Nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon

Claims

What is claimed is:

1. An oligonucleotide for use as a primer in detecting a target nucleotide sequence, said oligonucleotide comprising:

(a) a first nucleotide sequence complementary to a sequence flanking said target sequence;

(b) a second nucleotide sequence at the 5' end of said first sequence;

(c) a third nucleotide sequence at the 5' end of said second sequence;

(d) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and

(e) means for emitting a detectable signal when the strands of said duplex are separated.

2. The oligonucleotide according to claim 1 wherein said signal emitting means comprises an energy donor moiety and an energy acceptor moiety, each bound to said oligonucleotide and spaced such that said signal is detectable only when the strands of said duplex are separated.

3. The oligonucleotide according to claim 2 wherein said energy donor moiety is a fluorophore and said energy acceptor moiety is a fluorophore quencher.

4. A method for the amplification and detection of a target nucleotide sequence in a sample comprising the steps of:

(a) providing a pair of primers each complementary to said target nucleotide sequence, at least one member of said primer pair comprising the detecting oligonucleotide of claim 1;

(b) separating the strands of the nucleic acid containing the target nucleotide sequence;

(c) annealing said pair of primers to the opposite strands of said separated nucleic acid;

(d) synthesizing new strands of nucleic acid complementary to the strands of said separated nucleic acid;

(e) separating said new strands from their complementary strands; and

(f) repeating steps (c)-(e) wherein the synthesis of new strands separates the duplex strands of said oligonucleotide, thereby causing said detectable signal to be emitted.

5. A kit for use in detecting a target nucleotide sequence comprising:

(a) first and second oligonucleotide primers at least one of which comprises:

(i) a 3' nucleotide sequence that is complementary to a sequence flanking said target nucleotide sequence;

(ii) a 5' nucleotide sequence that is not complementary to a sequence flanking said target sequence; and

(b) a third oligonucleotide primer comprising:

(i) a first sequence identical to said 5' sequence;

(ii) a second sequence at the 5' end of said first sequence;

(iii) a third nucleotide sequence at the 5' end of said second sequence;

(iv) a fourth nucleotide sequence at the 5' end of said third sequence, said fourth sequence being complementary to said second sequence so as to form a double stranded duplex, and

(v) means for emitting a detectable signal when the strands of said duplex are separated.

6. The kit according to claim 5 wherein said 5' nucleotide sequence is not a naturally occurring sequence.

7. The oligonucleotide of claim 2 wherein said energy donor and acceptor moieties are spaced a distance in the range of about 10-40 nucleotides.

8. The oligonucleotide of claim 2 wherein said acceptor moiety is a fluorophore that emits fluorescent light at a wavelength different than that emitted by said donor moiety.

9. The oligonucleotide of claim 1 wherein said target nucleotide sequence is selected from the group consisting of genomic DNA, cDNA, mRNA, and chemically synthesized DNA.

10. The oligonucleotide of claim 1 wherein said target nucleotide sequence is a sequence of an infectious disease agent.

11. The oligonucleotide of claim 1 wherein said target nucleotide sequence is a wild-type human genomic sequence, mutation of which is implicated in the presence of a human disease or disorder.

12. The oligonucleotide of claim 2 wherein said donor moiety is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, and Reactive Red 4, and said acceptor moiety is selected from the group consisting of DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, fluorescein, Malachite green, and Texas Red.

13. The oligonucleotide of claim 12 wherein said donor moiety is fluorescein or a derivative thereof, and said acceptor moiety is DABCYL.

14. The oligonucleotide of claim 1 wherein said first or third nucleotide sequence further comprises a restriction endonuclease recognition site.

15. The oligonucleotide of claim 2 wherein said energy donor moiety and said energy acceptor moiety are situated on complementary nucleotides that are opposite each other in said duplex.

16. The oligonucleotide of claim 2 wherein said energy donor moiety and said energy acceptor moiety are situated on opposite strand nucleotides that are five nucleotides apart in said duplex.

17. A kit comprising in one or more containers:

(a) a first oligonucleotide; and

(b) a second oligonucleotide, wherein said first and second oligonucleotides are primers for use in a nucleic acid amplification reaction to amplify a preselected target nucleic acid sequence, and at least one of said first and second oligonucleotides is the oligonucleotide of claim 1.

18. The kit of claim 17 which further comprises a blocking oligonucleotide comprising a sequence complementary and hybridizable to a sequence of said first or said second oligonucleotide.

19. The kit of claim 17 which further comprises in one or more containers:

(c) an optimized buffer for said amplification reaction;

(d) a control nucleic acid comprising the preselected target sequence; and

(e) a DNA polymerase.

