U.S. patent number 5,866,336 [Application Number 08/778,487] was granted by the patent office on 1999-02-02 for nucleic acid amplification oligonucleotides with molecular energy transfer labels and methods based thereon.
This patent grant is currently assigned to Oncor, Inc.. Invention is credited to Satish K. Bhatnagar, Robert J. Hohman, Irina A. Nazarenko, Emily S. Winn-Deen.
United States Patent |
5,866,336 |
Nazarenko , et al. |
February 2, 1999 |
Nucleic acid amplification oligonucleotides with molecular energy
transfer labels and methods based thereon
Abstract
The present invention provides labeled nucleic acid
amplification oligonucleotides, which can be linear or hairpin
primers or blocking oligonucleotides. The oligonucleotides of the
invention are labeled with donor and/or acceptor moieties of
molecular energy transfer pairs. The moieties can be fluorophores,
such that fluorescent energy emitted by the donor is absorbed by
the acceptor. The acceptor may be a fluorophore that fluoresces at
a wavelength different from the donor moiety, or it may be a
quencher. The oligonucleotides of the invention are configured so
that a donor moiety and an acceptor moiety are incorporated into
the amplification product. The invention also provides methods and
kits for directly detecting amplification products employing the
nucleic acid amplification primers. When labeled linear primers are
used, treatment with exonuclease or by using specific temperature
eliminates the need for separation of unincorporated primers. This
"closed-tube" format greatly reduces the possibility of carryover
contamination with amplification products, provides for high
throughput of samples, and may be totally automated.
Inventors: |
Nazarenko; Irina A.
(Gaithersburg, MD), Bhatnagar; Satish K. (Gaithersburg,
MD), Winn-Deen; Emily S. (Potomac, MD), Hohman; Robert
J. (Gaithersburg, MD) |
Assignee: |
Oncor, Inc. (Gaithersburg,
MD)
|
Family
ID: |
27103159 |
Appl.
No.: |
08/778,487 |
Filed: |
January 3, 1997 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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683667 |
Jul 16, 1996 |
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Current U.S.
Class: |
435/6.12;
435/91.2; 536/22.1; 536/25.32; 536/24.3 |
Current CPC
Class: |
C12Q
1/686 (20130101); C12Q 1/6818 (20130101); C12Q
1/6844 (20130101); C12Q 1/686 (20130101); C12Q
2565/1015 (20130101); C12Q 2525/301 (20130101); C12Q
1/6844 (20130101); C12Q 2565/101 (20130101); C12Q
2525/301 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 001/68 (); C12P 017/34 ();
C07H 021/06 (); C07H 021/00 () |
Field of
Search: |
;435/91.2,6
;536/25.32,22.1,24.3 |
References Cited
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EP |
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EP |
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628 640 A1 |
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EP |
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EP |
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EP |
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JP |
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JP |
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WO |
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WO |
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95/32306 |
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Nov 1995 |
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WO |
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Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Tung; Joyce
Attorney, Agent or Firm: Cohen; Jonathan M. Oncor, Inc.
Parent Case Text
This application is a continuation-in-part of application Ser. No.
08/683,667 filed Jul. 16, 1996, now abandoned, which is
incorporated by reference herein in its entirety.
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.
Description
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
method is generated in a manner similar to that disclosed by Heller
et al. (see PCT '845 FIG. 1). Other publications have disclosed the
use of energy transfer in a method for the estimation of distances
between specific sites in DNA (Ozaki and McLaughlin, 1992, Nucl.
Acids Res., 20: 5205-5214), in a method for the analysis of
structure of four way DNA junction (Clegg et al. 1992, Biochem.,
31: 4846-4856), and in a method for observing the helical geometry
of DNA (Clegg et al., 1993, Proc. Natl. Acad. Sci. USA, 90:
2994-2998).
2.2. METHODS OF MONITORING NUCLEIC ACID AMPLIFICATION
Prior to the present invention, application of energy transfer to
the direct detection of genetic amplification products had not been
attempted. In prior art methods of monitoring amplification
reactions using energy transfer, a label is not incorporated into
the amplification product. As a result, these methods have relied
on indirect measurement of the amplification reaction.
Commonly used methods for detecting nucleic acid amplification
products require that the amplified product be separated from
unreacted primers. This is commonly achieved either through the use
of gel electrophoresis, which separates the amplification product
from the primers on the basis of a size differential, or through
the immobilization of the product, allowing washing away of free
primer. However, three methods for monitoring the amplification
process without prior separation of primer have been described. All
of them are based on FRET, and none of them detect the amplified
product directly. Instead, all three methods detect some event
related to amplification. For that reason, they are accompanied by
problems of high background, and are not quantitative, as discussed
below.
One method, described in Wang et al. (U.S. Pat. No. 5,348,853; Wang
et al., 1995, Anal. Chem., 67: 1197-1203), uses an energy transfer
system in which energy transfer occurs between two fluorophores on
the probe. In this method, detection of the amplified molecule
takes place in the amplification reaction vessel, without the need
for a separation step. This method results in higher sensitivity
than methods that rely on monolabeled primers.
The Wang et al. method uses an "energy-sink" oligonucleotide
complementary to the reverse primer. The "energy-sink" and
reverse-primer oligonucleotides have donor and acceptor labels,
respectively. Prior to amplification, the labeled oligonucleotides
form a primer duplex in which energy transfer occurs freely. Then,
asymmetric PCR is carried out to its late-log phase before one of
the target strands is significantly overproduced.
A primer duplex complementary to the overproduced target strand is
added to prime a semi-nested reaction in concert with the excess
primer. As the semi-nested amplification proceeds, the primer
duplex starts to dissociate as the target sequence is duplicated.
As a result, the fluorophores configured for energy transfer are
disengaged from each other, causing the energy transfer process
preestablished in all of the primer duplexes to be disrupted for
those primers involved in the amplification process. The measured
fluorescence intensity is proportional to the amount of primer
duplex left at the end of each amplification cycle. The decrease in
the fluorescence intensity correlates proportionately to the
initial target dosage and the extent of amplification.
This method, however, does not detect the amplified product, but
instead detects the dissociation of primer from the "energy-sink"
oligonucleotide. Thus, this method is dependent on detection of a
decrease in emissions; a significant portion of labeled primer must
be utilized in order to achieve a reliable difference between the
signals before and after the reaction. This problem was apparently
noted by Wang et al., who attempted to compensate by adding a
preliminary amplification step (asymmetric PCR) that is supposed to
increase the initial target concentration and consequently the
usage of labeled primer, but also complicates the process.
A second method for detection of amplification product without
prior separation of primer and product is the 5' nuclease PCR assay
(also referred to as the TaqMan.RTM. assay) (Holland et al., 1991,
Proc. Natl. Acad. Sci. USA, 88: 7276-7280; Lee et al., 1993,
Nucleic Acids Res., 21: 3761-3766). This assay detects the
accumulation of a specific PCR product by hybridization and
cleavage of a doubly labeled fluorogenic probe (the "TaqMan" probe)
during the amplification reaction. The fluorogenic probe consists
of an oligonucleotide labeled with both a fluorescent reporter dye
and a quencher dye. During PCR, this probe is cleaved by the
5'-exonuclease activity of DNA polymerase if, and only if, it
hybridizes to the segment being amplified. Cleavage of the probe
generates an increase in the fluorescence intensity of the reporter
dye.
In the TaqMan assay, the donor and quencher are preferably located
on the 3' and 5'-ends of the probe, because the requirement that
5'-3 hydrolysis be performed between the fluorophore and quencher
may be met only when these two moieties are not too close to each
other (Lyamichev et al., 1993, Science, 260:778-783). However, this
requirement is a serious drawback of the assay, since the
efficiency of energy transfer decreases with the inverse sixth
power of the distance between the reporter and quencher. In other
words, the TaqMan assay does not permit the quencher to be close
enough to the reporter to achieve the most efficient quenching. As
a consequence, the background emissions from unhybridized probe can
be quite high.
Furthermore, the TaqMan assay does not measure the amplification
product directly, because the amplification primers are not
labeled. This assay measures an event related to amplification: the
hydrolysis of the probe that hybridizes to the target DNA between
the primer sequences. As a result, this assay method is accompanied
by significant problems.
First, hybridization will never be quantitative unless the labeled
oligonucleotide is present in great excess. However, this results
in high background (because the quenching is never quantitative).
In addition, a great excess of oligonucleotide hybridized to the
middle of the target DNA will decrease PCR efficiency. Furthermore,
not all of the oligonucleotides hybridized to the DNA will be the
subject of 5'-3' exonuclease hydrolysis: some will be displaced
without hydrolysis, resulting in a loss of signal.
Another method of detecting amplification products that relies on
the use of energy transfer is the "beacon probe" method described
by Tyagi and Kramer (1996, Nature Biotech., 14:303-309) which is
also the subject of U.S. Pat. Nos. 5,119,801 and 5,312,728 to
Lizardi et al. This method employs oligonucleotide hybridization
probes that can form hairpin structures. On one end of the
hybridization probe (either the 5' or 3' end) there is a donor
fluorophore, and on the other end, an acceptor moiety. In the case
of the Tyagi and Kramer method, this acceptor moiety is a quencher,
that is, the acceptor absorbs energy released by the donor, but
then does not itself fluoresce. Thus when the beacon is in the open
conformation, the fluorescence of the donor fluorophore is
detectable, whereas when the beacon is in hairpin (closed)
conformation, the fluorescence of the donor fluorophore is
quenched. When employed in PCR, the molecular beacon probe, which
hybridizes to one of the strands of the PCR product, is in "open
conformation," and fluorescence is detected, while those that
remain unhybridized will not fluoresce (Tyagi and Kramer, 1996,
Nature Biotechnol., 14: 303-306. As a result, the amount of
fluorescence will increase as the amount of PCR product increases,
and thus may be used as a measure of the progress of the PCR.
However, since this method is based on hybridization of the probe
to template between the primer sequences, it has a number of
problems associated with it, some of which are similar to those
described above in connection with the TaqMan method. First, it is
unlikely that the beacon probes will hybridize quantitatively to
one strand of double-stranded PCR product, especially when the
amplification product is much longer than the beacon probe. Even
those probes that are hybridized could be displaced by the second
DNA strand over a short period of time; as a result, this method
cannot be quantitative.
Efforts to increase the hybridization efficiency by increasing the
concentration of beacon probe will result in decreased
amplification efficiency, since the necessity for DNA polymerase to
displace hybridized beacons during the reaction will slow down the
rate of polymerization. An excess of probe will also increase the
background. In addition, the ratio between the amplification
product and the beacon probes will change as amplification
proceeds, and so will change the efficiency of hybridization. Thus
the detection of the amplified product may not be quantitative.
Therefore, in view of the deficiencies in prior art methods of
detecting amplification products, it is clear that there exists in
the art a need for an improved method of detecting amplification
products rapidly, sensitively, reliably and quantitatively. The
present invention solves this problem by providing nucleic acid
amplification primers that are detectably labeled with
energy-transfer labels. It also solves this problem by providing
methods for detecting amplification products that are adaptable to
many methods for amplification of nucleic acid sequences and that
greatly decrease the possibility of carryover contamination with
amplification products.
Citation of references herein shall not be construed as an
admission that such references are prior art to the present
invention.
3. SUMMARY OF THE INVENTION
The present invention relates to oligonucleotides for amplification
of nucleic acids that are detectably labeled with molecular energy
transfer (MET) labels. One or more oligonucleotides of the
invention containing a donor and/or acceptor moiety of a MET pair
are incorporated into the amplified product of an amplification
reaction, such that the amplified product contains both a donor and
acceptor moiety of a MET pair. When the amplified product is
double-stranded, the MET pair incorporated into the amplified
product may be on the same strand or, when the amplification is
triamplification, on opposite strands. In certain instances wherein
the polymerase used in amplification has 5'-3' exonuclease
activity, one of the MET pair moieties may be cleaved from at least
some of the population of amplified product by this exonuclease
activity. Such exonuclease activity is not detrimental to the
amplification methods of the invention.
The invention also relates to methods for detecting the products of
nucleic acid amplification using these labeled oligonucleotides of
the invention. 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.
The nucleic acid amplification oligonucleotides of the invention
utilize the principle of molecular energy transfer (MET) between a
donor moiety and an acceptor moiety. In a preferred embodiment, the
MET is fluorescence resonance energy transfer (FRET), in which the
oligonucleotides are labeled with donor and acceptor moieties,
wherein the donor moiety is a fluorophore and the acceptor moiety
may be a fluorophore, such that fluorescent energy emitted by the
donor moiety is absorbed by the acceptor moiety. In one embodiment
of the present invention, the acceptor moiety is a fluorophore that
releases the energy absorbed from the donor at a different
wavelength; the emissions of the acceptor may then be measured to
assess the progress of the amplification reaction. In another
embodiment, the acceptor moiety is a quencher.
In a preferred embodiment, the amplification primer is a hairpin
primer that contains both donor and acceptor moieties, and is
configured such that the acceptor moiety quenches the fluorescence
of the donor. When the primer as incorporated into the
amplification product its configuration changes, quenching is
eliminated, and the fluorescence of the donor moiety may be
detected.
In one embodiment, the present invention provides nucleic acid
amplification primers that form a hairpin structure in which MET
will occur when the primer is not incorporated into the
amplification product. In a preferred embodiment, a primer forms a
hairpin structure in which the energy of a donor fluorophore is
quenched by a non-fluorescing fluorophore when the primer is not
incorporated into the amplification product.
In another embodiment, the present invention provides
oligonucleotides that are linear (non-duplex) and that are
separately labeled with donor and acceptor moieties, such that MET
will occur when the oligonucleotides are incorporated into the
amplification product. For example, the blocking oligonucleotide
and the reverse primer complementary to the blocking
oligonucleotide can be so labeled in a triamplification
reaction.
In yet another embodiment, the donor moiety and acceptor moiety are
on a single, linear oligonucleotide used in an amplification
reaction.
The present invention also provides a method of directly detecting
amplification products. This improved technique meets two major
requirements. First, it permits detection of the amplification
product without prior separation of unincorporated
oligonucleotides. Second, it allows detection of the amplification
product directly, by incorporating the labeled oligonucleotide into
the product.
The present invention provides a method of directly detecting
amplification products through the incorporation of labeled
oligonucleotide(s) (e.g., primers, blocking oligonucleotides)
wherein instead of separating unincorporated oligonucleotides from
amplification product, as in prior art approaches, signal from the
remaining free oligonucleotide(s) is eliminated in one (or more) of
the following ways:
a) by treatment with a 3'-5' exonuclease;
b) by heating the amplification product to a temperature such that
the primer-oligonucleotide duplex dissociates and, as a result,
will not generate any signal; or
c) by using a primer labeled with both donor and acceptor moieties
and that can form a hairpin structure, in which the energy transfer
from donor to acceptor will occur only when the primer is not
incorporated into the amplification product.
In a further embodiment, the present invention provides a method
for the direct detection of amplification products in which the
detection may be performed without opening the reaction tube. This
embodiment, the "closed-tube" format, reduces greatly the
possibility of carryover contamination with amplification products
that has slowed the acceptance of PCR in many applications. The
closed-tube method also provides for high throughput of samples and
may be totally automated. The present invention also relates to
kits for the detection or measurement of nucleic acid amplification
products. Such kits may be diagnostic kits where the presence of
the nucleic acid being amplified is correlated with the presence or
absence of a disease or disorder.
3.1. DEFINITIONS
As used herein, the following terms shall have the abbreviations
indicated.
ASP, allele-specific polymerase chain reaction bp, base pairs
DAB or DABCYL, 4-(4'-dimethylaminophenylazo) benzoic acid
EDANS, 5-(2'-aminoethyl) aminonapthalene-1-sulfonic acid
FAM or Flu, 5-carboxyfluorescein
FRET, fluorescence resonance energy transfer
JOE, 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein
HPLC, high-performance liquid chromatography
MET, molecular energy transfer
NASBA, nucleic acid sequence-based amplification
PSA, prostate specific antigen
Rhod, rhodamine
ROX, 6-carboxy-X-rhodamine
R6G, 6-carboxyrhodamine
SDA, strand displacement amplification
TAMRA, N,N,N',N'-tetramethyl-6-carboxyrhodamine
4. DESCRIPTION OF THE FIGURES
The present invention may be understood more fully by reference to
the following detailed description of the invention, examples of
specific embodiments of the invention and the appended figures
described below:
FIGS. 1A-B illustrate schematically the structure of the hairpin
primers of the invention in the (A) closed (quenched) and (B) open
(emitting signal) states. .smallcircle., donor fluorophore;
.circle-solid., quencher fluorophore.
