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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the field of nucleic acid chemistry.
More specifically, it relates to methods of controlling the fluorescence
of fluorescently labeled oligonucleotides in solution using a DNA binding
compound. Additionally, it relates to methods for detecting degradation of
fluorescently labeled single-stranded oligonucleotides in solution.
Additionally, the invention relates to methods for detecting nucleic acid
sequences by hybridization with a complementary oligonucleotide probe.
2. Description of the Related
Nucleic acid detection using oligonucleotide probes has become a standard
method for specific target detection. Numerous modifications of the method
have been described. Generally, a DNA sample is immobilized on a solid
support and then hybridized to a labeled target-specific probe (see, for
example, Falkow et al., U.S. Pat. No. 4,358,535, incorporated herein by
reference).
Several nucleic acid detection methods have been described which involve
selective cleavage of oligonucleotide probes following formation of
probe-target hybridization duplexes. Detection of cleaved probes indicates
the occurrence of hybridization and, hence, the presence of target
sequences. For example, Saiki et al., 1985, Biotechnology 3:1008-1012,
incorporated heroin by reference, describe "oligomer restriction"
detection methods, in which hybridization of the target-specific probe
generates a restriction site which is then cleaved by the corresponding
restriction enzyme. PCT Patent Publication No. WO 89/09284, incorporated
herein by reference, describes methods in which RNA probes are used to
detect DNA target sequences. RNA probes hybridized to DNA target are
cleaved using RNaseH, which selectively cleaves RNA in RNA-DNA hybrid
duplexes. U.S. Pat. No. 5,210,015, incorporated herein by reference,
describes methods which use the 5' to 3' exonuclease activity of a nucleic
acid polymerase to cleave probes hybridized to target sequences and
thereby release labeled oligonucleotide fragments for detection. These
methods require an additional oligonucleodde hybridized upstream of the
probe hybridization site to act as a primer for the polymerase-mediated
extension reaction. Probe cleavage occurs concomitant with primer
extension.
The invention of the polymerase chain reaction (PCR), a process for
amplifying nucleic acids, enabled the detection of nucleic acids with
greatly increased sensitivity and specificity. Using PCR, segments of
single copy genomic DNA can be selectively amplified to an easily
detectable level prior to detection. PCR methods are disclosed in U.S.
Pat. No. 4,683,202, incorporated herein by reference. PCR and methods for
detecting PCR products using an oligonucleotide probe capable of
hybridizing with the amplified target nucleic acid are described in U.S.
Pat. No. 4,683,195, and European Patent Publication No. 237,362, both
incorporated herein by reference.
Similar to the methods for detecting unamplified nucleic acid described
above, methods for detecting amplification product have been described
which involve selective cleavage of hybridization probes following
formation of probe-target hybridization duplexes. Saiki et al., 1985,
Science 230:1350-1353, incorporated herein by reference, describe the
application of "oligomer restriction" to the detection of amplified
product. U.S. Pat. No. 5,210,015 also describes the analysis of PCR
amplification products using the 5' to 3' exonuclease activity of a
nucleic acid polymerase to cleave labeled probes hybridized to target
sequences (see also Holland et al., 1991, Proc. Natl. Acad. Sci. U.S.A.
88:7276-7280, incorporated herein by reference). Probes that hybridize to
a region of the target nucleic acid bounded by the amplification primers
are incorporated into the amplification reaction mixture. Hybridized
probes are cleaved by the 5' to 3' nuclease activity of the polymerase
during primer extension. Detection of labeled fragments indicates the
occurrence of both primer extension and probe hybridization, and,
therefore, amplification of the specific target sequence.
A number of agents have been described for labeling nucleic acids, whether
probe or target, for facilitating detection of target nucleic acid. Labels
have been described that provide signals detectable by fluorescence,
radioactivity, colorimetry, X-ray diffraction or absorption, magnetism,
and enzymatic activity and include, for example, fluorophores,
chromophores, radioactive isotopes (particularly .sup.32 P and .sup.125
I), electron-dense reagents, enzymes, and ligands having specific binding
partners. Labeling can be achieved by a number of means, such as chemical
modification of a primer or probe to incorporate a label or the use of
polymerizing agents to incorporate a modified nucleoside triphosphate into
an extension product.
