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Description  |
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FIELD OF THE INVENTION
The present invention relates to methods for detecting amplification of
nucleic acid target sequences and in particular to detection of
amplification by fluorescence polarization.
BACKGROUND OF THE INVENTION
Strand Displacement Amplification (SDA) utilizes the ability of a
restriction enzyme to nick a hemimodified recognition site and the ability
of a polymerase to displace a downstream DNA strand to amplify a target
nucleic acid (U.S. Pat. No. 5,270,184, hereby incorporated by reference;
Walker, et al. 1992. Proc. Natl. Acad. Sci. USA 89, 392-396; Walker, et
al. 1992. Nucl. Acids Res. 20, 1691-1696). The target for SDA may be
present on fragments of nucleic acid generated by treatment with a
restriction endonuclease, or targets appropriate for SDA may be generated
by extension and displacement of primers. This second type of target
generation and the subsequent steps of the SDA reaction are illustrated in
FIG. 1. The target generation process (left side of FIG. 1) produces
copies of the target sequence flanked by the nickable restriction sites
required for SDA. These modified target sequences are exponentially
amplified by repeated nicking, strand displacement, and repriming of
displaced strands (right side of FIG. 1). Despite the apparent complexity
of FIG. 1, SDA operates under a very simple protocol: double-stranded
target DNA is heat denatured in the presence of all reagents except the
restriction enzyme and polymerase. Exponential amplification then proceeds
at a constant, reduced temperature upon addition of the enzymes, without
any further manipulation of the reaction. SDA is capable of 10.sup.8 -fold
amplification of target sequences in 2 hours at a constant reaction
temperature, usually about 35.degree.-42.degree. C.
Fluoresence Polarization (FP) is a measure of the time-average rotational
motion of fluorescent molecules. It has been known since the 1920's and
has been used in both research and clinical applications for sensitive
determination of molecular volume and microviscosity. The FP technique
relies upon changes in the rotational properties of molecules in solution.
That is, molecules in solution tend to "tumble" about their various axes.
Larger molecules (e.g., those with greater volume or molecular weight)
tumble more slowly and along fewer axes than smaller molecules. They
therefore move less between excitation and emission, causing the emitted
light to exhibit a relatively higher degree of polarization. Conversely,
fluorescence emissions from smaller fluorescent molecules, which exhibit
more tumbling between excitation and emission, are more multiplanar (less
polarized). When a smaller fluorescent molecule takes a larger or more
rigid conformation its tumbling decreases and the emitted fluorescence
becomes relatively more polarized. This change in the degree of
polarization of emitted fluorescence can be measured and used as an
indicator of increased size and/or rigidity of the fluorescent molecule.
In fluorescence polarization techniques, the fluorescent molecule is first
excited by polarized light. The polarization of the emission is measured
by measuring the relative intensities of emission (i) parallel to the
plane of polarized excitation light and (ii) perpendicular to the plane of
polarized excitation light. A change in the rate of tumbling due to a
change in size and/or rigidity is accompanied by a change in the
relationship between the plane of excitation light and the plane of
emitted fluorescence, i.e., a change in fluorescence polarization. Such
changes can occur, for example, when a single stranded oligonucleotide
probe becomes double stranded or when a nucleic acid binding protein binds
to an oligonucleotide. Fluorescence anisotropy is closely related to FP.
This technique also measures changes in the tumbling rates of molecules
but is calculated using a different equation. It is to be understood that
polarization and anisotropy are interchangeable techniques for use in the
present invention. The term fluorescence polarization is generally used
herein but should be understood to include fluorescence anisotropy
methods. In steady state measurements of polarization and anisotropy,
these values are calculated according to the following equations:
##EQU1##
where Ipa is the intensity of fluorescence emission parallel to the plane
of polarized excitation light and Ipe is the intensity of fluorescence
emission perpendicular to the plane of polarized excitation light.
As FP is homogenous, this technique is ideal for studying molecular
interactions in solution without interference by physical manipulation.
Fluorescence polarization is therefore a convenient method for monitoring
conversion of single-stranded fluorescently labelled DNA to
double-stranded form by hybridization (Murakami, et al. 1991. Nucl. Acids
Res. 19, 4097-4102). The ability of FP to differentiate between single and
double-stranded nucleic acid conformations without physical separation of
the two forms has made this technology an attractive alternative for
monitoring probe hybridization in diagnostic formats. European Patent
Publication No. 0 382 433 describes fluorescence polarization detection of
amplified target sequences by hybridization of a fluorescent probe to the
amplicons or by incorporation of a fluorescent label into the
amplification products by target-specific extension of a
fluorescently-labeled amplification primer. PCT Patent Publication No. WO
92/18650 describes similar methods for detecting amplified RNA or DNA
target sequences by the increase in fluorescence polarization associated
with hybridization of a fluorescent probe.
