C)(2) hybridizationprobes.

FIG. 6 shows the chemical structure of the monovalentN-hydroxysuccinimide ester of CyS T M .

FIG. 7 shows the chemical structure of the monovalentN-hydroxysuccinimide ester of C ~ 5 . 5 ~ ~ .

FIG. 8 shows the emission spectrum of fluorescein (solidline) and the excitation spectrum of Cy5 (broken line).

FIG. 9 shows resonance energy transfer occurring

between fluorescein- and Cy5-labeled adjacent hybridiza-tion probes at each cycle during PCR.

FIG. 1 0 shows the effect of varying the ratio of the Cy5probe to the fluorescein probe on the resonance energytransfer signal generated during PCR.

FIG. 11shows the effect of varying the probe concentra-tion at a given probe ratio on the resonance energy transfersignal generated during PCR.

FIG. 12 shows the effect of spacing between the labeledoligonucleotides on the resonance energy transfer signalgenerated during PCR.

FIG. 13shows the time course of adjacent probe hybrid-

ization by fluorescence energy transfer immediately after 30

cycles of amplification with Taq DNA polymerase (exo+;solid line) and the Stoffel fragment of Taq DNApolymerase

(exo-; dotted line) of temperature cycling and the type of polymerase on fluorescence development during PCR withadjacent hybridization probes; temperature is shown with abold line.

FIG. 14 is a fluorescence ratio v. cycle number plot foramplification with Taq DNA polymerase (exo+; solid line),Stoffel fragment of Taq DNA polymerase (exo-; brokenline), and KlenTaq DNApolymerase (exo-; dotted line): toppanel-cycling isbetween 94 " C. and 60" C. with a 20 secondhold at 60" C.; middle panel-cycling is between 94 " C. and60" C. with a 120 second hold at 60" C.; bottom panel-cycling is between 94 " C. and 60" C. with a slow increasefrom 60" C. to 75" C.

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17FIG. 15 is a fluorescence v. cycle number plot for a

number of different initial template copy reactions moni-tored with SybrTMGreen I: 0, (A); 1, W) ;10, (-) ; o 2 , (-); o 3 , (+I; i o 4 , ( 0 ) o5 , (01; i o 6 , XI; i o7 , (A ) ; os , (0);

l o 9 , (+ I

P(.I;o 2 , (*)I; i o3 , (0 ) ;o 4 ,(0);i o ,+I; i o 6 , w); i o7 , ( 0 1; os , ; o 9 , (+I.

(.I;o 2 , (*)I; i o3 , (0 ) ;o 4 ,(0);o 5 , (+I; i o 6 , w); i o , 0 1; os , i o9 , (+I.

FIG. 16 is a fluorescence ratio v. cycle number plot for anumber of different initial template copy reactions moni-tored with a dual-labeled hydrolysis robe: 0,(-); 1, (A); 10,

FIG. 17 is a fluorescence ratio v. cycle number plot for anumber of different initial template copy reactions moni-tored with adjacent hybridization probes: 0, (-); 1, (A); 10,

FIG. 18 is a fluorescence ratio V. cycle number plotdistinguishing two hybridization probe designs monitoredby resonance energy transfer: (0)two hybridization probeslabeled respectively with fluorescein and Cy5; and (+) aprimer labeled with Cy5 and a probe labeled with fluores-cein.

FIGS. 19A-C provide a comparison of three fluorescencemonitoring techniques for PCR, including the double-strandspecific DNA dye SYBR Green I (A), a dual-labeled

fluoresceinirhodamine hydrolysis probe (B), and afluorescein-labeled hybridization probe with a Cy5-labeledprimer (C);

FIG. 19D shows the coefficient of variation for the threemonitoring techniques represented in FIGS. 19A-C.

FIG. 20 shows a typical log fluorescence vs cycle numberplot of a standard amplification monitored with SYBRGreen I.

FIG. 21 shows an expontial curve fit to cycles 20-27 of the data from FIG. 20.

FIG. 22 shows an exponential fit to an unknown to

determine initial copy number from amplification data.

FIG. 23 shows a typical fluorescence v. cycle number plotof five standards monitored each cycle with adjacent hybrid-ization probes, wherein initial copy numbers are represented

as follows: l o3 , (0) o 4 , (0) o 5 , (A) ; o 6 ,(0)

o 7 , (+).FIG. 24 shows a curve fit to the standard data of FIG. 23.

FIG. 25 shows a typical fluorescence vs cycle number plotof five standards monitored each cycle with a hydrolysisprobe, wherein initial copy numbers are represented asfollows: 1.5, (0) 15, (0);150, (A); 1500,(0);15,000,

FIG. 26 shows a curve fit to the standard data of FIG. 25.

FIG. 27 shows a typical log fluorescence vs cycle numberplot of three standard amplifications monitored with SYBRGreen I, wherein: (W); (0);(A).

FIG. 28 shows different curve fit to the standard data of FIG. 27.

FIGS. 29A&B show plots of (A) time v. fluorescence and(B) time v. temperature demonstrating the inverse relation-

ship between temperature and fluorescence.FIG. 30 is a chart showing 2D plots of temperature v.

time, fluorescence v. time, and fluorescence v. temperature,also shown as a 3D plot, for the amplification of a 180 basepair fragment of the hepatitis B genome in the presence of SYBR Green

FIG. 31is a fluorescence v. temperature projection for theamplification of a 536 base pair fragment of the humanbeta-globin gene in the presence of SYBR Green I.

FIGS. 32A&B provide a plot showing (A) a linear changein fluorescence ratio with temperature for hydrolysis probes,and (B) a radical change with temperature for hybridizationprobes.

(+I.

S

10

1s

20

2s

30

3s

40

4s

so

5s

60

65

18FIG. 33 shows a fluorescence ratio v. temperature plot of

amplification with an exo- polymerase in the presence of adjacent hybridization probes.

FIG. 34 shows a fluorescence ratio v. temperature plot of amplification with an exo+ polymerase in the presence of adjacent hybridization probes.

FIG. 35 shows a 3-dimensional plot of temperature, time

and fluorescence during amplification with an exo- poly-merase in the presence of adjacent hybridization probes.

FIG. 36 shows a 3-dimensional plot of temperature, time,and fluorescence during amplification with an exo+ poly-

merase in the presence of adjacent hybridization probes.

FIG. 37 shows melting curves for PCR-amplified prod-ucts of hepatitis B virus (0;50% GC, 180 bp); beta-globin(A; 53.2% GC, 536 bp); and prostate specific antigen (x;

60.3% GC, 292 bp).

FIG. 38shows melting curves for PCR-amplified productof hepatitis B virus at heating rates of 0.10" C. to 5.0" C.

FIG. 39 shows melting curves for PCR-amplified productof hepatitis B virus at various S YBRT MGreen I concentra-tions.

FIGS. 40A&B show (A) melting curves and (B) electro-

phoretically fractionated bands of products of a beta-globinfragment amplified with (a) no added template, (b) l o 6copies of added template under low stringency conditions,and (c) l o 6copies of added template under higher stringencyconditions.