FIG. 2 illustrates schematically the use of hairpin primers to
directly measure the amplification products from a PCR in which the
employed DNA polymerase lacks 5'-3' exonuclease activity. An energy
transfer signal is generated upon the incorporation of the hairpin
primer into the double-stranded PCR product. (a) and (b),
complementary strands of the target sequence to be amplified;
.smallcircle. donor fluorophore; .circle-solid., quencher; F,
forward primer; R, reverse primer.
FIG. 3 (Steps A-D) illustrates the amplification products from a
PCR in which the employed DNA polymerase has 5'-3' exonuclease
activity. (a) and (b), complementary strands of the target sequence
to be amplified; .smallcircle. donor fluorophore; .circle-solid.,
quencher; F, forward primer; R, reverse primer.
FIG. 4 gives a schematic example of a selected target sequence (SEQ
ID NO:1) ligated to a universal hairpin primer (SEQ ID NO:2). (d)
is the selected primer sequence of 8-40 nucleotides, preferably
.about.15 nucleotides, that is complementary to the target nucleic
acid sequence to be amplified. (d') is the 5' cohesive end of the
selected primer sequence. The cohesive end is 1-10 nucleotides,
preferably 3-4 nucleotides, and complementary to the 5' cohesive
end (a') of the universal hairpin primer. (b) is a loop on the
universal hairpin primer that is long enough provide a distance of
15-25 nucleotides, preferably 20 nucleotides, between the donor (F,
FAM) and the quencher (D, DABCYL) when the hairpin is in the "open"
configuration. (a) and (c) are the two strands of the stem of the
universal hairpin primer. When the selected primer sequence is
ligated to the universal hairpin primer, the quencher (DABCYL) will
be located on a nucleotide that is internal to the 3' end. The
donor (FAM) may be located on a nucleotide either at the 5' end (as
shown) or internal to the 5' end. The only requirement is that the
donor and quencher are close enough to enable quenching when the
hairpin is in the "closed" ("silent") conformation.
FIG. 5 illustrates schematically the use of a FRET
donor-acceptor-labeled hairpin primer in PCR. See Section 5.2.1 for
a detailed description of Cycles 1-4.
FIG. 6 illustrates schematically the use of a FRET
donor-acceptor-labeled hairpin primer in triamplification. In this
embodiment of triamplification, unlike in PCR, a third
oligonucleotide ("blocker") is ligated to the extended hairpin
primer. The fluorescent signal is generated as a result of
replication, however, as occurs in PCR.
FIG. 7 illustrates schematically triamplification using two linear
primers, each labeled with a FRET moiety. BL, blocker; R, reverse
primer; F, forward primer; .box-solid., a commercially available 3'
modifying group able to protect the oligonucleotide from extension
by DNA polymerase or hydrolysis by 3'-5' exonuclease on the 3' end
of the blocker; X, 2'-O-methyl-modification in reverse primer; D,
donor fluorophore; A.smallcircle., acceptor fluorophore.
FIGS. 8A-B illustrate the effect of (A) 3'-5' exonuclease and (B)
elevated temperature on unincorporated FRET-labeled primers during
triamplification. BL, blocker; R, reverse primer; F, forward
primer; P, 5' phosphate; .box-solid., protection group on 3'-end of
blocker; X, 2'-O-methyl-modification in reverse primer; D, donor
fluorophore; A.smallcircle., acceptor fluorophore.
FIG. 9 illustrates schematically the use of hairpin primers in
nucleic acid sequence-based amplification (NASBA). NASBA depends on
continuous cycling of the reverse transcription and RNA
transcription reactions at one temperature. See Section 5.2.3 for a
detailed description of Steps 1-9.
FIG. 10 illustrates schematically the use of hairpin primers in
strand displacement amplification (SDA) of a double-stranded DNA
target. Primers 1 and 2 differ, being forward and reverse primers,
respectively. SDA depends on continuous cycling of the nicking and
the polymerization/displacement steps at one temperature. See
Section 5.2.4 for a detailed description of Steps 1-4. pol,
polymerase; restrictase, restriction endonuclease.
FIGS. 11A-B illustrate a two-chamber amplification tube in
"closed-tube" format. The tube can be inverted (FIG. 11B) and used
to mix 3'-5' exonuclease with amplification product only when
desired, without opening the tube after amplification takes place
(see Section 12, Example 6).
FIG. 12 illustrates portions of the two strands (upper strand: SEQ
ID NO:3 and SEQ ID NO:4; lower strand: SEQ ID NO:8 and SEQ ID NO:9)
of the template, and the oligonucleotides, PSA-I (SEQ ID NO:5),
PSA-P (SEQ ID NO:6), and PSA-B (SEQ ID NO:7), used in the
amplification of human prostate specific antigen (PSA) DNA as
described in all the examples except those employing hairpin
primers, the sequences of which are provided in Section 12.
FIGS. 13A-C FIG. 13A illustrates schematically the PCR
amplification procedure used in the experiment described in Section
7 (Example 1). The left portion of FIG. 13A illustrates a PCR
amplification using a rhodamine-modified reverse primer. The right
portion of FIG. 13A illustrates a PCR amplification using a
non-modified reverse primer. The results are shown on the
accompanying denaturing 6% polyacrylamide gel (FIG. 13B) and
agarose gel (FIG. 13C). FIG. 13B compares the sizes of the DNA
strands that were amplified with [.sup.32 P]-labeled forward primer
when non-modified reverse primer (Lane 1) or rhodamine-modified
reverse primer (Lane 2) was used. FIG. 13C compares the amounts of
double-stranded PCR amplification product obtained with
non-modified reverse primer (Lane 1) and rhodamine-modified reverse
primer (Lane 2).
FIGS. 14A-B FIG. 14A illustrates schematically the experimental
procedure used in Section 8 (Example 2). The results are shown in
the accompanying denaturing 6% polyacrylamide gel (FIG. 14B). Lane
1 of the gel represents a strand of amplified DNA with incorporated
[.sup.32 P]-and rhodamine-labeled reverse primer, while Lane 2
represents a strand of amplified DNA with incorporated [.sup.32
P]-labeled forward (F) primer.
FIGS. 15A-B FIG. 15A illustrates schematically the experimental
procedure used in Section 9 (Example 3). The results are shown on
the accompanying denaturing 15% polyacrylamide gel (FIG. 15B). Lane
1 of the gel represents [.sup.32 P]- and rhodamine-labeled reverse
primer, Lanes 2-4 represent [.sup.32 P]- and rhodamine-labeled
reverse primer after incubation with T4 DNA polymerase that has
3'-5' exonuclease activity for 2 minutes (Lane 2), 5 minutes (Lane
3), and 15 minutes (Lane 4).
FIG. 16 illustrates the detection of amplification product by FRET
after nuclease treatment (Section 10, Example 4). Emission spectrum
1 was obtained after triamplification with DNA template and
exonuclease treatment. Spectrum 2 was obtained after
triamplification without DNA template and exonuclease treatment (no
DNA control).
FIGS. 17A-B illustrates the effect of elevated temperatures
(75.degree. C.) on FRET following triamplification (A) without and
(B) with DNA template (Section 11, Example 5).
FIGS. 18A-B FIG. 18A depicts the structure of the PSA cDNA upstream
hairpin primer (SEQ ID NO:10). The portion of the sequence
complementary to the target DNA is shown in bold. FIG. 18B shows an
emission spectrum of the fluorescein-labeled hairpin primer in the
absence (1) and presence (2) of a DABCYL moiety. The spectra
obtained from 0.5 ml of a 40 nM sample of oligonucleotide were
measured as described in Section 6.4 using a 488 nm excitation
wavelength.
FIG. 19 shows the efficiency of amplification with the hairpin
primers. Products of amplification were separated on an MDE gel. An
ethidium-bromide stained gel is shown. Lanes 1-3 show the products
of amplification of 10.sup.-9 M PSA cDNA with unlabeled control
linear primer (Lane 1), FAM-hairpin primer (Lane 2), and
FAM/DABCYL-hairpin primer (Lane 3). Lanes 4-6 show the products of
amplification of 10.sup.-11 M PSA CDNA with control primer (Lane
4), FAM-hairpin primer (Lane 5), and FAM/DABCYL-hairpin primer
(Lane 6). Lane M contains a 100 bp marker (Gibco BRL).
FIGS. 20A-B illustrates schematically and shows the results,
respectively, of a PCR amplification in the presence of hairpin
primers. PCR amplification of PSA cDNA was performed with two
primers: an upstream hairpin primer labeled with FAM and DABCYL,
and a downstream primer labeled with .sup.32 P on its 5' end (FIG.
20A). An upstream primer without the hairpin structure was used as
a control. The structure of the hairpin primer is presented in FIG.
18A and the sequences of the regular primers are presented in
Section 12.3. FIG. 20B is an autoradiogram that shows the size of
the PCR product synthesized. [.sup.32 P]-labeled strands of the PCR
products were synthesized in the presence of the unlabeled control
linear primer (Lane 1) or FAM/DABCYL--labeled hairpin primer (Lane
2) and analyzed on a 6% denaturing polyacrylamide gel.
FIGS. 21A-B FIG. 21A shows the fluorescence spectra of the
amplification reactions performed with the hairpin primers labeled
with FAM/DABCYL. The structure of the FAM/DABCYL labeled hairpin
primer is presented in FIG. 18A and the sequence of the regular
downstream primer is presented in Section 12.3. Spectra 1-6 show
the fluorescence intensity of the amplified PSA cDNA after 0 (1),
20 (2), 25 (3), 30 (4), 35 (5) or 40 (6) cycles. FIG. 21B shows the
fluorescence intensity of the amplification reaction mixtures and
the fraction of the [.sup.32 P]-labeled primers incorporated into
the PCR products plotted against the number of cycles. The
incorporation of the [.sup.32 P]-labeled primers into the PCR
products was determined by electrophoresis on a 6% denaturing gel
and quantitated using the PhosphorImager.
FIG. 22 shows the sensitivity of PCR with hairpin primers. Spectra
1-6 show the results of the amplification when 0 (1), 10 (2),
10.sup.2 (3), 10.sup.3 (4), 10.sup.4 (5) , 10.sup.5 (6) or 10.sup.6
(7) molecules of cloned PSA CDNA per reaction were used as template
DNA for the 40 cycles of PCR. The structure of the FAM/DABCYL
labeled hairpin primer is presented in FIG. 18A and the sequence of
the regular downstream primer is presented in Section 12.3.
FIG. 23 shows the visible fluorescence of PCR products synthesized
with hairpin primers. 10.sup.6 (Tube 1), 10.sup.4 (Tube 2),
10.sup.3 (Tube 3) and 0 (Tube 4) molecules of the cloned PSA cDNA
template were used as template DNA for the 40 cycles of PCR with
FAM/DABCYL labeled hairpin primers. DNA fluorescence was visualized
in 0.2 ml thin-walled PCR tubes using an UV transilluminator image
analysis system.
FIGS. 24A-G show the fluorescence intensity of PSA cDNA amplified
with different FAM/DABCYL-labeled hairpin primers (FIGS. 24A-G
correspond to SEQ ID NOS:13-18, and 25, respectively). All primers
had at least an 18-nucleotide sequence complementary to the target,
wnich consisted of a 3' single-stranded priming sequence, a 3' stem
sequence and part of the loop. Sequences complementary to the
target DNA are shown in shadowed bold italics. f, FAM; d, DABCYL;
nucl, nucleotide number; rel. (%), percent intensity of
fluorescence relative to DNA amplified with Primer A.
FIG. 25 illustrates schematically the use of linear primers to
directly measure the amplification products from a PCR. An energy
transfer signal is generated upon the incorporation of the primer
into the double-stranded PCR product. After amplification, the
signal from unincorporated primer is eliminated by 3'-5'
exonuclease hydrolysis. D, donor moiety; A, acceptor moiety; F,
forward primer; R, reverse primer.
FIG. 26 illustrates the three sets of PCR primers used in the
experiments in Section 13, Example 7. Uup (SEQ ID NO:19) and Ud
(SEQ ID NO:20), are the upstream and downstream primers,
respectively, for sequences of bisulfite-treated unmethylated DNA.
Mup (SEQ ID NO:21) and Md (SEQ ID NO:22), are the upstream and
downstream primers, respectively, for sequences of
bisulfite-treated methylated DNA. Wup (SEQ ID NO:23) and Wd (SEQ ID
NO:24), are the upstream and downstream primers, respectively, for
DNA not treated with bisulfite. One of the two primers in each set
has a hairpin structure at its 5' end, labeled with a FAM/DAB
(DABCYL) FRET pair at the positions illustrated.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to oligonucleotides for amplification
of nucleic acids that are detectably labeled with molecular energy
transfer (MET) labels. One or more oligonucleotides of the
invention containing a donor and/or acceptor moiety of a MET pair
are incorporated into the amplified product of an amplification
reaction, such that the amplified product contains both a donor and
acceptor moiety of a MET pair. When the amplified product is
double-stranded, the MET pair incorporated into the amplified
product may be on the same strand or, when the amplification is
triamplification, on opposite strands. In certain instances wherein
the polymerase used in amplification has 5'-3' exonuclease
activity, one of the MET pair moieties may be cleaved from at least
some of the population of amplified product by this exonuclease
activity. Such exonuclease activity is not detrimental to the
amplification methods of the invention.
The invention also relates to methods for detecting the products of
nucleic acid amplification using these labeled oligonucleotides of
the invention. 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.
The nucleic acid amplification oligonucleotides of the invention
utilize the principle of MET between a donor moiety and an acceptor
moiety. In a preferred embodiment, the MET is fluorescence
resonance energy transfer (FRET), in which the oligonucleotides are
labeled with donor and acceptor moieties, wherein the donor moiety
is a fluorophore and the acceptor moiety may be a fluorophore, such
that fluorescent energy emitted by the donor moiety is absorbed by
the acceptor moiety. In one embodiment of the present invention,
the acceptor moiety is a fluorophore that releases the energy
absorbed from the donor at a different wavelength; the emissions of
the acceptor may then be measured to assess the progress of the
amplification reaction.
In a preferred embodiment, the amplification primer is a hairpin
primer that contains both donor and acceptor moIeties and is
configured such that the acceptor moiety quenches the fluorescence
of the donor. When the primer is incorporated into the
amplification product its configuration changes, quenching is
eliminated, and the fluorescence of the donor moiety may be
detected.
In one embodiment, the present invention provides nucleic acid
amplification primers that form a hairpin structure in which MET
will occur when the primer is not incorporated into the
amplification product. In a preferred embodiment, a primer forms a
hairpin structure in which the energy of a donor fluorophore is
quenched by a non-fluorescing fluorophore when the primer is not
incorporated into the amplification product.
In another embodiment, the present invention provides
oligonucleotides that are linear (non-duplex) and that are
separately labeled with donor and acceptor moieties, such that MET
will occur when the oligonucleotides are incorporated into the
amplification product. For example, the blocking oligonucleotide
and the primer complementary to the blocking oligonucleotide can be
so labeled in a triamplification reaction.
In yet another embodiment, using a pair of linear primers, the
donor moiety and acceptor moiety are on a single linear primer used
in the amplification reaction. Where the amplification reaction is
triamplification, the oligonucleotide labeled with both the donor
and acceptor moieties is not the blocking oligonucleotide.
The invention provides 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
oligonucleotides, a first one of said oligonucleotides comprising a
sequence complementary to a preselected target sequence that may be
present in said sample, and said first one and a second of said
oligonucleotides being a pair of primers adapted for use in said
amplification reaction such that said primers are incorporated into
an amplified product of said amplification reaction when said
target sequence is present in the sample; at least one of said
primers being labeled with a first moiety selected from the group
consisting of a donor moiety and an acceptor moiety of a molecular
energy transfer pair; and wherein the same or a different
oligonucleotide is labeled with a second moiety selected from the
group consisting of said donor moiety and said acceptor moiety,
said second moiety being the member of said group that is not said
first moiety, wherein said primer labeled with said first moiety
and said oligonucleotide labeled with said second moiety are
configured so as to be incorporated into said amplified product,
wherein the donor moiety emits energy of one or more particular
wavelengths when excited, and the acceptor moiety absorbs energy at
one or more particular wavelengths emitted by the donor moiety; (b)
conducting the amplification reaction; (c) stimulating light
emission from said donor moiety; and (d) detecting or measuring
energy emitted by said donor moiety or acceptor moiety.
The nucleic acids in the sample may be purified or unpurified.