A variety of fluorescent DNA binding compounds are known. These include
intercalating agents which bind non-covalently to the stacked bases of
nucleic acids and display a change in fluorescence, either an increase or
shift to a different wavelength, as a result. U.S. Pat. No. 4,582,789,
incorporated herein by reference, describes several intercalating moieties
including psoralens. Ethidium bromide (EtBr) is an intercalating compound
that displays increased fluorescence when bound to double-stranded DNA
rather than when in free solution (Sharp et al., 1973, Biochemistry
12:3055, incorporated herein by reference).. Although EtBr can be used to
detect both single- and double-stranded nucleic acids, the affinity of
EtBr for single-stranded nucleic acid is relatively low. EtBr is routinely
used to non-specifically detect nucleic acids following gel
electrophoresis. Following size fractionation on an appropriate gel
matrix, for example, agarose or acrylamide, the gel is soaked in a dilute
solution of EtBr. The DNA is then visualized by examining the gel under UV
light (see Maniatis et al., 1982 eds., Molecular Cloning: A Laboratory
Manual, New York, Cold Spring Harbor Laboratory, incorporated herein by
reference).
A homogeneous assay for PCR and concurrent PCR product detection based on
the increased fluorescence that ethidium bromide (EtBr) and other DNA
binding labels exhibit when bound to double-stranded DNA is described in
Higuchi et al., 1992, Bio/Techniques 10:413-417; Higuchi et al., 1993,
Bio/Techniques 11:1026-1030; and European Patent Publication Nos. 487,218
and 512,334, each incorporated herein by reference. The methods allow
direct detection of the increase of double-stranded DNA during an
amplification reaction, most significantly from the increase in amplified
target. However, these methods detect only the total amount of
double-stranded DNA in the reaction and do not distinguish specific
nucleic acid sequences; assay specificity depends on the specificity of
the amplification reaction.
The use of oligonucleotide probes labeled with interacting fluorescent
labels in nucleic acid hybridization assays is described in Morrison,
1992, in Nonisotopic DNA Probe Techniques, Kricka, ed., Academic Press,
Inc., San Diego, Calif., chapter 13; and Heller and Morrison, 1985, in
Rapid Detection and Identification of Infections Agents, Academic Press,
Inc., San Diego, Calif., pages 245-256; both incorporated herein by
reference. The methods rely on the change in fluorescence that occurs when
suitable fluorescent labels are brought into close proximity, described in
the literature as fluorescence energy transfer (FET), fluorescence
resonance energy transfer, nonradiative energy transfer, long-range energy
transfer, dipole-coupled energy transfer, or Forster energy transfer. A
number of suitable fluorescent labels are known in the art and
commercially available from, for example, Molecular Probes (Eugene,
Oreg.).
Morrison, 1992, supra, described FET-based assay formats in which
interacting fluorescent labels are bound to separate oligonucleotides that
are either brought together or separated by probe hybridization. These
assay formats, which require two probes, are described as either
non-competitive or competitive, depending on whether probe-probe
hybridization competes with probe-target hybridization. In an alternative
assay format, one fluorescent label is bound to the hybridization probe,
and the second fluorescent label is brought into close proximity by
intercalating into the double-stranded hybridization duplex. No
significant interaction occurs between the intercalating label and the
unhybridized probe in solution. Because the intercalating label can
intercalate into any double-stranded nucleic acid, this format is
practical only for the detection of single stranded target nucleic acid.
In one embodiment of the nucleic acid detection methods described in U.S.
Pat. No. 5,210,015, described above, a probe is used which is labeled with
interacting fluorescent labels in close proximity. The labels are attached
to the probe separated by one or more nucleotides such that probe
degradation during amplification separates the labels, thereby producing a
detectable change in fluorescence. Such multiply-labeled probes are
difficult and costly to synthesize.
Conventional techniques of molecular biology and nucleic acid chemistry,
which are within the skill of the art, are fully explained fully in the
literature. See, for example, Sambrook et al., 1985, Molecular Cloning--A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid
Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series,
Methods in Enzymology (Academic Press, Inc.), all of which are
incorporated herein by reference. All patents, patent applications, and
publications mentioned herein, both supra and infra, are incorporated
herein by reference.
SUMMARY OF THE INVENTION
The present invention provides methods for controlling the light emission
of a oligonucleotide probe labeled with a light-emitting label in solution
using a DNA binding compound which can interact with the label to modify
the light emission of the label.