Fluorescence polarization may be monitored as either transient state FP or
steady state FP. In transient state FP, the excitation light source is
flashed on the sample and polarization of the emitted light is monitored
by turning on the photomultiplier tube after the excitation light source
is turned off. This reduces interference from light scatter, as
fluorescence lasts longer than light scatter, but some fluorescence
intensity is lost. In steady state FP, excitation light and emission
monitoring are continuous (i.e., the excitation source and photomultiplier
tube are on continuously). This results in measurement of an average
tumbling time over the monitoring period and includes the effects of
scattered light.
The present invention provides FP or fluorescence anisotropy detection
methods for use with nucleic acid amplification methods such as SDA.
Previously, SDA-amplified target sequences were detected following
amplification using .sup.32 P-probes (Walker, et al. 1992 Nucl. Acids
Res., supra) or by a sandwich hybridization assay with chemiluminescent
signal generation (Spargo, et al. 1993. Molec. Cell. Probes 7, 395-404).
Both of these detection formats require separation of free and bound
detector probe before the signal can be measured. However, the ability to
differentiate free and bound probe using FP without physical separation
enables performance of SDA and detection of amplification in a
homogeneous, closed system. Furthermore, it has been discovered that SDA
and FP detection can be combined in a single step (i.e., real-time
amplification and detection), in part as a result of the isothermal nature
of SDA. A closed, homogeneous assay reduces operating steps and procedural
complexity, as well as providing improved control of the dispersal of
amplification products in the laboratory, thereby reducing the potential
for false positives due to accidental contamination of samples with target
DNA.
Certain of the terms and phrases used herein are defined as follows:
An amplification primer is a primer for amplification of a target sequence
by primer extension or ligation of adjacent primers hybridized to the
target sequence. For amplification by SDA, the oligonucleotide primers are
preferably selected such that the GC content is low, preferably less than
70% of the total nucleotide composition of the probe. Similarly, for SDA
the target sequence preferably has a low GC content to minimize secondary
structure. The 3' end of an SDA amplification primer (the target binding
sequence) hybridizes at the 3' end of the target sequence. The target
binding sequence confers target specificity on the amplification primer.
The SDA amplification primer further comprises a recognition site for a
restriction endonuclease near its 5' end. The recognition site is for a
restriction endonuclease which will nick one strand of a DNA duplex when
the recognition site is hemimodified, as described by Walker, et al.
(1992. Proc. Natl. Acad. Sci. and Nucl. Acids Res., supra). The SDA
amplification primer generally also comprises additional sequences 5' to
the restriction endonuclease recognition site to allow the appropriate
restriction endonuclease to bind to its recognition site and to serve as a
primer for the polymerase after nicking, as is known in the art. For the
majority of the SDA reaction, the amplification primer is responsible for
exponential amplification of the target sequence. The SDA amplification
primer may also be referred to as the "S" primer, e.g., S.sub.1 and
S.sub.2 when a pair of amplification primers is used for amplification of
a double stranded sequence. For other amplification methods which do not
require attachment of specialized sequences to the ends of the target, the
amplification primer generally consists of only the target binding
sequence.
A bumper or external primer is a primer used in SDA which anneals to a
target sequence upstream of the amplification primer such that extension
of the bumper primer displaces the downstream primer and its extension
product. Bumper primers may also be referred to as "B" primers, e.g.,
B.sub.1 and B.sub.2 when a pair of bumper primers is used to displace the
extension products of a pair of amplification primers. Extension of bumper
primers is one method for displacing the extension products of
amplification primers, but heating is also suitable.
The terms target or target sequence refer to nucleic acid sequences
amplifiable by amplification primers. These include the original nucleic
acid sequence to be amplified, the complementary second strand of the
original nucleic acid sequence to be amplified, and either strand of a
copy of the original sequence which is produced by the amplification
reaction. These copies also serve as amplifiable target sequences by
virtue of the fact that they also contain copies of the original target
sequences to which the amplification primers hybridize.
Copies of the target sequence which are generated during the amplification
reaction are referred to as amplification products, amplimers or
amplicons.
