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Claims  |
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What is claimed is:
1. A method for detecting the presence of a nucleic acid target sequence
comprising:
a) hybridizing to the target sequence a detector oligonucleotide comprising
a single-stranded target binding sequence and an intramolecularly
base-paired secondary structure 5' to the target binding sequence, wherein
at least a portion of the target binding sequence forms a single-stranded
tail which binds to the target sequence, the secondary structure having
linked thereto a donor fluorophore and an acceptor dye such that
fluorescence of the donor fluorophore is quenched;
b) in a primer extension reaction, synthesizing a complementary strand
using the base-paired secondary structure as a template, thereby
linearizing or unfolding the base-paired secondary structure and producing
a change in a fluorescence parameter, and;
c) detecting the change in the fluorescence parameter as an indication of
the presence of the target sequence.
2. The method of claim 1 wherein the base-paired secondary structure is
selected from the group consisting of stem-loop structures, pseudoknots
and triple helices.
3. The method of claim 1 wherein the complementary strand is synthesized in
a target amplification reaction.
4. The method of claim 1 wherein the complementary strand is synthesized by
extension of the target sequence using the detector oligonucleotide as a
template.
5. The method of claim 1 wherein the base-paired secondary structure
comprises a totally or partially single-stranded restriction endonuclease
recognition site.
6. The method of claim 1 wherein a change in fluorescence intensity is
detected as an indication of the presence of the target sequence.
7. The method of claim 6 wherein an increase in donor fluorescence
intensity or a decrease in acceptor fluorescence intensity is detected as
an indication of the presence of the target sequence.
8. The method of claim 6 wherein the change in fluorescence intensity is
detected as a) an increase in a ratio of donor fluorophore fluorescence
after linearizing or unfolding to donor fluorophore fluorescence in the
detector oligonucleotide prior to linearizing or unfolding, or b) as a
decrease in a ratio of acceptor dye fluorescence after linearizing or
unfolding to acceptor dye fluorescence in the detector oligonucleotide
prior to linearizing or unfolding.
9. The method of claim 1 wherein a change in fluorescence lifetime is
detected as an indication of the presence of the target sequence.
10. The method of claim 1 wherein the change in the fluorescence parameter
is detected in real-time.
11. The method of claim 1 wherein the change in the fluorescence parameter
is detected at an endpoint.
12. The method of claim 1 wherein the donor fluorophore and the acceptor
dye are separated by about 7-75 nucleotides in the linearized or unfolded
secondary structure.
13. The method of claim 1 wherein the secondary structure is a hairpin.
14. The method of claim 1 wherein the donor fluorophore is fluorescein and
the acceptor dye is Rhodamine X or the donor fluorophore is Rhodamine X
and the acceptor dye is Cy5.
15. The method of claim 1 wherein the intramolecularly base-paired
secondary structure comprises a portion of the target binding sequence.
16. A method for detecting amplification of a target sequence comprising,
in an amplification reaction:
a) hybridizing to the target sequence a detector oligonucleotide comprising
a single-stranded target binding sequence and an intramolecularly
base-paired secondary structure 5' to the target binding sequence, wherein
at least a portion of the target binding sequence forms a single-stranded
tail which binds to the target sequence, the secondary structure having
linked thereto a donor fluorophore and an acceptor dye such that
fluorescence of the donor fluorophore is quenched;
b) extending the hybridized detector oligonucleotide on the target sequence
with a polymerase to produce a detector oligonucleotide extension product
and separating the detector oligonucleotide extension product from the
target sequence;
c) hybridizing a primer to the detector oligonucleotide extension product
and extending the primer with the polymerase, thereby linearizing or
unfolding the secondary structure and producing a change in a fluorescence
parameter, and;
d) detecting the change in the fluorescence parameter as an indication of
amplification of the target sequence.
17. The method of claim 16 wherein the target sequence is amplified by
Strand Displacement Amplification.
18. The method of claim 16 wherein the secondary structure further
comprises a partially or entirely single-stranded restriction endonuclease
recognition site.
19. The method of claim 18 wherein the restriction endonuclease recognition
site is for BsoBI or AvaI.