A pair of primers, consisting of a forward primer and a reverse
primer, for use in PCR or strand displacement amplification,
consists of primers that are each complementary with a different
strand of two complementary nucleic acid strands, such that when an
extension product of one primer in the direction of the other
primer is generated by a nucleic acid polymerase, that extension
product can serve as a template for the synthesis of the extension
product of the other primer. A pair of primers, consisting of a
forward primer and a reverse primer, for use in triamplification,
consists of primers that are each complementary with a different
strand of two complementary nucleic acid strands, such that when an
extension-ligation product of one primer in the direction of the
other primer is generated by a nucleic acid polymerase and a
nucleic acid ligase, that extension-ligation product can serve as a
template for the synthesis of the extension-ligation product of the
other primer. The amplified product in these instances is that
content of a nucleic acid in the sample between and including the
primer sequences.
As referred to herein, nucleic acids that are "complementary" can
be perfectly or imperfectly complementary, as long as the desired
property resulting from the complementarity is not lost, e.g.,
ability to hybridize.
In a specific embodiment, the invention provides 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 a hairpin primer of the invention
labeled with a donor moiety and an acceptor moiety; (b) conducting
the amplification reaction; (c) stimulating energy emission from
said donor moiety; and (d) detecting or measuring energy emitted by
said donor moiety.
The present invention also provides a method of directly detecting
amplification products. This improved technique meets two major
requirements. First, it permits detection of the amplification
product without prior separation of unincorporated
oligonucleotides. Second, it allows detection of the amplification
product directly, by incorporating the labeled oligonucleotide(s)
into the product.
The present invention provides a method of directly detecting
amplification products through the incorporation of labeled
oligonucleotide(s) (e.g., primers, blocking oligonucleotides)
wherein instead of separating unreacted oligonucleotides from
amplification product, as in prior art approaches, signal from the
remaining free oligonucleotide(s) is eliminated in one (or more) of
the following ways:
a) by treatment with a 3'-5' exonuclease;
b) by heating the amplification product to a temperature such that
the primer-oligonucleotide duplex dissociates and, as a result,
will not generate any signal; or
c) by using a primer labeled with both donor and acceptor moieties
and that can form a hairpin structure, in which the energy transfer
from donor to acceptor will occur only when the primer is not
incorporated into the amplification product.
In a further embodiment, the present invention provides a method
for the direct detection of amplification products in which the
detection may be performed without opening the reaction tube. This
embodiment, the "closed-tube" format, reduces greatly the
possibility of carryover contamination with amplification products
that has slowed the acceptance of PCR in many applications. The
closed-tube method also provides for high throughput of samples and
may be totally automated. The present invention also relates to
kits for the detection or measurement of nucleic acid amplification
products. Such kits may be diagnostic kits where the presence of
the nucleic acid being amplified is correlated with the presence or
absence of a disease or disorder.
For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections set forth below.
5.1. OLIGONUCLEOTIDES
The present invention provides oligonucleotides for nucleic acid
amplification that are incorporated into the amplified product and
that utilize the principle of molecular energy transfer (MET) and,
preferably, fluorescence resonance energy transfer (FRET). The
oligonucleotides of the invention are labeled with a donor and/or
an acceptor moiety, i.e., a "MET pair." The acceptor moiety may
simply quench the emission of the donor moiety, or it may itself
emit energy upon excitation by emission from the donor moiety. In a
preferred embodiment, the donor moiety is a fluorophore and the
acceptor moiety may or may not be a fluorophore, such that
fluorescent energy emitted by the donor moiety is absorbed by the
acceptor moiety. The labeled oligonucleotides are forward and/or
reverse primers, and/or, in the case of triamplification, a
blocking oligonucleotide. The oligonucleotides used in the
amplification reaction are labeled such that at least one MET pair
is incorporated into the amplified product (although 5'-3'
exonuclease activity, if present, may subsequently remove a moiety
from at least some of the amplified product population).
In one embodiment of the present invention, the acceptor moiety is
a fluorophore that releases the energy absorbed from the donor at a
different wavelength; use of the emissions of the donor and/or
acceptor may then be measured to assess the progress of the
amplification reaction, depending on whether the donor and acceptor
moieties are incorporated into the amplification product close
enough for MET to occur. In another embodiment, the acceptor moiety
is a quencher that quenches the fluorescence of the donor when the
donor and acceptor moieties are incorporated into the amplification
product close enough for MET to occur.
In a further specific embodiment (see Section 5.1.1 infra), an
oligonucleotide primer is used that forms a hairpin structure in
which FRET will occur, when the primer is not incorporated into the
amplification product. In a preferred embodiment, the hairpin
primer is labeled with a donor-quencher FRET pair. When the hairpin
primer is incorporated into the amplification product, its
configuration changes (i.e., it is linearized), quenching is
eliminated, and the fluorescence of the donor may be detected.
In yet another embodiment (see Section 5.1.2 infra), the labeled
oligonucleotide, that can be a primer or, in the case of
triamplification, a blocking oligonucleotide, is a linear molecule
that does not form a hairpin configuration. In one embodiment, the
donor-acceptor FRET pair is located on the same, single-stranded
oligonucleotide primer. In another embodiment, the donor moiety is
located on a first oligonucleotide and the acceptor is located on a
second oligonucleotide. In a specific embodiment, one of the two
FRET-labeled oligonucleotides is a primer for triamplification, and
the other FRET-labeled oligonucleotide is a blocker for
triamplification (see Section 5.4.2).
The oligonucleotides for use in the amplification reactions of the
invention can be any suitable size, and are preferably in the range
of 10-100 or 10-80 nucleotides, more preferably 20-40
nucleotides.
The oligonucleotide can be DNA or RNA or chimeric mixtures or
derivatives or modified versions thereof, so long as it is still
capable of priming the desired amplification reaction, or, in the
case of a blocking oligonucleotide, functioning as a blocking
oligonucleotide. In addition to being labeled with a MET moiety,
the oligonucleotide can be modified at the base moiety, sugar
moiety, or phosphate backbone, and may include other appending
groups or labels, so long as it is still capable of priming the
desired amplification reaction, or functioning as a blocking
oligonucleotide, as the case may be.
For example, the oligonucleotide may comprise at least one modified
base moiety which is selected from the group including but not
limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
In another embodiment, the cligonucleotide comprises at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the oligonucleotide comprises at least
one modified phosphate backbone selected from the group consisting
of a phosphorothioate, a phosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
Oligonucleotides of the invention may be synthesized by standard
methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
The oligonucleotides of the present invention may be derived by
standard methods known in the art, e.g., by de novo chemical
synthesis of polynucleotides using an automated DNA synthesizer
(such as is commercially available from Biosearch, Applied
Biosystems, etc.) and standard phosphoramidite chemistry; or by
cleavage of a larger nucleic acid fragment using non-specific
nucleic acid cleaving chemicals or enzymes or site-specific
restriction endonucleases.
A preferable method for synthesizing oligonucleotides is conducted
using an automated DNA synthesizer by methods known in the art. As
examples, phosphorothioate oligonucleotides may be synthesized by
the method of Stein et al. (1988, Nucl. Acids Res. 16:3209-3221),
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. Once the desired
oligonucleotide is synthesized, it is cleaved from the solid
support on which it was synthesized and treated, by methods known
in the art, to remove any protecting groups present. The
oligonucleotide may then be purified by any method known in the
art, including extraction and gel purification. The concentration
and purity of the oligonucleotide may be determined by examining
cligonucleotide that has been separated on. an acrylamide gel, or
by measuring the optical density at 260 nm in a
spectrophotometer.
Oligonucleotides of the invention may be labeled with donor and
acceptor moieties during chemical synthesis or the label may be
attached after synthesis by methods known in the art. In a specific
embodiment, the donor moiety is a fluorophore. In another specific
embodiment, both donor and acceptor moieties are fluorophores.
Suitable moieties that can be selected as donor or acceptors in
FRET pairs are set forth in Table 1.
TABLE 1 ______________________________________ Suitable moieties
that can be selected as donor or acceptors in FRET pairs
______________________________________
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid acridine
and derivatives: acridine acridine isothiocyanate
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate
(Lucifer Yellow VS) N-(4-anilino-1-naphthyl)maleimide
anthranilamide Brilliant Yellow coumarin and derivatives: coumarin
7-amino-4-methylcoumarin (AMC, Coumarin 120)
7-amino-4-trifluoromethylcouluarin (Coumaran 151) cyanosine
4',6-diaminidino-2-phenylindole (DAPI) 5',
5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin
diethylenetriamine pentaacetate
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride) 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL)
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC) eosin and
derivatives: eosin eosin isothiocyanate erythrosin and derivatives:
erythrosin B erythrosin isothiocyanate ethidium fluorescein and
derivatives: 5-carboxyfluorescein (FAM)
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) fluorescein
fluorescein isothiocyanate QFITC (XRITC) fluorescamine IR144 IR1446
Malachite Green isothiocyanate 4-methylumbelliferone ortho
cresolphthalein nitrotyrosine pararosaniline Phenol Red
B-phycoerythrin o-phthaldialdehyde pyrene and derivatives: pyrene
pyrene butyrate succinimidyl 1-pyrene butyrate Reactive Red 4
(Cibacron .RTM. Brilliant Red 3B-A) rhodamine and derivatives:
6-carboxy-X-rhodamine (ROX) 6-carboxyrhodamine (R6G) lissamine
rhodamine B sulfonyl chloride rhodamine (Rhod) rhodamine B
rhodamine 123 rhodamine X isothiocyanate sulforhodamine B
sulforhodamine 101 sulfonyl chloride derivative of sulforhodamine
101 (Texas Red) N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA)
tetramethyl rhodamine tetramethyl rhodamine isothiocyanate (TRITC)
riboflavin rosolic acid terbium chelate derivatives
______________________________________
One of ordinary skill in the art can easily determine, using
art-known techniques of spectrophotometry, which fluorophores will
make suitable donor-acceptor FRET pairs. For example, FAM (which
has an emission maximum of 525 nm) is a suitable donor for TAMRA,
ROX, and R6G (all of which have an excitation maximum of 514 nm) in
a FRET pair. Primers are preferably modified during synthesis, such
that a modified T-base is introduced into a designated position by
the use of Amino-Modifier C6 dT (Glen Research), and a primary
amino group is incorporated on the modified T-base, as described by
Ju et al. (1995, Proc. Natl. Acad. Sci., USA 92:4347-4351). These
modifications may be used for subsequent incorporation of
fluorescent dyes into designated positions of the
oligonucleotides.
The optimal distance between the donor and acceptor moieties will
be that distance wherein the emissions of the donor moiety are
absorbed by the acceptor moiety. This optimal distance varies with
the specific moieties used, and may be easily determined by one of
ordinary skill in the art using techniques known in the art. For
energy transfer in which it is desired that the acceptor moiety be
a fluorophore that emits energy to be detected, the donor and
acceptor fluorophores are preferably separated by a distance of up
to 30 nucleotides, more preferably from 3-20 nucleotides, and still
more preferably from 6-12 nucleotides. For energy transfer wherein
it is desired that the acceptor moiety quench the emissions of the
donor, the donor and acceptor moieties are preferably separated by
a distance of less than one nucleotide (e.g., on the opposite
strand, complementary nucleotides of a duplex structure), although
a 5 nucleotide distance (one helical turn) is also advantageous for
use.
In yet another embodiment, the oligonucleotides may be further
labeled with any other art-known detectable marker, including
radioactive labels such as .sup.32 P, .sup.35 S, .sup.3 H, and the
like, or with enzymatic markers that produce detectable signals
when a particular chemical reaction is conducted, such as alkaline
phosphatase or horseradish peroxidase. Such enzymatic markers are
preferably heat stable, so as to survive the denaturing steps of
the amplification process.
Oligonucleotides may also be indirectly labeled by incorporating a
nucleotide linked covalently to a hapten or to a molecule such as
biotin, to which a labeled avidin molecule may be bound, or
digoxygenin, to which a labeled anti-digoxygenin antibody may be
bound. Oligonucleotides may be supplementally labeled during
chemical synthesis or the supplemental label may be attached after
synthesis by methods known in the art.
The oligonucleotides of the invention have use in nucleic acid
amplification reactions, as primers, or, in the case of
triamplification, blocking oligonucleotides, to detect or measure a
nucleic acid product of the amplification, thereby detecting or
measuring a target nucleic acid in a sample that is complementary
to a 3' primer sequence. Accordingly, the oligonucleotides of the
invention can be used in methods of diagnosis, wherein a 3' primer
sequence is complementary to a sequence (e.g., genomic) of an
infectious disease agent, e.g. of human disease including but not
limited to viruses, bacteria, parasites, and fungi, thereby
diagnosing the presence of the infectious agent in a sample of
nucleic acid from a patient. The target nucleic acid can be genomic
or cDNA or mRNA or synthetic, human or animal, or of a
microorganism, etc. In another embodiment that can be used in the
diagnosis or prognosis of a disease or disorder, the target
sequence is a wild type human genomic or RNA or cDNA sequence,
mutation of which is implicated in the presence of a human disease
or disorder, or alternatively, can be the mutated sequence. In such
an embodiment, optionally, the amplification reaction can be
repeated for the same sample with different sets of primers that
amplify, respectively, the wild type sequence or the mutated
version. By way of example, the mutation can be an insertion,
substitution, and/or deletion of one or more nucleotides, or a
translocation.
5.1.1. HAIRPIN PRIMERS
The present invention provides oligonucleotide primers that form a
hairpin structure in which MET will occur when the primer is not
incorporated into the amplification product.
Accordingly, in a specific embodiment, the invention provides a
hairpin primer that is an oligonucleotide comprising, or
alternatively consisting of, the following contiguous sequences in
5' to 3' order: (a) a first nucleotide sequence of 6-30
nucleotides, wherein a nucleotide within said first nucleotide
sequence is labeled with a first moiety selected from the group
consisting of a donor moiety and an acceptor moiety of a molecular
energy transfer pair, wherein the donor moiety emits energy of one
or more particular wavelengths when excited, and the acceptor
moiety absorbs energy at one or more particular wavelengths emitted
by the donor moiety; (b) a second, single-stranded nucleotide
sequence of 3-20 nucleotides; (c) a third nucleotide sequence of
6-30 nucleotides, wherein a nucleotide within said third nucleotide
sequence is labeled with a second moiety selected from the group
consisting of said donor moiety and said acceptor moiety, and said
second moiety is the member of said group not labeling said first
nucleotide sequence, wherein said third nucleotide sequence is
sufficiently complementary in reverse order to said first
nucleotide sequence for a duplex to form between said first
nucleotide sequence and said third nucleotide sequence such that
said first moiety and second moiety are in sufficient proximity
such that, when the donor moiety is excited and emits energy, the
acceptor moiety absorbs energy emitted by the donor moiety; and (d)
at the 3' end of said oligonucleotide, a fourth, single-stranded
nucleotide sequence of 8-40 nucleotides that comprises at its 3'
end a sequence sufficiently complementary to a preselected target
sequence so as to be able to prime synthesis by a nucleic acid
polymerase of a nucleotide sequence complementary to a nucleic acid
strand comprising said target sequence; wherein when said duplex is
not formed, said first moiety and said second moiety are separated
by a distance that prevents molecular energy transfer between said
first and second moiety.
In a specific embodiment wherein the donor and acceptor moieties
are a FRET pair, separation of the first and second moiety by a
distance that prevents FRET is observed by the failure of the
second moiety to quench the fluorescence of the first moiety (when
the second moiety is a quencher), or the failure of the second
moiety to absorb the fluorescence of the first moiety and then
itself to fluoresce (when the second moiety is a fluorophore).
In a specific embodiment, the second nucleotide sequence (the loop
structure) and/or the first nucleotide sequence (of the duplex)
and/or third nucleotide sequence (of the duplex) do not contain a
sequence complementary to the target sequence. Alternatively, the
second nucleotide sequence and/or the first nucleotide sequence
and/or the third nucleotide sequence or any portion of the
foregoing sequences may also contain a sequence complementary to
the target sequence.
In a preferred embodiment, a primer forms a hairpin structure in
which the energy of a donor fluorophore is quenched by a
non-fluorescing acceptor moiety when the primer is not incorporated
into the amplification product. One of ordinary skill in the art
can easily determine, from the known structures and
hydrophobicities of a given FRET pair, the steric arrangement that
will bring the pair into closest proximity for MET.