The term extension product refers to the single-stranded copy of a target
sequence produced by hybridization of a primer and extension of the primer
by polymerase using the target sequence as a template.
SUMMARY OF THE INVENTION
The present invention provides methods for detection of nucleic acid
amplification using FP and a detector probe comprising a fluorescent dye.
Amplification is detected as an increase in FP associated with
target-dependent formation of double-stranded fluorescent products from
the single-stranded detector probe. In one embodiment, the invention also
relates to a novel technique that can be used to enhance the magnitude of
the increase in FP which results from formation of double-stranded DNA.
Enhancing the magnitude of this change is advantageous because there is
otherwise only about a 20 mP difference between single-stranded FP values
and double-stranded FP values for a fluorescein labelled oligonucleotide.
It has been found that double-stranded DNA binding proteins can be used to
enhance the increase in FP associated with the single- to double-stranded
conversion. In one example, the double-stranded DNA binding protein is the
restriction endonuclease EcoRI, which in its natural form requires
magnesium to cut the DNA to which it binds. In the absence of magnesium,
EcoRI binds but does not cut, and under these conditions can be used to
enhance the increase in FP associated with single- to double-stranded
conversion. In a second example, the DNA binding protein is a mutant form
of EcoRI, designated EcoRI (Gln 111) and described by Wright, et. al.
(1989. J. Biol. Chem. 264, 11816-11821 ). EcoRI (Gln 111) binds more
tightly than natural EcoRI to double-stranded DNA but does not cut the
nucleic acid even in the presence of magnesium. Binding of this protein
also enhances the increase in FP which accompanies conversion to
double-stranded form.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the target generation scheme for SDA (left side) and the
reaction steps for exponential amplification of a target sequence by SDA
(right side).
FIG. 2A illustrates the various forms of the fluorescent detector probe
generated in a target-dependent manner during SDA when the detector probe
is entirely homologous to the target sequence. FIG. 2B illustrates the
various forms of the fluorescent detector probe generated in a
target-dependent manner during SDA when the detector probe is partially
homologous to the target sequence, e.g., when the detector probe contains
a recognition site for a double-stranded DNA binding protein (i.e., the
nucleotide sequence to which the DNA binding protein binds).
FIG. 3 is a graph of the increase in FP associated with SDA of an M.
tuberculosis target sequence.
FIG. 4 is a graph of fluorescence polarization values obtained after SDA of
various initial amounts of an M. tuberculosis target sequence.
FIG. 5 is a graph showing enhancement of the increase in FP by addition of
EcoRI or EcoRI (Gln 111) when a single-stranded oligonucleotide is
converted to double-stranded form by hybridization to its complement.
FIG. 6 is a graph showing enhancement of the increase in FP by addition of
EcoRI (Gln 111) when the detector probe is converted to double stranded
form during amplification of a target sequence.
FIG. 7 is a graph illustrating FP values obtained upon amplification of
various initial amounts of target sequence with post-amplification
addition of EcoRI (Gln 111), using a detector probe which does not contain
an EcoRI recognition site.
FIG. 8 is a graph illustrating FP values obtained upon amplification of
various initial amounts of target sequence with post-amplification
addition of EcoRI (Gln 111), using a detector probe which contains an
EcoRI recognition site.
FIG. 9 is a graph showing the increase in FP associated with conversion of
the detector probe to double-stranded form during amplification of an M.
tuberculosis target sequence, with EcoRI (Gln 111) present during
amplification.
DETAILED DESCRIPTION OF THE INVENTION
The Strand Displacement Amplification target generation and amplification
reaction schemes are illustrated in FIG. 1. The target DNA is heat
denatured in the presence of an excess of four primers (B.sub.1, B.sub.2,
S.sub.1 and S.sub.2). S.sub.1 and S.sub.2 are amplification primers
containing target binding sequences at their 3' ends and a recognition
site for a restriction endonuclease (e.g., HincII - .sup.5' GTTGAC) at a
position 5' to the target binding sequences. The restriction endonuclease
recognition sites are designated by the raised boxes. For convenience, the
following description will use HincII and exo.sup.-- Klenow as examples,
however, any of the restriction enzymes and exonuclease deficient
polymerases known for use in SDA may be substituted.
S.sub.1 and S.sub.2 hybridize to the opposite, complementary strands of the
double stranded target sequence, flanking the region to be amplified.
B.sub.1 and B.sub.2 are external or bumper primers which consist only of
target binding sequences and hybridize at positions 5' to S.sub.1 and
S.sub.2. After annealing of the primers to the target at about 40.degree.