20. The method of claim 16 wherein the target sequence is amplified by the
Polymerase Chain Reaction.
21. The method of claim 16 wherein the target sequence is amplified by 3SR,
TMA or NASBA.
22. The method of claim 16 wherein a change in fluorescence intensity is
detected.
23. The method of claim 22 wherein an increase in donor fluorophore
fluorescence intensity or a decrease in acceptor dye fluorescence
intensity is detected as an indication of amplification of the target
sequence.
24. The method of claim 22 wherein the change in fluorescence intensity is
detected as a) an increase in a ratio of donor fluorophore fluorescence
after linearizing or unfolding and donor fluorophore fluorescence in the
detector oligonucleotide prior to linearizing or unfolding, or b) as a
decrease in a ratio of acceptor dye fluorescence after linearizing or
unfolding and acceptor dye fluorescence in the detector oligonucleotide
prior to linearizing or unfolding.
25. The method of claim 22 wherein the change in fluorescence intensity is
detected in real-time.
26. The method of claim 22 wherein the change in fluorescence intensity is
detected at a selected end-point in the amplification reaction.
27. The method of claim 16 wherein the donor fluorophore and the acceptor
dye are separated by about 7-75 nucleotides in the linearized or unfolded
secondary structure.
28. The method of claim 16 wherein the donor fluorophore is fluorescein and
the acceptor dye is Rhodamine X or the donor fluorophore is Rhodamine X
and the acceptor dye is Cy5.
29. The method of claim 16 wherein the intramolecularly base-paired
secondary structure comprises a portion of the target binding sequence.
30. A method for detecting a target sequence comprising:
a) providing a detector oligonucleotide comprising a single-stranded target
binding sequence and an intramolecularly base-paired secondary structure
adjacent to the target binding sequence, wherein at least a portion of the
target binding sequence forms a single-stranded 5' or 3' tail which binds
to the target sequence, the secondary structure having linked thereto a
donor fluorophore and an acceptor dye such that fluorescence of the donor
fluorophore is quenched;
b) hybridizing the detector oligonucleotide to the target sequence, thereby
reducing donor fluorophore quenching and producing a change in a
fluorescence parameter, and;
c) detecting the change in the fluorescence parameter as an indication of
the presence of the target sequence.
31. The method of claim 30 wherein the secondary structure comprises a
portion of the target binding sequence.
32. The method of claim 30 wherein the intramolecularly base-paired
secondary structure is 5' to the target binding sequence.
33. The method of claim 30 wherein the intramolecularly base-paired
secondary structure is 3' to the target binding sequence.
34. The method of claim 30 wherein the change in the fluorescence parameter
is detected in real-time.
35. The method of claim 30 wherein the change in the fluorescence parameter
is detected at an endpoint.
36. The method of claim 30 wherein the change in the fluorescence parameter
is a change in fluorescence intensity.
37. The method of claim 30 wherein the secondary structure is a hairpin.
38. The method of claim 30 wherein the detector oligonucleotide is
immobilized on a solid phase.
39. An oligonucleotide comprising a single-stranded target binding sequence
and an intramolecularly base-paired secondary structure adjacent to the
target binding sequence, wherein at least a portion of the target binding
sequence forms a single-stranded 5' or 3' tail which binds to a target
sequence, the secondary structure having linked thereto a first dye and a
second dye such that fluorescence of the first or the second dye is
quenched in the intramolecularly base-paired secondary structure and a
change in a fluorescence parameter is detectable upon linearization or
unfolding of the secondary structure.
40. The oligonucleotide of claim 39 wherein the secondary structure
comprises a partially or entirely single-stranded restriction endonuclease
recognition site.
41. The oligonucleotide of claim 39 wherein the intramolecularly
base-paired secondary structure is 5' to the target binding sequence.
42. The oligonucleotide of claim 41 wherein the intramolecularly
base-paired secondary structure comprises a portion of the target binding
sequence.
43. The oligonucleotide of claim 39 wherein the intramolecularly
base-paired secondary structure is 3' to the target binding sequence.
44. The oligonucleotide of claim 43 wherein the intramolecularly
base-paired secondary structure comprises a portion of the target binding
sequence.
