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BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to fluorescent probes which include a
fluorescent reporter molecule and a fluorescent quencher molecule. More
specifically, the invention relates to fluorescent probes which include a
fluorescent reporter molecule and a fluorescent quencher molecule which
may be used in hybridization assays and in nucleic acid amplification
reactions, especially polymerase chain reactions (PCR).
2. Description of Related Art
Fluorescent reporter molecule - quencher molecule pairs have been
incorporated onto oligonucleotide probes in order to monitor biological
events based on the fluorescent reporter molecule and quencher molecule
being separated or brought within a minimum quenching distance of each
other. For example, probes have been developed where the intensity of the
reporter molecule fluorescence increases due to the separation of the
reporter molecule from the quencher molecule. Probes have also been
developed which lose their fluorescence because the quencher molecule is
brought into proximity with the reporter molecule. These reporter -
quencher molecule pair probes have been used to monitor hybridization
assays and nucleic acid amplification reactions, especially polymerase
chain reactions (PCR), by monitoring either the appearance or
disappearance of the fluorescence signal generated by the reporter
molecule.
As used herein, a reporter molecule is a molecule capable of generating a
fluorescence signal. A quencher molecule is a molecule capable of
absorbing the fluorescence energy of an excited reporter molecule, thereby
quenching the fluorescence signal that would otherwise be released from
the excited reporter molecule. In order for a quencher molecule to quench
an excited fluorophore, the quencher molecule must be within a minimum
quenching distance of the excited reporter molecule at some time prior to
the reporter molecule releasing the stored fluorescence energy.
Probes containing a reporter molecule - quencher molecule pair have been
developed for hybridization assays where the probe forms a hairpin
structure, i.e., where the probe hybridizes to itself to form a loop such
that the quencher molecule is brought into proximity with the reporter
molecule in the absence of a complementary nucleic acid sequence to
prevent the formation of the hairpin structure. WO 90/03446; European
Patent Application No. 0 601 889 A2. When a complementary target sequence
is present, hybridization of the probe to the complementary target
sequence disrupts the hairpin structure and causes the probe to adopt a
conformation where the quencher molecule is no longer close enough to the
reporter molecule to quench the reporter molecule. As a result, the probes
provide an increased fluorescent signal when hybridized to a target
sequence than when unhybridized. Probes including a hairpin structure have
the disadvantage that they can be difficult to design and may interfere
with the hybridization of the probe to the target sequence.
Assays have also been developed for identifying the presence of a hairpin
structure using two separate probes, one containing a reporter molecule
and the other a quencher molecule. Mergney, et al., Nucleic Acids
Research, 22:6 920-928 (1994). In these assays, the fluorescence signal of
the reporter molecule decreases when hybridized to the target sequence due
to the quencher molecule being brought into proximity with the reporter
molecule.
One particularly important application for probes including a reporter -
quencher molecule pair is their use in nucleic acid amplification
reactions, such as polymerase chain reactions (PCR), to detect the
presence and amplification of a target nucleic acid sequence. In general,
nucleic acid amplification techniques have opened broad new approaches to
genetic testing and DNA analysis. Arnheim and Erlich, Ann. Rev. Biochem.,
61: 131-156 (1992). PCR, in particular, has become a research tool of
major importance with applications in, for example, cloning, analysis of
genetic expression, DNA sequencing, genetic mapping and drug discovery.
Arnheim and Erlich, Ann. Rev. Biochem., 61: 131-156 (1992); Gilliland et
al., Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan et al., PCR
Methods and Applications, 1: 222-228 (1992); Green et al., PCR Methods and
Applications, 1: 77-90 (1991); Blackwell et al., Science, 250: 1104-1110
(1990).
The widespread applications of nucleic acid amplification techniques has
driven the development of instrumentation for carrying out the
amplification reactions under a variety of circumstances. Important design
goals for such instrument development have included fine temperature
control, minimization of sample-to-sample variability in multi-sample
thermal cycling, automation of pre- and post-reaction processing steps,
high speed temperature cycling, minimization of sample volumes, real time
measurement of amplification products and minimization of cross
contamination, for example, due to "sample carryover". In particular, the
design of instruments permitting amplification to be carried out in closed
reaction chambers and monitored in real time would be highly desirable for
preventing cross-contamination. Higuchi et al., Biotechnology, 10: 413-417
(1992) and 11: 1026-1030 (1993); and Holland et al., Proc. Natl. Acad.
Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a
design goal would be especially desirable in the analysis of diagnostic
samples, where a high frequency of false positives and false negatives,
for example caused by "sample carryover", would severely reduce the value
of an amplification procedure. Moreover, real time monitoring of an
amplification reaction permits far more accurate quantification of
starting target DNA concentrations in multiple-target amplifications, as
the relative values of close concentrations can be resolved by taking into
account the history of the relative concentration values during the
reaction. Real time monitoring also permits the efficiency of the
amplification reaction to be evaluated, which can indicate whether
reaction inhibitors are present in a sample.
Holland et al. (cited above), U.S. Pat. No. 5,210,015 to Gelfand, et al.
and others have proposed fluorescence-based approaches to provide real
time measurements of amplification products during PCR. Such approaches
have either employed intercalating dyes (such as ethidium bromide) to
indicate the amount of double-stranded DNA present, or they have employed
probes containing fluorescence-quencher pairs (also referred to as the
"Taq-Man" approach) where the probe is cleaved during amplification to
release a fluorescent molecule whose concentration is proportional to the
amount of double-stranded DNA present. During amplification, the probe is
digested by the nuclease activity of a polymerase when hybridized to the
target sequence to cause the fluorescent molecule to be separated from the
quencher molecule, thereby causing fluorescence from the reporter molecule
to appear.
The Taq-Man approach, illustrated in FIG. 1, uses an oligonucleotide probe
containing a reporter molecule - quencher molecule pair that specifically
anneals to a region of a target polynucleotide "downstream", i.e. in the
direction of extension of primer binding sites. The reporter molecule and
quencher molecule are positioned on the probe sufficiently close to each
other such that whenever the reporter molecule is excited, the energy of
the excited state nonradiatively transfers to the quencher molecule where
it either dissipates nonradiatively or is emitted at a different emission
frequency than that of the reporter molecule. During strand extension by a
DNA polymerase, the probe anneals to the template where it is digested by
the 5'.fwdarw.3' exonuclease activity of the polymerase. As a result of
the probe being digested, the reporter molecule is effectively separated
from the quencher molecule such that the quencher molecule is no longer
close enough to the reporter molecule to quench the reporter molecule's
fluorescence. Thus, as more and more probes are digested during
amplification, the number of reporter molecules in solution increases,
thus resulting in an increasing number of unquenched reporter molecules
which produce a stronger and stronger fluorescent signal.
Three main factors influence the utility of reporter-quencher molecule pair
probes in hybridization and amplification assays. The first factor is the
effectiveness of the quencher molecule on the probe to quench the reporter
molecule. This first factor, herein designated "RQ.sup.- ", can be
characterized by the ratio of the fluorescent emissions of the reporter
molecule to the quencher molecule when the probe is not hybridized to a
complementary polynucleotide. That is, RQ.sup.- is the ratio of the
fluorescent emissions of the reporter molecule to the fluorescence of the
quencher molecule when the oligonucleotide probe is in a single-stranded
state. Influences on the value of RQ.sup.- include, for example, the
particular reporter and quencher molecules used, the spacing between the
reporter and quencher molecules, nucleotide sequence-specific effects, and
the degree of flexibility of structures, e.g., linkers, to which the
reporter and quencher molecules are attached, and the presence of
impurities. Wo et al., Anal. Biochem., 218: 1-13 (1994); and Clegg, Meth.
Enzymol., 211: 353-388 (1992). A related quantity RQ.sup.+, refers to the
ratio of fluorescent emissions of the reporter molecule to the quencher
molecule when the oligonucleotide probe is hybridized to a complementary
polynucleotide.