C., HincII is added along with an exonuclease deficient form of the Klenow
fragment of E. coli DNA polymerase I (exo.sup.-- Klenow). At this point
the remaining target generation steps on the left side of FIG. 1 proceed
as a single cascade. Exo.sup.-- Klenow, which is present in large molar
excess over the number of target sequences, simultaneously extends all
four primers using dGTP, dCTP, dUTP (or TTP) and dATP.alpha.S
(deoxyadenosine 5'-[.alpha.-thio]triphosphate). S.sub.1 and S.sub.2 are
extended and their extension products are displaced by extension of
B.sub.1 and B.sub.2. The displaced extension products of S.sub.1 and
S.sub.2 (S.sub.1 -ext and S.sub.2 -ext) serve as targets for binding of
the opposite amplification primers. Further rounds of extension and
displacement produce two target fragments with a hemiphosphorothioate
HincII site at each end and two longer target fragments with a
hemiphosphorothioate HincII site at only one end (bottom left side of FIG.
1). Incorporation of dATP.alpha.S in place of dATP in one of the two
strands by the polymerase causes the restriction endonouclease to nick one
strand rather than cleave both strands of the duplex. "Nicking" refers to
cleavage of one strand of double stranded DNA as opposed to double
stranded cleavage. HincII nicks the unmodified primer strands of the
hemiphosphorothioate recognition sites, leaving intact the modified
complementary strands. Exo.sup.-- Klenow then extends from the 3'-end of
the nick and displaces the downstream newly synthesized strand. New
S.sub.1 and S.sub.2 primers bind to the displaced strands and are
extended. This is followed by additional nicking and strand displacement
steps until the four duplexes at the bottom left side of FIG. 1 converge
into the "steady-state" amplification cycle illustrated on the fight side
of FIG. 1. During each SDA cycle, the 3' end of S.sub.1 hybridizes to the
3'-end of the displaced target strand T.sub.2, forming a duplex with
5'-overhangs. Likewise, S.sub.2 binds to displaced T.sub.1. Exo.sup.--
Klenow extends the recessed 3'-ends of the duplexes producing
hemiphosphorothioate recognition sites which are nicked by HincII. These
nicking and extension/displacement steps cycle continuously (short curved
arrows on the right side of FIG. 1) because extension at a nick
regenerates a nickable HincII recognition site. The strand displaced from
the S.sub.1 -T.sub.2 duplex is identical to T.sub.1. Likewise, the
displaced strand from the S.sub.2 -T.sub.1 duplex is identical to T.sub.2.
Consequently, target amplification is exponential because each displaced
T.sub.2 binds a new S.sub.1 primer and each displaced T.sub.1 binds a new
S.sub.2 (long curved arrows on the fight side of FIG. 1). Sense and
antisense strands are differentiated by thin and thick lines. Intact and
nicked HincII recognition sites are represented by and . The partial
HincII recognition sites (5'-GAC and its complement 5'-GTC) are present at
the 5'-and 3'-ends of displaced strands and are represented by and ,
respectively.
In the inventive amplification detection system, a single-stranded
oligodeoxynucleotide detector probe comprising a fluorescent label is
converted to double-stranded form in a target-dependent manner during SDA
(FIG. 2A and FIG. 2B). FIG. 2A and FIG. 2B illustrate the same process,
except that in FIG. 2A the entire nucleotide sequence of the detector
probe is complementary to the target sequence and in FIG. 2B a portion of
the nucleotide sequence of the detector probe is not complementary to the
target sequence. As discussed below, the non-complementary portion of the
detector probe may comprise a recognition site for a DNA binding protein
or may be a structural feature which results in an increase in FP. In FIG.
2B, the non-complementary sequence or structural feature is depicted as .
In both FIG. 2A and FIG. 2B, the fluorescent label is depicted as .
The following references to structures relate to both FIG. 2A and FIG. 2B.