45. The oligonucleotide of claim 39 wherein the first dye is Cy5 or
fluorescein and the second dye is Rhodamine X.
46. The oligonucleotide of claim 39 wherein the first dye is fluorescein
and the second dye is Dabcyl.
47. The oligonucleotide of claim 39 which is immobilized on a solid phase.
48. The method of claim 3 wherein the detector oligonucleotide is an
amplification primer.
49. The method of claim 48 wherein the amplification primer is an
amplification primer for use in PCR, SDA, NASBA, TMA or 3SR.
50. The method of claim 3 wherein the detector oligonucleotide is a signal
primer.
51. The method of claim 3 wherein the target amplification reaction is
selected from the group consisting of PCR, NASBA, 3SR, TMA and SDA.
52. The method of claim 1 further comprising quantitating an amount of the
target sequence initially present.
53. The method of claim 52 wherein the amount of the target sequence is
quantitated by a rate of change in the fluorescence parameter.
54. The method of claim 52 wherein the amount of the target sequence is
quantitated by monitoring time to positivity.
55. The method of claim 5 wherein the restriction endonuclease recognition
site is rendered double-stranded by the primer extension reaction and the
double-stranded restriction endonuclease recognition site is cleaved or
nicked by a restriction endonuclease.
56. The method of claim 16 wherein the detector oligonucleotide is an
amplification primer.
57. The method of claim 16 wherein the detector oligonucleotide is a signal
primer.
58. The method of claim 16 further comprising quantitating an amount of the
target sequence initially present.
59. The method of claim 58 wherein the amount of the target sequence is
quantitated by a rate of change in the fluorescence parameter.
60. The method of claim 58 wherein the amount of the target sequence is
quantitated by monitoring time to positivity.
61. The method of claim 16 wherein the secondary structure is a hairpin.
62. The method of claim 18 wherein the restriction endonuclease recognition
site is rendered double-stranded by extension of the primer and the
double-stranded restriction endonuclease recognition site is cleaved or
nicked by a restriction endonuclease.
63. The method of claim 31 wherein the detector oligonucleotide hybridizes
to the target sequence with at least one mismatch, thereby reducing the
change in the fluorescence parameter as compared to hybridization of the
detector oligonucleotide without the mismatch.
64. The method of claim 30 further comprising quantitating an amount of the
target sequence initially present.
65. The method of claim 64 wherein the amount of the target sequence is
quantitated by a rate of change in the fluorescence parameter.
66. The method of claim 64 wherein the amount of the target sequence is
quantitated by monitoring time to positivity.
67. The method of claim 30 wherein the secondary structure further
comprises a totally or partially single-stranded restriction endonuclease
recognition site.
68. The method of claim 67 wherein the restriction endonuclease recognition
site is rendered double-stranded by hybridizing to the target sequence and
the double-stranded restriction endonuclease recognition site is cleaved
or nicked by a restriction endonuclease.
69. A method for detecting a nucleic acid target sequence comprising:
a) hybridizing to the target sequence a detector oligonucleotide comprising
a single-stranded target binding sequence and an intramolecularly
base-paired secondary structure 5' to the target binding sequence, wherein
at least a portion of the target binding sequence forms a single-stranded
tail which binds to the target sequence and the intramolecularly
base-paired secondary structure comprises a partially or entirely
single-stranded restriction endonuclease recognition site, the secondary
structure having linked thereto a donor fluorophore and an acceptor dye
such that fluorescence of the donor fluorophore is quenched;
b) in a primer extension reaction, synthesizing a complementary strand
using the base-paired secondary structure as a template, thereby
linearizing or unfolding the base-paired secondary structure, rendering
the restriction endonuclease recognition site partially or entirely
double-stranded and producing a change in a fluorescence parameter;
c) optionally, cleaving or nicking the partially or entirely
double-stranded restriction endonuclease recognition site, and;
d) detecting the change in the fluorescence parameter as an indication of
the presence of the target sequence.
70. The method of claim 69 wherein the complementary strand is synthesized
in a target amplification reaction.
71. The method of claim 69 wherein a change in fluorescence intensity is
detected as an indication of the presence of the target sequence.