The present invention also provides methods for detecting degradation of
oligonucleotides in solution. The oligonucleotides are labeled with a
light-emitting label. Oligonucleotide cleavage is carried out in the
presence of a DNA binding compound that can interact with the label to
modify the light emission of the label. Oligonucleotide degradation is
detected by measuring the resulting change in light emission of the label.
The present invention provides conditions under which significant
in-solution quenching by a DNA binding compound of a light-emitting bound
to a oligonucleotide occurs. This quenching occurs without hybridization
of the labeled oligonucleotide to its complementary sequence. The methods
of the present invention utilize the dependence of this quenching on the
length of the labeled oligonucleotide. The quenching of a light-emitting
label bound to a short oligonucleotide (about 6 nucleotides or less) is
detectably less than the quenching of the light-emitting label bound to a
longer oligonucleotide.
Both the occurrence of in-solution quenching by a DNA binding compound of a
light-emitting label bound to a single-stranded oligonucleotide and the
dependence on the length of the oligonucleotide are surprising in view of
the prior art. Previously-described assays (see Morrison, 1992, supra)
based on the quenching by an intercalating compound of a fluorescent label
bound to a probe rely on the intercalation of the quencher into a
double-stranded hybridization duplex to bring the quencher and label into
close proximity. The prior art teaches that in-solution quenching of label
bound to unhybridized single-stranded probes is insignificant. It is well
known that quenching by fluorescence energy transfer requires that the
interacting labels be in close proximity, and that the two molecules in
solution are not maintained in close enough proximity to cause significant
quenching. Furthermore, the intercalating quenchers described in the prior
art do not bind single-stranded DNA significantly, and, therefore, no
appreciable quenching of a label bound to a single-stranded DNA in
solution was expected. In contrast, the present invention relies on the
quenching of a fluorescent label bound to a single-stranded nucleic acid
by a DNA binding compound that occurs in solution.
The present invention further provides improved methods for detecting a
target nucleic acid in a sample by hybridization to an oligonucleotide
probe. The methods rely on the selective cleavage of probes hybridized to
target nucleic acid. Detection of cleaved probes using the methods of the
present invention indicates the presence of target nucleic acid.
Thus, the present invention provides a method for detecting a target
nucleic acid in a sample, wherein the method comprises:
(a) providing a reaction mixture that comprises the sample, a DNA binding
compound, and an oligonucleotide probe labeled with a light-emitting
label, wherein said probe contains a sequence that is capable of
hybridizing to the target nucleic acid, and wherein the DNA binding
compound is capable of modifying the light emission of the label;
(b) measuring the light emission of the label;
(c) treating said mixture under conditions under which said oligonucleotide
probe hybridizes to said target sequence and is cleaved;
(d) measuring the light emission of the label; and
(e) determining if the target sequence is present by the difference in
light emission between step (b) and step (d).
The selective cleavage of probes hybridized to target nucleic acid can be
achieved by any of a number of known methods. Examples of suitable
reactions that selectively cleave probes hybridized to a target sequence
are described above in Saiki et al., 1985, supra; PCT Patent Publication
No. WO 89/09284; and U.S. Pat. No. 5,210,015.
The methods of the present invention for detecting nucleic acids are
particularly suited for use in conjunction with amplification processes.
Thus, in one embodiment of the invention, the target sequence is amplified
prior to step (c).
In a preferred embodiment, the present invention provides improvements to
the homogeneous PCR amplification and PCR product detection assay
described in U.S. Pat. No. 5,210,015, that use a single nucleic acid
polymerase both for primer extension and for cleavage of hybridized
labeled probes. The improvements provided by the present invention allow
the use of a probe labeled with a single light-emitting label without
requiring post-reaction manipulations to separate cleaved and uncleaved
probes.
Thus, the present invention provides a method for detecting a target
nucleic acid sequence in a sample using a polymerase chain reaction (PCR),
wherein the method comprises:
(a) providing a PCR reaction mixture comprising said sample, a pair of
oligonucleotide primers, a nucleic acid polymerase having 5' to 3'
nuclease activity, a DNA binding compound, and an oligonucleotide probe
capable of hybridizing to a region of the target nucleic acid bounded by
the oligonucleotide primers, and wherein the probe is labeled with a
light-emitting label, and wherein the DNA binding compound is capable of
modifying the light emission of the label;
(b) measuring the light emission of the label;
(c) treating the PCR reaction mixture under conditions for PCR, wherein the
5' to 3' nuclease activity of the nucleic acid polymerase cleaves probes
hybridized to the target sequence;
(d) measuring the light emission of the label;
(e) determining if the target sequence is present by the difference in
light emission between step (b) and step (d).