A second factor is the efficiency of the probe to hybridize to a
complementary polynucleotide. This second factor depends on the probe's
melting temperature, T.sub.m, the presence of a secondary structure in the
probe or target polynucleotide, the annealing temperature, and other
reaction conditions.
A third factor is the efficiency with which the DNA polymerase 5'.fwdarw.3'
exonuclease activity cleaves the bound probe between the reporter molecule
and quencher molecule. This efficiency depends on such factors as the
proximity of the reporter or quencher to the 5' end of the probe, the
"bulkiness" of the reporter or quencher, and the degree of complementarity
between the probe and target polynucleotide. Lee et al., Nucleic Acids
Research, 21: 3761-3766 (1993).
Since quenching depends on the physical proximity of the reporter molecule
to the quencher molecule, it was previously assumed that the quencher and
reporter molecules must be attached to the probe such that the quencher
molecule remains at all times within the maximum distance at which the
quencher molecule can quench the reporter molecule, this distance
generally being a separation of about 6-16 nucleotides. Lee et al. Nucleic
Acids Research, 21: 3761-3766 (1993); Mergny et al., Nucleic Acids
Research 22: 920-928 (1994); Cardullo et al., Proc. Natl. Acad. Sci., 85:
8790-8794 (1988); Clegg et al., Proc. Natl. Acad. Sci., 90: 2994-2998
(1993); and Ozaki et al., Nucleic Acids Research, 20: 5205-5214 (1992).
This short separation between the reporter molecule and the quencher
molecule is typically achieved by attaching one member of the
reporter-quencher pair to the 3' or 5' end of the probe and the other
member to an internal base 6-16 nucleotides away.
There are at least two significant disadvantages associated with attaching
a reporter or quencher molecule to an internal base. Attaching a reporter
or quencher molecule to an internal nucleotide typically involves more
difficult chemistry than the chemistry required to attach the molecule to
a terminal nucleotide. In addition, attachment of a reporter or quencher
molecule to an internal nucleotide can adversely affect the hybridization
efficiency of the probe. Ward et al., U.S. Pat. No. 5,328,824; and Ozaki
et al. Nucleic Acids Research, 20: 5205-5214 (1992).
A need currently exists for effective oligonucleotide probes containing a
fluorescent reporter molecule and a quencher molecule for use in
hybridization and nucleic acid amplification assays. Accordingly, a need
exists for probes which exhibit distinguishable fluorescence
characteristics when hybridized and not hybridized to a target nucleic
acid sequence. A further need exists for probes where the reporter
molecule and quencher molecule are positioned on the probe such that the
quencher molecule can effectively quench the fluorescence of the reporter
molecule. A further need exists for probes which are efficiently
synthesized. Yet a further need exists for the reporter molecule and
quencher molecule to be positionable on the probe such that the reporter
and quencher molecules do not adversely impact the hybridization
efficiency of probe. These and further objectives are provided by the
probes and methods of the present invention.
SUMMARY OF THE INVENTION
The present invention relates to an oligonucleotide probe which includes a
fluorescent reporter molecule and a quencher molecule capable of quenching
the fluorescence of the reporter molecule. According to the present
invention, the oligonucleotide probe is constructed such that the probe
exists in at least one single-stranded conformation when unhybridized
where the quencher molecule is near enough to the reporter molecule to
quench the fluorescence of the reporter molecule. The oligonucleotide
probe also exists in at least one conformation when hybridized to a target
polynucleotide where the quencher molecule is not positioned close enough
to the reporter molecule to quench the fluorescence of the reporter
molecule. By adopting these hybridized and unhybridized conformations, the
reporter molecule and quencher molecule on the probe exhibit different
fluorescence signal intensities when the probe is hybridized and
unhybridized. As a result, it is possible to determine whether the probe
is hybridized or unhybridized based on a change in the fluorescence
intensity of the reporter molecule, the quencher molecule, or a
combination thereof. In addition, because the probe can be designed such
that the quencher molecule quenches the reporter molecule when the probe
is not hybridized, the probe can be designed such that the reporter
molecule exhibits limited fluorescence until the probe is either
hybridized or digested.
