U.S. patent application number 09/894796 was filed with the patent office on 2002-08-01 for probes and methods for detection of nucleic acids.
Invention is credited to Hellyer, Tobin J., Nadeau, James G..
Application Number | 20020102574 09/894796 |
Document ID | / |
Family ID | 24360721 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102574 |
Kind Code |
A1 |
Nadeau, James G. ; et
al. |
August 1, 2002 |
Probes and methods for detection of nucleic acids
Abstract
The invention employs an unlabeled signal primer comprising a 5'
adapter sequence for detection of nucleic acid target sequences.
The detection system further comprises a reporter probe, the 3' end
of which hybridizes to the complement of the 5' adapter sequence of
the signal primer to produce a 5' overhang. Polymerase is used to
fill in the overhang and synthesize the complement of the 5'
overhang of the reporter probe. Synthesis of the reporter probe
complement is detected, either directly or indirectly, as an
indication of the presence of the target.
Inventors: |
Nadeau, James G.; (Ellicott
City, MD) ; Hellyer, Tobin J.; (Owings Mills,
MD) |
Correspondence
Address: |
Becton, Dickinson and Company
1 Becton Drive
Franklin Lakes
NJ
07417
US
|
Family ID: |
24360721 |
Appl. No.: |
09/894796 |
Filed: |
June 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09894796 |
Jun 28, 2001 |
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09590061 |
Jun 8, 2000 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
C12Q 2525/191 20130101;
C12Q 2561/101 20130101; C12Q 2561/101 20130101; C12Q 2525/191
20130101; C12Q 2565/549 20130101; C12Q 1/6844 20130101; C12Q 1/6818
20130101; C12Q 2565/549 20130101; C12Q 1/6818 20130101; C12Q 1/6855
20130101; C12Q 1/6844 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is:
1. A method for detecting a nucleic acid target sequence
comprising: a) hybridizing a signal primer comprising an adapter
sequence to the target sequence, whereby a complement of the
adapter sequence is produced; b) hybridizing a reporter probe
comprising a reporter moiety to the complement of the adapter
sequence, whereby a double-stranded reporter moiety is produced,
and; c) detecting synthesis of the complement of the reporter
moiety as an indication of the presence of the target sequence.
2. The method of claim 1 wherein the double-stranded reporter
moiety is produced upon hybridization of the reporter moiety to the
complement of the adapter sequence.
3. The method of claim 2 wherein the reporter moiety is a molecular
beacon.
4. The method of claim 1 wherein the double-stranded reporter
moiety is produced upon synthesis of a complement of the reporter
moiety.
5. The method of claim 1 wherein the complement of the adapter
sequence is synthesized concurrently with target amplification.
6. The method of claim 5 wherein target amplification is by SDA,
3SR, NASBA, TMA or PCR.
7. The method of claim 1 wherein the complement of the adapter
sequence is synthesized without amplification of the target
sequence.
8. The method of claim 7 wherein the complement of the adapter
sequence is displaced from the signal primer by extension of an
upstream primer prior to hybridization to the reporter probe.
9. The method of claim 1 wherein the reporter probe is
non-extendible.
10. The method of claim 1 wherein a change in fluorescence is
detected.
11. The method of claim 10 wherein the change in the fluorescence
results directly from unfolding of a secondary structure.
12. The method of claim 10 wherein the change in fluorescence
results from cleavage or nicking of a restriction endonuclease
recognition site in the double-stranded reporter moiety.
13. The method of claim 10 wherein the change in fluorescence is
detected in real-time.
14. The method of claim 10 wherein the change in fluorescence is
detected at a selected endpoint in the reaction.
15. The method of claim 1 wherein the reporter moiety is labeled
with a fluorescent donor/quencher dye pair.
16. The method of claim 1 wherein the reporter moiety is selected
from the group consisting of secondary structures and specialized
sequences.
17. The method of claim 16 wherein the double-stranded reporter
moiety is detected by unfolding of a hairpin structure, unfolding
of a G-quartet structure or nicking or cleavage of a restriction
endonuclease recognition site.
18. The method of claim 1 which comprises multiple signal primers,
each signal primer having a separately detectable adapter
sequence.
19. The method of claim 18 wherein each signal primer hybridizes to
a different sequence variant of the target sequence.