In another embodiment of the homogeneous PCR amplification/detection assay,
the DNA binding compound provided in the reaction mixture is characterized
as providing a detectable signal when bound to double-stranded DNA, which
signal is greater than the amount of said signal provided by said compound
when it is unbound, and the signal of the DNA binding compound is
monitored in order to measure the total increase in double-stranded DNA
resulting from the amplification process. In this embodiment, the DNA
binding compound functions both as a quencher of unbound probe light
emission and as a signal-generating compound as used in the methods
described in Higuchi et al., 1992, supra. In this embodiment of the
present invention, the change in signal generated by the DNA binding
compound indicates that amplification has taken place, and the change in
light emission of the probe label indicates amplification of the specific
target sequence. Hence, the methods provide separate measures of the
success of the amplification in a homogenous assay without requiring
additional reagents.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 relates to the dependence of in-solution fluorescent quenching on
the length of the oligonucleotide to which the fluorophore is bound.
FIG. 2 relates to the dependence of in-solution fluorescent quenching on
temperature.
FIG. 3 relates to the dependence of in-solution fluorescent quenching on
temperature and the augmentation of quenching observed resulting from the
presence of a hairpin secondary structure within the labeled
single-stranded oligonucleotide.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
To aid in understanding the invention, several terms are defined below.
The terms "nucleic acid" and "oligonucleotide" refer to probes and oligomer
fragments to be detected, and shall be generic to polydeoxyribonucleotides
(containing 2-deoxy-D-ribose), to polyribonucleotides (containing
D-ribose), and to any other type of polynucleotide which is an N glycoside
of a purine or pyrimidine base, or modified purine or pyrimidine base.
There is no intended distinction in length between the terms "nucleic
acid" and "oligonucleotide", and these terms will be used interchangeably.
These terms refer only to the primary structure of the molecule. Thus,
these terms include double- and single-stranded DNA, was well as double-
and single-stranded RNA.
The terms "target region", "target sequence", and "target nucleic acid
sequence" refer to a region of a nucleic acid which is to be detected.
The term "probe" refers to an oligonucleotide, typically labeled, that
forms a duplex structure with a sequence of a target nucleic acid due to
complementary base pairing. The probe will comprise a "hybridizing
region", preferably consisting of 10 to 50 nucleotides, more preferably 20
to 30 nucleotides, corresponding to a region of the target sequence.
"Corresponding" means identical to or complementary to the designated
nucleic acid. In the present invention, probe oligonucleotides are labeled
with, i.e., bound to, a fluorescent label to enable detection.
The term "hybridization" refers the formation of a duplex structure by two
single-stranded nucleic acids due to complementary base pairing.
Hybridization can occur between fully complementary nucleic acid strands
or between nucleic acid strands that contain minor regions of mismatch.
Conditions under which only fully complementary nucleic acid strands will
hybridize are referred to as "stringent hybridization conditions". Two
single-stranded nucleic acids that are complementary except for minor
regions of mismatch are referred to as "substantially complementary".
Stable duplexes of substantially complementary sequences can be achieved
under less stringent hybridization conditions. Those skilled in the art of
nucleic acid technology can determine duplex stability empirically
considering a number of variables including, for example, the length and
base pair concentration of the oligonucleotides, ionic strength, and
incidence of mismatched base pairs.
The terms "sequence-specific oligonucleotide" and "SSO" refer to
oligonucleotide probes wherein the hybridizing region is exactly
complementary to the sequence to be detected. The use of stringent
hybridization conditions under which the probe will hybridize only to that
exactly complementary target sequence allows the detection of the specific
target sequence. Stringent hybridization conditions are well known in the
art (see, e.g., Sambrook et al., 1985, Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
incorporated herein by reference). Stringent conditions are sequence
dependent and will be different in different circumstances. Generally,
stringent conditions are selected to be about 5.degree. C. lower than the
thermal melting point (Tm) for the specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength
and pH) at which 50% of the base pairs have dissociated. Relaxing the
stringency of the hybridizing conditions will allow sequence mismatches to
be tolerated; the degree of mismatch tolerated can be controlled by
suitable adjustment of the hybridization conditions.