72. A method for detecting amplification of a target sequence comprising,
in an amplification reaction:
a) hybridizing to the target sequence a detector oligonucleotide comprising
a single-stranded target binding sequence and an intramolecularly
base-paired secondary structure 5' to the target binding sequence, wherein
at least a portion of the target binding sequence forms a single-stranded
tail which binds to the target sequence and the intramolecularly
base-paired secondary structure comprises a partially or entirely
single-stranded restriction endonuclease recognition site, the secondary
structure having linked thereto a donor fluorophore and an acceptor dye
such that fluorescence of the donor fluorophore is quenched;
b) extending the hybridized detector oligonucleotide on the target sequence
with a polymerase to produce a detector oligonucleotide extension product
and separating the detector oligonucleotide extension product from the
target sequence;
c) hybridizing a primer to the detector oligonucleotide extension product
and extending the primer with a polymerase, thereby linearizing or
unfolding the secondary structure, rendering the restriction endonuclease
recognition site partially or entirely double-stranded and producing a
change in a fluorescence parameter;
d) optionally, cleaving or nicking the partially or entirely
double-stranded restriction endonuclease recognition site, and;
e) detecting the change in the fluorescence parameter as an indication of
the presence of the target sequence.
73. The method of claim 72 wherein the amplification reaction is SDA, PCR,
3SR, TMA or NASBA.
74. The method of claim 72 wherein a change in fluorescence intensity is
detected. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The invention relates to methods for detecting nucleic acid target
sequences, and in particular to detection methods employing fluorescence
quenching.
BACKGROUND OF THE INVENTION
Sequence-specific hybridization of oligonucleotide probes has long been
used as a means for detecting and identifying selected nucleotide
sequences, and labeling of such probes with fluorescent labels has
provided a relatively sensitive, nonradioactive means for facilitating
detection of probe hybridization. Recently developed detection methods
employ the process of fluorescence energy transfer (FET) for detection of
probe hybridization rather than direct detection of fluorescence
intensity. Fluorescence energy transfer occurs between a donor fluorophore
and an acceptor dye (which may or may not be a fluorophore) when the
absorption spectrum of one (the acceptor) overlaps the emission spectrum
of the other (the donor) and the two dyes are in close proximity. The
excited-state energy of the donor fluorophore is transferred by a
resonance dipole-induced dipole interaction to the neighboring acceptor.
This results in quenching of donor fluorescence. In some cases, if the
acceptor is also a fluorophore, the intensity of its fluorescence may be
enhanced. The efficiency of energy transfer is highly dependent on the
distance between the donor and acceptor, and equations predicting these
relationships have been developed by Forster (1948. Ann. Phys. 2, 55-75).
The distance between donor and acceptor dyes at which energy transfer
efficiency is 50% is referred to as the Forster distance (R.sub.o). Other
mechanisms of fluorescence quenching are also known including, for
example, charge transfer and collisional quenching.
Energy transfer and other mechanisms which rely on the interaction of two
dyes in close proximity to produce quenching are an attractive means for
detecting or identifying nucleotide sequences, as such assays may be
conducted in homogeneous formats. Homogeneous assay formats are simpler
than conventional probe hybridization assays which rely on detection of
the fluorescence of a single fluorophore label, as heterogeneous assays
generally require additional steps to separate hybridized label from free
label. Typically, FET and related methods have relied upon monitoring a
change in the fluorescence properties of or both dye labels when they are
brought together by the hybridization of two complementary
oligonucleotides. In this format, the change in fluorescence properties
may be measured as a change in the amount of energy transfer or as a
change in the amount of fluorescence quenching, typically indicated as an
increase in the fluorescence intensity of one of the dyes. In this way,
the nucleotide sequence of interest may be detected without separation of
unhybridized and hybridized oligonucleotides. The hybridization may occur
between two separate complementary oligonucleotides, one of which is
labeled with the donor fluorophore and one of which is labeled with the
acceptor. In double-stranded form there is decreased donor fluorescence
(increased quenching) and/or increased energy transfer as compared to the
single-stranded oligonucleotides. Several formats for FET hybridization
assays are reviewed in Nonisotopic DNA Probe Techniques (1992. Academic
Press, Inc., pgs. 311-352).