20. A method for detecting amplification of a target sequence
comprising, in an amplification reaction: a) hybridizing a signal
primer comprising an adapter sequence to the target sequence; b)
extending the signal primer on the target sequence to produce an
extension product; c) hybridizing an amplification primer to the
extension product and extending the amplification primer to
synthesize a complement of the adapter sequence; d) hybridizing to
the complement of the adapter sequence a reporter probe comprising
a reporter moiety, whereby a double-stranded reporter moiety is
produced; e) detecting the double-stranded reporter moiety as an
indication of amplification of the target sequence.
21. The method of claim 20 wherein the double-stranded reporter
moiety is produced upon hybridization of the reporter moiety to the
complement of the adapter sequence.
22. The method of claim 21 wherein the reporter is a molecular
beacon.
23. The method of claim 20 wherein the double-stranded reporter
moiety is produced upon synthesis of a complement of the reporter
moiety
24. The method of claim 20 wherein the target sequence is amplified
by SDA, PCR, 3SR, TMA or NASBA.
25. The method of claim 20 wherein a change in fluorescence is
detected.
26. The method of claim 25 wherein the change in fluorescence is
detected in real-time.
27. The method of claim 25 wherein the change in fluorescence is
detected at a selected end-point in the amplification reaction.
28. The method of claim 20 wherein the reporter moiety is labeled
with a fluorescent donor/quencher dye pair.
29. The method of claim 20 wherein the reporter moiety is selected
from the group consisting of secondary structures and specialized
sequences.
30. The method of claim 29 wherein the double-stranded reporter
moiety is detected by unfolding of a hairpin structure, unfolding
of a G-quartet or by nicking or cleavage of a restriction
endonuclease recognition site.
31. The method of claim 29 wherein a change in the fluorescence
results directly from unfolding of a secondary structure.
32. The method of claim 29 wherein a change in fluorescence results
from cleavage or nicking of a restriction endonuclease recognition
site in the double-stranded reporter moiety.
33. The method of claim 20 wherein the reporter probe is
non-extendible.
34. The method of claim 20 which comprises multiple signal primers,
each signal primer having a separately detectable adapter
sequence.
35. The method of claim 34 wherein each signal primer hybridizes to
a different sequence variant of the target sequence.
36. A method for detecting a nucleic acid target sequence
comprising: a) hybridizing a signal primer comprising an adapter
sequence to the target sequence such that the adapter sequence
produces a 5' overhang; b) synthesizing a complement of the adapter
sequence by extension of the hybridized target sequence; c)
hybridizing a reporter probe comprising a reporter moiety to the
complement of the adapter sequence, whereby a double-stranded
reporter moiety is produced, and; d) detecting the double-stranded
reporter moiety as an indication of the presence of the target
sequence.
37. The method of claim 36 wherein the double-stranded reporter
moiety is produced upon hybridization of the reporter moiety to the
complement of the adapter sequence.
38. The method of claim 37 wherein the reporter is a molecular
beacon.
39. The method of claim 36 wherein the double-stranded reporter
moiety is produced upon synthesis of a complement of the reporter
moiety
40. The method of claim 36 wherein the double-stranded reporter
moiety is detected by unfolding of a secondary structure or by
means of a specialized sequence.
41. The method of claim 40 wherein unfolding of a hairpin structure
or a G-quarter structure is detected.
42. The method of claim 40 wherein cleavage or nicking of a
restriction endonuclease recognition site is detected.
43. The method of claim 36 wherein a change in fluorescence is
detected.
44. The method of claim 43 wherein the reporter moiety is labeled
with a donor/quencher dye pair.
45. The method of claim 36 wherein the signal primer or the
reporter probe is non-extendible.
46. The method of claim 36 which comprises multiple signal primers,
each signal primer having a separately detectable adapter
sequence.
47. The method of claim 46 wherein each signal primer hybridizes to
a different sequence variant of the target sequence.
48. A set of oligonucleotides for detecting a target sequence
comprising: a) an unlabeled signal primer comprising a single
oligonucleotide having a 3' target binding sequence and a 5'
adapter sequence, and; b) a reporter probe comprising a 5' reporter
moiety and 3' sequence which is substantially identical to the
adapter sequence.