FIGS. 41A&B show (A) melting curves and (B) meltingpeaks of hepatitis B virus fragment (HBV), 0-globin, and amixture thereof.

FIGS. 42A-D show (A) a relative fluorescence v. cyclenumber plot for PCR amplified products from variousamounts of 0-globin template, (B) melting peaks and (C)electrophoretic bands of the various products, and (D)corrected fluorescence of the data of (A).

FIGS. 43A&B show (A) melting curves and (B) meltingpeaks from PCR amplified products of a mixture of the

cystic fibrosis gene and the c-erbB-2 oncogene.FIG. 44 show melting peaks at various cycle numbers for

the cystic fibrosis gene (CFTR) and c-erbB-2 (neu).

FIG. 45 shows a graph of integrated melting peaks of CFTR and neu PCT products.

FIGS. 46A&B show (A) melting curves and (B) meltingpeaks for PCR products of a person heterozygous for thefactor V Leiden mutation (solid line), homozygous for thefactor V Leiden mutation (dotted line), homozygous wildtype (broken line), and no DNA control (alternating dot anddash).

FIG. 47 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 wild type (alternating dot anddash).

FIG. 48 shows melting peaks of a homozygous mutant of the methylenetatrahydrofolate gene (solid line), homozy-

gous wild type (broken line), heterozygous mutant (dottedline), and no DNA control (alternating dot and dash).

FIG. 49 shows the shape of reannealing curves of ampli-fied 0-globin PCR products from various initial templateamounts.

FIG. 50 shows the determination of a second order rateconstant for determining initial template concentration.

FIG. 51 shows a block diagram for controlling thermalcycling from fluorescence data.

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27an approach rate of 20" C ./sec, and 75 " C. for 0 se c with anapproach rate of 1" C./sec in a capillary fluorescence rapidtemperature cycler. During temperature cycling, fluoresceinand Cy5 fluorescence were acquired each cycle at the end of the annealingiextension segment. Resonance energy transferwas observed as both a decrease in fluorescein fluorescence,and an increase in Cy5 fluorescence beginning around cycle26 of amplification (FIG. 9). In general, observing thefluorescence ratio of Cy5 to fluorescein fluorescence is

perferred.

The unexpectedly good results with the fluoresceiniCy5pair can at least partly be rationalized. The overlap integral,JDA depends not only on spectral overlap, but also on theextinction coefficient of the acceptor (Cy5 has an extinctioncoefficient of 250,000 M-lcm-l at 650 nm), and on the 4thpower of the wavelength. Both of these factors will favor ahigh JDA for Cy5, even given low spectral overlap. Recently,phycoerythrin and Cy7 were shown to be an effectivetandem probe for immunofluorescence, despite low spectraloverlap. In a later example, the utility of fluorescein andCy5.5 as labels on hybridization probes is demonstrated.Fluorescence resonance energy transfer can be used tomonitor nucleic acid hybridization even when the interacting

dyes have low spectral overlap. The use of fluorescein withCy5, Cy5.5 and other red or infrared emitting dyes asresonance energy transfer pairs for monitoring hybridizationhas not been previously recognized. Fluorescein has a longemission "tail" that goes out to 600 nm, 700 nm and beyondthat can be used to excite these far red and infrared dyes. Therate of energy transfer is dependent on the overlap integral,but is also effected by the 6th power of the distance betweenthe fluorophores. If the probes are designed so that theresonance energy transfer dyes are in close proximity, thetransfer rate is high. At least with fluoresceiniCy.5,

fluoresceiniCy5.5 and like pairs, resonance energy transferappears to predominate over collisional quenching and otherforms of energy loss when the fluorophores are closetogether, 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 thechange in the ratio of light intensity in the donor andacceptor windows at minimal and maximal resonanceenergy transfer. One way to obtain minimal and maximaltransfer is to attach both fluorophores to the same oligo-

nucleotide and measure fluorescence ratio before and afterdigestion with phospodiesterase.

EXAMPLE 4

The dual-labeled fluoresceiniCy5 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 phosphoryla-tion 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-carboninternucleotide 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 hydrolysiswith phosphodiesterase (Sigma, St. Louis, Mo.). The changein the fluorescence ratio was 220-fold after hydrolysis. Ad u a l - l a b e l e d f l u o r e s c e i n i r h o d a m i n e p r o b eF-ATGCCCT*CCC CCATGCCATC CTGCGTp (SEQ IDNO:20) was purchased from Perkin Elmer (Foster City,Calif.), where F is fluorescein and * is a rhodamine attachedto a modified T residue by an amino-linker arm. The change

28in the fluorescence ratio (rhodamine to fluorescein) was22-fold after hydrolysis with phosphodiesterase.

The potential signal from the fluoresceiniCy5 pair was10-fold that of the fluoresceinirhodamine pair.

5

EXAMPLE 5

The effect of the ratio, concentration, and spacing of

fluorescein and Cy5-labeled adjacent hybridization probesduring PCR was studied. Amplification of the beta globin

l o locus and probe pair of Example 3 was used and themaximum change in the fluorescence ratio of Cy5 to fluo -rescein was observed. The maximal signal occurred whenthe ratio of Cy5 to fluorescein-labeled probes was 2 : l (FIG.10).At this 2 : l ratio, the best signal occurred at a fluoresceinprobe concentration of 0.2 p M and a Cy5-labeled probeconcentration of 0.4 pM (FIG. 11). The optimal number of intervening bases between adjacent hybridization probesduring PCR was also determined. Several probes of the samelength but slightly shifted in their hybridization position

2o were synthesized according to Example 3 so that when theyhybridized to the beta globin target, 0, 1, 2, 3, 4, or 6 bases

remained between the probes. The highest signal duringPCR occurred with one intervening base (FIG. 12).Although some resonance energy transfer was detected at a

25 spacing of 15 and even 25 bases, much better transfer

occurred at e 5 bases.

Heller et al. (U.S. Pat. No. 4,996,143), found that energytransfer efficiency decreased as the number of nucleotides

3o between fluorophores decreased from 4 to 0 units. Incontrast, the best energy transfer with the fluoresceiniCy5

pair was seen at 0 to 2 intervening nucleotides.

Hybridization probe method. If 2 probes are synthesizedthat hybridize adjacently on a target and each is labeled with

35 one fluorophore of a resonance energy transfer pair, theresonance energy transfer increases when hybridizationoccurs (FIG. 5C). The fluoresceinirhodamine pair is mostcommonly used for nucleic acid detection.

One aspect of this invention is to provide a sequence-40 specific homogeneous hybridization method for detection of

PCR products. It is not obvious how to achieve this. Usinghybridization probes during amplification is counterintui-

tive. It does not seem that both probe hybridization andpolymerase extension can occur. To get sequence specific

45 fluorescence, the probes must be hybridized, but the probescannot be hybridized if the polymerase is to complete primerextension and exponentially amplify DNA.