Alternatively, the donor and acceptor may be linked to a single
oligonucleotide such that there is a detectable difference in the
fluorescence properties of one or both when the oligonucleotide is
unhybridized vs. when it is hybridized to its complementary sequence. In
this format, donor fluorescence is typically increased and energy
transfer/quenching are decreased when the oligonucleotide is hybridized.
For example, a self-complementary oligonucleotide labeled at each end may
form a hairpin which brings the two fluorophores (i.e., the 5' and 3'
ends) into close proximity where energy transfer and quenching can occur.
Hybridization of the self-complementary oligonucleotide to its complement
on a second oligonucleotide disrupts the hairpin and increases the
distance between the two dyes, thus reducing quenching. A disadvantage of
the hairpin structure is that it is very stable and conversion to the
unquenched, hybridized form is often slow and only moderately favored,
resulting in generally poor performance. Tyagi and Kramer (1996. Nature
Biotech. 14, 303-308) describe a hairpin labeled as described above with a
detector sequence in the loop between the self-complementary arms of the
hairpin which form the stem. The base-paired stem must melt in order for
the detector sequence to hybridize to the target and cause a reduction in
quenching. A "double hairpin" probe and methods of using it are described
by B. Bagwell, et al. (1994. Nucl. Acids Res. 22, 2424-2425; U.S. Pat. No.
5,607,834). These structures contain the target binding sequence within
the hairpin and therefore involve competitive hybridization between the
target and the self-complementary sequences of the hairpin. Bagwell solves
the problem of unfavorable hybridization kinetics by destabilizing the
hairpin with mismatches, thus favoring hybridization to the target. In
contrast to these publications, the detector oligonucleotides of the
invention have the target binding sequence wholly or partially in a
single-stranded "tail" region rather than fully contained within the
intramolecularly base-paired secondary structure. The secondary structure
(e.g., a hairpin) therefore need not unfold in order to initially
hybridize to the target. Hybridization of the single-stranded tail is not
competitive so the kinetics of the reaction favor hybridization to the
target. Hybridization of the detector oligonucleotide through the
single-stranded tail also increases the local concentration of target,
thereby driving any subsequent unfolding of the secondary structure. By
shifting the kinetics of the reaction to better favor unfolding in the
presence of target, the methods of the invention allow the use of
perfectly base-paired secondary structures which would otherwise be too
stable to be effective for target detection.
Homogeneous methods employing energy transfer or other mechanisms of
fluorescence quenching for detection of nucleic acid amplification have
also been described. R. Higuchi, et al. (1992. Biotechnology 10, 413-417)
disclose methods for detecting DNA amplification in real-time by
monitoring increased fluorescence for ethidium bromide as it binds to
double-stranded DNA. The sensitivity of this method is limited because
binding of the ethidium bromide is not target specific and background
amplification products are also detected. L. G. Lee, et al. (1993. Nuc.
Acids Res. 21, 3761-3766) disclose a real-time detection method in which a
doubly-labeled detector probe is cleaved in a target
amplification-specific manner during PCR. The detector probe is hybridized
downstream of the amplification primer so that the 5'-3' exonuclease
activity of Taq polymerase digests the detector probe, separating two
fluorescent dyes which form an energy transfer pair. Fluorescence
intensity increases as the probe is cleaved. Published PCT application WO
96/21144 discloses continuous fluorometric assays in which enzyme-mediated
cleavage of nucleic acids results in increased fluorescence. Fluorescence
energy transfer is suggested for use in the methods, but only in the
context of a method employing a single fluorescent label which is quenched
by hybridization to the target. There is no specific disclosure of how a
restriction endonuclease would be used in a fluorescence energy transfer
system.
Energy transfer and other fluorescence quenching detection methods have
also been applied to detecting a target sequence by hybridization of a
specific probe. Japanese Patent No. 93015439 B discloses methods for
measuring polynucleotides by hybridizing the single-stranded target to a
single-stranded polynucleotide probe tagged with two labels which form an
energy transfer pair. The double-stranded hybrid is cleaved by a
restriction enzyme between the labels and fluorescence of one of the
labels is measured. A shortcoming of this method is that the restriction
site in the probe must also be present in the target sequence being
detected. The patent does not describe adaptation of the probe for use in
assays where the target sequence does not contain an appropriate
restriction site or where cleavage of the target is not desired. S. S.