49. The set of oligonucleotides of claim 48 further comprising a
second signal primer having an adapter sequence which is
substantially identical to an adapter sequence of a first signal
primer.
50. The set of oligonucleotides of claim 48 further comprising a
second signal primer having an adapter sequence which is different
from an adapter sequence of a first signal primer.
51. The set of oligonucleotides of claim 48 wherein the reporter
moiety is labeled.
52. The set of oligonucleotides of claim 51 wherein the reporter
moiety is labeled with a fluorescent donor/quencher dye pair.
53. The set of oligonucleotides of claim 48 wherein the reporter
moiety is selected from the group consisting of secondary
structures and specialized sequences.
54. The set of oligonucleotides of claim 53 wherein the reporter
moiety is selected from the group consisting of hairpins,
G-quartets and restriction endonuclease recognition sites.
55. The set of oligonucleotides of claim 48 where in the reporter
probe is non-extendible.
56. An oligonucleotide comprising a reporter moiety and nucleotides
15-37 of SEQ ID NO:2, nucleotides 10-34 of SEQ ID NO:15,
nucleotides 16-40 of SEQ ID NO:16, nucleotides 16-35 of SEQ ID
NO:17, nucleotides 16-30 of SEQ ID NO:18, nucleotides 16-40 of SEQ
ID NO:19 or nucleotides 19-43 of SEQ ID NO:20.
57. The oligonucleotide of claim 56 selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.
58. An oligonucleotide comprising the target binding sequence of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14 and an adapter
sequence.
59. The oligonucleotide of claim 58 selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
Description
FIELD OF THE INVENTION
[0001] The invention relates to materials and methods for detecting
nucleic acid target sequences.
BACKGROUND OF THE INVENTION
[0002] Sequence-specific hybridization of labeled 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) rather than direct
detection of fluorescence intensity for detection of probe
hybridization. Fluorescence energy transfer occurs between a donor
fluorophore and a quencher dye (which may or may not be a
fluorophore) when the absorption spectrum of one (the quencher)
overlaps the emission spectrum of the other (the donor) and the two
dyes are in close proximity. Dyes with these properties are
referred to as donor/quencher dye pairs or energy transfer dye
pairs. The excited-state energy of the donor fluorophore is
transferred by a resonance dipole-induced dipole interaction to the
neighboring quencher. This results in quenching of donor
fluorescence. In some cases, if the quencher (also referred to as
an "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 quencher,
and equations predicting these relationships have been developed by
Forster (1948. Ann. Phys. 2, 55-75). The distance between donor and
quencher 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. In these cases the
quencher may be a fluorescent dye but it need not be. Fluorescence
quenching mechanisms that are not based on FET typically do not
require appreciable overlap between the absorption spectrum of the
quencher and the emission spectrum of the donor fluorophore.
[0003] 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 one 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 quencher. 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 quencher 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, an oligonucleotide
labeled with donor and quencher dyes may contain self-complementary
sequences that base-pair to form a hairpin which brings the two
dyes into close spatial proximity where energy transfer and
quenching can occur. Hybridization of this oligonucleotide to its
complementary sequence in a second oligonucleotide disrupts the
hairpin and increases the distance between the two dyes, thus
reducing quenching. See Tyagi and Kramer (1996. Nature Biotech. 14,
303-308) and B. Bagwell, et al. (1994. Nucl. Acids Res. 22,
2424-2425; U.S. Pat. No. 5,607,834). Homogeneous methods employing
energy transfer or other mechanisms of fluorescence quenching for
detection of nucleic acid amplification have also been described.
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.
[0004] Signal primers (sometimes 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 homogeneous detection of nucleic acid amplification (U.S. Pat.
No. 5,547,861 which is incorporated herein by reference). 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. Examples of
homogeneous detection methods for use with single-stranded signal
primers are described in U.S. Pat. No. 5,550,025 (incorporation of
lipophilic dyes and restriction sites) and U.S. Pat. No. 5,593,867
(fluorescence polarization detection). More recently signal primers
have been adapted for detection of nucleic acid targets using FET
methods which employ unfolding of secondary structures (e.g., U.S.
Pat. 5,691,145 and U.S. Pat. No. 5,928,869). Partially
single-stranded, partially double-stranded signal primers labeled
with donor/quencher dye pairs have also recently been described.