In a specific embodiment, the hairpin primer comprises four parts
(FIG. 1): Part (d) is a 3' terminal sequence and comprises a
sequence complementary to the target sequence; it is a primer for
DNA polymerase. Part (c) is a first stem sequence on the 5' end of
the primer sequence. Part (b) forms a single-stranded loop of
nucleotides. Part (a) is a second stem sequence, which is
complementary to the first stem sequence. Parts (a), (b), and (c)
or portions thereof may or may not be complementary to the target
DNA to be amplified. Part (d) is preferably 8-30 nucleotides long;
Part (c) is preferably 6-30 nucleotides long; Part (b) is
preferably 3-20 nucleotides long.
The first stem sequence, Part (c), contains the donor fluorophore
and the second stem sequence, Part (a), contains the acceptor
(e.g., quencher), or it can be opposite. In a non-incorporated
hairpin primer, the emission of the donor will be transferred to
the acceptor, since the two moieties will be in close proximity to
each other when two stem sequences are in duplex.
The donor and acceptor moieties can be located on either terminal
nucleotides of the hairpin stem (duplex region), or internally
located. Thus, in one embodiment of the invention, the donor and
acceptor (or quencher) moieties are respectively located on the 5'
end of the hairpin primer sequence that is complementary to the
target and located on the complementary nucleotide residue on the
hairpin stem (FIG. 1), or vice versa. Each moiety may alternatively
be located on a nucleotide internal within a complementary stem
sequence. Alternatively, one of the moieties may be located on an
internal nucleotide and the other on the terminal nucleotide at the
5' end. One or both of the moieties may alternatively be located at
the other end of the duplex region.
Preferably, donor and acceptor moieties are attached to the
complementary strands of the stem, one moiety on the 5' end and the
other moiety 5 bp apart on the complementary strand. For example,
the two moieties can be offset by a 5 bp (180.degree.) turn of the
double helix formed by the two complementary strands of the stem,
and will therefore be in closest proximity sterically, and the
emission of the donor will be transferred to (and, e.g., quenched
by) the acceptor.
Alternatively, the two moieties can be on complementary strands of
the stem separated by a distance of less than 1 nucleotide (3.4
.ANG.) when the hairpin is in the closed configuration. Most
preferably, the two moieties are on complementary nucleotides on
the stem, directly opposite from one another when the hairpin is in
the closed configuration.
When a hairpin primer is linearized, the donor moiety must be
separated from the acceptor (e.g., quencher) moiety by an
intervening sequence that is long enough to substantially prevent
MET. Where a FRET pair that consists of donor and acceptor
fluorophores is used, the two FRET moieties are separated by an
intervening sequence, comprising (a) at least a portion of the
first stem sequence, (b) the loop, and (c) at least a portion of
the second stem sequence; the intervening sequence being preferably
15-25 nucleotides in length, and more preferably, 20 nucleotides in
length.
In one embodiment, the acceptor moiety is a fluorophore that will
re-emit the energy provided by the donor at a different wavelength;
that is, when the primer is in the closed state, emissions from the
acceptor, but not from the donor, will be detected. In a preferred
embodiment, the acceptor moiety is a quencher and absorbs the
energy emitted by the donor without fluorescing. In either case,
the fluorescence of donor may be detected only when the primer is
in the linearized, open state i.e., is incorporated into a
double-stranded amplification product. Energy transfer in this
state will be minimal and the strong emission signal from the donor
will be detected.
A critical aspect of the invention is that the transition from the
closed to the open state occurs only during amplification. FIGS. 2
and 3 schematically illustrate the use of the hairpin primers of
the present invention in PCR. In FIG. 2, the DNA polymerase used in
PCR lacks 5'-3' exonuclease activity, whereas in FIG. 3, it has
5'-3' activity. For PCR, either one or both PCR primers can be a
hairpin primer.
In FIGS. 2 and 3, (a) and (b) are two complementary strands of the
target sequence to be amplified and "R" and "F" are the reverse and
forward primers, respectively, for PCR amplification. By way of
example and not limitation, the reverse hairpin primer is designed
such that there is a donor fluorophore and quencher incorporated
into it. Reverse hairpin primer that is not incorporated into the
PCR product will have fluorophore and quencher in close proximity;
thus the fluorescence from the free reverse primer will be
quenched. See Section 5.2.1 infra for methods of use of hairpin
primers in PCR.
5.1.1.1. UNIVERSAL HAIRPIN PRIMERS
In one embodiment, the oligonucleotide primer of the invention is a
"universal" hairpin primer that can be ligated, either chemically
(e.g., using cyanogen bromide) or enzymatically (e.g., using
ligase) to any selected primer sequence and used to amplify a
target nucleic acid sequence that contains the complement of the
primer sequence. The invention provides a "universal" hairpin
primer that is an oligonucleotide, the nucleotide sequence of which
consists of the following contiguous sequences in 5' to 3' order:
(a) a first single-stranded nucleotide sequence of 1 to 10
nucleotides; (b) a second nucleotide sequence of 2-30 nucleotides,
wherein a nucleotide within said first nucleotide sequence or said
second nucleotide sequence is labeled with a first moiety selected
from the group consisting of a donor moiety and an acceptor moiety
of a molecular energy transfer pair, wherein the donor moiety emits
energy of one or more particular wavelengths when excited, and the
acceptor moiety absorbs energy at one or more particular
wavelengths emitted by the donor moiety; (c) a third,
single-stranded nucleotide sequence of 3-20 nucleotides; (d) a
fourth nucleotide sequence of 2-30 nucleotides, wherein a
nucleotide within said fourth nucleotide sequence is labeled with a
second moiety selected from the group consisting of said donor
moiety and said acceptor moiety, and said second moiety is the
member of said group not labeling said first or second nucleotide
sequence, wherein said fourth nucleotide sequence is sufficiently
complementary in reverse order to said second nucleotide sequence
for a duplex to form between said second nucleotide sequence and
said fourth nucleotide sequence such that said first moiety and
second moiety are in sufficient proximity such that, when the donor
moiety is excited and emits energy, the acceptor moiety absorbs
energy emitted by the donor moiety.
An example of a universal hairpin primer is shown in FIG. 4. The
universal hairpin primer of the invention comprises a first stem
sequence on the 3' end (2-30 nucleotides long, preferably 4-6
nucleotides long), a loop (3-20 nucleotides long, preferably 4-6
nucleotides long), a second stem sequence essentially complementary
to the first stem sequence (2-30 nucleotides long, preferably 4-6
nucleotides long), and a 5' single-stranded cohesive ("sticky") end
sequence (e.g., 1-10 nucleotides long, preferably 3-4 nucleotides
long). In a specific embodiment, the "sticky" end sequence is
5'GGC-3'.
Selected primer sequences that are complementary to a target DNA
sequence and that are suitable for ligation to the universal
hairpin primer may be derived by standard methods known in the art,
e.g., by de novo chemical synthesis of polynucleotides using an
automated DNA synthesizer and standard phosphoramidite chemistry;
or by cleavage of a larger nucleic acid fragment using non-specific
nucleic acid cleaving chemicals or enzymes or site-specific
restriction endonucleases.
In order to join a universal hairpin primer to the selected primer
sequence, the selected primer sequence should contain a cohesive
sequence on the 5' end essentially complementary to the cohesive
sequence of the universal hairpin primer (FIG. 4). In one
embodiment, the 5' cohesive end on the selected primer sequence is
chemically synthesized to complement the 5' cohesive end on the
universal hairpin primer. In another embodiment, the 5' cohesive
end on the selected primer sequence is produced by the staggered
cut of a restriction endonuclease.
A labeling moiety on the universal hairpin primer must not be
situated so as to substantially interfere with subsequent ligation
at its 3' end to the selected primer sequence. Thus, preferably, a
labeling moiety is not located on the 3' terminal nucleotide of the
universal hairpin primer (FIG. 4). At the 5' end of the hairpin, a
labeling moiety may be located either on the terminal nucleotide at
the 5' end (as shown in FIG. 4) or on a nucleotide internal to the
5' end.
The donor (fluorescent) and acceptor (quencher) moieties of a
universal hairpin primer such as shown in FIG. 4 must be separated
by a distance such that the emissions of the donor moiety are
quenched by the acceptor moiety. Preferably, the donor and acceptor
moieties are separated by a distance of less than 1 nucleotide (3.4
.ANG.) when the hairpin is in the closed configuration.
In one embodiment, the two FRET moieties are separated by an
intervening sequence, comprising a portion of the first stem
sequence, the loop, and a portion of the second stem sequence, that
is preferably 15-25 nucleotides in length. More preferably, the
loop on the universal hairpin is long enough provide a distance of
20 nucleotides between a donor (e.g., FAM) and a quencher (e.g.,
DABCYL) when the hairpin is in the "open" configuration.
FIG. 4 gives a schematic example of a selected target sequence
(8-40 nucleotides, preferably .about.15 nucleotides) and a
universal hairpin primer prior to their ligation to each other.
5.1.2. LINEAR OLIGONUCLEOTIDES
In another embodiment, the oligonucleotide primers are both linear
molecules that cannot form a hairpin configuration. In a specific
embodiment, a donor-acceptor FRET pair are both fluorophores
located on the same, single-stranded oligonucleotide primer, within
distance of each other so that FRET can occur. In this embodiment,
the double-labeling with a FRET pair increases the separation
between the excitation and the emission frequencies of a label.
This increased separation decreases background fluorescence that
can interfere with accurate quantitation of the emission
signal.
For example, in a specific embodiment, fluorescein may serve as the
donor moiety and rhodamine as the acceptor moiety. Fluorescein
exhibits peak excitation at 488 nm, but the excitation spectrum is
broad and it exhibits some excitation at its emission frequency at
520 nm. This contributes to an emission artifact at 520 nm that
decreases the accuracy and sensitivity of quantitative
spectrophotometry when using fluorescein as a single label. If a
fluorescein moiety is used as a donor and a rhodamine moiety as an
acceptor (rhodamine has peak excitation at 520 nm and peak emission
at 605 nm), however, excitation will occur at 488 nm and emission
will occur at 605 nm, greatly decreasing background artifact.
In another specific embodiment, the donor moiety is located on a
first oligonucleotide primer and the acceptor is located on a
second, complementary oligonucleotide. In a referred aspect of this
embodiment, one of the two FRET-labeled primers is a primer for
triamplification, and the other FRET-labeled oligonucleotide is a
blocking oligonucleotide (blocker) for triamplification.
5.2. METHODS FOR DETECTION OF AMPLIFICATION PRODUCTS USING HAIRPIN
PRIMERS
In a specific embodiment of a hairpin primer of the invention, the
acceptor moiety is a fluorophore or quencher that absorbs the
energy transmitted by the donor moiety. In a preferred embodiment,
the acceptor moiety is a quencher; the primer is configured such
that the acceptor moiety on free primer quenches the fluorescence
from the donor. When the primer is incorporated into the
amplification product, its configuration changes, quenching is
eliminated, and the fluorescence of the donor moiety is
detected.
The detection method of the present invention may be applied to any
amplification system in which an oligonucleotide is incorporated
into an amplification product e.g., polymerase chain reaction (PCR)
systems (U.S. Pat. Nos. 4,683,195 and 4,683,202), triamplification
systems (TriAmp.TM., Oncor Inc.; U.S. application Ser. No.
08/461,823, filed Jun. 5, 1995, which is incorporated by reference
herein in its entirety; PCT International Publication No. WO
9417206 A1, dated Aug. 4, 1994; PCT International Publication No.
WO 9417210 A1, dated Aug. 4, 1994), nucleic acid sequence-based
amplification (NASBA) systems (U.S. Pat. No. 5,409,818; Compton,
1991, Nature 350:91-92), and strand displacement amplification
(SDA) systems (Walker et al., 1992, Nucl. Acids Res. 20:1691-1696).
As a result of amplification, the hairpin primers are incorporated
into the double-stranded polynucleotide amplification products.
Although various specific embodiments involving a FRET pair are
described hereinbelow as involving a preferred FRET pair consisting
of a donor fluorophore moiety and a quencher acceptor moiety, it
will be understood that such embodiments could also have been
described in terms of the acceptor moiety being a fluorophore
rather than a quencher.
5.2.1. METHODS OF USE OF HAIRPIN PRIMERS IN POLYMERASE CHAIN
REACTION (PCR)
In one embodiment, the hairpin primers of the invention are used to
prime a polymerase chain reaction (PCR), thereby becoming
incorporated into the amplification product (examples being
illustrated in FIGS. 2 and 3A-D). The PCR primers contain hairpin
structures on their 5' ends with FRET donor and acceptor moieties
located in close proximity (30 nucleotides or less) on the hairpin
stem. The primers are designed in such a way that a fluorescent
signal from the donor moiety is generated only when the primers are
incorporated into an amplification product. The modified hairpin
primers do not interfere with the activity of DNA polymerase, and
in a preferred aspect, thermostable Pfu polymerase or Taq
polymerase can be used. The forward and/or reverse primers can be
hairpin primers.
In the example shown in FIG. 3, the hairpin primer has a quencher
on its 5' terminal nucleotide, and contains a donor fluorophore on
the opposite strand of its duplex, the fluorophore and quencher
being a FRET pair. In the first cycle of PCR (FIG. 3B), both
primers will hybridize to the respective target strands and will be
extended by DNA polymerase. In the second cycle (FIG. 3C) the
extended product from the reverse primer will become a template for
the forward primer and extended product from the forward primer
will become a template for the reverse primer. When the forward
primer is extended to the 5' end of the hairpin structure, either
of two things can happen, depending on the DNA polymerase used:
either the 5'-3' exonuclease activity of the DNA polymerase will
hydrolyze the 5' nucleotides with quencher, and/or DNA polymerase
will displace the 5'-end of the hairpin and copy the template. In
both cases, the quencher and the fluorophore will be separated from
each other and a signal will be generated (FIG. 3D).
Hairpin primers may be employed in any amplification method in
which the hairpin primer is not complementary to any other
oligonucleotide used in the reaction mixture, and in which the
hairpin primer is incorporated into a double-stranded DNA
amplification product, e.g., PCR, triamplification, nucleic acid
sequence-based amplification (NASBA), and strand displacement
amplification (SDA) (see infra). Thus, for example, in
triamplification involving the use of a hairpin primer, the other,
non-hairpin primer is complementary to the blocking
oligonucleotide.
In another specific embodiment (FIG. 5), a universal hairpin primer
is used, along with two selected linear primers, Primer 1 and
Primer 2, to prime a PCR. In this case, the universal hairpin
primer is incorporated into the amplification product and is not
ligated to one of the two linear primer sequences. In this
embodiment, the 3' sequence of the universal hairpin primer is
identical to the 5' sequence of one of the pair of linear forward
and reverse primers used in the amplification, and this 5' sequence
(sequence "A" on Primer 2 in FIG. 5) must not be complementary to
the target sequence.
During the first cycle of PCR, Primer 1, which is complementary to
a target DNA (+) strand is extended. Primer 2 has a 3' portion that
has a sequence complementary to the target (-) strand and a 5'
portion, designated "A" in FIG. 5, that has a sequence that is not
complementary to the target. Sequence A is preferably 10-25
nucleotides, and more preferably, 12-15 nucleotides in length.
During the second cycle, the product of the extension of Primer 2
(shown by the arrow) becomes a template for Primer 1. Primer 1 is
extended and the amplification product now includes a sequence,
designated "A'," complementary to sequence A.
During the third cycle, the A sequence of the hairpin primer
anneals to the A' sequence of the amplification product from the
previous cycle.
During the fourth cycle, the extended hairpin primer becomes a
template for Primer 1. During the extension of Primer 1, the
hairpin unfolds, the quencher and fluorophore are separated, and a
fluorescent signal is emitted from the amplification product. In a
similar way, the method can be applied to triamplification. In this
case, the hairpin primer is the primer not complementary to the
blocker.
5.2.1.1. METHODS OF USE OF HAIRPIN PRIMERS IN ALLELE-SPECIFIC PCR
(ASP)
In another embodiment, primers of the invention are used to prime
an allele-specific PCR (ASP). In this embodiment, one or both
amplification primers may be hairpin primers. In ASP, a target DNA
is preferentially amplified if it is completely complementary to
the 3' end of a PCR amplification primer. The 3' end of the hairpin
primer should terminate at or within one or 2 bases of a known
mutation site in a gene (target DNA) to which it has a
complementary sequence. Under the appropriate reaction conditions,
the target DNA is not amplified if there is a base mismatch (e.g.,
a nucleotide substitution caused by a mutation) or a small deletion
or insertion, at the 3' end of the primer (Okayama et al, 1989, J.
Lab. Clin. Med. 114:105-113; Sommer et al., 1992, BioTechniques
12:82-87). Thus, ASP can be used to detect the presence or absence
of at least a single mismatch between the hairpin sequence that is
complementary to the preselected target sequence and a nucleic acid
in the sample; amplification indicates the absence of such a single
mismatch.