The term "subsequence" refers herein to a nucleotide sequence contained
within another sequence.
The term "label", as used herein, refers to any atom or molecule which can
be attached to a nucleic acid, and which can be used either to provide a
detectable signal or to interact with a second label to modify the
detectable signal provided by the second label. Preferred labels are
light-emitting compounds which generate a detectable signal by
fluorescence, chemiluminescence, or bioluminescence.
The term "chromophore" refers to a non-radioactive compound that absorbs
energy in the form of light. Some chromophores can be excited to emit
light either by a chemical reaction, producing chemiluminescence, or by
the absorption of light, producing fluorescence.
The term "fluorophore" refers to a compound which is capable of
fluorescing, i.e. absorbing light at one frequency and emitting light at
another, generally lower, frequency.
The term "bioluminescence" refers to a form of chemiluminescence in which
the light-emitting compound is one that is found in living organisms.
Examples of bioluminescent compounds include bacterial luciferase and
firefly luciferase.
The term "quenching" refers to a decrease in fluorescence of a first
compound caused by a second compound, regardless of the mechanism.
Quenching typically requires that the compounds be in close proximity. As
used herein, either the compound or the fluorescence of the compound is
said to be quenched, and it is understood that both usages refer to the
same phenomenon.
The term "intercalator" refers to an agent or moiety capable of
non-covalent insertion between stacked base pairs in a nucleic acid double
helix.
The term "homogeneous", as used herein applied to multi-step processes,
refers to methods for carrying out the steps of the process, wherein the
need for sample handling and manipulation between steps is minimized or
eliminated. For example, a "homogeneous" amplification/detection assay
refers to a coupled amplification and detection assay wherein the need for
sample handling and manipulation between the amplification and detection is
minimized or eliminated.
The term "reaction mixture" refers to a solution containing reagents
necessary to carry out the reaction. An "amplification reaction mixture",
which refers to a solution containing reagents necessary to carry out an
amplification reaction, typically contains oligonucleotide primers and a
DNA polymerase in a suitable buffer. Reaction mixtures for specific
reactions are well-known in the literature.
The present invention provides methods for controlling the light emission
of an oligonucleotide label with a light-emitting label in solution. The
methods of the invention are applicable to the detection of cleavage of
single-stranded oligonucleotides labeled with a single light-emitting
label. Detection of the cleaved oligonucleotide is carried out in a
solution containing a DNA binding compound that can interact with the
label to decrease the light emission of the label. The change in the
length of the labeled oligonucleotide from cleavage results in a
detectable increase in the light emission of the attached label. Suitable
light-emitting labels and DNA binding compounds that can interact to
modify the light emission of the label are described below.
Mechanisms by which the light emission of a compound can be quenched by a
second compound are described in Morrison, 1992, in Nonisotopic DNA Probe
Techniques (Kricka ed., Academic Press, Inc. San Diego, Calif.), Chapter
13. One well known mechanism is fluorescence energy transfer (FET), also
referred to in the literature as fluorescence resonance energy transfer,
nonradiative energy transfer, long-range energy transfer, dipole-coupled
energy transfer, and Forster energy transfer. The primary requirement for
FET is that the emission spectrum of one of the compounds, the energy
donor, must overlap with the absorption spectrum of the other compound,
the energy acceptor. Styer and Haugland, 1967, Proc. Natl. Acad. Sci.
U.S.A. 98:719, incorporated herein by reference, show that the energy
transfer efficiency of some common emitter-quencher pairs can approach
100% when the separation distances are less than 10 angstroms. The energy
transfer rate decreases proportionally to the sixth power of the distance
between the energy donor and energy acceptor molecules. Consequently,
small increases in the separation distance greatly diminish the energy
transfer rate, resulting in an increased fluorescence of the energy donor
and, if the quencher chromophore is also a fluorophore, a decreased
fluorescence of the energy acceptor.
In the exemplified methods of the present invention, the emission of
fluorescent label bound to the single-stranded oligonucleotide is
detected. A DNA binding compound quenches the label fluorescence to a
degree that depends on the length of the attached oligonucleotide.
Although FET quenching is well known, both the occurrence of in-solution
quenching by a DNA binding compound of a fluorescent label bound to a
single-stranded oligonucleotide and the dependence of the quenching on the
length of the oligonucleotide are unexpected in view of the prior art.