For example, U.S. Pat. No. 5,846,726 discloses signal primers with
donor/quencher dye pairs flanking a single-stranded restriction
endonuclease recognition site. In the presence of the target, the
restriction site becomes double-stranded and cleavable by the
restriction endonuclease. Cleavage separates the dye pair and
decreases donor quenching.
[0005] U.S. Pat. No. 5,866,336 describes use of a fluorescently
labeled hairpin on an amplification primer in PCR. The 3' end of
the hairpin primer hybridizes to the complement of a non-target
sequence appended to the target by a second primer. In this system,
the hairpin primer plays an integral part in amplification of the
target sequence and must be extendible. In contrast, in the present
invention it is not necessary for the reporter probe to be
extendible, as it does not participate in amplification of the
target sequence but generates signal in a separate series of
reaction steps which occur concurrently with target amplification.
In further contrast, the signal primers of the invention hybridize
to an internal sequence of the target (i.e., between the
amplification primers), so that the signal generation reaction
detects a subsequence of the target, not the amplification product
itself.
[0006] Other fluorescence quenching methods for detection of a
target sequence employ cleavage of a restriction site produced by
direct hybridization of a probe containing the single-stranded
restriction site to the single-stranded target. 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 between the
labels by a restriction enzyme and fluorescence of one of the
labels is measured. A disadvantage of this method is that the
restriction site in the probe must also be present in the target
sequence being detected. S. S. Ghosh, et al. (1994. Nucl. Acids
Res. 22, 3155-3159) describe restriction enzyme catalyzed cleavage
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, with one of the fluorescent
labels linked to each of the two strands.
SUMMARY OF THE INVENTION
[0007] The present invention employs a signal primer for detection
of nucleic acid target sequence amplification. The signal primer of
the invention is similar in structure to the signal primer
described in U.S. Pat. No. 5,547,861 (which is incorporated by
reference herein) but it is unlabeled. The detection system further
comprises a labeled reporter probe, the 3' end of which hybridizes
to the complement of the 5' tail sequence of the signal primer to
produce a 5' overhang. The region of the reporter probe which forms
the 5' overhang (the reporter moiety) comprises a structure or
sequence which is labeled in a manner which permits detection of
synthesis of the complement of the overhang. Preferably, the
reporter moiety is fluorescently labeled such that fluorescence is
quenched prior to extension of the signal primer and synthesis of a
complementary strand. The presence of the complement of the
reporter moiety, rendering it double-stranded, reduces fluorescence
quenching directly and/or allows a subsequent reaction to take
place to reduce quenching. In either mechanism, the complement of
the labeled structure or sequence leads to an increased distance
between the dyes. An associated increase in donor fluorescence or a
change in another fluorescence parameter associated with decreased
fluorescence quenching can be detected as an indication of
amplification of the target sequence.
[0008] The 5' tail sequence of the signal primer comprises a
sequence which does not hybridize to the target (the adapter
sequence). The adapter sequence may be selected such that it is the
same in a variety of signal primers which have different 3' target
binding sequences (i.e., a "universal" 5' tail sequence). This
allows a single reporter probe sequence to be used for detection of
any desired target sequence, which is an advantage in that
synthesis of the reporter probe is more complex due to the
labeling. Further, the invention simplifies the synthesis of the
target-specific signal primer. As the signal primer is not labeled,
signal primers with different target binding sequences specific for
different targets may be more easily and efficiently
synthesized.
DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1A illustrates detection of a nucleic acid target
sequence in a Strand Displacement Amplification (SDA) reaction
according to the method of the invention. FIG. 1B illustrates the
additional reaction steps which may occur when the fluorescently
labeled sequence in the reporter probe is a nickable RERS.
[0010] FIG. 2 illustrates the results of Example 1.
[0011] FIG. 3A and FIG. 3B illustrate the results of Example 2.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Certain terms used herein are defined as follows:
[0013] 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. 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.
[0014] 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 sequence downstream of the primer binding
site. Extension products result from hybridization of a primer to a
template containing a complementary sequence and extension of the
primer by polymerase using the template.
[0015] 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. A target sequence may
also be referred to as a template for extension of hybridized
primers.