5.2.2. METHODS OF USE OF HAIRPIN PRIMERS IN TRIAMPLIFICATION
5.2.2.1. GENERAL STEPS IN TRIAMPLIFICATION REACTIONS
Both hairpin primers and linear primers (see Section 5.3.4) can be
used in triamplification reactions.
A triamplification reaction is based on three oligonucleotides: two
primers and a blocking oligonucleotide (blocker). An example is
shown in FIG. 6. The two primers, a forward and a reverse
"extending" primers, are complementary to the two strands of a
selected target (template) DNA. A third oligonucleotide, a blocker,
is partially complementary to one of the two extending primers.
Triamplification utilizes two thermostable enzymes: DNA polymerase
and DNA ligase. During the repeated steps of polymerization and
ligation, one of the extended primers is ligated to the
blocker.
In one version of triamplification (the "gap" version), the forward
oligonucleotide is a primer substantially complementary to a first
segment at a first end of the target sequence to be amplified. The
reverse oligonucleotide is a primer substantially complementary to
a second segment at a second end of the target nucleic acid
sequence on a different strand of the target nucleic acid. The
third oligonucleotide (the "blocker" or "blocking oligonucleotide")
is substantially complementary to at least a portion of the forward
or reverse primer.
A schematic illustration of gap triamplification, which consists of
repeated elongation and ligation of the amplification product, is
shown in FIG. 7. Blocker may be used at the same or higher
concentration than the concentration of forward and reverse
primers. Preferably, blocker is used at a 1.2 to 2-fold higher
concentration than the concentration of forward and reverse
primers. The primer complementary to the blocker preferably is
modified to prevent strand displacement during amplification; in a
preferred embodiment, this primer contains 2'-O-methyl at the
position complementary to the 5' end of the blocker in order to
prevent strand displacement.
In the case where linear primers of the invention are used (Section
5.3.4), the blocker is preferably modified in order to protect it
from exonuclease hydrolysis (which is used with amplification
methods using linear, but not hairpin primers) and from undesirable
extension during amplification. In a preferred embodiment, the
blocker has biotin on its 3' end in order to protect it from
exonuclease hydrolysis and from undesirable extension during
amplification.
An alternate version of triamplification, the "non-gap version," is
substantially similar to the gap version described above, with the
difference that the 5' end of the forward primer is adjacent to the
3' end of the reverse primer.
5.2.2.2. USE OF HAIRPIN PRIMERS IN TRIAMPLIFICATION REACTIONS
In one embodiment of the invention, hairpin primers are used to
prime a triamplification reaction, thereby becoming incorporated
into the amplification product. When using hairpin primers in
triamplification, the hairpin structure is part of whichever
primer, either the forward or the reverse primer, that is not
complementary to the blocker (FIG. 6). It cannot be used on the
primer complementary to the blocker, because, in this case, the
blocker will interfere with the formation of the hairpin on the
primer that is not incorporated into the amplification product.
The hairpin primer is preferably labeled with a FRET donor-acceptor
pair on its stem. During the first cycle of triamplification, the
hairpin primer will be extended and ligated to the blocker. During
the second cycle, the extended hairpin primer will become a
template for the second primer. In the course of extension of the
second primer, the hairpin will open, the quencher will be
separated from the fluorophore and the donor will emit a
fluorescence signal.
5.2.3. METHODS OF USE OF HAIRPIN PRIMERS IN NUCLEIC ACID
SEQUENCE-BASED AMPLIFICATION (NASBA)
The primers of the invention may be used to prime nucleic acid
sequence-based amplification (NASBA), an example of which is shown
in FIG. 9. NASBA uses continuous cycling of reverse transcription
and RNA transcription reactions and is conducted at one
temperature. It uses three enzymes (reverse transcriptase, RNase H,
and T7 RNA polymerase). In one embodiment, the method uses two
primers, one of which is a hairpin primer of the invention that is
labeled with FRET donor and acceptor (e.g., quencher) moieties. In
an alternative embodiment, both primers are hairpin primers of the
invention.
Primer 1 has preferably about 20 bases on its 3' end that are
complementary to a target RNA and a promoter sequence 5' to the
target-complementary sequence that is recognized by T7 RNA
polymerase. Primer 2 is a hairpin primer of the invention that is
complementary to the RNA (-) sequence and has a hairpin structure
on its 5' end that is labeled with energy transfer moieties such as
is illustrated by way of example in FIG. 9.
The non-cycling NASBA phase proceeds as follows (FIG. 9). In Step
1, Primer 1 anneals to the RNA target sequence. Reverse
transcriptase uses dNTPs to extend the 3' end of the Primer 1,
forming a RNA/DNA hybrid. In Step 2, RNase H hydrolyzes the RNA
strand of the hybrid. In Step 3, hairpin Primer 2 anneals to the
single DNA strand remaining from the hybrid. Reverse transcriptase
synthesizes the second DNA strand, rendering the promoter region
double-stranded. In Step 4, the third enzyme in the mixture, T7 RNA
polymerase, binds to the promoter sequence and generates up to 100
RNA copies from each template molecule.
The cycling NASBA phase then proceeds as follows. In Step 5,
hairpin Primer 2 binds to the RNA template through its 3' end
priming sequence, and reverse transcriptase extends it and
generates a RNA/DNA hybrid. The 5' end of the hairpin is displaced
and copied as a result of replication. The quencher and the
fluorophore are now spaced far enough apart that the fluorophore is
no longer quenched and its fluorescence will be detectable. In Step
6, RNase H hydrolyzes the RNA strand. The resulting single-stranded
DNA is now "silent" (fluorescence is quenched) because the hairpin
structure is formed again. In Step 7, Primer 1 binds to the
single-stranded DNA. Reverse transcriptase binds to the 3' ends of
both the primer and the DNA template. In Step 8, the 3' end of the
single-stranded DNA is extended, yielding a double-stranded,
transcriptionally active promoter. Simultaneously, the 3' end of
Primer 1 is extended. The 5' end of the hairpin is displaced and
copied as a result of replication. The quencher and the fluorophore
are now spaced far enough apart that the fluorophore is no longer
quenched and its fluorescence will be detectable. In Step 9, T7 RNA
polymerase generates multiple RNA copies from each template
molecule.
Hence in this embodiment, the amplification products of steps 5 and
8 will have incorporated the FRET-labeled hairpin primer and will
give a fluorescent signal during the cyclic phase.
In the above example, a hairpin primer is employed in the NASBA
process as described by Compton (1991, Nature 350:91-92). However,
if polymerase-specific 5'-3' exonuclease activity is present in
addition to reverse transcriptase, T7 RNA polymerase and RNase H,
the 5' end of the hairpin-primer will be hydrolyzed during
replication. A fluorescence signal will be generated not only at
steps 5 and 8, but also at steps 6 and 7, since there will be no
quencher attached to the DNA template.
5.2.4. METHODS OF USE OF HAIRPIN PRIMERS IN STRAND DISPLACEMENT
AMPLIFICATION (SDA)
The hairpin primers of the invention may be used in strand
displacement amplification (SDA) of a double-stranded DNA target.
The forward and/or reverse primers can be hairpin primers. SDA
depends on the continuous cycling of nicking and
polymerization/displacement steps and is conducted at one
temperature.
In a specific embodiment (FIG. 10), Primer 1 and Primer 2 are both
hairpin primers of the invention. Each has a single-stranded
priming sequence on the 3' end, a recognition site for the
restriction endonuclease, and a FRET-labeled hairpin structure on
the 5' end.
SDA proceeds as follows. In Step 1, the target DNA is denatured and
Primer 1 and Primer 2 anneal through their 3' sequences. In Step 2:
The 3' ends of the primers are extended using dNTPs, one of which
is a 5'-[.alpha.-thio]triphosphate. A double stranded restriction
site is formed with one modified strand (the thio-modified strand
is resistant to endonuclease hydrolysis). At the same time, the 5'
end of the hairpin primer is displaced and copied as a result of
replication. The quencher and the fluorophore are now spaced far
enough apart that the fluorophore is no longer quenched and its
fluorescence will be detectable. In Step 3, the non-modified strand
of the double-stranded DNA is nicked by the restriction
endonuclease. In Step 4, DNA polymerase that lacks 5'-3'
exonuclease activity extends the 3' end of the nick, displacing the
single-stranded DNA target, which will go through the same cycle
again.
Hence in this embodiment, the amplification products of Steps 2, 3
and 4 will have incorporated the FRET-labeled hairpin primer and
will give a fluorescent signal.
5.3. METHODS OF DETECTION OF AMPLIFICATION PRODUCTS USING 3'-5'
EXONUCLEASE AND/OR ELEVATED TEMPERATURE
The methods of the invention described in the following Section
(5.3) may be also combined with those methods described in Section
5.4 (employing linear primers) for use during nucleic acid
amplification reactions including PCR, triamplification, NASBA and
SDA. Since the use of 3'-5' exonuclease or elevated temperature
allows detection of amplified product without the need for
separation of unincorporated primers (thus allowing a "closed tube"
format), such procedures are preferred for use with linear primers.
Since the use of hairpin primers allows one to distinguish between
amplified produce and unincorporated primers based on type of
signal detected, exonuclease treatment or heat is not necessary for
use in procedures employing the hairpin primers of the
invention.
5.3.1. USE OF 3'-5' EXONUCLEASE IN AMPLIFICATION REACTIONS
As described in certain of the embodiments in Section 5.4 relating
to PCR and triamplification, and also for use with NASBA and SDA,
after an amplification reaction is complete, 3'-5' exonuclease can
be introduced into the reaction vessel to cleave all free primer.
Then, the donor label is stimulated with light of the appropriate
wavelength. When the acceptor moiety is a fluorophore, the only
acceptor label that will emit is that which remains on uncleaved
primer that has been incorporated into the amplified product, thus
giving an indication of the extent of amplification. The further
amplification has proceeded, the greater the signal will be. When
the acceptor moiety does not fluoresce and dissipates transfer
energy as heat (i.e., quenches), the progress of the amplification
reaction may be measured as a decrease in the emissions of the
donor.
In one embodiment, wherein triamplification is employed (Section
5.4.2), single-strand-specific 3'-5' exonuclease is added to the
amplification vessel after the amplification is complete. As shown
in FIG. 8, 3'-5' exonuclease treatment hydrolyzes the
non-base-paired end of the reverse primer. The 3'-end of the
blocker is protected and remains intact.
The interaction of the FRET fluorophores inside the amplified
product will not be affected by this treatment for two reasons.
First, the 3'-end of the amplified product will be base-paired and
thus will not be a good substrate for the exonuclease. Second, the
primer that is incorporated into the amplification product is
extended on its 3' end and its labeled nucleotide residue will be
relatively far from the unprotected 3'-hydroxyl. Therefore, it will
take much longer for the nuclease to reach the modified residue. As
a result, the only detectable FRET signal will come from the
amplified product and will be free of background. Preferably the
donor should be on the forward primer, and the acceptor on the
blocker, but the converse is also possible.
The use of 3'-5' exonuclease in nucleic acid amplifications using
linear primers eliminates the necessity of separating the
amplification product from the non-incorporated oligonucleotides
after the reaction. In a preferred embodiment, the method of the
present invention may be carried out in the vessel in which the
amplification reaction proceeds, without opening the vessel in
order to allow for separation of amplification product. Polymerase
and exonuclease may be mechanically separated during amplification,
for example, in a two-chamber reaction tube as shown in FIG. 11A.
After amplification, the reaction tube is inverted, as in FIG. 11B,
allowing exonuclease to mix with the amplification mixture,
resulting in hydrolysis of unreacted labeled primer. This provides
for a greatly decreased chance of carryover contamination, and
consequently, fewer false positive results in clinical studies.
This "closed-tube" format is also readily amenable to
automation.
In another embodiment, triamplification or PCR amplification can be
performed as described in Sections 5.4.1, 5.4.2 and 6, with the
exception that thermostable DNA polymerase is present as a
combination of two enzymes, with and without 3'-5' exonuclease
activity. The ratio of polymerase to exonuclease can be adjusted
such that polymerization predominates during the amplification
cycles. After amplification, when the cycling is over,
single-stranded template will no longer be generated to which
primers can bind. Hence there will be no template/primer complex
for DNA polymerase to bind for dNTP incorporation. Therefore, the
DNA polymerase will have a chance to bind and digest the unreacted
primers using its 3'-5' exonuclease activity.
5.3.2. USE OF TEMPERATURE ELEVATION IN AMPLIFICATION REACTIONS
Background fluorescence of an amplification reaction such as a
triamplification reaction can be decreased greatly by increasing
the temperature of the amplification vessel, as an alternative to
using exonuclease. During detection, the temperature in the vessel
is raised sufficiently high enough to cause the short duplex formed
between the unused blocker and the reverse primer to dissociate,
preventing FRET. At the same time, the much longer amplification
product remains double-stranded and generates a FRET signal (see,
e.g., Example 5). In this embodiment, detection will preferably be
carried out using a thermostable-cuvette or plate-reader
fluorimeter. This embodiment also has the advantage that separation
of the amplification product from unused primer is not required.
Thus, as in the previous embodiment that uses exonuclease
treatment, amplification products may be detected directly, without
opening the reaction vessel.
5.4. METHODS FOR DETECTION OF AMPLIFICATION PRODUCTS USING LINEAR
PRIMERS
Linear primers of the invention can be employed, for example, in
PCR, NASBA, strand displacement, and triamplification. When using
linear primers in closed-tube format amplification reactions, 3'-5'
exonuclease treatment and/or temperature elevation (Section 5.3) is
preferably used to distinguish the primers from the amplification
product.
5.4.1. METHODS OF USE OF LINEAR PRIMERS IN POLYMERASE CHAIN
REACTION (PCR)
In one embodiment, the primers of the invention are used to prime a
polymerase chain reaction (PCR) (an example of which is shown in
FIG. 25), thereby becoming incorporated into the amplification
product. A donor fluorophore moiety is attached to the primer, and
an acceptor moiety that is either a fluorophore or a quencher is
attached a short distance away from the donor (30 nucleotides or
less) on the same primer.
After the PCR amplification is complete, 3'-5' exonuclease is
introduced into the reaction vessel. The exonuclease cleaves all
free primer in the reaction vessel. The reaction mixture is then
exposed to light of the appropriate wavelength to excite the donor
moiety.
When the acceptor moiety is a fluorophore, the only acceptor label
that will emit light is that which remains on uncleaved primer that
has been incorporated into the amplified product, thus giving an
indication of the extent of amplification. The further
amplification has proceeded, the greater the signal from the
acceptor moiety will be. When the acceptor moiety does not
fluoresce and dissipates transfer energy as heat (i.e., it
quenches), the progress of the reaction may be measured as a
decrease in the emissions of the donor.
5.4.1.1. METHODS OF USE OF LINEAR PRIMERS IN ALLELE-SPECIFIC PCR
(ASP)
In another embodiment, linear primers of the invention are used to
prime an allele-specific PCR (ASP) as is described in Section
5.2.1.1 supra. In this embodiment, one or both amplification
primers can be linear primers.
5.4.2. METHODS OF USE OF LINEAR OLIGONUCLEOTIDES IN
TRIAMPLIFICATION
In one embodiment, a pair of linear primers of the invention is
used in triamplification (the general steps for which are described
in Section 5.2.2.1).
As applied to the gap version of triamplification, and in an
embodiment wherein the donor and acceptor moieties, respectively,
of a MET pair are situated on separate linear oligonucleotides,
either the forward or the reverse extending primer, and the third
or blocking oligonucleotide are labeled. However, one of the pair
of MET donor-acceptor labels should be on the blocker, and the
other should be on a single-stranded 3' end of the primer that is
complementary to the blocker (see, e.g., FIGS. 7 and 8). In such a
specific embodiment employing a FRET pair consisting of donor and
acceptor fluorophores, the primer and blocking oligonucleotide are
labeled with the donor and acceptor fluorophores, respectively,
such that when both oligonucleotides are in close proximity
(hybridized to each other) and the donor label is stimulated, FRET
occurs and a fluorescence signal is produced at the emission
wavelength of the acceptor fluorophore. (Alternatively, the
acceptor moiety may be a quencher.) In a specific embodiment, the
primer that is not complementary to the blocker is unlabeled with
either the donor or acceptor moieties of the FRET pair, or
alternatively, is labeled with both moieties (see paragraph below).