One solution to this problem is to use a dual-labeled singleprobe and utilize the 5'-exonuclease activity of common heat

SO stable DNA polymerases to cleave the probe duringextension, thereby separating the 2 fluorophores. In thiscase, the fluorescence signal arises from separation of theresonance energy transfer pair upon probe hydrolysis (FIG.5B), rather than approximation of the fluorophores by adja-

ss cent hybridization (FIG. 5C). However, dual-labeled probesare difficult to make, requiring manual addition of at leastone fluorophore to the oligo and usually require extensivepurification. The probes are expensive, and two dual-labeledprobes are necessary for competitive quantification of a

60 target or for mutation detection. Afurther concern is that theobserved fluorescence depends on the cumulative amount of probe hydrolyzed, not directly on the amount of productpresent at any given cycle. This results in a continuedincrease in fluorescence even after the PCR plateau has been

65 reached. Finally and most importantly, probe hydrolysisdoes not always occur during polymerase extension, aneffect that is not well understood. For example, the dual-

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47 48dropped from the denaturation temperature and held con-stant at a lower temperature that is still high enough to

prevent primer annealing (FIG. 2). The rate of productreannealing follows second order kinetics (see B. Young &

M. Anderson, Quantitative analysis of solutionhybridization, In: Nucleic Acid Hybridization: A PracticalApproach 47-71 (B. Hames & S. Higgins, eds., (1985),

which is now incorporated herein by reference). For anygiven PCR product and temperature, a second order rateconstant can be measured. Once the rate constant isknown,any unknown DNA concentration can be determined fromexperimental reannealing data. Cooling is neverinstantaneous, and some reannealing occurs before a con-stant temperature is reached. Rapid cooling will maximizethe amount of data available for rate constant and DNAconcentration determination. The technique requires purePCR product, but such can be assured by melting curves alsoobtained during temperature cycling using the present inven-tion. This method of quantification by the present inventionis advantageously independent of any signal intensity varia-tions between samples.

EXAMPLE 24

A 536 base pair fragment of the beta-globin gene wasamplified from human genomic DNA (Example 7) andpurified (see Example 2). Different amounts of the purifiedDNA were mixed with a 1:30,000 dilution of SYBRTM

Green I in 5p l of 50 mM Tris, pH 8.3 and 3 mM MgC1,. Thesamples were denatured at 94 " C. and then rapidly cooled to

85" C. The fluorescence at 52Ck550 nm was monitored at85" C. over time. When different concentrations of DNAwere tested, the shape of the reannealing curve was char-

acteristic of the DNA concentration (See FIG. 49). For anygiven PCR product and temperature, a second order rateconstant can be determined. FIG. 50 shows the determina-tion of a second order reannealing rate constant for 100 ngof the 536 base pair fragment in 5p l at 85" C. The curve wasfit by non-linear least squares regression with F,,,, Fmi,, to

and k as the floating parameters using the second order rateequation shown in FIG. 50. Analysis programs for this kind

of curve fitting are well known in the art (for example, theuser defined curve fit of Delta Graph, Deltapoint, Inc,Monteray, Calif.). Once the rate constant is known, anunknown DNA concentration can be determined fromexperimental reannealing data.

With the rate constant (k) defined, DNA concentrationsare determined on unknown samples. The fluorescence vstime curves of unknown samples are fit by non-linear leastsquares regression, preferably during temperature cycling inreal time (for example, using the nonlinear Levenberg-

Marquardt method described in the LabView programmingenvironment, National Instruments, Austin, Tex.). For thisfit, F,,,, Fmi,, to, and [DNA] are the floating parameters and

k is constant.Since some fluorescent dyes affect reannealing in a con-

centration dependent manner, the assumption of secondorder kinetics for product reannealing is checked by deter-

mining the rate constant at different standard DNA concen-trations. The relationship isdefined and alternate formula forfitting incorporated as necessary.

Also within the scope of the present invention is to useprobe annealing rates to determine product concentrations.The rate of fluorescence resonance energy transfer is fol-lowed over time after a quick drop to a probe annealingtemperature that is greater than the primer annealing tem-

perature (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 usingtwo labeled probes, the rate of fluorescence development isthird order. These two arrangements are shown in FIG. 18.

5 When the concentration of the probe(s) ismuch greater thanthe product concentration, pseudo-first order and pseudo-second order equations are adequate to describe the possi-bilities. The appropriate rate equations for these different

conditions are well known in the art (see Young, B. andAnderson, M., supra). For the purposes of this invention, itis adequate that the prior art suggests appropriate rateequations that are tested experimentally and corrected if necessary.

When probe annealing rates are used to determine productconcentrations, possible interfering effects include productreannealing (with probe displacement by branch migration)and primer annealing and extension through the probe. Thelater is minimized when the probe Tm's are higher than theprimer Tm's and a probe annealing temperature is chosen tominimize primer annealing. FIG. 13 shows that even if

20 extension occurs, the fluorescence increases with time forabout 20 sec. During this period, the fluorescence increasedepends on product concentration.

Probe annealing rates are used to determine product

concentration similar to the method described above for2s determining product concentration by product reannealing.

The steps are summarized as follows: (1) choosing theappropriate rate equation for the system, (2) running knownDNA standards to determine the rate constant, (3) checkingthe validity of the rate equation by comparing different rate

3o constants derived from different concentrations, and (4)using the rates constants to determine the DNA concentra-tion of unknowns from their probe annealing data.

Fluorescence Feedback for Control of TemperatureCycling. In contrast to endpoint and cycle-by-cycle analysis,

35 the present invention can also monitor fluorescence through-

out each temperature cycle. Continuous fluorescence moni-toring can be used to control temperature cycling param-eters. The present invention uses fluorescence feedback forreal time control and optimization of amplification. Con-

40 tinuous fluorescence monitoring of PCR samples containinga double-strand-specific DNA dye or fluorescently labeledoligonucleotide probes can be used to monitor hybridizationand melting during individual amplification cycles. Thisinformation can be used by the temperature control algo-

45 rithms within the temperature cycling apparatus to improveand customize thermal cycling conditions. ConventionalPCR is performed by programming all cycling parametersbefore amplification. With continuous monitoring, determi-nation of temperature cycling requirements can occur during

so amplification, based on continuous observation of annealing, extension, and denaturation. The potential ben-

efits of using hybridization information to control tempera-ture cycling include:

1. Ensuring complete denaturation of the PCR product

5s each cycle while:a. Minimizing exposure to excessively high denatur-

ation temperatures thus avoiding heat-induced dam-age to the amplification products and polymerase.Limiting the time product isexposed to denaturationtemperatures is especially useful for amplification of long products.

b. Increasing reaction specificity by minimizing thedenaturation temperature. This selects against prod-

ucts with a Tm higher than the intended amplifica-

2. Maximizing the amplification efficiency by ensuringadequate time for primer annealing each cycle while:

10 .

60

65 tion product.