According to the present invention, the fluorescence intensity of the
reporter molecule is preferably greater than the fluorescence intensity of
the quencher molecule when the probe is hybridized to the target
polynucleotide. The fluorescence intensity of the reporter molecule is
more preferably at least about a factor of 3.5 greater than the
fluorescence intensity of the quencher molecule when the probe is
hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the
target polynucleotide is also preferably at least about a factor of 6
greater than the fluorescence intensity of the oligonucleotide probe when
not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher molecule by
at least about 15 nucleotides, more preferably at least about 18
nucleotides. The reporter molecule is preferably separated from the
quencher molecule by between about 15 and 60 nucleotides, more preferably
between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5'
terminal nucleotides of the probe. The quencher molecule is also
preferably attached to either the 3' or 5' terminal nucleotides of the
probe. More preferably, the reporter and quencher molecules are attached
to the 3' and 5' or 5' and 3' terminal nucleotides of the probe
respectively.
The reporter molecule is preferably a fluorescein dye and the quencher
molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe of the present invention is
immobilized on a solid support. The oligonucleotide probe may be attached
directly to the solid support, for example by attachment of the 3' or 5'
terminal nucleotide of the probe to the solid support. More preferably,
however, the probe is attached to the solid support by a linker. The
linker serves to distance the probe from the solid support. The linker is
most preferably at least 30 atoms in length, more preferably at least 50
atoms in length.
A wide variety of linkers are known in the art which may be used to attach
the oligonucleotide probe to the solid support. The linker most preferably
includes a functionalized polyethylene glycol because it does not
significantly interfere with the hybridization of probe to the target
oligonucleotide, is commercially available, soluble in both organic and
aqueous media, easy to functionalize, and completely stable under
oligonucleotide synthesis and post-synthesis conditions.
The linkages between the solid support, the linker and the probe are
preferably not cleaved during removal of base protecting groups under
basic conditions at high temperature. Examples of preferred linkages
include carbamate and amide linkages.
The present invention also relates to the use of the oligonucleotide probe
as a hybridization probe to detect target polynucleotides. Accordingly,
the present invention relates to a hybridization assay for detecting the
presence of a target polynucleotide in a sample. In one embodiment of the
method, the hybridization probe is immobilized on a solid support.
According to the method, an oligonucleotide probe of the present invention
is contacted with a sample of polynucleotides under conditions favorable
for hybridization. The fluorescence signal of the reporter molecule before
and after being contacted with the sample is compared. Since the reporter
molecule on the probe exhibits a greater fluorescence signal when
hybridized to a target sequence, an increase in the fluorescence signal
after the probe is contacted with the sample indicates the hybridization
of the probe to target sequences in the sample, thereby indicating the
pressure of target sequences in the sample. Quantification of the change
in fluorescence intensity as a result of the probe being contacted with
the sample can be used to quantify the amount of target sequences present
in the sample.
The present invention also relates to the use of the oligonucleotide probe
for monitoring nucleic acid amplification. Accordingly, the present
invention relates to a method for monitoring nucleic acid amplification by
performing nucleic acid amplification on a target sequence using a nucleic
acid polymerase having 5'.fwdarw.3' nuclease activity, a primer capable of
hybridizing to the target sequence and an oligonucleotide probe according
to the present invention which is capable of hybridizing to the target
sequence 3' relative to the primer. According to the method, the nucleic
acid polymerase digests the oligonucleotide probe during amplification
when it is hybridized to the target sequence, thereby separating the
reporter molecule from the quencher molecule. As the amplification is
conducted, the fluorescence of the reporter molecule is monitored, the
generation of fluorescence corresponding to the occurrence of nucleic acid
amplification. Accordingly, the amount of amplification performed can be
quantified based on the change in fluorescence observed. It is noted that
the fluorescence of the quencher molecule may also be monitored, either
separately or in combination with the reporter molecule, to detect
amplification.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates a method for real-time monitoring nucleic acid
amplification utilizing a probe which is degraded by the 5'.fwdarw.3'
exonuclease activity of a nucleic acid polymerase.
FIG. 2 illustrates a probe according to the present invention immobilized
to a solid support in hybridized and unhybridized conformations.