After triamplification, exonuclease treatment and/or temperature
elevation are preferably used to allow detection of amplified
product without the need for separation of unincorporated primers
(see Sections 5.3.1 and 5.3.2).
In another embodiment using triamplification wherein it is desired
to use linear oligonucleotide(s) doubly labeled with both acceptor
and donor moieties of a MET pair, and wherein exonuclease treatment
(but not temperature elevation) is to be used after the
triamplification reaction so as to avoid the need for separation of
unincorporated labeled oligonucleotides, the forward and/or the
reverse primer can each be labeled with both the donor and acceptor
moieties of the FRET pair (within FRET distance of each other) if
one of the moieties is on a 3' single stranded extension.
5.5. METHODS OF USE OF HAIRPIN OR LINEAR PRIMERS IN MULTIPLEX
ASSAYS
Through the use of several specific sets of primers, amplification
of several nucleic acid targets can be performed in the same
reaction mixture. In a preferred embodiment, one or both primers
for each target can be hairpin primers labeled with a fluorescent
moiety and a quenching moiety that can perform FRET. Amplification
of several nucleic acid targets requires that a different
fluorescent acceptor moiety, with a different emission wavelength,
be used to label each set of primers.
During detection and analysis after an amplification, the reaction
mixture is illuminated and read at each of the specific wavelengths
characteristic for each of the sets of primers used in the
reaction. It can thus be determined which specific target DNAs in
the mixture were amplified and labeled. In a specific embodiment,
two or more primer pairs for amplification of different respective
target sequences are used.
5.6. ASSAYING THE METHYLATION STATUS OF DNA USING AMPLIFICATION
REACTIONS OF THE INVENTION
Methylation of cytosine located 5' to guanosine is known to have
profound effects on the expression of several eukaryotic genes
(Bird, 1992, Cell 70: 5-8). In normal cells, methylation occurs
predominantly in CG-poor regions, while CG-rich areas, called
"CpG-islands," remain unmethylated. The exception is extensive
methylation of CpG islands associated with transcriptional
inactivation of regulatory regions of imprinted genes (Li et al.,
1993, Nature 366: 362-365) and with entire genes on the inactive
X-chromosome of females (Pfeifer et al., 1989, Science 246:
810-813).
Aberrant methylation of normally unmethylated CpG islands has been
documented as a relatively frequent event in immortalized and
transformed cells (Antequera et al., 1990, Cell 62: 503-514), and
has been associated with transcriptional inactivation of defined
tumor suppressor genes in human cancers (Herman et al., 1996,
Proc.Natl. Acad. Sci., USA, 93: 9821-9826). Sensitive detection of
CpG island methylation has the potential to define tumor suppressor
gene function and provides a new strategy for early tumor
detection.
Methylation specific PCR is a sensitive detection method for
abnormal gene methylation in small DNA samples (Herman et al.,
1996, Proc. Natl. Acad. Sci., USA, 93: 9821-9826). Methylation
specific PCR employs an initial bisulfite reaction to modify DNA.
All unmethylated cytosines are dominated in a bisulfite reaction
and converted to uracils. Methylated cytosines are unaffected by
the bisulfite reaction. Consequently, a sequence of DNA that is
methylated will differ in sequence, after bisulfite treatment, from
an identical sequence that is unmethylated. Hence, different sets
of primers may be designed to specifically amplify each of those
sequences (e.g, a pair of primers to amplify unmethylated,
bisulfite treated DNA will have one or more G residues replaced by
an A residue (to be complementary to nucleotides that were formerly
unmethylated cytosines), and one or more C residues replaced by a T
residue, respectively, for the two primers of the pair, relative to
the primer pair for the methylated or untreated DNA).
As in any other PCR-based technique, this method is very sensitive.
Any carry-over contamination from sources external to the PCR will
cause false positive results. The use of the MET-labeled hairpin
primers of the present invention eliminates the risk of carry-over
contamination, since the reaction may be performed and monitored
(in real time, if necessary) in a closed-tube format.
The use of bisulfite treatment in the methods of the invention is
not limited to those methods employing PCR; other amplification
methods may alternatively be employed. The invention thus provides
a method of assaying the methylation status of DNA using an
amplification reaction of the invention, with hairpin or linear
primers. The method comprises: prior to conducting an amplification
reaction, contacting a sample containing purified nucleic acids
with an amount of bisulfite sufficient to convert unmethylated
cytosines in the sample to uracil; and conducting the amplification
reaction in the presence of a primer pair specific for preselected
target sequences. Pairs of primers, used in separate reaction
vessels, are preferably specific for bisulfite-treated methylated,
bisulfite-treated unmethylated, and nonbisulfite-treated (wild
type) nucleic acids, respectively. Conclusions about the
methylation status of the nucleic acids in the sample can be drawn
depending on which primer pair(s) give amplification product. In a
preferred embodiment, the amplification reaction is PCR sing one or
more hairpin primers.
5.7. KITS FOR THE AMPLIFICATION AND DETECTION OF SELECTED TARGET
DNA SEQUENCES
An additional aspect of the present invention relates to kits for
the detection or measurement of nucleic acid amplification
products. In specific embodiments, the kits comprise one or more
primer oligonucleotides of the invention, such as a hairpin primer,
including but not limited to a universal hairpin primer, and/or
linear primers, in one or more containers. The kit can further
comprise additional components for carrying out the amplification
reactions of the invention. Where the target nucleic acid sequence
being amplified is one implicated in disease or disorder, the kits
can be used for diagnosis or prognosis. In a specific embodiment, a
kit is provided that comprises, in one or more containers, forward
and reverse primers of the invention for carrying out
amplification, and optionally, a DNA polymerase or two DNA
polymerases respectively with and without exonuclease activity. A
kit for triamplification can further comprise, in one or more
containers, a blocking oligonucleotide, and optionally DNA
ligase.
Oligonucleotides in containers can be in any form, e.g.,
lyophilized, or in solution (e.g., a distilled water or buffered
solution), etc. Oligonucleotides ready for use in the same
amplification reaction can be combined in a single container or can
be in separate containers. Multiplex kits are also provided,
containing more than one pair of amplification (forward and
reverse) primers, wherein the signal being detected from each
amplified product is of a different wavelength, e.g., wherein the
donor moiety of each primer pair fluoresces at a different
wavelength. Such multiplex kits contain at least two such pairs of
primers.
In a specific embodiment, a kit comprises, in one or more
containers, a pair of primers preferably in the range of 10-100 or
10-80 nucleotides, and more preferably, in the range of 20-40
nucleotides, that are capable of priming amplification [e.g., by
polymerase chain reaction (see e.g., Innis et al., 1990, PCR
Protocols, Academic Press, Inc., San Diego, Calif.), for example,
competitive PCR and competitive reverse-transcriptase PCR (Clementi
et al., 1994, Genet. Anal. Tech. Appl. 11(1):1-6; Siebert et al.,
1992, Nature 359:557-558); triamplification, NASBA, strand
displacement, or other methods known in the art, under appropriate
reaction conditions, of at least a portion of a selected target
nucleic acid.
In another embodiment, a kit for the detection of a selected target
DNA target sequence comprises in one or more containers (a) PCR
primers, one or both of which are hairpin primers labeled with
fluorescent and quenching moieties that can perform MET; and
optionally: (b) a control DNA target sequence; (c) an optimized
buffer for amplification; (d) appropriate enzymes for the method of
amplification contemplated, e.g., a DNA polymerase for PCR or
triamplification or SDA, a reverse transcriptase for NASBA; (d) a
set of directions for carrying out amplification, e.g., describing
the optimal conditions, e.g., temperature, number of cycles for
amplification. Optionally, the kit provides (e) means for
stimulating and detecting fluorescent light emissions, e.g., a
fluorescence plate reader or a combination
thermocycler-plate-reader to perform the analysis.
In yet another embodiment, a kit for triamplification is provided.
The kit comprises forward and reverse extending primers, and a
blocking oligonucleotide. Either the forward or reverse primer is
labeled with one moiety of a pair of MET moieties, and the blocking
oligonucleotide is labeled with the other MET moiety of the pair.
One embodiment of such a kit comprises, in one or more containers:
(a) a first oligonucleotide; (b) a second oligonucleotide, wherein
said first and second oligonucleotides are linear primers for use
in a triamplification reaction; (c) a third oligonucleotide that is
a blocking oligonucleotide that comprises a sequence complementary
and hybridizable to a sequence of said first oligonucleotide, said
first and third oligonucleotides being labeled with a first and
second moiety, respectively, that are members of a molecular energy
transfer pair consisting of a donor moiety and an acceptor moiety,
such that when said first and third oligonucleotides are hybridized
to each other and the donor moiety is excited and emits energy, the
acceptor moiety absorbs energy emitted by the donor moiety; and (d)
in a separate container, a nucleic acid ligase.
Another embodiment of a kit comprises in a container a universal
hairpin primer, optionally also comprising a second container
containing cyanogen bromide or a nucleic acid ligase (e.g., DNA
ligase, for example, T4 DNA ligase).
A kit for carrying out a reaction such as that shown in FIG. 5
comprises in one or more containers: (a) a first oligonucleotide
primer; (b) a second oligonucleotide primer, wherein the first and
second oligonucleotide primers are forward and reverse primers for
DNA synthesis in an amplification reaction to amplify a nucleic
acid sequence, and wherein said second oligonucleotide primer
comprises (i) a 5' sequence that is not complementary to a
preselected target sequence in said nucleic acid sequence, and (ii)
a 3' sequence that is complementary to said preselected target
sequence; and (c) a third oligonucleotide primer that comprises in
5' to 3' order (i) a first nucleotide sequence of 6-30 nucleotides,
wherein a nucleotide within said first nucleotide sequence is
labeled with a first moiety selected from the group consisting of a
donor moiety and an acceptor moiety of a molecular energy transfer
pair, wherein the donor moiety emits energy of one or more
particular wavelengths when excited, and the acceptor moiety
absorbs energy at one or more particular wavelengths emitted by the
donor moiety; (ii) a second, single-stranded nucleotide sequence of
3-20 nucleotides; (iii) a third nucleotide sequence of 6-30
nucleotides, wherein a nucleotide within said third nucleotide
sequence is labeled with a second moiety selected from the group
consisting of said donor moiety and said acceptor moiety, and said
second moiety is the member of said group not labeling said first
nucleotide sequence, wherein said third nucleotide sequence is
sufficiently complementary in reverse order to said first
nucleotide sequence for a duplex to form between said first
nucleotide sequence and said third nucleotide sequence such that
said first moiety and second moiety are in sufficient proximity
such that, when the donor moiety is excited and emits energy, the
acceptor moiety absorbs energy emitted by the donor moiety; (iv) at
the 3' end of said third oligonucleotide primer, a fourth
nucleotide sequence of 10-25 nucleotides that comprises at its 3'
end a sequence identical to said 5' sequence of said second
oligonucleotide primer. Where such kit is used for
triamplification, a blocking oligonucleotide can also provided.
Another kit of the invention comprises in one or more containers:
(a) a first oligonucleotide; (b) a second oligonucleotide, said
first and second oligonucleotide being hybridizable to each other;
said first oligonucleotide being labeled with a donor moiety said
second oligonucleotide being labeled with an acceptor moiety, said
donor and acceptor moieties being a molecular energy transfer pain,
wherein the donor moiety emits energy of one or more particular
wavelengths when excited, and the acceptor moiety absorbs energy at
one or more particular wavelengths emitted by the donor moiety; and
(c) in a separate container, a nucleic acid ligase.
6. EXAMPLES: GENERAL EXPERIMENTAL METHODS
The following experimental methods were used for all of the
experiments detailed below in the Examples, Sections 7-13, except
as otherwise noted. In all of the Examples, the experiments were
carried out using either triamplification or PCR.
6.1. OLIGONUCLEOTIDE SEQUENCES: SYNTHESIS AND MODIFICATION
Three oligodeoxynucleotides complementary to segments of human
prostate specific antigen (PSA) DNA were synthesized (FIG. 12).
Reverse primer contained a 2'-O-methyl moiety at a position
complementary to the 5'-end of the blocker. This modification was
essential for prevention of strand displacement during the
amplification process (see Section 5.2.2.1) The blocker had biotin
on its 3' end, in order to protect it from 3'-5' exonuclease
hydrolysis and from undesirable extension during amplification.
During the synthesis of blocker and forward primer, the primary
amino group was incorporated on the modified T-base (Amino-Modifier
C6 dT) as described by Ju et al. (1995, Proc. Natl. Acad. Sci. USA
92:4347-4351). These modifications were used for subsequent
incorporation of fluorescent dyes into designated positions of the
oligonucleotides. Synthesized oligonucleotides were desalted and
FAM (as a donor) and rhodiamine (as an acceptor) were attached to a
modified thymidine residue of the reverse primer and blocker,
respectively, by the method published by Ju et al. (1995, Proc.
Natl. Acad. Sci. USA 92:4347-4351). Labeled oligonucleotides were
purified on a 15% denaturing polyacrylamide gel.
The absorption spectra of the primers were measured on a Hewlett
Packard 8452A diode array spectrophotometer and fluorescence
emission spectra were taken on a Shimadzu RF-5000
spectrofluorophotometer.
6.2. AMPLIFICATION OF PROSTATE SPECIFIC ANTIGEN (PSA) TARGET
DNA
Triamplification was performed in 120 .mu.l of 20 mM tris-HCl (pH
8.5), 10 mM (NH.sub.4).sub.2 SO.sub.4, 0.1 mg/ml BSA, 2 mM NAD 0.1%
Triton X100, 2 mM MgCl.sub.2, 200 .mu.M each dNTP, 10.sup.-11 M
template, 250 nM forward primer, 250 nM reverse primer labeled with
FAM, 500 nM blocker labeled with Rhod, 6 units of Pfu-exo.sup.- DNA
polymerase (polymerase without 3'-5' exonuclease activity;
Stratagene) and 30 units of Amp DNA ligase (Epicentre Tech). PCR
amplification was performed using the same conditions, except that
blocker and ligase omitted from the PCR reaction mixture. Thermal
cycling was performed using denaturation for 5 min at 94.degree.
C., followed by 35 cycles of 30 sec at 95.degree. C. and 2 min
60.degree. C. The PCR was completed with a final 6 min extension at
60.degree. C.
As a first control, a similar triamplification reaction was
performed in the absence of DNA template. As a second control, the
reaction mixture was not incubated in the thermocycler.
6.3. 3'-5' EXONUCLEASE TREATMENT
Four units of T4 DNA polymerase that had 3'-5' exonuclease activity
were added to the amplified DNA or control probe in 120 .mu.l of
the amplification buffer and incubated at 37.degree. C. for 15 min,
unless otherwise indicated.
6.4. ENERGY TRANSFER MEASUREMENTS
Energy transfer measurements were made on a Shimadzu RF-5000
spectrofluorophotometer. The excitation wavelength was 488 nm and
the emission spectra were taken between 500 and 650 nm.
7. EXAMPLE 1: DNA POLYMERASE COPIES A DNA TEMPLATE WITH RHODAMINE
MODIFICATION
This experiment (FIG. 13A) was conducted to determine the effects
of modification of a DNA template with rhodamine on the activity of
DNA polymerase. If rhodamine labeling of the reverse primer were to
block the incorporation of dNTP, elongation of the forward primer
would stop at the base opposite the modification. In this case, the
two strands of amplified product would be of different sizes: the
one with incorporated forward primer would be shorter.
A PCR amplification (FIG. 13A) was performed using the conditions
for triamplification described in Section 6, but without using
blocker. As illustrated in FIG. 13B, the strands synthesized in the
presence of modified and unmodified reverse primer were of the same
size, indicating that rhodamine-labeling did not interfere with
amplification.
The effects of rhodamine labeling on the yield of the amplification
reaction were also estimated. PCR amplification was performed and
as a control, unmodified reverse primer was used. As shown on the
agarose gel of FIG. 13C, the amount of product was similar when
rhodamine-reverse primer or non-modified reverse primer was
present.
These results lead to the conclusion that the modifications in the
DNA template do not affect the elongation reaction catalyzed by DNA
polymerase.
8. EXAMPLE 2: MODIFICATION OF A REVERSE PRIMER DOES NOT AFFECT THE
REACTION CATALYZED BY DNA LIGASE
Since triamplification uses thermostable DNA-ligase for
amplification, it was important to determine whether the
modification of primers affects ligation efficiency.
Triamplification was performed as described in Section 6 with
rhodamine-labeled reverse primer. As shown in FIG. 14A, the blocker
had four nucleotides plus biotin on its 3'-end that extended it
beyond the reverse primer sequence.