[0016] A signal primer according to the present invention comprises
a 3' target binding sequence which hybridizes to a complementary
sequence in the target and further comprises a 5' tail sequence
which is not complementary to the target (the adapter sequence).
The adapter sequence is selected such that its complementary
sequence will hybridize to the 3' end of the reporter probe
described below. In some embodiments of the invention the adapter
sequence is selected such that its complementary sequence binds to
both the 3' end of the reporter probe and to a sequence within the
reporter moiety of the reporter probe, as described below. In
preferred embodiments of the invention, the signal primer does not
comprise a detectable label.
[0017] A reporter probe according to the present invention
comprises a label which is preferably at least one donor/quencher
dye pair, i.e., a fluorescent donor dye and a quencher for the
donor fluorophore. The label is linked to a sequence or structure
in the reporter probe (the reporter moiety) which does not
hybridize directly to the target sequence. The sequence of the
reporter probe 3' to the reporter moiety is selected to hybridize
to the complement of the signal primer adapter sequence. In
general, the 3' end of the reporter probe does not contain
sequences with any significant complementarity to the target
sequence. In some instances, however, the reporter probe may
contain the sequence that hybridizes to the adapter complement and
another short sequence at the 3' end that hybridizes to a short
segment of the target complement. In this case, the region of
target complementarity is not large enough to permit significant
hybridization without concurrent hybridization of the
adapter-specific region of the reporter probe. The label of the
reporter probe is detected as an indication of the presence of a
complement of the reporter moiety which renders it double-stranded,
thereby indicating the presence of or the amplification of the
target. The 3' terminus of the reporter probe may be capped to
prevent extension by polymerase or it may be extendible. Capping
may enhance performance by reducing background signal and the
nonproductive consumption of reagents in spurious side-reactions
resulting from the formation of primer dimers and other errant
priming events.
[0018] Any nucleic acid sequence or structure which can be labeled
such that the presence of its complement, generated according to
the methods of the invention, indicates the presence of the target
sequence can serve as the reporter moiety of the reporter probe.
Preferably, the reporter moiety is labeled with a donor/quencher
dye pair such that donor fluorescence is quenched prior to
detection of a target and such that quenching of donor fluorescence
is reduced as an indication of the presence of the target. The
reporter moiety may be a secondary structure at the 5' end of the
reporter probe, such as a stem-loop (or hairpin) as described in
U.S. Pat. No. 5,928,869 or a G-quartet as described in U.S. Pat.
No. 5,691,145. The secondary structure is labeled such that the
donor and quencher are in close proximity when the secondary
structure is folded, resulting in quenching of donor fluorescence.
In the presence of target, the secondary structure is unfolded in a
target-dependent primer extension reaction so that the distance
between the donor and quencher is increased. This decreases
quenching and produces an increase in donor fluorescence which can
be detected as an indication of the presence of the target
sequence. Alternatively, the reporter moiety may be a
single-stranded sequence at the 5' end of the reporter probe which
is labeled with the donor and quencher in sufficiently close
proximity to produce quenching and which contains a single-stranded
restriction endonuclease recognition site (RERS) as described in
U.S. Pat. No. 5,846,726 and U.S. Pat. No. 5,919,630. In the
single-stranded reporter probe, the RERS is not cleavable. However,
in the presence of target, the single-stranded RERS is converted to
double-stranded form in a target-dependent primer extension
reaction and thereby becomes cleavable. Treatment with the
appropriate restriction endonuclease cleaves the RERS between the
two dyes, separating them into separate nucleic acid fragments. The
associated increase in distance between the dyes results in reduced
quenching of donor fluorescence which can be detected as an
indication of the presence of the target sequence. In a further
embodiment, an RERS reporter moiety may be rendered nickable in the
target-dependent primer extension reaction, as taught in U.S. Pat.
No. 5,846,726 and U.S. Pat. No. 5,919,630. In this embodiment, when
the RERS is rendered double-stranded the restriction endonuclease
nicks the strand to which the donor and quencher are linked.
Polymerase extends from the nick, displacing from the reporter
probe a single-stranded fragment linked to one of the dyes. This
also increases the distance between the donor and quencher and
results in an increase in donor fluorescence due to decreased
quenching.