Ghosh, et al. (1994. Nucl Acids Res. 22, 3155-3159) describe restriction
enzyme catalyzed cleavage reactions of fluorophore-labeled
oligonucleotides which are analyzed using fluorescence resonance energy
transfer. In these assays, the complementary oligonucleotides are
hybridized to produce the double-stranded restriction site, and one of the
fluorescent labels is linked to each of the two strands (i.e., they are
not linked to the same strand, see FIG. 1 of Ghosh, et al.). S. P. Lee, et
al. (1994. Anal Biochem. 220, 377-383) describe fluorescence "dequenching"
techniques using restriction endonucleases to cleave double-stranded DNA.
However, these methods relate to assays employing only a single
fluorescent label which is quenched by interaction with the DNA, not by
fluorescence energy transfer from a second fluorescent label.
Hybridization of the labeled oligonucleotide to its complement and
cleavage of the double-stranded restriction site relieved non-transfer
quenching of the label and quenched fluorescence was totally recovered.
Signal primers (also referred to as detector probes) which hybridize to the
target sequence downstream of the hybridization site of the amplification
primers have been described for use in detection of nucleic acid
amplification (U.S. Pat. No. 5,547,861). The signal primer is extended by
the polymerase in a manner similar to extension of the amplification
primers. Extension of the amplification primer displaces the extension
product of the signal primer in a target amplification-dependent manner,
producing a double-stranded secondary amplification product which may be
detected as an indication of target amplification. The secondary
amplification products generated from signal primers may be detected by
means of a variety of labels and reporter groups, restriction sites in the
signal primer which are cleaved to produce fragments of a characteristic
size, capture groups, and structural features such as triple helices and
recognition sites for double-stranded DNA binding proteins. Examples of
detection methods for use with signal primers are described in U.S. Pat.
No. 5,550,025 (incorporationof lipophilic dyes and restriction sites) and
U.S. Pat. No. 5,593,867 (fluorescence polarization detection).
SUMMARY OF THE INVENTION
The present invention employs a detector oligonucleotide for detection of
nucleic acid target sequences by fluorescence quenching mechanisms. The
otherwise single-stranded detector oligonucleotide is selected such that
it forms an intramolecularly base-paired secondary structure under the
selected reaction conditions for primer extension or hybridization to the
target. The detector oligonucleotide is further modified by linkage to two
dyes which form a donor/acceptor dye pair. The two dyes are positioned on
the detector oligonucleotide such that they are in close spatial proximity
in the base-paired, folded secondary structure and the fluorescence of the
donor dye is quenched. The detector oligonucleotide may further comprise a
restriction endonuclease recognition site (RERS) between the two dyes
which remains partially or entirely single-stranded in the base-paired
secondary structure. As the detector oligonucleotide is initially
single-stranded except for the base-paired portion of the secondary
structure and remains single-stranded with the secondary structure folded
in the absence of target, donor fluorescence is quenched. In the presence
of target, however, the detector oligonucleotide is unfolded or
linearized, increasing the distance between the donor and acceptor dyes
and causing a change in fluorescence. If an RERS is present in the portion
of the detector oligonucleotide between the two dyes, it is uncleavable or
unnickable in the absence of target. The RERS becomes double-stranded in
the presence of target, however, allowing cleavage or nicking by the
restriction endonuclease and separation of the two dyes onto separate
nucleic acid fragments. Cleavage or nicking further contributes to the
change in fluorescence which indicates target amplification or the
presence of the target sequence.
In alternative exemplary embodiments, the invention employs the detector
oligonucleotide as a signal primer in target amplification reactions for
detecting target sequence amplification, in non-amplification based primer
extension methods for detection of target sequences and in hybridization
reactions for detection of target sequences.
DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the reaction scheme wherein the detector oligonucleotide
is employed as a signal primer according to the invention.