Because of the extremely rapid decrease in interaction of fluorescent
labels with increasing distance, it was believed that labels in solution
do not significantly interact. The general lack of in-solution interaction
is evident in the previously-described assays based on the quenching by an
intercalating compound of a fluorescent label bound to a probe (see
Morrison, 1992, supra). These previously-described assays rely on the
intercalation of the quencher into a double-stranded hybridization duplex
to bring the quencher and label into close proximity and thereby increase
the quenching relative to the background unquenched state, which consists
of the unhybridized labeled single-stranded probes in solution with the
intercalating quencher. The intercalating quenchers described do not
significantly bind single-stranded DNA, i.e., the unhybridized probe. As
expected from the distance dependence of FET, the prior art does not
report significant in-solution quenching of the unhybridized probe. In
contrast to the teaching of the prior art, the present invention provides
conditions under which significant quenching of a fluorescent label bound
to a single-stranded nucleic acid by a DNA binding compound occurs in
solution.
Many fluorophores and DNA-binding chromophores described in the art are
suitable for use in the methods of the present invention. Suitable
fluorophore and DNA-binding chromophore pairs are chosen such that the
emission spectrum of the fluorophore overlaps with the absorption spectrum
of the chromophore. Ideally, the fluorophore should have a high Stokes
shift (a large difference between the wavelength for maximum absorption
and the wavelength for maximum emission) to minimize interference by
scattered excitation light.
Suitable labels which are well known in the art include, but are not
limited to, fluoroscein and derivatives such as FAM, HEX, TET, and JOE;
rhodamine and derivatives such as Texas Red, ROX, and TAMRA; Lucifer
Yellow, and coumarin derivatives such as 7-Me.sub.2 N-coumarin-4-acetate,
7-OH-4-CH.sub.3 -coumarin-3-acetate, and 7-NH.sub.2 -4-CH.sub.3
-coumarin-3-acetate (AMCA). FAM, HEX, TET, JOE, ROX, and TAMRA are
marketed by Perkin Elmer, Applied Biosystems Division (Foster City,
Calif.). Texas Red and many other suitable compounds are marketed by
Molecular Probes (Eugene, Oreg.). Examples of chemiluminescent and
bioluminescent compounds that may be suitable for use as the energy donor
include luminol (aminophthalhydrazide) and derivatives, and Luciferases.
In a preferred embodiment, the DNA binding agent is an intercalating agent.
Suitable well-known intercalating agents include ethidium bromide and
acridine orange.
Non-intercalating DNA binding agents are also suitable. For example,
members of a class of DNA-binding compounds commonly referred to as
"groove binders" are suitable. These compounds recognize and bind the
minor groove of duplex DNA. Malachite Green is an example of this class of
compounds that was demonstrated to function in the present methods.
In one embodiment of the invention, the DNA binding compound also provides
a signal which is detectably altered upon intercalation into
double-stranded DNA. Ethidium bromide, like other DNA binding labels, such
as acridines, proravine, acridine orange, acriflavine, fluorcoumarin,
ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium,
mithramycin, ruthenium polypyridyls, and anthramycin, exhibits altered
fluorescence emissions when bound to double-stranded DNA. Preferably, a
DNA binding compound which does not inhibit an amplification reaction is
used to allow monitoring of the accumulation of amplified sequences.
An oligonucleotide can be prepared by any suitable method, including, for
example, cloning and isolation of appropriate sequences using restriction
enzymes and direct chemical synthesis by a method such as the
phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99;
the phosphodiester method of Brown et al, 1979, Meth. Enzymol. 68:109-151;
the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron
Lett. 22:1859-1862; and the solid support method of U.S. Pat. No.
4,458,066, each incorporated herein by reference. Methods for synthesizing
labeled oligonucleotides are described in Agrawal and Zamecnik, 1990, Nucl.
Acids. Res. 18(18):5419-5423; MacMillan and Vetdine, 1990, J. Org. Chem.
55:5931-5933; Pieles et al., 1989, Nucl. Acids. Res. 17(22):8967-8978;
Roger et al., 1989, Nucl. Acids. Res. 17(19):7643-7651; and Tesler et al.,
1989, J. Am. Chem. Soc. 111:6966-6976, each incorporated herein by
reference. A review of synthesis methods is provided in Goodchild, 1990,
Bioconjugate Chemistry 1(3):165-187, incorporated herein by reference.