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53 54When multiplex analysis in one PCR reaction is desired, The present invention may be embodied in other specific

it is common practice to use different fluorescent labels with forms without departing from its spirit or essential charac-distinguishable emission spectra to identify the multiple teristics. The described embodiments are to be considered inproducts. The analysis is complicated by the limited number all respects only as illustrative and not restrictive. The scopeof fluorophores available and the overlapping emission s of the invention is, therefore, indicated by the appendedspectra of those fluorophores that are available (see HM claims rather than by the foregoing description. All changesShapiro, supra). Analysis of product or probe hybridization which come within the meaning and range of equivalency of with melting curves is another method to distinguish mul- the claims are to be embraced within their scope.tiple PCR products. By following hybridization during tem- Programming code for carrying out melting curve andperature cycling, the number of probes and/or spectral colors i o other analyses is found in the Programming Code Appendixneeded to distinguish multiple products can be minimized. (Microfiche).

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(iii) NUMBER OF SEQUENCES:27

( 2 ) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 0 base pairs(B) TYPE: nucleic acid

(C) STRANDEDNESS: single-stranded

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CGTGGTGGAC TTCTCTCAAT

( 2 ) INFORMATION FOR SEQ ID NO:2:


(A) LENGTH: 2 0 base pairs

(B) TYPE: nucleic acid



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

AGAAGATGAG GCATAGCAGC



(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid




CAAACAGACA CCATGGTGCA CCTGACTCCT GAGGA



(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid




AAGTCTGCCG TTACTGCCCT GTGGGGCAAG

20

20

35

30



(A) LENGTH: 27 base pairs

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55 56

- continued

(B) TYPE: n u c l e i c a c i d

(C) STRANDEDNESS: s ing le- s t randed

(D) TOPOLOGY: l i n e a r

( x i ) SEQUENCE DESCRIPTION: SEQ ID NO:5:

TCTGCCGTTA CTGCCCTGTG GGGCAAG

(2) INFORMATION FOR SEQ ID NO:6:

(i)SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 b ase p a i r s





CAACTTCATC CACGTNCACC








CTGTCCGTGA CGTGGATT








AAGTCCTCCG AGTATAGC




( B ) TYPE: n u c l e i c a c i d




TAATCTGTAA GAGCAGATCC CTGGACAGGC GAGGAATACA GGTATT



(A) LENGTH: 46 b ase p a i r s( B ) TYPE: n u c l e i c a c i d




TAATCTGTAA GAGCAGATCC CTGGACAGGC AAGGAATACA GGTATT

(2) INFORMATION FOR SEQ ID N0:ll:




27

20

18

18

46

46

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57 58

- continued



( x i ) SEQUENCE DESCRIPTION: SEQ ID N0:ll:

TAATCTGTAA GAGCAGATCC








TGTTATCACA CTGGTGCTAA





(C) STRANDEDNESS: s ing le-

s t randed



AATACCTGTA TTCCTCGCCT GTC








ATGCCTGGCA CCATTAAAGA








GCATGCTTTG ATGACGCTTC




(B) TYPE: n u c l e i c a c i d(C) STRANDEDNESS: s ing le- s t randed



CGGATCTTCT GCTGCCGTCG






20

20

23

20

20

20

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59 60

- continued



CCTCTGACGT CCATCATCTC








GAAGTCTGCC GTTACTGCCC TGTGGGGCAA G



(A) LENGTH: 2 6 b ase p a i r s





CTGCCGTACT GCCCTGTGGG GCAAGG








ATGCCCTCCC CCATGCCATC CTGCGT








CAACTTCATC CACGTTCACC





(C) STRANDEDNESS: s ing le-

s t randed(D) TOPOLOGY: l i n e a r


GTCTGCCGTT ACTGCCCTGT GGGGCAA







20

31

26

26

20

27

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61 62

- continued


CCTCAAACAG ACACCATGGT GCACCTGACT CC








GAAGTCTGCC GTTACTGCCC TGTGGGGCAA








TGAAGGAGAA GGTGTCTGCG GGA








CCTCGGCTAA ATAGTAGTGC GTCGA








AGGACGGTGC GGTGAGAGTG

32

30

23

25

20

soWe claim:1. A method for analyzing a target DNA sequence of a


exciting the biological sample with light at a wavelengthabsorbed by the donor fluorophore and detecting fluo-

rescent emission from the fluorescence energy transferpair.

2. A method for analyzing a target DNA sequence of a


tion in the presence of two nucleic acid probes thathybridize to adjacent regions of the target sequence,One Of said probes being labeled with an acceptorfluorophore and the other probe labeled with a donorfluorophore of a fluorescence energy transfer pair suchthat upon hybridization of the two probes with thetarget sequence, the donor and acceptor fluorophoresare within 25 nucleotides of one another, said poly-merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targeted

biological sample, said method comprising the steps of

tion in the Presence of two nucleic acid Probes that 5shybridize to adjacent regions of the target sequence,

One Of said probes being labeled with an acceptorfluorophore and the other probe labeled with a donorfluorophore of a fluorescence energy transfer pair suchthat upon hybridization of the two probes with thetarget sequence, the donor and acceptor fluorophores 6o

are within 25 nucleotides of one another, said poly-merase chain reaction comprising the steps of adding athermostable polymerase and primers for the targetednucleic acid sequence to the biological sample andthermally cycling the biological sample between at 65

least a denaturation temperature and an elongationtemperature;

biological sample, said method comprising the steps of

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63 64nucleic acid sequence to the biological sample andthermally cycling the biological sample between atleast a denaturation temperature and an elongation

temperature;

by the donor fluorophore; and

fluorescence energy transfer pair.

probes being labeled with an acceptor fluorophore and theother probe labeled with a donor fluorophore of a fluores-cence energy transfer pair.

13. The method of claim 12 wherein a donor fluorophore

spectrum overlap less than 25%, and the acceptor fluoro-phore has a peak extinction coefficient greater than 100,000M -i cm-i an d upon hybridization of the two probes, the

donor and acceptor fluorophores are within 15nucleotides of one another,

energy

transfer pair comprises fluorescein as the donor and Cy5 or~ ~ 5 . 5s the acceptor,

15.A method for detecting a target nucleic acid sequenceof a biological sample, said method comprising the steps of

exciting the sample with light at a wavelength absorbed 5 emission spectrum an d an acceptor fluoroph ore absorption

monitoring temperature dependent fluorescence from the

3. The method of claims 1 or 2 wherein the donor andacceptor fluorophores are at a distance of about 0-5 nucle-

otides.4. The method of claim 3 wherein the donor and acceptor

fluorophores are at a distance of about e 2 nucleotides.5 . The method of claim 4 wherein the donor and acceptor

14,The method of claim 13wherein the

fluorophores are at a distance of 1nucleotide. 1s

6.Amethod of real time monitoring of a polymerase chainreaction amplification of a target nucleic acid sequence in abiological sample, said method comprising the steps of

(a) adding to the biological sample an effective amount of two nucleic acid primers and a nucleic acid probe, 2o

wherein one of said primers and the probe are eachlabeled with one member of a fluorescence energytransfer pair comprising an acceptor fluorophore and adonor fluorophore, and wherein the labeled probehybridizes to an amplified copy of the target nucleic 2s

acid sequence within 15 nucleotides of the labeledprimer;

(b) amplifying the target nucleic acid sequence by poly-

merase chain reaction;

(c) illuminating the biological sample with light of aselected wavelength that is absorbed by said donorfluorophore; and

30

(a) adding to the biological sample an effective amount of a pair of oligonucleotide probes that hybridize to thetarget nucleic acid sequence, one of said probes beinglabeled with an acceptor fluorophore and the otherprobe labeled with a donor fluorophore of a fluores-

cence energy transfer pair, wherein an emission spec-trum of the donor fluorophore and an absorption spec-

trum of the acceptor fluorophore overlap less than 25%,

the acceptor fluorophore has a peak extinction coeffi-cient greater than 100,000 M-' cm-I and upon hybrid-ization of the two probes, the donor and acceptorfluorophores are within 25 nucleotides of one another;

(b) illuminating the biological sample with a selectedwavelength of light that is absorbed by said donorfluorophore; and

(c) detecting fluorescent emission of the biologicalsample.