Hybridization, extension and displacement of a fluorescent detector probe
for FP detection of amplification occurs concurrently with the SDA cycle
shown on the right side of FIG. 1. The fluorescent detector probe (D)
hybridizes to one of the two complementary strands of a double stranded
target sequence or to a displaced single-stranded copy of the target
strand downstream from one of the SDA primers (e.g., S.sub.1). This
provides one source of double-stranded detector probe (structure I). The
amplification primer/target complex is identical to the complex shown at
the top left of the SDA cycle illustrated on the right side of FIG. 1, but
in FIG. 1 it is depicted without the detector probe. The primer and
detector probe in structure I are then simultaneously extended by
exo.sup.-- Klenow polymerase (structure II), resulting in displacement of
the detector probe extension product (structure III by extension of the
upstream amplification primer S.sub.1 in a manner analogous to the strand
displacement reaction intrinsic to SDA. The displaced, single-stranded
probe extension product (structure III) hybridizes to the other SDA primer
(S.sub.2) forming a complex (structure IV) which becomes fully
double-stranded by exo.sup.-- Klenow polymerase extension, again
providing a source of double-stranded fluorescent detector probe
(structure V). Structure V is a template for linear SDA, due to the
nickable HincII site on S.sub.2. Nicking, polymerase extension and strand
displacement using structure V as a template produces single-strands to
which additional fluorescent detector probes hybridize (structure VI) and
are extended to generate structure VII. Structure I, structure II,
structure V and structure VII are all forms of double-stranded fluorescent
detector probe which are indicative of target-specific SDA. Each of these
structures contributes in a target-dependent manner to the increase in FP
associated with double-stranded conversion of the single-stranded
fluorescent detector probe. FIG. 2B illustrates how this same process also
results in conversion of the DNA binding protein recognition site or
structural feature to double-stranded form (structures V, VI and VII).
These structures may then bind the double-stranded DNA binding protein to
enhance the increase in FP associated with double-strandedness of the
detector probe.
Any protein which binds to a specific double-stranded DNA sequence may be
used to enhance the increase in FP associated with conversion of the
detector probe to double-stranded form. This is accomplished by including
the nucleotide sequence of the appropriate recognition site for the
double-stranded DNA binding protein in the nucleotide sequence of the
detector probe at a position near the site of attachment of the
fluorescent label. Recognition sites for restriction endonucleases (e.g.,
EcoRI and mutant forms of EcoRI) are useful for this purpose. In addition,
the recognition site for the Trp repressor protein (i.e., the Trp
repressor operator sequence) or the recognition sequence for the DNA
binding domain of the estrogen receptor protein (i.e., the estrogen
responsive element sequence) may be included in the sequence of the
detector probe to enhance the increase in FP upon binding to Trp repressor
or estrogen receptor. Changes in FP may also be enhanced by designing a
detector probe comprising a nucleotide sequence which results in a
structural feature which further slows the tumbling of double-stranded
DNA, for example a recognition site for a third oligonucleotide which in
double-stranded form is capable of hybridizing to the third
oligonucleotide to form a triple helical structure.
It is not a requirement of the invention that the detector probe carry the
fluorescent label, as in the foregoing illustrative example. It will be
apparent to one skilled in the art that, in certain embodiments of the
invention, the detector probe may be unlabeled. That is, if a
double-stranded DNA binding protein is included to increase the magnitude
of the difference in FP between single- and double-stranded forms, the
fluorescent label may be linked to the double-stranded DNA binding protein
rather than to the detector probe. Similarly, if formation of a triple
helix is used to increase the magnitude of the change in FP, the
fluorescent label may be linked to the third oligonucleotide which
hybridizes to the double-stranded structure containing the unlabeled
detector probe. In this embodiment, the presence of the unlabeled detector
probe in the amplification reaction would result in unlabeled versions of
structures V, VI, and VII (of FIG. 2B), produced in a target-dependent
manner. As these structures contain double-stranded recognition sites for
double-stranded DNA binding proteins or triple helix forming
oligonucleotides, specific binding of the fluorescently labeled protein or
oligonucleotide to double-stranded detector probe structures will increase
the magnitude of the change in FP and provide the fluorescent label needed
for detection by FP. As smaller double-stranded DNA binding agents have a
greater effect on the change in FP, triple helix formation is likely to be
a more sensitive detection system than protein binding in many cases.
FIG. 2A, FIG. 2B and the foregoing description of the invention use SDA as
an example, however, the invention may also be applied to any
amplification method in which a strand-displacing polymerase is used or
can be substituted for a polymerase which has 5'-3' exonuclease activity.
The ability of the polymerase to displace a downstream strand of DNA
without digesting it is the essential feature of the amplification method
which provides target-specific generation of double-stranded fluorescent
detector probe. The other features of such amplification methods, such as
the nature of the target sequence and the structural features of the
amplification primers, are not critical to the present invention. The
inventive methods may therefore be used in isothermal amplification
reactions other than SDA, e.g., Self-Sustained Sequence Replication (3SR),
as the detection method is independent of whether the target sequence is
RNA or DNA. In 3 SR, target-dependent generation of double-stranded
detector probe occurs generally as illustrated in FIG. 2A and 2B for SDA.