The methods of the present invention are particularly suitable for the
detection of amplified nucleic acids, either DNA or RNA. Suitable
amplification methods in addition to the PCR (U.S. Pat. Nos. 4,683,195;
4,683,202; and 4,965,188), include, but are not limited to, the following:
Ligase Chain Reaction (LCR, Wu and Wallace, 1989, Genomics 4:560-569 and
Barany, 1991, Proc. Natl. Acad. Sci. U.S.A. 88:189-193); Polymerase Ligase
Chain Reaction (Barany, 1991, PCR Methods and Applic. 1:5-16); Gap-LCR (PCT
Patent Publication No. WO 90/01069); Repair Chain Reaction (European Patent
Publication No. 439,182 A2), 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
U.S.A. 86:1173-1177; Guatelli et al., 1990, Proc. Natl. Acad. Sci. U.S.A.
87:1874-1878; PCT Patent Publication No. WO 92/0880A), and NASBA (U.S.
Pat. No. 5,130,238). All of the above references are incorporated herein
by reference. This invention is not limited to any particular
amplification system. As other systems are developed, those systems may
benefit by practice of this invention. A recent survey of amplification
systems was published in Abramson and Myers, 1993, Current Opinion in
Biotechnology 4:41-47, incorporated herein by reference.
A preferred embodiment of the invention provides improvements to the
process described in U.S. Pat. No. 5,210,015, and Holland et al., 1991,
Proc. Natl. Acad. Sci. U.S.A. 88:7276-7280, incorporated herein by
reference. The process uses the 5' to 3' exonuclease activity of a
thermostable DNA polymerase to cleave annealed labeled oligonucleotide
probes from hybridization duplexes and release labeled fragments for
detection. Cleavage of the labeled probes of the present invention by the
5' to 3' exonuclease activity of the DNA polymerase frees the labels into
the reaction mixture. The in-solution signal quenching by the DNA binding
compound is significantly greater when the fluorophore is bound to the
full-length uncleaved oligonucleotide probe than when bound to the
shortened cleaved fragment. The resulting increase in observed
fluorescence indicates probe cleavage, which necessarily indicates both
the presence of target sequences and the occurrence of probe/target
hybridization.
The present homogeneous PCR/detection assay is suitable for use in
conjunction with the methods described in Higuchi et al, 1992, supra. In
this embodiment, the fluorescence of the DNA binding compound is also
measured. Thus, the fluorescence of the DNA binding agent enables
detection that amplification has occurred, and the fluorescence of the
cleaved hybridized probe indicates target specific amplification.
The detection methods of the present invention are applicable to a number
of assays. Each assay requires a target sample in a buffer that is
compatible with the assay reagents. If the target is amplified either
before or simultaneously with detection of probe cleavage, the target
nucleic acid must be in a buffer compatible with the enzymes used to
amplify the target. The target nucleic acid can be isolated from a variety
of biological materials including tissues, body fluids, feces, sputum,
saliva, plant cells, bacterial cultures, and the like. Sample preparation
methods suitable for each assay are described in the art.
In general, the nucleic acid in the sample will be a sequence of DNA, most
usually genomic DNA. However, the present invention can also be practiced
with other nucleic acids, such as messenger RNA, ribosomal RNA, viral RNA,
or cloned DNA. Suitable nucleic acid samples include single or
double-stranded DNA or RNA for use in the present invention. Those of
skill in the art will recognize that, depending on which reaction is used
to cleave the labeled oligonucleotide probes, whatever the nature of the
nucleic acid, the nucleic acid can be detected merely by making
appropriate and well recognized modifications to the method being used.
Sample preparation will vary depending on the source of the sample, the
target to be detected, and the reaction used. Suitable sample preparation
protocols are known in the art and described in the literature cited above
(e.g., see Sambrook et al., supra). Simple and rapid methods of preparing
samples for the PCR amplification of target sequences are described in
Higuchi, 1989, in PCR Technology (Erlich ed., Stockton Press, New York),
and in PCR Protocols, Chapters 18-20 (Innis et al., ed., Academic Press,
1990), both incorporated herein by reference. One of skill in the art
would be able to select and empirically optimize a suitable protocol.