16. The method of claim 15 wherein the resonance enerpv,(d) detecting the fluorescence emission of the sample.7. The method of claim 6 further comprising the step of 35

transfer pair comprises fluorescein as the donor.17. The method of claim 16 wherein the resonance energy

monitoring temperature dependent fluorescence of the transfer pair comprises Cy5 or Cy5.5 as the acceptor.sample. 18. The method of claim 15 wherein the donor and

8. The method of claims 6 wherein the donor and acceptor acceptor fluorophores are within about 0-5 nucleotides of fluorophores are at a distance of about 4-6 nucleotides from one another upon hybridization of the two probes with the

one another.9. An improved method of amplifying a target nucleic 19. The method of claim 18 wherein the donor and

acid sequence biological sample, said method comprising acceptor fluorophores are within about 0-2 nucleotides of the steps of each other.

(a) adding to the biological sample an effective amount of 20. The method of claim 19 wherein the donor and

a nucleic-acid-binding fluorescent entity; 45 acceptor fluorophores are within 1nucleotide of each other.

(b) amplifying the target nucleic acid sequence using 21. A method of real time monitoring of a Polymerasepolymerase chain reaction, comprising thermal ly chain reaction amplification of a target nuclcic acidcycling the biological sample using initial predeter- sequence in a biological sample, said method comprising themined temperature and time parameters, and then steps of

(i) illuminating the biological sample with a selected so amplifying the target sequence by polymerase chain reac-wavelength of light that is absorbed by said fluores- tion in the presence of two nucleic acid probes thatcent entity during the polymerase chain reaction; hybridize to adjacent regions of the target sequence,

(ii) monitoring fluorescence from said sample to deter- one of said probes being labeled with an acceptormine the optimal temperature and time parameters fluorophore and the other probe labeled with a donor

for the polymerase chain reaction; and fluorophore of a fluorescence energy transfer pair such(iii) adjusting the initial temperature and time param- that upon hybridization of the two probes with the

eters in accordance with the fluorescence, target sequence, the donor and acceptor fluorophoreswherein the monitoring fluorescence step consists of moni - are within 25 nucleotides of one another, said poly-

toring an amplification dependent emission of the fluores- merase chain reaction comprising the steps of addingcent entity. 60 the two nucleic acid probes, a thermostable

10. The method of claim 9 wherein the fluorescent entity polymerase, and primers for the targeted nucleic acidcomprises a double strand specific nucleic acid binding dye. sequence to the biological sample to create an ampli-

11.The method of claim 9 wherein the fluorescent entity fication medium and thermally cycling the amplifica-

comprises a fluorescently labeled oligonucleotide probe that tion medium between at least a denaturation tempera-

hybridizes to the targeted nucleic acid sequence. ture and an elongation temperature; and

12. The method of claim 9 wherein the fluorescent entity illuminating the amplification medium with light having acomprises a pair of oligonucleotide probes, one of said wavelength absorbed by the donor fluorophore and

40 target nucleic acid sequence.

5s

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65 66detecting fluorescent emission from the amplificationmedium at a plurality of temperatures;

to monitor the temperature dependent fluorescent emis-sion.

22.The method of claim 21wherein the resonance energy s

33. The method of claim 29 wherein the donor andacceptor fluorophores are at a distance of about 4-6 nucle-otides from one another when the labeled probe is hybrid-

ized to an amplified copy of the target nucleic acid sequence.34. A method of detecting a difference at a selected locus

transfer pair comprises fluorescein as the donor and Cy5 or in a first nucleic acid as compared to a secon d nucleic acid,Cy5.5 as the acceptor.23' The method Of 21 wherein th e donor an d

(a) providing a pair of oligonucleotide primers configuredacceptor fluorophores are within about 0-

5 nucleotides of for amplifying, by polymerase chain reaction, aone another.

selected segment of the first nucleic acid and a corre-24. The method of claim 23 wherein the donor and

acceptor fluorophores are within about 0-2 nucleotides of sponding segment of the second nucleic acid, wherein

one another. the selected segment and corresponding segment each

25. The method of claim 24 wherein the donor and comprises the selected locus, to result in amplifiedacceptor fluorophores are within 1nucleotide of each other. products containing a copy of the selected locus;

26. The method of claim 21 wherein said monitoring step (b) providing a pair of oligonucleotide probes, one of saidcomprises determining melting profiles of the probes melt- probes being labeled with an acceptor fluorophore anding from said target sequence. the other probe being labeled with a donor fluorophore

27. A method of real time monitoring of a polymerase of a fluorogenic resonance energy transfer pair suchchain react ion amplification of a target nucleic acid that upon hybridization of the two probes with thesequence in a biological sample, said method comprising the 20 amplified products the dono r and acceptor ar e in reso-

nance energy transfer relationship, wherein one of thesteps of

amplifying the target sequence by polymerase chain reac- probes is configured for hybridizing to the amplified

products such that said one of the probes spans thetion in the presence of a fluorescent entity, said poly -

selected locus and exhibits a first melting profile whenmerase chain reaction comprising the steps of adding

primers for the target nucleic acid sequence to thedistinguishable from a second melting profile of the

biological sample to create an amplification mediumsecond nucleic acid;

and thermally cycling the amplification mediumbetween at least a denaturation temperature and an (C) amplifying the selected segment of first nucleic acid

elongation temperature; and the corresponding segment of the second nucleic

exciting the fluorescent entity with light at a wavelength acid by polymerase chain reaction in the presence of absorbed by the fluorescent entity and detecting fluo- effective amounts of probes to result in an amplifiedrescent emission from the fluorescent entity; and selected segment and an amplified corresponding

monitoring temperature dependent fluorescence from the segment, at least a portion thereof having both thefluorescent entity in the amplification medium; probes hybridized thereto with the fluorogenic reso-

wherein the monitoring step consists of monitoring an nance energy transfer pair in resonance energy transferamplification dependent signal of the fluorescent entity, and relationship;

wherein the fluorescent entity is SYBRTMGreen I. (d) illuminating the amplified selected segment and the28. The method of claim 27 further comprising the step of amplified corresponding segment with the probes

determining a melting profile of the amplified target 4o hybridized thereto with light to elicit fluorescence bysequence. the fluorogenic resonance energy transfer pair;

29. A method for analyzing a target DNA sequence of a (e) measuring fluorescence emission as a function of biological sample, said method comprising the steps of temperature to determine the first melting profile of

(a) adding to the biological sample an effective amount of said one of the probes melting from the amplified

two nucleic acid primers and a nucleic acid probe to 45 selected segment of first nucleic acid and the secondform an amplification medium, wherein one of said melting profile of said one of the probes melting fromprimers and the probe are each labeled with one mem- the amplified corresponding segment of second nucleicber of a fluorescence energy transfer pair comprising an acid; and

acceptor fluoroPhore and a donor fluorophore, and ( f ) comparing the first melting profile to the secondwherein the labeled Probe Hybridizes to an amplified SO melting profile, wherein a difference therein indicatescopy of the target nucleic acid sequence within 15 the presence of the difference in the sample nucleicnucleotides of the labeled primer; acid.