In cases in which the extended forward primer was ligated to the
blocker, the resulting strand would be expected to be approximately
4 nucleotides longer than the opposite strand, which would have
incorporated the extended reverse primer. If no ligation took place
and instead the blocker was displaced, then both strands would be
expected to be of the same length. By using [.sup.32 P]-labeled
forward or reverse primer in parallel experiments, the efficiency
of ligation was estimated.
As shown in FIG. 14B, most of the product with labeled forward
primer was longer than the strand with labeled reverse primer,
indicating that there was no significant effect of modification on
the ligation reaction.
9. EXAMPLE 3: EXONUCLEASE CAN REMOVE A NUCLEOTIDE RESIDUE LABELED
WITH RHODAMINE
Exonuclease hydrolysis of a [.sup.32 P]-labeled reverse primer
labeled with rhodamine (FIG. 15A) was performed in an amplification
reaction mixture in a PCR amplification using the methods described
in Section 6. T4 DNA polymerase with 3'-5' exonuclease activity was
used. Products of hydrolysis were analyzed on a 15% denaturing
polyacrylamide gel. The results presented in FIG. 15B demonstrate
nearly quantitative hydrolysis of the modified oligonucleotide
after 5 minutes. Similar results were obtained when a [.sup.32
P]-labeled reverse primer labeled with rhodamine was in complex
with blocker.
10. EXAMPLE 4: DETECTION OF AMPLIFICATION PRODUCT BY ENERGY
TRANSFER AFTER NUCLEASE TREATMENT
To detect the triamplification product blip FRET between the
reverse primer labeled with FAM and the blocker labeled with
rhodamine, the triamplification and the subsequent exonuclease
treatment were performed as described in Section 6. As a control,
the triamplification reaction was also performed in the absence of
DNA template.
Emission spectra are presented in FIG. 16. The FRET signal at 605
nm was emitted by the double-stranded amplification product (FIG.
16, Spectrum 1) whereas no FRET signal was emitted from the control
reaction run without DNA template (FIG. 16, Spectrum 2).
11. EXAMPLE 5: DETECTION OF AMPLIFICATION PRODUCT BASED ON
DIFFERENT THERMOSTABILITY OF AMPLIFIED PRODUCT AND BLOCKER/REVERSE
PRIMER COMPLEX
The goal of this experiment was to determine whether a specific
temperature could be found at which free blocker and reverse primer
were no longer in duplex, so that no energy transfer could occur
between them. At this temperature, however, the double-stranded
triamplification product would still remain in duplex, so that the
primers incorporated into it would generate a FRET signal.
Triamplification was performed as described in Section 6. A control
reaction was run in the absence of DNA template. After
amplification, reaction mixtures were heated to 75.degree. C. and
emission spectra were taken. The results indicate that at this
temperature, there was no signal from non-amplified primers (FIGS.
17A-B). However, emission of rhodamine at 605 nm (i.e., a FRET
signal) from the amplified product could be clearly detected.
12. EXAMPLE 6: CLOSED-TUBE FORMAT USING HAIRPIN PRIMERS FOR
AMPLIFICATION AND DETECTION OF DNA BASED ON ENERGY TRANSFER
12.1. SUMMARY
A new method for the direct detection of PCR-amplified DNA in a
closed system is described. The method is based on the
incorporation of fluorescence resonance energy transfer-labeled
primers into the amplification product. The PCR primers contain
hairpin structures on their 5' ends with donor and acceptor
moieties located in close proximity on the hairpin stem. The
primers are designed in such a way that a fluorescent signal is
generated only when the primers are incorporated into an
amplification product. A signal to background ratio of 35:1 was
obtained using the hairpin primers labeled with FAM as a donor and
DABCYL as a quencher. The modified hairpin primers do not interfere
with the activity of DNA polymerase, and both thermostable Pfu and
Taq polymerase can be used. This method was applied to the
detection of cDNA for prostate specific antigen. The results
demonstrate that the fluorescent intensity of the amplified product
correlates with the amount of incorporated primers, and as little
as ten molecules of the initial template can be detected. This
technology eliminates the risk of carry-over contamination,
simplifies the amplification assay, and opens up new possibilities
for the real-time quantification of the amplified DNA over an
extremely wide dynamic range.
12.2. INTRODUCTION
Polymerase chain reaction (PCR) and other nucleic acid
amplification techniques provide a tool for the geometric
amplification of minute amounts of initial target sequences
(reviewed in Mullis and Faloona, 1987, Methods in Enzymology, 155:
335-350; Landegren, 1993, Trends Genet. 9: 199-204). The extreme
sensitivity of DNA/RNA amplification methods has encouraged the
development of diagnostics for the early detection of cancer and
infectious agents. However, drawbacks to the clinical use of
nucleic acid amplification include the possibility of
false-positive results due to carry-over contamination, and
false-negative results caused by unsuccessful reactions and/or
nonstandardized reaction conditions (Orrego, 1990, in Innis et al.
(eds.), PCR Protocols, A guide to methods and applications,
Academic Press, San Diego, Calif., pp. 447-454).
A major source of carry-over contamination are amplification
products from previous amplification reactions. Due to the extreme
sensitivity of PCR, even minimal contamination can generate a false
positive result, and accordingly, several approaches have been
devised to deal with this problem. These include incorporation of
dUTP with subsequent treatment with uracil N-glycosylase (Longo et
al., 1990, Gene 93: 125-128), incorporation of ribonucleotides into
the PCR primers followed by base treatment (Walder et al., 1993,
Nucleic Acids Res. 21: 4339-4343) or the use of isopsoralen
derivatives which undergo a cycloaddition reaction with thymidine
residues upon exposure to UV light (Cimino et al., 1991, Nucleic
Acids Res., 19: 88-107). However, a simpler and more certain
solution to the problem would be a closed system, where both the
amplification reaction and the detection step take place in the
same vessel, so that the reaction tube is never opened after
amplification. In addition, the "closed tube" format significantly
simplifies the detection process, eliminating the need for
post-amplification analysis by such methods as gel electrophoresis
or dot blot analysis.
The method described infra is designed to measure directly
amplified DNA by incorporation of labeled oligonucleotide primers
into the reaction product. The conformational transitions that the
primers undergo serve as switches for energy transfer between two
labels. In this method, the donor and acceptor (quencher) moieties
are both attached to a hairpin structure on the 5' end of the
amplification primer. The primers are designed in such a way that
the fluorescent signal is generated only when the labeled
oligonucleotides are incorporated into the double-stranded
amplification product. This highly sensitive method may be used to
obtain quantitative or qualitative results. Applications for this
system to the detection of a specific DNA sequence include, in
addition to PCR, triamplification, nucleic acid sequence-based
amplification (NASBA), and strand displacement amplification.
12.3. MATERIALS AND METHODS
Oligonucleotide primers
The following oligodeoxynucleotides complementary to the 172 bp
segment of human prostate specific antigen (PSA) cDNA were
chemically synthesized: 5'-CCCTCAGAAGGTGACCAAGTTCAT (SEQ ID NO:11),
as an upstream primer, and 5'-GGTGTACAGGGAAGGCCTTTCGGGAC (SEQ ID
NO:12), as a downstream primer. The structures of the upstream
hairpin primers with energy transfer labels are shown in FIGS.
24A-G. FAM was incorporated into the 5' end of hairpin primers by
using FAM phosphoramidite in the last step of the chemical
synthesis. A modified T-base was introduced into a designated
position by the use of Amino-Modifier C6 dT (Glen Research), and
the DABCYL was attached to the primary amino group as described by
Ju et al. (1995, Proc. Natl. Acad. Sci. USA, 92: 4347-4351).
Labeled oligonucleotides were purified by HPLC.
Preparation of PSA cDNA
The human PSA-expressing LNCaP cell line (American Type Culture
Collection) was used in the experiments. LNCaP cells were diluted
with lymphocytes isolated from whole blood at ratios ranging from 1
LNCaP cell to 10.sup.2 lymphocytes to 1 LNCaP cell to 10.sup.6
lymphocytes. Messenger RNA was isolated using the Dynal
purification kit. cDNA was synthesized from the isolated mRNA using
reverse transcriptase (Appligene) and oligodT.sub.12-18 primers
(Pharmacia) according to the recommended protocol.
PCR conditions
Amplification of the PSA cDNA was performed in 100 .mu.l of 20 mM
Tris-HCl (pH 8.5), 50 mM KCl, 2 mM MgCl.sub.2, 200 .mu.M each dNTP,
500 nM each of the upstream and the downstream primers, and 5 units
of the Pfu.sup.exo- DNA polymerase (which lacks 3'-5' exonuclease
activity; Stratagene). Thermal cycling was performed with a 5 min
denaturation at 94.degree. C., followed by 20-40 cycles of 30 sec
at 95.degree. C., 45 sec at 60.degree. C. and 1.5 min at 72.degree.
C., and completed with a final 5 min extension at 72.degree. C.
The PCR product was purified using QIAquick Spin PCR Purification
Kit (Qiagen) and cloned into pUC19 plasmid. MDE.TM. gels (FMC
BioProducts) were used for the gel-based detection of the PCR
products. Electrophoresis in a 6% polyacrylamide gel with 7M urea,
and subsequent quantification on a PhosphorImager-SP (Molecular
Dynamics) was used to estimate the amount of primer incorporated
into the amplification product.
Fluorescence detection
A Shimadzu RF-5000 spectrofluorophotometer was used to measure the
fluorescence spectra of the individual samples. The 100 .mu.l
reaction mixture was diluted to 500 .mu.l with 20 mM Tris-HCl, pH
8.5, 50 mM NaCl, and 2 mM MgCl.sub.2, and placed into a 10.times.3
cuvette (NSG Precision Cells, Inc.) at room-temperature. For the
FAM/DABCYL (4-(4'-dimethylaminophenylazo) benzoic acid) FRET pair,
a 488 nm excitation wavelength was used and a spectrum was taken
between 500 and 650 nm. The fluorescent PCR product was also
visualized by placing the tube directly against a UV
transilluminator image analysis system (Appligene), and
photographed with a mounted camera using a D540/40 filter (Chroma
Technology).
12.4. RESULTS
Experimental design of PCR with hairpin primers
In this method, a hairpin structure is present on the 5' end of one
(or both) of the FCR primers (FIG. 1). The sequence of the hairpin
stem and loop may be partially complementary to the target DNA
sequence, but this is not necessary. There are two moieties
attached to the stem sequence of the hairpin: a quencher on the 5'
end of the hairpin and a fluorophore on the opposite side of the
hairpin stem. The positions of the fluorophore and the quencher may
be switched, depending on the availability of the commercial
precursors of these moieties. DABCYL is a non-fluorescent
chromophore whose absorption spectrum overlaps with the emission
spectrum of FAM. When stimulated by light of peak wavelength of 488
nm, FAM emits fluorescence of peak wavelength 516 nm. However, when
DABCYL is located sufficiently close to the donor fluorophore, the
energy can be transferred to DABCYL and dissipated as heat.
Therefore, when the modified primer is in a "closed" configuration
(hairpin), the FAM and DABCYL are in close proximity, and the
emission of the fluorescein is quenched by DABCYL.
During the first cycle of PCR (FIG. 2), the primers are extended
and become templates during the second cycle. Since the hairpin
structures are very stable (Varani, 1995, Annu. Rev. Biophys.
Biomol. Struct., 24: 379-404), the stems are unlikely to be melted
during the annealing step of the PCR on every target molecule. In
this case, when the DNA polymerase lacking 5'-3' exonuclease
activity reaches the 5' end of the hairpin stem, it will displace
it and copy the sequence. Thus, the hairpin primer will be
linearized by incorporation into the double-stranded helical
structure during PCR, the donor and acceptor will be about 20
nucleotides (.about.70 .ANG.) apart, resulting in no significant
energy transfer between them (Selvin, 1995, Methods Enzymol., 246:
300-334), and the fluorescence from the FAM will be markedly
enhanced.
Sequence and spectroscopic properties of the hairpin primer
The structure of the hairpin primer for the amplification of cDNA
for prostate specific antigen (PSA) is shown in FIG. 18A (SEQ ID
NO:10). The primer consists of a 12 nucleotide long single-stranded
priming sequence, a 7 bp stem, and a 6 nucleotide loop. The
fluorescent moiety (FAM) is located on the 5' end of the primer and
a quencher (DABCYL) is across from FAM on the opposite strand of
the stem sequence. FIG. 18B presents the emission spectra of the
FAM labeled hairpin primer before and after the incorporation of
DABCYL. With no quencher present, FAM that is excited at a
wavelength of 488 nm emits a peak wavelength of 516 nm. When the
same oligonucleotide is also labeled with DABCYL, the fluorescence
energy is transferred to the quencher and a much lower peak is
detected at 516 nm. The residual fluorescence of the
FAM/DABCYL-labeled oligonucleotide is partially caused by the
presence of small quantities of oligonucleotides labeled with FAM
alone. Therefore an extensive HPLC purification of the labeled
oligonucleotides was very important for the low background in
subsequent experiments.
Similar results were obtained with rhodamine as a quencher (data
not presented). As a quencher, however, DABCYL has an advantage of
being a non-fluorescent chromophore: it absorbs the energy of the
fluorescein without emitting light itself. As a result, the
emission of the fluorescein may be detected more precisely, without
interference from the emission of the acceptor.
Use of hairpin-oligonucleotides as PCR primers
PCR of the fragment of PSA cDNA was performed using thermostable
pfu.sup.exo- DNA polymerase. Total cDNA from human PSA-expressing
LNCaP cells mixed with lymphocytes was used for amplification. The
preliminary experiments using ethidium bromide-stained gels for the
assay showed that one PSA cell per 10.sup.5 lymphocytes could be
detected. For quantification purposes, the PCR product was cloned
and used to compare the efficiency of amplification in the presence
of the hairpin primer with that for the control primer, which lacks
the hairpin structure and modifications. FIG. 19 shows that the
amount of amplified product was similar for the control primer, the
hairpin primer containing FAM alone and the hairpin primer labeled
with the FAM/DABCYL FRET pair.
A crucial requirement for the method is the linearization of the
hairpin primer during amplification. Therefore DNA polymerase must
be able to synthesize the strand complementary to the hairpin
primer all the way through the hairpin to its 5' end. The following
experiment was conducted to determine whether modifications of the
structure of the hairpin primer affect the subsequent synthesis of
the full-length PCR product. PCR amplification of PSA cDNA was
performed with two primers: an upstream FAM/DABCYL-labeled hairpin
primer and a downstream primer labeled with .sup.32 P on its 5' end
(FIG. 20A). An upstream primer without the hairpin structure was
used as a control.
If the structure and/or the modifications of the hairpin primer
creates an obstacle for DNA polymerase, this primer will not be
copied all the way to its 5' end, and the [.sup.32 P]-labeled
strand will be shorter than the corresponding strand synthesized in
the presence of the control primer.
To estimate the length of the individual strands, denaturing gel
electrophoresis was performed. As illustrated by the results in
FIG. 20B, the [.sup.32 P]-labeled strand that was synthesized in
the presence of the hairpin primer was longer than the
corresponding strand made with the control primer, indicating that
DNA polymerase was able to read through the hairpin structure and
synthesize a full-length product.
Another important aspect of this method is the thermostability of
the hairpin primer. If the oligonucleotide phosphodiester bonds or
the linker arms through which donor and/or acceptor are tethered to
the oligonucleotide are cleaved as a result of high temperature,
the quencher will be separated from the fluorophore and the
background will increase. Indeed, when 50 pmoles of the hairpin
primer was incubated in a 100 .mu.l reaction for 40 cycles, the
background signal increased from 3.8 units to 12 units of
fluorescence intensity. However, the observed background was still
very low: it comprised only 6% of the fluorescence emitted by 50
pmoles of fluorescein-labeled oligonucleotides (200 units), which
was the amount used in the assays.
Monitoring of PCR with hairpin primers
To demonstrate that the fluorescence of the PCR product could be
used to monitor the reaction, total cDNA from the mixture of 1
human PSA-expressing LNCaP cell per 10.sup.4 lymphocytes was
amplified with the FAM/DABCYL-labeled hairpin primer. After
different numbers of cycles, the fluorescence intensity of the
amplified product was determined using a spectrofluorophotometer
(FIG. 21A). The results show that after only 20 cycles, the
fluorescence intensity increased five times compared to the
non-amplified reaction mixture, and a thirty-five-fold increase was
detected after 40 cycles of amplification. The same samples were
also analyzed by denaturing gel electrophoresis with subsequent
quantification on the PhosphorImager to determine the fraction of
[.sup.32 P]-labeled primers incorporated into the product. The
results in FIG. 21B demonstrate that the fluorescence intensity of
the reaction mixture correlates with the amount of primers
incorporated into the product.