[0019] One embodiment of the method of the invention as applied to
SDA is illustrated schematically in FIG. 1A. The initial steps of
the reaction correspond to the signal primer reaction described in
U.S. Pat. No. 5,547,861. A signal primer having a 3' target binding
sequence (B) and a noncomplementary 5' tail (A) hybridizes to the
target downstream from an amplification primer (S.sub.1) (Step 1).
As illustrated, the entire hybridization site of the signal primer
is downstream from the hybridization site of the amplification
primer. However, the hybridization sites of the signal primer and
the amplification primer on the target may also partially overlap
(typically only by several nulceotides) without significantly
affecting the methods of the invention. As used herein, the term
"downstream from" with respect to the hybridization sites of the
signal primer and the amplification primer on the target is
intended to encompass nonoverlapping and partially overlapping
sites in the target. Following hybridization to the target, the
amplification primer and the signal primer are simultaneously
extended on the target sequence, and extension of the amplification
primer displaces the single-stranded signal primer extension
product (Step 2). The second amplification primer (S.sub.2)
hybridizes to the signal primer extension product (Step 3) and both
the signal primer extension product and the amplification primer
are extended to produce a double-stranded secondary amplification
product with a hemimodified RERS at one end (Step 4). In SDA,
nicking of the unmodified S.sub.2 strand of the RERS (shown as an
arrow in Step 4) and displacement of the strand downstream from the
nick produces a single-stranded oligonucleotide which comprises the
complement of the signal primer (Step 5). The complement of the
signal primer and the double-stranded secondary amplification
product are produced only when the target is present and amplified.
They may therefore be detected as an indication of target
amplification.
[0020] In the detection method taught in U.S. Pat. No. 5,547,861,
the double-stranded secondary amplification product is detected. In
contrast, the present invention detects the single-stranded
oligonucleotide which is displaced from the double-stranded
secondary amplification product after nicking. As this
oligonucleotide comprises the complement of the signal primer, the
3' end of the reporter probe hybridizes to it (Step 6). The 5' end
of the reporter probe, containing the labeled structure or
sequence, forms an overhang with two recessed 3' ends which are
appropriate substrates for polymerase. If the reporter probe is not
capped to prevent extension, both the reporter probe and the
single-stranded oligonucleotide are extended to produce a
completely double-stranded molecule (Step 7). If the reporter probe
is not extendible, only the recessed 3' end of the single-stranded
oligonucleotide (which comprises the complement of the signal
primer) is extended and the product is partially single-stranded
and partially double-stranded. In either case, the sequence
complementary to the labeled structure or sequence of the reporter
probe is synthesized, rendering it double-stranded FIG. 1A
exemplifies the invention using a hairpin reporter moiety labeled
with a donor/quencher dye pair such that donor fluorescence is
quenched. It will be appreciated from this example that it may not
be necessary for the reporter moiety to be rendered entirely
double-stranded to be detected. For example, a partial complement
of the hairpin structure can be sufficient to keep the arms of the
stem from hybridizing to each other. As used herein,
"double-stranded reporter moiety" is intended to encompass both
fully and partially double-stranded reporter moieties provided they
are sufficiently double-stranded to render the reporter moiety
detectable. When the reporter moiety is rendered double-stranded in
the primer extension reaction, the hairpin is unfolded. Upon
unfolding, the two dyes become sufficiently spatially separated to
reduce or eliminate quenching of donor fluorescence by the
quencher. The resulting increase in donor fluorescence, or a change
in another fluorescence parameter associated with a change in
fluorescence quenching (such as fluorescence lifetime, fluorescence
polarization or a change in emission of the quencher/acceptor dye),
may be detected as an indication of amplification of the target
sequence. In addition, as illustrated in FIG. 1A, multiple reporter
moieties may be combined in a single reporter probe, for example a
labeled hairpin may comprise a single-stranded RERS in the
single-stranded "loop." In this embodiment synthesis of the
complement of the reporter moiety not only unfolds the hairpin to
produce an increase in fluorescence, the RERS concurrently becomes
cleavable or nickable, generally producing an additional
fluorescence increase.