20. A kit comprising in one or more containers:

(a) a first oligonucleotide;

(b) a second oligonucleotide, wherein said first and second oligonucleotides are primers for use in a nucleic acid amplification reaction to amplify a first preselected target nucleic acid sequence, and at least one of said first and second oligonucleotides is the oligonucleotide of claim 3;

(c) a third oligonucleotide, and

(d) a fourth oligonucleotide, wherein said third and fourth oligonucleotides are primers for use in said nucleic acid amplification reaction to amplify a second preselected target sequence, and at least one of said third and fourth oligonucleotides is an oligonucleotide of claim 3, and wherein said donor moiety of said first and second oligonucleotide emits fluorescent light of a different wavelength than said donor moiety of said third or fourth oligonucleotide.

21. The kit of claim 17 wherein said amplification reaction is selected from the group consisting of the polymerase chain reaction, strand displacement, triamplification and NASBA.

22. An oligodeoxynucleotide, the sequence of which consists of: 5'-ACCTTCTACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:13), wherein fluorescein or a derivative thereof is attached to the 5' A and DABCYL is attached to the T at nucleotide number 20.

23. An oligodeoxynucleotide, the sequence of which consists: 5'-CACCTTCACCCTCAGAAGGTGACCAAGTTCAT-3' (SEQ ID NO:18), wherein fluorescein or a derivative thereof is attached to the 5' C and DABCYL is attached to the T at nucleotide number 20.

24. The kit of claim 17 wherein said first and second oligonucleotides are oligodeoxynucleotides.

25. A method for detecting or measuring a product of a nucleic acid amplification reaction comprising:

(a) contacting a sample comprising nucleic acids with at least two oligonucleotide primers, said oligonucleotide primers being adapted for use in said amplification reaction such that said primers are incorporated into an amplified product of said amplification reaction when a preselected target sequence is present in the sample; at least one of said oligonucleotide primers being the oligonucleotide of claim 2;

(b) conducting the amplification reaction;

(c) stimulating energy emission from said donor moiety; and

(d) detecting or measuring energy emitted by said acceptor moiety.

26. The method of claim 25 wherein said donor moiety is a fluorophore.

27. The method of claim 26 wherein said acceptor moiety is a quencher of light emitted by said fluorophore.

28. The method of claim 26 wherein said acceptor moiety emits fluorescent light of a wavelength different from that emitted by said donor moiety.

29. The method of claim 25 wherein said preselected target sequence is selected from the group consisting of genomic DNA, cDNA and mRNA.

30. The method of claim 25 wherein said donor moiety is selected from the group consisting of fluorescein, 5-carboxyfluorescein (FAM), rhodamine, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid (EDANS), anthranilamide, coumarin, terbium chelate derivatives, and Reactive Red 4; and said acceptor moiety is selected from the group consisting DABCYL, rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine, ethidium, Malachite green, fluorescein and Texas Red.

31. The method of claim 25 wherein said donor moiety is fluorescein or a derivative thereof, and said acceptor moiety is DABCYL.

32. The method of claim 25 wherein the oligonucleotide is a oligodeoxynucleotide.

33. The method of claim 30 wherein said donor moiety and said acceptor moiety are situated on complementary nucleotides that are opposite each other in said duplex.

34. The method of claim 30 wherein said donor moiety and said acceptor moiety are situated on opposite strand nucleotides that are five nucleotides apart in said duplex.

35. The method of claim 30 wherein said oligonucleotide primers comprise a plurality of different oligonucleotides, each oligonucleotide comprising at its 3' end a said sequence complementary to different preselected target sequence whereby said different oligonucleotides are incorporated into different amplified products when each said target sequence is present in said sample, each said oligonucleotide being labeled with a donor moiety that emits light of a different wavelength than that emitted by the other donor moieties, and wherein step (d) of said method comprises detecting or measuring light emitted by each of the donor moieties.

36. The method of claim 30 wherein said amplification reaction is selected from the group consisting of polymerase chain reaction, allele-specific polymerase chain reaction, triamplification, strand displacement, and NASBA.

37. The kit of claim 17 which further comprises in a separate container DNA ligase.

38. The method of claim 25 which further comprises prior to said conducting step, contacting said nucleic acids with an amount of bisulfite sufficient to convert unmethylated cytosines in the sample to uracil.