In another experiment, the sensitivity of this method was explored.
For quantification purposes, cloned PSA cDNA was used as a
template. 40 cycles of PCR were performed with 0, 10, 10.sup.2,
10.sup.3, 10.sup.4, 10.sup.5, or 10.sup.6 molecules of cloned PSA
cDNA per reaction. The results in FIG. 22 demonstrate that the
method is sensitive enough to detect 10 molecules of the initial
DNA template with a spectrofluorophotometer. The fluorescent PCR
product was also visualized by placing the tube directly on a UV
transilluminator equipped with a mounted camera and D540/40 filter.
This filter permits the detection of the emission in a narrow
wavelength window: between 515 and 560 nm. As shown in FIG. 23, the
fluorescence of the PCR reaction performed with 10.sup.4, 10.sup.5
and 10.sup.6 molecules of the initial template could easily be
detected by visual inspection of the tubes.
Effect of the structure of labeled hairpin primer on the
amplification and detection
Several hairpin primers with varying sizes of stem, loop and 3'
single-stranded sequences were synthesized to estimate how these
parameters might affect the efficiency of the PCR and the
signal-to-background ratio. The structures and the relative
fluorescent intensities are presented in FIGS. 24A-G. All primers
tested had at least an 18-nucleotide sequence complementary to the
target, which comprised a 3' single-stranded priming sequence, a 3'
stem sequence and part of the loop (highlighted in bold in FIGS.
24A-G).
The length of the 3' single-stranded priming sequence was found to
be very important for the efficiency of the hairpin primers in the
PCR reaction. Almost no product was detected when the length of the
priming sequence was decreased from twelve nucleotides in Structure
A to six nucleotides in Structure G (FIG. 24). A possible
explanation for this result is that the hairpin structure is the
preferred conformation of this oligonucleotide, even at the
60.degree. C. annealing temperature, and that the nucleotides in
the stem and loop of the hairpin are not available for
hybridization to the target DNA. In this case, the only part of the
molecule not involved in the secondary structure is the 3'
single-stranded sequence; however, the six nucleotide sequence on
the 3' end of Structure G is not long enough to be an efficient PCR
primer.
Only minor variations in the amount of product generated were found
when the sizes of stem and loop were changed slightly. The PCR was
slightly less efficient when the length of the stem was greater
than 7 bp. Stabilization of the stem by replacement of an AT-base
pair at the 3' end with GC increased the signal-to-background ratio
by 10%.
12.5. DISCUSSION
The method for detection of amplification products in a "closed
tube" format is an important step towards a PCR-based automated
diagnostic system, since it not only reduces the complexity of the
reaction, but also eliminates the chances of carry-over
contamination and, consequently minimizes the chances of
false-positive results. The amplification primer contains a hairpin
structure with two labels on its stem that can undergo fluorescence
resonance energy transfer. One label is a fluorophore donor and
another is a quencher that can absorb energy emitted by the
fluorophore. A thirty-five-fold quenching of the fluorescence was
observed when the oligonucleotide primers were in the hairpin
conformation, so that less than 3% of maximum fluorescence is
detected when the primers are not incorporated into the product.
The switch from the hairpin to linearized conformation occurs as a
result of replication: the 5' end of the stem is displaced by DNA
polymerase, a complementary strand is synthesized and the hairpin
can no longer be formed. In the incorporated primers, the distance
between the fluorophore and the quencher is around 20 base pairs,
which is close to 70 .ANG., the distance at which energy transfer
is negligible (Selvin, 1995, Methods Enzymol. 246: 300-334) and so
the quantitative emission of the fluorophore can be detected.
The main advantage of this method is the generation of the
fluorescent signal by the product itself, rather than by the
hybridized probe, as in previous methods (Holland, et al., 1991,
Proc. Natl. Acad. Sci. USA, 88: 7276-7280; Lee et al., 1993,
Nucleic Acids Res., 21; 3761-3766; Tyagi and Kramer, 1996, Nature
Biotechnol., 14: 303-309). This keeps background low and allows the
real-time quantification of the amplified DNA over an extremely
wide dynamic range. In addition, the detection does not require
special buffer or temperature conditions that are necessary for
methods involving hybridization. The discrimination between a long
double-stranded DNA product and the short hairpin primer is so
efficient that the signal-to-background ratio will be the same over
a wide temperature range under a variety of reaction
conditions.
This method can be applied to many amplification systems in which a
single-stranded oligonucleotide is incorporated into the
double-stranded product, and is compatible with any thermostable
DNA polymerase. The present example used Pfu.sup.exo- DNA
polymerase, an enzyme without 5'-3' and 3'-5' exonuclease activity.
Similar results were obtained with Taq polymerase, which has 5'-3'
exonuclease activity (data not shown). 5'-3' exonuclease activity
is a part of the excision-repair function of some DNA polymerases,
and it will not attack a free primer. However, if the extended
hairpin primer still maintains its hairpin conformation when
annealed to the template DNA, then DNA polymerase will hydrolyze
the 5' end of the hairpin stem, and the 5' nucleotide with the
tethered donor or acceptor will be released into the solution. In
either case, replication or hydrolysis, the donor fluorophore will
be separated from the acceptor, quenching will be eliminated, and
the fluorescence signal from the amplification product will be
detected, allowing any thermostable DNA polymerase to be used for
the proposed amplification/detection method.
The thirty-five-fold signal-to-background ratio presented in this
example can probably be increased even further. Published data
suggest that when the fluorophore and the quencher are covalently
linked to each other, 200-fold quenching may be achieved (Wang et
al., 1990, Tetrahedron Lett., 31: 6493-6496). This implies that
placing FRET labels in closer proximity to one another on the stem
structure will increase the efficiency of quenching. This goal may
be achieved by several approaches, such as variation of the linker
arms, changing the positions of the labels, or using FRET pairs in
which the donor and acceptor have some affinity to each other.
Another way to improve the system is to increase the
thermostability of the FRET-labeled oligonucleotides to prevent an
increase in the background during amplification due to the
spontaneous release of the labels into the solution.
The method described presented in this example can be applied to
any diagnostic procedure in which the presence of the target
nucleic acid is to be detected either qualitatively or
quantitatively. It may be applied to the detection of infectious
disease agents and microorganism contamination of food or water, as
well as to the detection of some forms of cancer. An important step
in the development of any application of this method is
optimization of the structure of the primers and cycling
conditions, since any side product can give a signal. However,
optimization is facilitated by the fact that the size and purity of
the product can be confirmed by gel electrophoresis, since the DNA
amplified with the labeled hairpin primers can be analyzed by any
of the traditional methods.
The present example demonstrates the utility of this method for the
detection of cDNA of prostate specific antigen. The results show
that the specificity and the sensitivity of detection are
comparable to that of other amplification-based methods: as few as
ten molecules of the initial target can be detected. This method
can also be used for a "multiplex" analysis in which several
targets are amplified in the same reaction. For this purpose,
hairpin primers labeled with different fluorophores can be used.
For clinical applications, in which a large number of samples are
to be tested, a fluorescence plate reader could be used to read the
assay results, either separately or coupled with the PCR
machine.
13. EXAMPLE 7: ASSAY FOR THE METHYLATION STATUS OF CpG ISLANDS
USING PCR WITH HAIRPIN PRIMERS
13.1. MATERIALS AND METHODS
Genomic DNA was obtained from OH3 (unmethylated P16 DNA) and HN 12
(methylated P16 DNA) cell lines (acquired from Drs. S. B. Baylin
and D. Sidransky, The Johns Hopkins Medical Institutions) and
treated with bisulfite (Herman et al., 1996, Proc. Natl. Acad.
Sci., USA, 93: 9821-9826).
Three sets. of PCR primers (FIG. 26) that amplify respectively
bisulfite-treated unmethylated DNA (Uup and Ud (SEQ ID NOS:19 and
20, respectively)), bisulfite-treated methylated DNA (Mup and Md)
(SEQ ID NOS:21 and 22, respectively), and the DNA not treated with
bisulfite (wild type, WT) (Wup and Wd) (SEQ ID NOS:23 and 24,
respectively) were chemically synthesized. One or the two primers
in each set had a hairpin structure at its 5' end, labeled with
FAM/DABCYL.
PCR was performed in 40 .mu.l of 10 mM Tris-HCl (pH 8.3), 50 mM
KCl, 2 mM MgCl.sub.2, 0.25 mM each dNTP, 0.5 .mu.M each primer, 100
ng of the corresponding DNA template and 1 unit of GoldTaq
polymerase (Perkin Elmer). Thermal cycling was performed using
denaturation for 12 min at 94.degree. C. (these conditions were
also required for the activation of the GoldTaq polymerase),
followed by 35 cycles of 45 sec at 95.degree. C., 45 sec at
65.degree. C. and 1 min at 72.degree. C. The PCR was completed with
a final 5 min extension at 72.degree. C.
13.2. RESULTS
The reaction products were analyzed as described in Section 6.
After PCR amplification, the fluorescence intensities of the
reaction mixtures were measured. The fluorescence intensity of the
reaction mixture amplified in the presence of DNA template (+)
differed significantly from the fluorescence intensity of the
reaction mixture amplified in the absence of DNA template (-)
(Table 2). For example, when a U-primer set (for amplification of a
sequence of U (bisulfite-treated unmethylated) DNA, see Table 2)
was used with U DNA, it was amplified and the intensity of signal
differed significantly from the intensity of the reaction mixture
with no template. Similarly, use of an M-primer set led to
amplification of M (bisulfite-treated methylated) DNA, and use of a
W-primer set led to amplification of W (wild-type chemically
unmodified) DNA.
TABLE 2 ______________________________________ The fluorescence
intensity (expressed as fluorescence units) in 20 .mu.l of the
reaction mixture after PCR in the presence (+) and in the absence
(-) of DNA template. U, unmethylated genomic DNA that underwent
chemical modification with bisulfite; M, methylated genomic DNA
that underwent chemical modification with bisulfite; W, genomic DNA
that did not undergo chemical modification.
______________________________________ U DNA M DNA W DNA + - + - +
- 18 6 20 6 23 9 ______________________________________
13.3. CONCLUSION
The results show that MET-labeled hairpin primers may be used in an
amplification reaction to detect, reliably and sensitively,
methylated or unmethylated DNA.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 25 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHER
INFORMATION: 5- carboxyfluorescein (FAM moiety) attached to 1st
nucleotide (A) NAME/KEY: Other (B) LOCATION: 22 (D) OTHER
INFORMATION: DABCYL attached to 22nd nucleotide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:1: GGCTACGAACCAGGTAAGCCGTA23 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:2: GCCGGTGACCAAGTTCAT18 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CTGGGGCAGCATTGAACCAGAGGAGTTCTT30 (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CATGTGCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTG40 (2) INFORMATION FOR SEQ
ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: PSA-I
(B) LOCATION: 1...26 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGTGTACAGGGAAGGCCTTTCGGGCA26 (2) INFORMATION FOR SEQ ID NO:6: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: PSA-P (B) LOCATION:
1...22 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCAGCATTGAACCAGAGGAGTT22 (2) INFORMATION FOR SEQ ID NO:7: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: PSA-B (B) LOCATION:
1...27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCGAAAGGCCTTCCCTGTACACCAAAA27 (2) INFORMATION FOR SEQ ID NO:8: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
AAGAACTCCTCTGGTTCAATGCTGCCCCAG30 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CACCTTGGTGTACAGGGAAGGCCTTTCGGGCAGGCACATG40 (2) INFORMATION FOR SEQ
ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other
(B) LOCATION: 1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM
moiety) attached to 1st nucleotide (A) NAME/KEY: Other (B)
LOCATION: 20 (D) OTHER INFORMATION: DABCYL attached to 20th
nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ACCTTCTACCCTCAGAAGGTGACCAAGTTCAT32 (2) INFORMATION FOR SEQ ID
NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCCTCAGAAGGTGACCAAGTTCAT24 (2) INFORMATION FOR SEQ ID NO:12: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGTGTACAGGGAAGGCCTTTCGGGAC26 (2) INFORMATION FOR SEQ ID NO:13: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION:
1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM moiety)
attached to 1st nucleotide (A) NAME/KEY: Other (B) LOCATION: 20 (D)
OTHER INFORMATION: DABCYL attached to 20th nucleotide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:13: ACCTTCTACCCTCAGAAGGTGACCAAGTTCAT32 (2)
INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE:
(A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHER INFORMATION: 5-
carboxyfluorescein (FAM moiety) attached to 1st nucleotide (A)
NAME/KEY: Other (B) LOCATION: 24 (D) OTHER INFORMATION: DABCYL
attached to 24th nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:14: ACCTTCTGTTCACCCTCAGAAGGTGACCAAGTTCAT36 (2) INFORMATION FOR
SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other
(B) LOCATION: 1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM
moiety) attached to 1st nucleotide (A) NAME/KEY: Other (B)
LOCATION: 24 (D) OTHER INFORMATION: DABCYL attached to 24th
nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
ACCTTCGATTCACCCTCAGAAGGTGACCAAGTTCAT36 (2) INFORMATION FOR SEQ ID
NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other (B)
LOCATION: 1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM
moiety) attached to 1st nucleotide (A) NAME/KEY: Other (B)
LOCATION: 22 (D) OTHER INFORMATION: DABCYL attached to 22nd
nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ACCTTCTGTACCCTCAGAAGGTGACCAAGTTCAT34 (2) INFORMATION FOR SEQ ID
NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other (B)
LOCATION: 1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM
moiety) attached to 1st nucleotide (A) NAME/KEY: Other (B)
LOCATION: 22 (D) OTHER INFORMATION: DABCYL attached to 22nd
nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
ACCTTCTATACCCTCAGAAGGTGACCAAGTTCAT34 (2) INFORMATION FOR SEQ ID
NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Other (B)
LOCATION: 1 (D) OTHER INFORMATION: 5- carboxyfluorescein (FAM
moiety) attached to 1st nucleotide (A) NAME/KEY: Other (B)
LOCATION: 20 (D) OTHER INFORMATION: DABCYL attached to 20th
nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CACCTTCACCCTCAGAAGGTGACCAAGTTCAT32 (2) INFORMATION FOR SEQ ID
NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B)
TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (ix) FEATURE: (A) NAME/KEY: Uup (upstream) (B) LOCATION: 1...27
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
TGGTTATTAGAGGGTGGGGTGGATTGT27 (2) INFORMATION FOR SEQ ID NO:20: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Ud (downstream) (B)
LOCATION: 1...46 (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHER
INFORMATION: 5- carboxyfluorescein (FAM moiety) attached to 1st
nucleotide (A) NAME/KEY: Other (B) LOCATION: 20 (D) OTHER
INFORMATION: DABCYL attached to 20th nucleotide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:20:
AGCTACTCTGATAAGTAGCTTACCCAACCCCAAACCACAACCATAA46 (2) INFORMATION
FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Mup
(upstream) (B) LOCATION: 1...24 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:21: TTATTAGAGGGTGGGGCGGATCGC24 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Md (downstream) (B)
LOCATION: 1...41 (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHER
INFORMATION: 5- carboxyfluorescein (FAM moiety) attached to 1st
nucleotide (A) NAME/KEY: Other (B) LOCATION: 20 (D) OTHER
INFORMATION: DABCYL attached to 20th nucleotide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:22:
AGCTACTCTGATAAGTAGCTGACCCCGAACCGCGACCGTAA41 (2) INFORMATION FOR SEQ
ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Wup
(upstream) (B) LOCATION: 1...20 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:23: CAGAGGGTGGGGCGGACCGC20 (2) INFORMATION FOR SEQ ID NO:24: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: Wd (downstream) (B)
LOCATION: 1...38 (A) NAME/KEY: Other (B) LOCATION: 1 (D) OTHER
INFORMATION: 5- carboxyfluorescein (FAM moiety) attached to 1st
nucleotide (A) NAME/KEY: Other (B) LOCATION: 20 (D) OTHER
INFORMATION: DABCYL attached to 20th nucleotide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:24: AGCTACTCTGATAAGTAGCTCCCGGGCCGCGGCCGTGG38
(2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE:
(A) NAME/KEY: OTHER (B) LOCATION: 1 (C) OTHER INFORMATION: 5-
carboxyfluorescein (FAM moiety) attached to 1st nucleotide (A)
NAME/KEY: OTHER (B) LOCATION: 20 (C) OTHER INFORMATION: DABCYL
attached to 20th nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:25: ACCTTCTACCCTCAGAAGGTGACCAA26
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