(b) amplifying the target nucleic acid sequence by poly- 35. The method of claim 34 wherein the resonance energymerase chain reaction; transfer pair comprises fluorescein as the donor and a

(c) illuminating the amplification medium with light hav- ss member selected from the group consisting of Cy5 anding wavelength that is absorbed by said donor fluoro- Cy5.5 as the acceptor.phore and detecting the fluorescence emission from the 36. The method of claim 34 wherein the donor and themedium. acceptor are coupled to the probes such that when both

30. The method of claim 29 further comprising the step of probes are hybridized to the amplified selected segment ormonitoring the temperature dependent fluorescence of the 60 the amplified corresponding segment, the donor and accep-sample. tor flurophores are separated by no more than about 25

31. The method of claim 30 wherein said monitoring step nucleotide residues.comprises determining a melting profile of the amplified 37. The method of claim 36 wherein the donor and thetarget sequence. acceptor are coupled to probes such that when the probes are

32. The method of claim 29 wherein the resonance energy 65 hybridized to the amplified selected segment or the ampli-transfer pair comprises fluorescein as the donor and Cy5 or fied corresponding segment, the donor and acceptor areCy5.5 as the acceptor. flurophores separated by about 0-5 nucleotide residues.

comprising the steps of

10

th e fluorescent entity, a polymerase, an d 25 the difference is present in the first nucleic acid that is

3o

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67 6838. The method of claim 37 wherein the resonance energy to the amplified selected segment or the amplified corre-

transfer donor and the resonance energy transfer acceptor sponding segment, the donor and acceptor are separated byflurophores are coupled to the probes such that when the no more than about 15nucleotide residues.probes are hybridized to the amplified selected segment or 43. The method of claim 42 wherein the donor and thethe amplified corresponding segment the donor and acceptor s acceptor are coupled to labeled primer and probe such thatflurophores are separated by about e 2 nucleotide residues. when the labeled primer and probe are hybridized to the

39. The method of claim 38 wherein the resonance energy amplified selected segment or the amplified correspondingtransfer donor and the resonance energy transfer acceptor segment, the donor and acceptor are separated by about 4-6

are coupled to the probes such that when the probes are nucleotide residues.hybridized to the amplified selected segment or the ampli- i o 44. A method of detecting heterozygosity at a selectedfied corresponding segment the donor and acceptor fluro- locus in the genome of an individual, wherein the genomephores are separated by 1nucleotide residue. comprises a mutant allele and a corresponding reference

40.A method of detecting a difference at a selected locus allele, each comprising the selected locus, comprising thein a first nucleic acid as compared to a second nucleic acid, steps of

comprising the steps of 1s

(a) providing a pair of oligonucleotide primers configuredfor amplifying, by polymerase chain reaction, aselected segment of the first nucleic acid and a corre -sponding segment of the second nucleic acid, whereinthe selected segment and corresponding segment each 20

comprises the selected locus, to result in amplifiedproducts containing a copy of the selected locus;

(b) providing an oligonucleotide probe, wherein one of said primers and the probe are each labeled with onemember of a fluorescence energy transfer pair com- 2s

prising an donor fluorophore and an acceptorfluorophore, and wherein the labeled probe and labeledprimer hybridize to the amplified products such that thedonor and acceptor are in resonance energy transferrelationship, and wherein the probe is configured for 30

hybridizing to the amplified products such that saidprobe spans the selected locus and exhibits a meltingprofile when the difference ispresent in the first nucleicacid that isdistinguishable from a melting profile of thesecond nucleic acid;

(c) amplifying the selected segment of first nucleic acidand the corresponding segment of the second nucleicacid by polymerase chain reaction in the presence of

effective amounts of primers and probe to result in an 4o

amplified selected segment and an amplified corre-

sponding segment, at least a portion thereof having theprimer and probe hybridized thereto with the fluoro-

genic resonance energy transfer pair in resonanceenergy transfer relationship;

(d) illuminating the amplified selected segment and theamplified corresponding segment with the labeledprimer and probe hybridized thereto with a selectedwavelength of light to elicit fluorescence by the fluo-

(e) measuring fluorescence emission as a function of temperature to determine in a first melting profile of said probe melting from the amplified selected segmentof first nucleic acid and a second melting profile of said

3s

4s

rogenic resonance energy transfer pair; so


(b) providing a pair of oligonucleotide primers configuredfor amplifying, by polymerase chain reaction, a firstselected segment of the mutant allele and a secondselected segment of the corresponding reference allelewherein both the first and second selected segmentscomprise the selected locus;

(c) providing a pair of oligonucleotide probes, one of saidprobes being labeled with an acceptor fluorophore andthe other probe being labeled with a donor fluorophoreof a fluorogenic resonance energy transfer pair suchthat upon hybridization of the two probes with theamplified first and second selected segments one of theprobes spans the selected locus and exhibits a firstmelting profile with the amplified first selected segmentthat is distinguishable from a second melting profilewith the amplified second selected segment;

(d) amplifying the first and second selected segments of sample genomic DNA by polymerase chain reaction inthe presence of effective amounts of probes to result inamplified first and second selected segments, at least aportion thereof having both the probes hybridizedthereto with the fluorogenic resonance energy transferpair in resonance energy transfer relationship;

(e) illuminating the amplified first and second selectedsegments having the probes hybridized thereto with aselected wavelength of light to elicit fluorescence bythe donor and acceptor;

( f ) measuring a fluorescence emission as a function of temperature to determine a first melting profile of saidone of the probes melting from the amplified firstselected segment and a second melting profile of saidone of the probes melting from the amplified secondselected segment; and

(g) comparing the first melting profile to the secondmelting profile, wherein distinguishable melting pro-files indicate heterozygosity in the sample genomicDNA.

45. The method of claim 44 wherein the fluoronenic

Probe melting from the amplified corr&ondi% seg- ss resonance energy transfer pair comprises fluorescein a thement of second nucleic acid; and donor and Cy5 or Cy5.5 as the acceptor.