FIG. 2 shows the change in fluorescence intensity occurring in real-time as
a target is amplified using the detector oligonucleotides of the invention
as signal primers.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs detector oligonucleotides to produce reduced
fluorescence quenching in a target-dependent manner. The detector
oligonucleotides contain a donor/acceptor dye pair linked such that
fluorescence quenching occurs in the absence of target. Unfolding or
linearization of an intramolecularly base-paired secondary structure in
the detector oligonucleotide in the presence of the target increases the
distance between the dyes and reduces fluorescence quenching. Unfolding of
the base-paired secondary structure typically involves intermolecular
base-pairing between the sequence of the secondary structure and a
complementary strand such that the secondary structure is at least
partially disrupted. It may be fully linearized in the presence of a
complementary strand of sufficient length. In a preferred embodiment, an
RERS is present between the two dyes such that intermolecular base-pairing
between the secondary structure and a complementary strand also renders
the RERS double-stranded and cleavable or nickable by a restriction
endonuclease. Cleavage or nicking by the restriction endonuclease
separates the donor and acceptor dyes onto separate nucleic acid
fragments, further contributing to decreased quenching. In either
embodiment, an associated change in a fluorescence parameter (e.g., an
increase in donor fluorescence intensity, a decrease in acceptor
fluorescence intensity or a ratio of fluorescence before and after
unfolding) is monitored as a indication of the presence of the target
sequence. Monitoring a change in donor fluorescence intensity is
preferred, as this change is typically larger than the change in acceptor
fluorescence intensity. Other fluorescence parameters such as a change in
fluorescence lifetime may also be monitored.
Certain terms used herein are defined as follows:
An amplification primer is a primer for amplification of a target sequence
by primer extension. For SDA, the 3' end of the amplification primer (the
target binding sequence) hybridizes at the 3' end of the target sequence.
The amplification primer comprises a recognition site for a restriction
endonuclease near its 5' end. The recognition site is for a restriction
endonuclease which will cleave one strand of a DNA duplex when the
recognition site is hemimodified ("nicking"), as described in U.S. Pat.
No. 5,455,166; U.S. Pat. No. 5,270,184 and; EP 0 684 315. A hemimodified
recognition site is a double stranded recognition site for a restriction
endonuclease in which one strand contains at least one derivatized
nucleotide which causes the restriction endonuclease to nick the primer
strand rather than cleave both strands of the recognition site. Usually,
the primer strand of the hemimodified recognition site does not contain
derivatized nucleotides and is nicked by the restriction endonuclease.
Alternatively, the primer may contain derivatized nucleotides which cause
the unmodified target strand to be protected from cleavage while the
modified primer strand is nicked. Such restriction endonucleases can be
identified in routine screening systems in which a derivatized dNTP is
incorporated into a restriction endonuclease recognition site for the
enzyme. Preferred hemimodified recognition sites are hemiphosphorothioated
recognition sites for the restriction endonucleases HincII, BsoBI and
BsrI. The amplification primer also comprises a 3'-OH group which is
extendible by DNA polymerase when the target binding sequence of the
amplification primer is hybridized to the target sequence. For the
majority of the SDA reaction, the amplification primer is responsible for
exponential amplification of the target sequence.
As no special sequences or structures are required to drive the
amplification reaction, amplification primers for PCR may consist only of
target binding sequences. Amplification primers for 3SR and NASBA, in
contrast comprise an RNA polymerase promoter near the 5' end. The promoter
is appended to the target sequence and serves to drive the amplification
reaction by directing transcription of multiple RNA copies of the target.
Extension products are nucleic acids which comprise a primer or a portion
of a primer and a newly synthesized strand which is the complement of the
target sequence downstream of the primer binding site. Extension products
result from hybridization of a primer to a target sequence and extension
of the primer by polymerase using the target sequence as a template.
The terms target or target sequence refer to nucleic acid sequences to be
amplified or detected. These include the original nucleic acid sequence to
be amplified, its complementary second strand and either strand of a copy
of the original sequence which is produced by replication or
amplification. The target sequence may also be referred to as a template
for extension of hybridized primers.