The T7 RNA polymerase lacks 5'-3' exonuclease activity. The degradative
activity of reverse transcriptase is an RNAse H activity which is active
only on RNA hybridized to DNA. In the 3 SR amplification scheme of
Guatelli, et al. (1990. 87, 1874-1878. See FIG. 1), the detector probe of
the invention hybridizes to the RNA target sequence and is displaced by
extension of the 3' amplification primer "A". The detector probe also
hybridizes to the DNA target sequences generated in the 3SR amplification
process. In either case, the extended detector probe is displaced by the
polymerase when the upstream 3' ("A") or 5' ("B") amplification primer is
extended. The opposite amplification primer then binds to the detector
probe extension product and is extended, converting the
fluorescently-labeled detector probe to double-stranded form. Detector
probe extension products which include the T7 RNA polymerase promoter
sequence are amplifiable by 3SR and provide a source of additional copies
of the detector probe.
The inventive methods may also be applied to monitoring of the Polymerase
Chain Reaction (PCR), although fluorescence polarization measurements must
be taken during the low temperature periods of the amplification cycle for
"real time" monitoring of amplification. Again, the mechanism for
target-dependent generation of double-stranded detector probe is generally
as illustrated in FIG. 2A and 2B. Using a 5'-3' exonuclease deficient
polymerase (e.g., exo.sup.-- Klenow, Bca polymerase or Bst polymerase),
extension of a PCR amplification primer hybridized to the target sequence
displaces the extended downstream detector probe. The opposite PCR
amplification primer hybridizes to the extension product of the detector
probe and is extended, resulting in conversion of the single-stranded
detector probe to double-stranded form. The double-stranded detector probe
is amplifiable by hybridization and extension of one amplification primers
and one detector probe in subsequent cycles, providing an additional
source of double-stranded detector probe. The increase in fluorescence
polarization or fluorescence anisotropy may then be detected after
conclusion of the PCR under conditions in which amplification products
remain double-stranded or during PCR at the low temperature points of the
cycling protocol.
Single- to double-stranded conversion of the detector probe is monitored by
measuring fluorescence polarization or fluorescence anisotropy. The change
in exclusion volume which accompanies the change in probe conformation
(primarily strandedness) results in a detectable increase in correlation
time (tumbling time) for the fluorescent label. The accompanying changes
in FP values may be monitored on a transient-state fluorometer (e.g., from
Diatron) or a steady state fluorometer (e.g., Jolley Instruments) designed
for detection of the selected fluorescent label. While the polarization
measurements may be taken post-amplification, as has been done previously,
the present methods also for the first time allow "real-time" monitoring
of fluorescence polarization during target sequence amplification.
Real-time monitoring of fluorescence provides significant advantages over
the prior art. That is, it provides an essentially immediate result, it is
quantitative, it improves sensitivity (analysis of a change in slope is
more accurate than a single endpoint), and the sample acts as its own
internal standard. This last advantage is particularly important for
analysis of clinical specimens, as sample viscosity may significantly
affect endpoint readings.
As hybridized and unhybridized (i.e., double and single stranded) probe are
not separated prior to measurement, FP-based detection of target
amplification requires appreciable conversion of the single-stranded
fluorescent detector probe to double-stranded form. Therefore, low
detector probe concentrations facilitate high sensitivity (i.e., detection
of amplification of initially low concentrations of target sequence)
because they result in a higher percentage of converted detector probe for
a given level of target amplification. However, low detector probe
concentrations present a kinetic challenge for SDA. The fluorescent
detector probe must hybridize to the displaced target strand before
hybridization and extension of the upstream amplification primer
(S.sub.1). The kinetics of S.sub.1 extension are controlled by S.sub.1
hybridization and polymerase binding rates. It is therefore useful in some
cases to modify conventional SDA reaction conditions (Walker et al. 1992.
Proc. Natl. Acad. Sci. and Nucl. Acids Res., supra; Walker. 1993. PCR
Methods and Applications 3, 1-6) to decrease the rate of S.sub.1 extension
and facilitate prior hybridization of the fluorescent detector probe when
low target concentrations are present. This may be achieved by adjusting
the concentration of S.sub.1 and polymerase to 10 nM-1 .mu.M and 0.1-10
unit, respectively. Lower S.sub.1 and polymerase concentrations result in
slower SDA rates, so it may also be necessary to extend the typical SDA
reaction time from 2 to 3 hours. If sensitivity is not essential, or for
quantitation of relatively high initial target concentrations, the
fluorescent detector probe concentration may be as high as the
concentration of amplification primer.