DETAILED DESCRIPTION
The present invention relates to an oligonucleotide probe which includes a
fluorescent reporter molecule and a quencher molecule capable of quenching
the fluorescence of the reporter molecule. According to the present
invention, the oligonucleotide probe is constructed such that the probe
exists in at least one single-stranded conformation when unhybridized
where the quencher molecule is near enough to the reporter molecule to
quench the fluorescence of the reporter molecule. The oligonucleotide
probe also exists in at least one conformation when hybridized to a target
polynucleotide such that the quencher molecule is not positioned close
enough to the reporter molecule to quench the fluorescence of the reporter
molecule. By adopting these hybridized and unhybridized conformations, the
reporter molecule and quencher molecule on the probe exhibit different
fluorescence signal intensities when the probe is hybridized and
unhybridized. As a result, it is possible to determine whether the probe
is hybridized or unhybridized based on a change in the fluorescence
intensity of the reporter molecule, the quencher molecule, or a
combination thereof. In addition, because the probe can be designed such
that the quencher molecule quenches the reporter molecule when the probe
is not hybridized, the probe can be designed such that the reporter
molecule exhibits limited fluorescence unless the probe is either
hybridized or digested.
According to the present invention, the fluorescence intensity of the
reporter molecule is preferably greater than the fluorescence intensity of
the quencher molecule when the probe is hybridized to the target
polynucleotide. The fluorescence intensity of the reporter molecule is
more preferably at least about a factor of 3.5 greater than the
fluorescence intensity of the quencher molecule when the probe is
hybridized to the target polynucleotide.
The fluorescence intensity of the oligonucleotide probe hybridized to the
target polynucleotide is also preferably at least about a factor of 6
greater than the fluorescence intensity of the oligonucleotide probe when
not hybridized to the target polynucleotide.
The reporter molecule is preferably separated from the quencher molecule by
at least about 15 nucleotides, more preferably at least about 18
nucleotides. The reporter molecule is preferably separated from the
quencher molecule by between about 15 and 60 nucleotides, more preferably
between about 18 and 30 nucleotides.
The reporter molecule is preferably attached to either the 3' or 5'
terminal nucleotides of the probe. The quencher molecule is also
preferably attached to either the 3' or 5' terminal nucleotides of the
probe. More preferably, the reporter and quencher molecules are attached
to the 3' and 5' or 5' and 3' terminal nucleotides of the probe
respectively.
The reporter molecule is preferably a fluorescein dye and the quencher
molecule is preferably a rhodamine dye.
In one embodiment, the oligonucleotide probe is attached to a solid
support. As illustrated in FIG. 2, when the probe is unhybridized, the
probe is able to adopt at least one single-stranded conformation such that
the quencher molecule is near enough to the reporter molecule to quench
the fluorescence of the reporter molecule. As further illustrated in FIG.
2, when the probe is hybridized to a target sequence, the probe adopts at
least one conformation where the quencher molecule is not positioned close
enough to the reporter molecule to quench the fluorescence of the reporter
molecule. As a result, the fluorescence intensity of the reporter molecule
increases when the probe hybridizes to a target sequence.
As illustrated in FIG. 2, different probes may be attached to the solid
support and may be used to simultaneously detect different target
sequences in a sample. Reporter molecules having different fluorescence
wavelengths can be used on the different probes, thus enabling
hybridization to the different probes to be separately detected.
Examples of preferred types of solid supports for immobilization of the
oligonucleotide probe include controlled pore glass, glass plates,
polystyrene, avidin coated polystyrene beads, cellulose, nylon, acrylamide
gel and activated dextran. CPG, glass plates and high cross-linked
polystyrene. These solid supports are preferred for hybridization and
diagnostic studies because of their chemical stability, ease of
functionalization and well defined surface area. Solid supports such as
controlled pore glass (CPG, 500 .ANG., 1000 .ANG.) and non-swelling high
cross-linked polystyrene (1000 .ANG.) are particularly preferred in view
of their compatibility with oligonucleotide synthesis.