( f ) comparing the first melting profile to the second 46. The method of claim 44 wherein the donor and themelting profile, wherein a difference therein indicates acceptor are coupled to the probes such that when the probethe presence of the difference in the sample nucleic and reference oligonucleotide are hybridized to the ampli-acid. 60 fied first selected segment or the amplified second selected

41. The method of claim 40 wherein the resonance energy segment, the donor and acceptor are separated by no moretransfer pair comprises fluorescein as the donor and a than about 25 nucleotide residues.member selected from the group consisting of Cy5 and 47. The method of claim 46 wherein the donor and theCy5.5 as the acceptor. acceptor are coupled to the probes such that when the probes

42. The method of claim 40 wherein the donor and the 65 are hybridized to the amplified first selected segment or theacceptor are coupled to the labeled primer and probe such amplified second selected segment, the donor and acceptorthat when both the labeled primer and probe are hybridized are separated by about 0-5 nucleotide residues.

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71(d) monitoring fluorescence emission; and

(e) determining a cycle when the fluorescence emissionreaches a plateau phase, indicating the completion of the reaction;

wherein the monitoring fluorescence emission step consistsof monitoring an amplification depend emission of thefluorescent dye.

57. The method of claim 56 wherein the nucleic-acid-binding fluorescent dye isa member selected from the groupconsisting of S YBRT MGREEN I, ethidium bromide, pic0

green, acridine orange, thiazole orange, YO-PRO-1, andchromomycin A3.

58. The method of claim 57 wherein the double-strand-

specific fluorescent dye is S YBRT MGREEN I.59.Amethod of determining a concentration of a selected

nucleic acid template by competitive quantitative poly-merase chain reaction comprising the steps of

(a) in a reaction mixture comprising:(i) effective amounts of each of a pair of oligonucle-

otide primers configured for amplifying, in a poly-merase chain reaction, a selected segment of the

selected template and a corresponding selected seg-ment of a competitive template to result in amplifiedproducts thereof,

(ii) an effective amount of an oligonucleotide probe

labeled with a resonance energy transfer donor or aresonance energy transfer acceptor of a fluorogenicresonance energy transfer pair, wherein the probe isconfigured for hybridizing to the amplified productssuch that the probe melts from the amplified productof the selected template at a melting temperature thatis distinguishable from the melting temperature atwhich the probe melts from the amplified product of the competitive template,

(iii) an effective amount of a reference oligonucleotidelabeled with the donor or the acceptor, with theproviso that as between the probe and referencetherefor; oligonucleotide one is labeled with the

donor and the other is labeled with the acceptor,wherein the reference oligonucleotide is configuredfor hybridizing to the amplified products such thatthe donor and the acceptor are in resonance energytransfer relationship when both the probe and thereference oligonucleotide hybridize to the amplifiedproducts;

amplifying, by polymerase chain reaction, an unknownamount of the selected template and a known amount of thecompetitive template to result in the amplified productsthereof;

(b) illuminating the reaction mixture with a selectedwavelength of light for eliciting fluorescence by thefluorogenic resonance energy transfer pair and deter-

mining a fluorescence emission as a function of tem-

perature as the temperature of the reaction mixture ischanged to result in a first melting curve of the probemelting from the amplified product of the selectedtemplate and a second melting curve of the probemelting from the competitive template;

(c) converting the first and second melting curves to firstand second melting peaks and determining relativeamounts of the selected template and the competitivetemplate from such melting peaks; and

(d) calculating the concentration of the selected templatebased on the known amount of the competitive tem-plate and the relative amounts of selected template andcompetitive template.

7260. The method of claim 59 wherein the reference oligo-

nucleotide is one of the pair of oligonucleotide primers.61. The method of claim 60 wherein the resonance energy

transfer pair comprises fluorescein as the donor and Cy5 or

62. The method of claim 60 wherein the donor and theacceptor are coupled to the probe and reference oligonucle-

otide such that when the probe and reference oligonucleotide

are hybridized to the amplified segment, the donor andacceptor are separated by no more than about 25 nucleotideresidues.

63. The method of claim 62 wherein the donor and theacceptor are coupled to the probe and the reference oligo-

nucleotide such that when the probe and reference oligo-1~ nucleotide are hybridized to the amplified segment, the

donor and acceptor are separated by about 0-5 nucleotideresidues.

64. The method of claim 63 wherein the donor and theacceptor are coupled to the probe and reference oligonucle-

2o otide such that when the probe and transfer oligonucleotideare hybridized to the amplified segment, the donor andacceptor are separated by about 0-2 nucleotide residues.

65. The method of claim 64 wherein the donor and the

acceptor are coupled to the probe and reference oligonucle-25 otide such that when the probe and transfer oligonucleotide

are hybridized to the amplified segment, the donor andacceptor are separated by 1nucleotide residue.

66. The method of claim 59 wherein the reference oligo-nucleotide is not one of the oligonucleotide primers.

67. The method of claim 66 wherein the resonance energytransfer pair comprises fluorescein as the donor and Cy5 orCy5.5 as the acceptor.

68. The method of claim 67 wherein the donor and theacceptor are coupled to the probe and the reference oligo-

35 nucleotide such that when the probe and reference oligo-

nucleotide are hybridized to the amplified segment, thedonor and acceptor are separated by no more than about 15nucleotide residues.


40 acceptor are coupled to the probe and the reference oligo-nucleotide such that when the probe and reference oligo-nucleotide are hybridized to the amplified segment, thedonor and acceptor are separated by about 4-6 nucleotideresidues.

70.Amethod of determining a concentration of a selectednucleic acid template in a polymerase chain reaction com-prising the steps of

5 Cy5.5 as the acceptor.

30

45

(a) in a reaction mixture comprising:(i) effective amounts of each of a first pair of oligo-

nucleotide primers configured for amplifying, in apolymerase chain reaction, a selected first segmentof the selected template to result in an amplified firstproduct thereof,

(ii) effective amounts of each of a second pair of

oligonucleotide primers configured for amplifying,in a polymerase chain reaction, a selected secondsegment of a reference template to result in anamplified second product thereof,

(iii) an effective amount of a nucleic-acid-binding

amplifying, by polymerase chain reaction, an unknownamount of the selected template to result in the amplifiedfirst product and a known amount of the reference templateto result in the amplified second product thereof;

(b) illuminating the reaction mixture with a selectedwavelength of light for eliciting fluorescence by thenucleic-acid-binding fluorescent dye;

so

55

60 fluorescent dye;

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73(c) continuously monitoring through a series of amplifi-

cation cycles the fluorescence emitted as a function of temperature and choosing one cycle to result in amelting curve of the amplified products wherein thefirst product and second product melt at differenttemperatures, wherein the monitoring consists of moni-toring the amplification dependent fluorescence emittedas a function of temperature;

(d) converting the melting curves to melting peaks anddetermining relative amounts of the selected templateand the reference template from such melting peaks;and

(e) calculating the concentration of the selected templatebased on the known amount of the reference templateand the relative amounts of selected template andreference template.

71. The method of claim 70 wherein the nucleic-acid-binding fluorescent dye is selected from the group consistingof S YBRT MGREEN I, ethidium bromide, pic0 green, acri-dine orange, thiazole orange, YO-PRO-1, and chromomycinA3.