Use of an unlabeled detector probe, as described above, may be employed in
the amplification reaction to allow rapid extension of the S.sub.1 primer
even when it is desired to keep the concentration of fluorescently-labeled
probe low, e.g., for increased sensitivity. In this embodiment, the
detector probe is not fluorescently labeled but includes near its 5' end a
sequence which, in double-stranded form, is capable of hybridizing to a
fluorescently labeled third oligonucleotide to form a triple helix.
Conversion of the unlabeled detector probe to double-stranded form
(following the reaction scheme of FIG. 2B--structures V, VI and VII)
allows hybridization to the fluorescently labeled third oligonucleotide
for detection of the associated increase in FP. The unlabeled detector
probe can be present in the amplification reaction at concentrations
comparable to S.sub.1 so that reaction kinetics are improved and its rate
of conversion to structure V, VI or VII is more rapid. Once these
target-specific double-stranded structures are formed during
amplification, the fluorescently labeled third oligonucleotide probe
hybridizes, enhancing the increase in FP and providing the fluorescent
label for specific detection. The third oligonucleotide with its
fluorescent label may be present at high or low concentration depending on
the sensitivity required, without interfering with the efficiency of
production of double-stranded detector probe products. In addition, in
this embodiment the fluorescent label may be linked to the 3' end of the
third oligonucleotide if desired, a configuration which is not desirable
when the label is linked to the detector probe.
The processes illustrated in FIG. 2A and FIG. 2B occur concurrently with
the SDA cycle depicted in FIG. 1, without interfering with the
amplification reaction. The presence of the fluorescent detector probe
does not increase SDA background reactions because any mispriming by the
detector probe and an amplification primer generates an extension product
which cannot be exponentially amplified due to the presence of only one
nickable HincII site (i.e., the fluorescent detector probe does not
contain a nickable HincII site). SDA requires two primers, each containing
a nickable HincII site, to support exponential amplification. This is in
contrast to the Polymerase Chain Reaction, in which any oligonucleotide
which hybridizes to any sequence and can be extended serves as an
amplification primer, allowing ntisprimed products to be exponentially
amplified. Background amplification in SDA is further reduced when the
fluorescent detector probe is used at low concentrations (e.g., 50 pM-10
nM).
Any fluorescent molecule known in the art for labeling nucleic acids may be
used in the methods of the invention (e.g., fluorescein and fluorescein
derivatives such as eosin; rhodamines such as Texas Red and
tetramethylrhodamine; cyanine dyes such as thiazole orange, oxazole yellow
and related dyes described in U.S. Pat. Nos. 4,957,870 and 4,888,867;
pyrene; porphyrin dyes such as La Jolla Blue.TM.). The fluorescent label
should be selected such that the fluorescent lifetime of the label is
comparable in magnitude to the correlation time being measured, taking
into account that temperature, viscosity, and the size of the
oligonucleotide to which the fluorescent dye is conjugated all affect
tumbling time. For example, fluorescein (lifetime approximately 4
nanosec.) and LaJolla Blue.TM. (lifetime approximately 2 nanosec.) are
both useful for correlation times of about 0.1-100 nanosec. If a nucleic
acid binding protein is used in conjunction with the fluorescent label,
the correlation time measured is generally increased. For example,
correlation time for a free fluorescein label is about 0.2 nanosec. The
correlation time increases to about 0.4 nanosec. when the fluorescein
label is conjugated to a single stranded oligonucleotide and increases
further to about 2 nanosec. when conjugated to a double-stranded
oligonucleotide. When FP is enhanced by binding the fluorescein-labeled
double-stranded oligonucleotide with EcoRI, the correlation time again
increases to about 20 nanosec. La Jolla Blue.TM. (Devlin, et al. 1993.
Clin. Chem. 39, 1939-1943) is particularly suitable for labeling the
fluorescent detector probe when biological samples are to be amplified, as
this dye absorbs and emits light in the near-infra red spectrum, a region
of relatively low background fluorescence with clinical specimens (peak
maxima at about 685 nm and 705 nm, respectively).