TABLE OF CONTENTS

1. INTRODUCTION

2. BACKGROUND OF THE INVENTION

2.1. FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET)

2.2. METHODS OF MONITORING NUCLEIC ACID AMPLIFICATION

3. SUMMARY OF THE INVENTION

3.1. DEFINITIONS

4. DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. OLIGONUCLECOTIDES

5.1.1. HAIRPIN PRIMERS

5.1.1.1. UNIVERSAL HAIRPIN PRIMERS

5.1.2. LINEAR OLIGONUCLEOTIDES

5.2. METHODS FOR DETECTION OF AMPLIFICATION PRODUCTS USING HAIRPIN PRIMERS

5.2.1. METHODS OF USE OF HAIRPIN PRIMERS IN POLYMERASE CHAIN REACTION (PCR)

5.2.1.1. METHODS OF USE OF HAIRPIN PRIMERS IN ALLELE-SPECIFIC PCR (ASP)

5.2.2. METHODS OF USE OF HAIRPIN PRIMERS IN TRIAMPLIFICATION

5.2.2.1. GENERAL STEPS IN TRIAMPLIFICATION REACTIONS

5.2.2.2. USE OF HAIRPIN PRIMERS IN TRIAMPLIFICATION REACTIONS

5.2.3. METHODS OF USE OF HAIRPIN PRIMERS IN NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION (NASBA)

5.2.4. METHODS OF USE OF HAIRPIN PRIMERS IN STRAND DISPLACEMENT AMPLIFICATION (SDA)

5.3. METHODS OF DETECTION OF AMPLIFICATION PRODUCTS USING 3'-5' EXONUCLEASE AND/OR ELEVATED TEMPERATURE

5.3.1. USE OF 3'-5' EXONUCLEASE IN AMPLIFICATION REACTIONS

5.3.2. USE OF TEMPERATURE ELEVATION IN AMPLIFICATION REACTIONS

5.4. METHODS FOR DETECTION OF AMPLIFICATION PRODUCTS USING LINEAR PRIMERS

5.4.1. METHODS OF USE OF LINEAR PRIMERS IN POLYMERASE CHAIN REACTION (PCR)

5.4.1.1. METHODS OF USE OF LINEAR PRIMERS IN ALLELE-SPECIFIC PCR (ASP)

5.4.2. METHODS OF USE OF LINEAR OLIGONUCLEOTIDES IN TRIAMPLIFICATION

5.5. METHODS OF USE OF HAIRPIN OR LINEAR PRIMERS IN MULTIPLEX ASSAYS

5.6. ASSAYING THE METHYLATION STATUS OF DNA USING AMPLIFICATION REACTIONS OF THE INVENTION

5.7. KITS FOR THE AMPLIFICATION AND DETECTION OF SELECTED TARGET DNA SEQUENCES

6. EXAMPLES: GENERAL EXPERIMENTAL METHODS

6.1. OLIGONUCLEOTIDE SEQUENCES: SYNTHESIS AND MODIFICATION

6.2. AMPLIFICATION OF PROSTATE SPECIFIC ANTIGEN (PSA) TARGET DNA

6.3. 3'-5' EXONUCLEASE TREATMENT

6.4. ENERGY TRANSFER MEASUREMENTS

7. EXAMPLE 1: DNA POLYMERASE COPIES A DNA TEMPLATE WITH RHODAMINE MODIFICATION

8. EXAMPLE 2: MODIFICATION OF A REVERSE PRIMER DOES NOT AFFECT THE REACTION CATALYZED BY DNA LIGASE

9. EXAMPLE 3: EXONUCLEASE CAN REMOVE A NUCLEOTIDE RESIDUE LABELED WITH RHODAMINE

10. EXAMPLE 4: DETECTION OF AMPLIFICATION PRODUCT BY ENERGY TRANSFER AFTER NUCLEASE TREATMENT

11. EXAMPLE 5: DETECTION OF AMPLIFICATION PRODUCT BASED ON DIFFERENT THERMOSTABILITY OF AMPLIFIED PRODUCT AND BLOCKER/REVERSE PRIMER COMPLEX

12. EXAMPLE 6: CLOSED-TUBE FORMAT USING HAIRPIN PRIMERS FOR AMPLIFICATION AND DETECTION OF DNA BASED ON ENERGY TRANSFER

12.1. SUMMARY

12.2. INTRODUCTION

12.3. MATERIALS AND METHODS

12.4. RESULTS

12.5. DISCUSSION

13. EXAMPLE 7: ASSAY FOR THE METHYLATION STATUS OF CpG ISLANDS USING PCR WITH HAIRPIN PRIMERS

13.1. MATERIALS AND METHODS

13.2. RESULTS

13.3. CONCLUSION

1. INTRODUCTION

The present invention relates to oligonucleotides for amplification of nucleic acids that are detectably labeled with molecular energy transfer (MET) labels. It also relates to methods for detecting the products of nucleic acid amplification using these oligonucleotides. It further relates to a rapid, sensitive, and reliable method for detecting amplification products that greatly decreases the possibility of carryover contamination with amplification products and that is adaptable to many methods for amplification of nucleic acid sequences, including polymerase chain reaction (PCR), triamplification, and other amplification systems.