72. The method of claim 71 wherein the double-strandspecific fluorescent dye is S YBRT MGREEN I.

73. A method of monitoring amplification of a selected

template in a polymerase chain reaction that also comprisesa positive control template comprising the steps of

(a) in a reaction mixture comprising:(i) effective amounts of each of a first pair of oligo-

nucleotide primers configured for amplifying, in apolymerase chain reaction, a selected first segmentof the selected template to result in an amplified firstproduct thereof,

(ii) effective amounts of each of a second pair of oligonucleotide primers configured for amplifying,in a polymerase chain reaction, a selected secondsegment of the positive control template to result inan amplified second product thereof,

(iii) an effective amount of a nucleic-acid-bindingfluorescent dye;

subjecting the selected template and the positive controltemplate to conditions for amplifying the selected templateand the positive control template by polymerase chainreaction; and

(b) illuminating the reaction mixture with a selected

wavelength of light for eliciting fluorescence by thenucleic-acid-binding fluorescent dye and continuouslymonitoring the fluorescence emitted as a function of temperature during an amplification cycle of the poly-

merase chain reaction to result in a first melting curveof the amplified first product, if the selected template is

amplified, and a second melting curve of the amplifiedsecond product, if the positive control template isamplified;wherein obtaining of the second melting curve indi-

cates that the polymerase chain reaction was

operative, obtaining the first melting curve indicatesthat the selected first segment was amplifiable, andabsence of the first melting curve indicates that theselected first segment was not amplifiable.

74. The method of claim 73 wherein the nucleic-acid-binding fluorescent dye is selected from the group consistingof S YBRT MGREEN I, ethidium bromide, pic0 green, acri-

dine orange, thiazole orange, YO-PRO-1, and chromomycinA3.

75. The method of claim 74 wherein the double-strandspecific fluorescent dye is S YBRT MGREEN I.

76. A method of analyzing nucleic acid hybridizationcomprising the steps of

74(a) providing a mixture comprising a nucleic acid sample

to be analyzed and a nucleic acid binding fluorescententity; and

(b) monitoring fluorescence while changing temperatureat a rate of 20.1" C./second.

77. The method of claim 76 wherein the nucleic acidbinding fluorescent entity is S YBRT MGreen I.

78. The method of claim 76 wherein the nucleic acidbinding fluorescent entity comprises a pair of oligonucle -10 otide probes wherein one of the probes is labeled with a

donor and the other probes is labeled with an acceptor of afluorogenic resonance energy transfer pair.

79. The method of claim 78 wherein one of the probes is

a primer for polymerase chain reaction amplification.80. A method of quantitating an initial copy number of a

sample containing an unknown amount of nucleic acidcomprising the steps of

(a) amplifying by polymerase chain reaction at least onestandard of known concentration in a mixture compris-ing the standard and a nucleic acid binding fluorescententity;

(b) measuring fluorescence as a function of cycle numberto result in a set of data points;

(c) fitting the data points to a given predetermined equa -tion describing fluorescence as a function of initialnucleic acid concentration and cycle number;

(d) amplifying the sample containing the unknownamount of nucleic acid in a mixture comprising thesample and the nucleic acid binding fluorescent entityand monitoring fluorescence thereof; and

(e) determining initial nucleic acid concentration from theequation determined in step (c).

81. The method of claim 80 wherein the nucleic acid35 binding fluorescent entity is S YBRT MGreen I.

82. The method of claim 80 wherein the nucleic acidbinding fluorescent entity comprises a pair of oligonucle -otide probes, one of which is labeled with a donor and theother of which is labeled with an acceptor of a fluorogenic

40 resonance energy transfer pair.83. The method of claim 82 wherein one of the probes is

a primer for amplification.84. The method of claim 27 wherein the temperature

dependent fluorescence is used to identify the amplified

85. The method of claim 84 wherein the amplified targetsequence is characterized by analysis of melting curves.

86. The method of claim 85 wherein the relative amountsof two or more amplified target sequences are determined by

87. The method of claim 85 wherein areas under themelting curves are found by none ear least squares regres-

sion of the sum of multiple gaussians.88. The method of claim 21, further comprising monitor-

5s ing the temperature dependent emission while changingtemperature at a rate of 20.1" C./second.

89. The method of claim 27, further comprising monitor-ing the amplification dependent signal of the fluorescententity while changing temperature at a rate of 2 0 . 1 "

90. The method of claim 30, further comprising monitor-ing the fluorescence while changing temperature at a rate of 20.1" C./second.

91. A method for analyzing a target DNA sequence of a


tion in the presence of two nucleic acid probes that

5

15

2o

25

3o

45 target sequence.

50 analysis of melting curves.

60 C./second.

65 biological sample, said method comprising the steps of

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77one of the probes melting from the amplified first amplified second selected segment, the donor and acceptorselected segment and a second melting profile of said are separated by 1nucleotide residue.one of the probes melting from the amplified second 104. The method of claim 14 wherein the resonanceselected segment; and energy transfer pair comprises fluorescein as the donor and

(g) comparing the first melting profile to the second 5 cY5.5 as the acceptor.

melting profile, wherein distinguishable melting pro- 105. The method of claim 22 wherein the resonancefiles indicate heterozygosity in the sample genomic energy transfer pair comprises fluorescein as the donor andDNA. Cy5.5 as the acceptor.

99 , The method of claim 98 wherein the fluorogenic 106. The method of claim 55 wherein the fluorogenic

107. An improved method of amplifying a target nucleicacid sequence of a biological sample, said method compris-

ing the steps of

(a) adding to the biological sample an effective amount of

resonance energy transfer pair comprises fluorescein as the 10 “I an ce energy transfer Pair is fluorescein and cY5.5.

donor and Cy5 or Cy5.5 as the acceptor.100, The method of claim 98 wherein the donor and the

acceptor are coupled to the probes such that when the probeand reference oligonucleotide are hybridized to the ampli-fied first selected segment or the amplified second selected 15

segment, the donor and acceptor are separated by no morethan about 25 nucleotide residues.

acceptor are coupled to the probes such that when the probesare hybridized to the amplified first selected segment or the 20

amplified second selected segment, the donor and acceptorare separated by about 0-5 nucleotide residues.


acceptor are coupled to the probes such that when the probesare hybridized to the amplified first selected segment or the 25

amplified second selected segment, the donor and acceptorare separated by about 0-2 nucleotide residues.

103. The method of claim 102 wherein the donor and theacceptor are coupled to the probes such that when the probes

a fluorescent label consisting of a nucleic-acid-bindingfluorescent entity;

(b) amplifying the target nucleic acid sequence using101.The method of claim 100 wherein the donor and the polymerase chain reaction, comprising thermally

cycling the biological sample using initial predeter-

mined temperature and time parameters, and then(i) illuminating the biological sample with a selected

wavelength of light that is absorbed by said fluores-

cent entity during the polymerase chain reaction;

(ii) monitoring fluorescence from said sample to deter-mine the optimal temperature and time parametersfor the polymerase chain reaction; and

(iii) adjusting the initial temperature and time param-

eters in accordance with the fluorescence.

are hybridized to the amplified first selected segment or the * * * * *

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