The oligonucleotide probe may be attached to the solid support in a variety
of manners. For example, the probe may be attached to the solid support by
attachment of the 3' or 5' terminal nucleotide of the probe to the solid
support. More preferably, however, the probe is attached to the solid
support by a linker which serves to distance the probe from the solid
support. The linker is most preferably at least 30 atoms in length, more
preferably at least 50 atoms in length.
The length and chemical stability of linker between solid support and the
first 3' unit of oligonucleotides play an important role in efficient
synthesis and hybridization of support bound oligonucleotides. The linker
arm should be sufficiently long so that a high yield (>97%) can be
achieved during automated synthesis. The required length of the linker
will depend on the particular solid support used. For example, a six atom
linker is generally sufficient to achieve a >97% yield during automated
synthesis of oligonucleotides when high cross-linked polystyrene is used
as the solid support. The linker arm is preferably at least 20 atoms long
in order to attain a high yield (>97%) during automated synthesis when CPG
is used as the solid support.
Hybridization of a probe immobilized to a solid support generally requires
that the probe be separated from the solid support by at least 30 atoms,
more preferably at least 50 atoms. In order to achieve this separation,
the linker generally includes a spacer positioned between the linker and
the 3' nucleoside. For oligonucleotide synthesis, the linker arm is
usually attached to the 3'-OH of the 3' nucleoside by an ester linkage
which can be cleaved with basic reagents to free the oligonucleotide from
the solid support.
A wide variety of linkers are known in the art which may be used to attach
the oligonucleotide probe to the solid support. The linker may be formed
of any compound which does not significantly interfere with the
hybridization of the target sequence to the probe attached to the solid
support. The linker may be formed of a homopolymeric oligonucleotide which
can be readily added on to the linker by automated synthesis.
Alternatively, polymers such as functionalized polyethylene glycol can be
used as the linker. Such polymers are preferred over homopolymeric
oligonucleotides because they do not significantly interfere with the
hybridization of probe to the target oligonucleotide. Polyethylene glycol
is particularly preferred because it is commercially available, soluble in
both organic and aqueous media, easy to functionalize, and completely
stable under oligonucleotide synthesis and post-synthesis conditions.
The linkages between the solid support, the linker and the probe are
preferably not cleaved during removal of base protecting groups under
basic conditions at high temperature. Examples of preferred linkages
include carbamate and amide linkages.
The oligonucleotide probe of the present invention may be used as a
hybridization probe to detect target polynucleotides. Accordingly, the
present invention relates to a hybridization assay for detecting the
presence of a target polynucleotide in a sample. According to the method,
an oligonucleotide probe of the present invention is contacted with a
sample of nucleic acids under conditions favorable for hybridization. The
fluorescence signal of the reporter molecule is measured before and after
being contacted with the sample. Since the reporter molecule on the probe
exhibits a greater fluorescence signal when hybridized to a target
sequence, an increase in the fluorescence signal after the probe is
contacted with the sample indicates the hybridization of the probe to
target sequences in the sample and hence the presence of target sequences
in the sample. Further, by quantifying the change in fluorescence
intensity as a result of the probe being contacted with the sample, the
amount of target sequences in the sample can be quantified.
According to one embodiment of the method, the hybridization probe is
immobilized on a solid support. The oligonucleotide probe is contacted
with a sample of nucleic acids under conditions favorable for
hybridization. The fluorescence signal of the reporter molecule is
measured before and after being contacted with the sample. Since the
reporter molecule on the probe exhibits a greater fluorescence signal when
hybridized to a target sequence, an increase in the fluorescence signal
after the probe is contacted with the sample indicates the hybridization
of the probe to target sequences in the sample. Immobilization of the
hybridization probe to the solid support enables the target sequence
hybridized to the probe to be readily isolated from the sample. In later
steps, the isolated target sequence may be separated from the solid
support and processed (e.g., purified, amplified) according to methods
well known in the art depending on the particular needs of the researcher.
The oligonucleotide probe of the present invention may also be used as a
probe for monitoring nucleic acid amplification. Accordingly, the present
invention re | | |