A detector oligonucleotide is an oligonucleotide which comprises a
single-stranded 5' or 3' "tail" which hybridizes to the target sequence
(the target binding sequence) and an intramolecularly base-paired
secondary structure adjacent to the target binding sequence. The detector
oligonucleotides of the invention further comprise a donor/acceptor dye
pair linked to the detector oligonucleotide such that donor fluorescence
is quenched when the secondary structure is intramolecularly base-paired
and unfolding or linearization of the secondary structure results in a
decrease in fluorescence quenching.
Cleavage of an oligonucleotide refers to breaking the phosphodiester bonds
of both strands of a DNA duplex or breaking the phosphodiester bond of
single-stranded DNA. This is in contrast to nicking, which refers to
breaking the phosphodiester bond of only one of the two strands in a DNA
duplex.
The detector oligonucleotides of the invention comprise a sequence which
forms an intramolecularly base-paired secondary structure under the
selected reaction conditions for primer extension or hybridization. The
secondary structure is positioned adjacent to the target binding sequence
of the detector oligonucleotide so that at least a portion of the target
binding sequence forms a single-stranded 3' or 5' tail. As used herein,
the term "adjacent to the target binding sequence" means that all or part
of the target binding sequence is left single-stranded in a 5' or 3' tail
which is available for hybridization to the target. That is, the secondary
structure does not comprise the entire target binding sequence. A portion
of the target binding sequence may be involved in the intramolecular
base-pairing of the adjacent secondary structure or the entire target
binding sequence may form a single-stranded 5' or 3' tail in the detector
oligonucleotide. If a portion of the target binding sequence of the
detector oligonucleotide is involved in intramolecular base-pairing in the
secondary structure, it may include all or part of a first sequence
involved in intramolecular base-pairing in the secondary structure but
preferably does not extend into its complementary sequence. For example,
if the secondary structure is a stem-loop structure (i.e., a "hairpin")
and the target binding sequence of the detector oligonucleotide is present
as a single-stranded 3' tail, the target binding sequence may also extend
5' through all or part of the first arm of the stem and, optionally,
through all or part of the loop. However, the target binding sequence
preferably does not extend into the second arm of the sequence involved in
stem intramolecular base-pairing. That is, it is desirable to avoid having
both sequences involved in intramolecular base-pairing in a secondary
structure capable of hybridizing to the target. Mismatches in the
intramolecularly base-paired portion of the detector oligonucleotide
secondary structure may reduce the magnitude of the change in fluorescence
in the presence of target but are acceptable if assay sensitivity is not a
concern. Mismatches in the target binding sequence of the single-stranded
tail are also acceptable but may similarly reduce assay sensitivity and/or
specificity. However, it is a feature of the present invention that
perfect base-pairing in both the secondary structure and the target
binding sequence do not compromise the reaction. Perfect matches in the
sequences involved in hybridization improve assay specificity without
negative effects on reaction kinetics.
The detector oligonucleotide further comprises a donor fluorophore and an
acceptor dye linked at positions in the detector oligonucleotide such that
the intramolecular base-pairing of the secondary structure brings the dyes
into close spatial proximity and results in fluorescence quenching.
Preferably neither dye is at the 3' terminus of the detector
oligonucleotide when it is used as a primer, as a 3' terminal label may
interfere with hybridization and/or extension of the oligonucleotide.
However, a selected donor fluorophore or acceptor dye may be linked at any
position which does not inhibit hybridization and/or extension, which
results in quenching in the folded secondary structure and which provides
a change in a fluorescence parameter upon unfolding or linearization. The
donor and acceptor dyes are also linked such that unfolding of the
secondary structure increases the distance between them and reduces
fluorescence quenching, resulting in a detectable change in a fluorescence
parameter.
The donor and an acceptor dye are linked to the detector oligonucleotide
such that donor fluorescence is totally or partially quenched when the
detector oligonucleotide forms the intramolecularly base-paired secondary
structure. The two dyes must be in sufficiently close proximity when the
secondary structure is folded so that quenching will occur. However, the
distance between them in the linear nucleotide sequence of the detector
oligonucleotide must provide for a change in proximity sufficient to
produce a detectable change in a fluorescence paramete | | |