2. BACKGROUND OF THE INVENTION

2.1. FLOURESCENCE RESONANCE ENERGY TRANSFER (FRET)

Molecular energy transfer (MET) is a process by which energy is passed non-radiatively between a donor molecule and an acceptor molecule. Fluorescence resonance energy transfer (FRET) is a form of MET. FRET arises from the properties of certain chemical compounds; when excited by exposure to particular wavelengths of light, they emit light (i.e., they fluoresce) at a different wavelength. Such compounds are termed fluorophores. In FRET, energy is passed non-radiatively over a long distance (10-100 .ANG.) between a donor molecule, which is a fluorophore, and an acceptor molecule. The donor absorbs a photon and transfers this energy nonradiatively to the acceptor (Forster, 1949, Z. Naturforsch., A4: 321-327; Clegg, 1992, Methods Enzymol., 211: 353-388).

When two fluorophores whose excitation and emission spectra overlap are in close proximity, excitation of one fluorophore will cause it to emit light at wavelengths that are absorbed by and that stimulate the second fluorophore, causing it in turn to fluoresce. In other words, the excited-state energy of the first (donor) fluorophore is transferred by a resonance induced dipole--dipole interaction to the neighboring second (acceptor) fluorophore. As a result, the lifetime of the donor molecule is decreased and its fluorescence is quenched, while the fluorescence intensity of the acceptor molecule is enhanced and depolarized. When the excited-state energy of the donor is transferred to a non-fluorophore acceptor, the fluorescence of the donor is quenched without subsequent emission of fluorescence by the acceptor. In this case, the acceptor functions as a quencher.

Pairs of molecules that can engage in fluorescence resonance energy transfer (FRET) are termed FRET pairs. In order for energy transfer to occur, the donor and acceptor molecules must typically be in close proximity (up to 70 to 100 .ANG.)(Clegg, 1992, Methods Enzymol., 211: 353-388; Selvin, 1995, Methods Enzymol., 246: 300-334). The efficiency of energy transfer falls off rapidly with the distance between the donor and acceptor molecules. According to Forster (1949, Z. Naturforsch., A4:321-327), the efficiency of energy transfer is proportional to D.times.10.sup.-6, where D is the distance between the donor and acceptor. Effectively, this means that FRET can most efficiently occur up to distances of about 70 .ANG..

Molecules that are commonly used in FRET include fluorescein, 5-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL), and 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Whether a fluorophore is a donor or an acceptor is defined by its excitation and emission spectra, and the fluorophore with which it is paired. For example, FAM is most efficiently excited by light with a wavelength of 488 nm, and emits light with a spectrum of 500 to 650 nm, and an emission maximum of 525 nm. FAM is a suitable donor fluorophore for use with JOE, TAMRA, and ROX (all of which have their excitation maximum at 514 nm).

In the 1970's, FRET labels were incorporated into immunofluorescent assays used to detect specific antigens (Ullman et al. U.S. Pat. Nos. 2,998,943; 3,996,345; 4,160,016; 4,174,384; and 4,199,559). Later, in the early 1980's, several patents were received by Heller and coworkers concerning the application of energy transfer for polynucleotide hybridization (U.S. Pat. Nos. 4,996,143, 5,532,129, and 5,565,322). In European Patent Application 82303699.1 (publication number EP 0 070 685 A2 dated Jan. 26, 1983), "Homogeneous nucleic acid hybridization diagnostics by non-radioactive energy transfer," the inventors claim that they can detect a unique single stranded polynucleotide sequence with two oligonucleotides: one containing the donor fluorophore, the other, an acceptor. When both oligonucleotides hybridize to adjacent fragments of analyzed DNA at a certain distance, energy transfer can be detected.

In European Patent Application 86116652.8 (publication number EP 0 229 943 A2 dated Jul. 29, 1987; "EP '943"), entitled "Fluorescent Stokes shift probes for polynucleotide hybridization assays," Heller et al. propose the same schema, but with specified distances between donor and acceptor for maximum FRET. They also disclose that the donor and acceptor labels can be located on the same probe (see, e.g., EP '943: Claim 2 and FIG. 1).

A similar application of energy transfer was disclosed by Cardullo et al. in a method of detecting nucleic acid hybridization (1988, Proc. Natl. Acad. Sci. USA, 85: 8790-8794). Fluorescein (donor) and rhodamine (acceptor) are attached to 5' ends of complementary oligodeoxynucleotides. Upon hybridization, FRET may be detected. In other experiments, FRET occurred after hybridization of two fluorophore-labeled oligonucleotides to a longer unlabeled DNA. This system is the subject of U.S. patent application Ser. No. 661,071, and PCT Application PCT/US92/1591, Publication No. WO 92/14845 dated Sep. 3, 1992 ("PCT '845," entitled "Diagnosing cystic fibrosis and other genetic diseases using fluorescence resonance energy transfer"). PCT '845 discloses a method for detection of abnormalities in human chromosomal DNA associated with cystic fibrosis by hybridization. The FRET signal used in this