Fluorescence of labels in solutions is measured in a spectrofluorometer,
such as a Hitachi/Perkin Elmer Model 650-40 (Perkin Elmer, Norwalk, Conn.)
or a PTI LS-100 Luminescence Spectrophotometer (Photon Technology
International, London, Ontario, Canada). A spectrofluorometer, depending
on the features of the particular machine utilized, offers the opportunity
to set the excitation and emission wavelength, as well as bandwidth. It
will be obvious to one of ordinary skill in the art how to determine the
wavelength and bandwidth settings for detecting the fluorescence from a
particular fluorescent label. General guidance is found in, for example,
The Merck Index, (eds. Budavari et al., 1989, Merck Co. Inc. Rahway, N.J.)
and the Molecular Probes, Inc. (Eugene, Oreg.) Catalog, 1990, by Haugland,
both incorporated herein by reference. Although each label has a discrete
fluorescence spectrum, a broad range of detection wavelengths are suitable
for practicing the invention.
Fluorescent measurements are carried out before and after the reaction that
results in probe cleavage, and the change in fluorescence is calculated
relative to the pre-reaction value. Equivalently, a portion of the
reaction mixture is not subject to the reaction conditions. In this
manner, the pre-reaction fluorescence can be measured, together with the
post-reaction fluorescence, after completion of the reaction. The use of
reaction vessels which are also suitable for use in measuring fluorescence
allows direct measurements of both pre- and post-reaction fluorescence
without opening the reaction vessel or other post-reaction manipulations.
In preferred methods in which the nucleic acid detection method is combined
with PCR amplification, as described above, the amplification reaction is
carded out as an automated process. Thermal cyclers are currently
available from Perkin Elmer (Norwalk, Conn.) that uses a heat block
capable of holding up to 48 or 96 reaction tubes. Consequently, up to 96
amplification reactions can be carded out simultaneously.
The present invention enables the automatic detection of PCR product in all
samples, without the need to handle the samples, open the tubes, or
interrupt the cycling reaction. Suitable optical systems, for example, are
described in Higuchi et al., 1992, supra, Higuchi et al., 1993, supra, and
copending and commonly assigned U.S. Ser. No. 08/113,168 and European
Patent Publication No. 512,334 all incorporated herein by reference. In
one such optical system, multiple fiber optic leads are used to transmit
the excitation light from the source to the reaction tube and measures the
emission light from each tube. Only a single fluorometer is needed to read
fluorescence from the reaction tubes, as each fiber optic can be read
rapidly one at a time. An alternative optical system uses a video camera
to measure the fluorescence of multiple reaction vessels simultaneously.
The use of transparent reaction vessel tops allows the measurement of
fluorescence without opening the vessel. It will be obvious to one of
skill in the art that the alternative detection apparatuses described in
the '168 application also are adaptable to the present methods.
An alternative suitable detection scheme is described that uses a 96-well
microliter format. This type of format is frequently desirable in clinical
laboratories for large scale sample screening, for example, for genetic
analysis such as screening for sickle-cell anemia or the AIDS virus in
blood bank screening procedures. The present invention is suitable for
this type of analysis and eliminates the need for the numerous washing and
extraction procedures that are required with known "in-well" assay
procedures such as ELISA type formats or other optical density-based
methods. (See Kolber et al., 1988, J. Immun. Meth. 108:255-264, Huschtscha
et al., 1989, In Vitro Cell and Dev. Biol. 25(1):105-108, and Voller et
al., 1979, The Enzyme Linked Immunosorbent Assay, Dynatech Labs,
Alexandria, Va.).
The present detection methods also allow direct fluorescence measurement
using an apparatus similar to ELISA plate reader, but designed to excite
and measure fluorescence. For example, the CytoFluor.TM. 2300 machine
manufactured by Millipore (Bedford, Mass.) is suitable in such a method.
Alternatively, an apparatus providing a continuous determination of
fluorescence is useful for monitoring the increase in PCR product during
the amplification reaction.
It will be obvious to one skilled in the art that the methods of the
present invention are not limited to a particular detection method,
thermal cycler or signal measuring machines, or number of reaction
vessels.
The methods of the present invention can be used to simultaneously detect
multiple target sequences. Probes specific to each target are present in
the reaction mixture. For each target nucleic acid present in the sample,
the corresponding probe will hybridize and be cleaved. In order to detect
the cleaved probes separately, each species of probe is labeled with a
label that fluoresces at a distinct wavelength. Each species of probe is
then detected separately by suitable selections of the measured
wavelength.
Thus, the methods of the present invention are useful for detecting the
amplification pro | | |