Background fluorescence may be further minimized by the use of
transient-state fluorescence spectroscopy rather than steady-state
fluorescence spectroscopy, thereby reducing the contribution from light
scattering. Lower background fluorescence allows the use of lower
concentrations of fluorescently labelled detector probe, which improves
detection sensitivity by ensuring that a greater percentage of
single-stranded detector probe is converted to double-stranded form for a
given concentration of amplified product. However, low detector probe
concentrations result in saturation of the detector probe over a broad
range of amplified product levels. End-point measurements of FP taken
under conditions of probe saturation after completion of SDA may not be
strictly quantitative with regard to the initial target levels. Monitoring
FP in "real-time," during SDA rather than after completion of the
amplification reaction, overcomes the problem of detector probe saturation
because samples containing higher target levels exhibit more rapid
increases in FP values than those containing less target. Of course, the
correlation between the rate of FP increase and initial target levels is
valid only when comparing samples in which the rate of amplification is
essentially identical. Again, for clinical specimens, each of which
contains varying levels of SDA inhibitors, the assay may not be strictly
quantitative. For example, it may be difficult to differentiate a sample
which contains a high amount of initial target and undergoes inefficient
SDA from a sample which contains a low amount of initial target but
undergoes SDA at a high rate. Nevertheless, realtime monitoring of FP
values during SDA provides at least a semi-quantitative estimate of
initial target levels. Quantitation may be improved by including an
additional target sequence at a known initial concentration as a positive
control (Walker, et al. 1994. Nucl. Acids Res. 22, 2670-2677). The
positive control target not only provides an indication of general SDA
performance for a sample (i.e., a control for false negatives), it also
provides a standard for quantitating the initial amount of target in the
sample.
The fluorescent label is covalently linked or conjugated to the detector
probe so as not to interfere with either emission of fluorescence from the
label or hybridization of the probe to the target sequence. As FP changes
occur when the label is near or involved in a conformational change, the
linkage should be in proximity to the site where the conformational change
is expected. This may be either the 5' end of the detector probe or an
internal site. In general, the label is not linked to the 3' end of the
detector probe, as the 3' end must be available for extension by
polymerase. A more rigid linkage or "tether", such as one containing
double bonds, slows the tumbling time of the fluorescent label and allows
measurement of longer correlation times. The fluorescent label is
covalently coupled to the detector probe via a linker or "tether" suitable
for use in conjugating labels to oligonucleotides, e.g., amino-ethyl,
amino-hexyl and amino-propyl linking arms (Applied Biosystems, Clontech,
Glen Research, Devlin, et al., supra.). Other amino linkers are described
in WO 92/18650. The label may also be conjugated to the oligonucleotide at
C5 of pyrimidines or C8 of purines, as generally described by Goodchild,
1990. Bioconj. Chem. 1, 165. Fluorescein may be linked internally by
synthesis of an oligonucleotide containing a phosphorothioate, and
subsequent reaction with iodoacetamidofluorescein.
FP may be used for detection of amplification of any target for which
appropriate amplification primers and detector probes can be designed. In
one representative embodiment, the inventive detection methods may be
applied to an SDA system previously developed for amplification of an M.
tuberculosis target sequence (IS6110 - Walker et al. 1992. Nucl. Acids
Res. and Proc. Natl. Acad. Sci., supra; Spargo et al. 1993, supra).
Samples containing different amounts of M. tuberculosis genomic DNA (which
includes the IS6110 target sequence) and a fluorescent detector probe were
amplified and amplification was simultaneously detected by the increase in
FP. The two samples containing target M. tuberculosis DNA exhibited an
increase in FP values over time while the negative control (zero M.
tuberculosis DNA) did not display any significant change in FP values.
Furthermore, the samples containing M. tuberculosis DNA exhibited
increasing FP values in a time dependent fashion which reflected initial
M. tuberculosis target levels (i.e., quantitative detection). This
experiment illustrated the utility of the system for real-time detection
of SDA. However, the current design of the Diatron fluorometer used for
detection of FP rendered careful execution of SDA reactions in the
fluorometer logistically difficult. Specifically, sample temperature
control on the current Diatron instrument was not optimum for the SDA
reaction. Consequently, SDA was also performed in the presence of the
detector probe in microcentrifuge tubes using a temperature controlled
water bath, with subsequent measurement of FP values in the fluorometer.
Samples containing M. tuberculosis DNA exhibited detectably higher FP
values than the samples lacking M. tuberculosis DNA even when only 1 M.
tuberculosis genome was present as a target. Although FP values generally
increased with increasing levels of initial M. tuberculosis DNA, FP values
tended to reach a maximum level over a range of target input levels. This
was due to the low | | |