[0021] As depicted in FIG. 1A, the folded reporter moiety (e.g., a
hairpin) of the reporter probe does not hybridize to the complement
of the adapter sequence. However, the adapter sequence may be
selected so that its complementary sequence will hybridize to all
or part of a folded reporter moiety of the reporter probe. In this
case, hybridization alone will unfold or partially unfold the
reporter moiety producing signal without the need for
polymerase-catalyzed extension following hybridization. The folded
reporter moiety in this embodiment may comprise all or part of the
reporter probe sequence. In an example of such an embodiment, the
reporter probe may be a molecular beacon as described by Tyagi and
Kramer, supra, in which the loop of the beacon hairpin comprises
all or part of the adapter sequence. As the complement of the
adapter sequence is synthesized during target amplification, it
binds to the molecular beacon and unfolds the structure, producing
increased fluorescence. In another embodiment the reporter probe
contains a single-stranded sequence 3' to the folded reporter
moiety such that both the single-stranded sequence and all or part
of the folded reporter moiety hybridize to the sequence
complementary to the adapter sequence as it is produced during
amplification.
[0022] In other alternative embodiments, other reporter moieties
may be substituted in the reaction scheme shown in FIG. 1A. For
example, other folded nucleic acid structures such as G-quartets
may be substituted and unfolded in a similar target-dependent
manner to reduce fluorescence quenching. Alternatively, a
specialized linear sequence may be used as the reporter moiety, for
example an RERS. When an RERS is used as the reporter moiety the
donor and quencher are linked flanking the cleavage site so that
when the RERS is rendered double-stranded and cleaved in a
target-dependent manner the two dyes are separated onto separate
nucleic acid fragments (Step 8, FIG. 1A). These alternative
secondary structures may also be combined with specialized
sequences, such as an RERS in a G-quartet. The RERS may
alternatively be rendered nickable rather than cleavable in its
double-stranded form. This is a particularly suitable embodiment
for use in SDA, as incorporation of modified nucleotides and
production of nickable RERS's are an integral part of the
amplification reaction. Generation of a nickable RERS in the
reporter probe adds some additional side reactions to the reaction
scheme of FIG. 1A (shown in Fig. 1B). Fig. 1B illustrates the
reaction if the RERS of the double-stranded molecule illustrated in
Step 7 of FIG. 1A is nicked rather than cleaved. Referring to Fig.
1B, as polymerase extends from the nick two products are produced:
the double-stranded molecule is regenerated (now carrying only one
of the two dyes) and the single-stranded molecule downstream from
the nick is displaced (Step 9, carrying the other of the two dyes).
The double-stranded molecule can be renicked with displacement of
additional single-stranded molecules and the displaced
single-stranded molecules hybridize to an amplification primer
(Step 10) and be extended to produce a nickable RERS in a fully
double-stranded molecule (Steps 11 and 12). Further nicking and
displacement produces single-stranded molecules with a partial RERS
derived from the previous reporter probe at one end and no label
(Step 13). This hybridizes to a new reporter probe (Step 14) and
the recessed end becomes extendible as the hairpin breathes and
allows the partial RERS to hybridize. Filling-in of the recessed
end renders the RERS nickable (Step 15) and the displaced
single-stranded molecule re-enters the reaction and the cycle
repeats. This amplifies the signal initially produced from a single
signal primer/target interaction by means of a separate reaction
occurring independently of any further target amplification.
[0023] In general, the length of the sequences involved in
intermolecular base-pairing between the complement of the adapter
sequence of the signal primer and the reporter probe is not
critical. For the signal primer, however, it has been observed that
in general the T.sub.m of the target binding sequence has a greater
influence on assay efficiency and that longer target binding
sequences generally produce more fluorescent signal in the assay.
This may be due to the competition between the signal primer and
the extension product of the upstream amplification primer for
hybridization to the target sequence. The appropriate length for
the signal primer and the reporter probe is determined by the
number of nucleotides required for stable base-pairing to maintain
a partially double-stranded molecule under the selected reaction
conditions and is within the ordinary skill in the art. For
convenience, the sequences involved in base-pairing are typically
between about 8 and 75 nucleotides in length. The maximum length is
limited only by practical concerns such as the ease and efficiency
of oligonucleotide synthesis and recovery.
[0024] Selection of the appropriate concentrations of signal primer
and reporter probe in the reaction is also within the ordinary
skill in the art. Preferably the concentration of signal primer and
reporter probe is relatively high and the concentration of upstream
amplification primer is relatively low, as this generally provides
higher fluorescent signal generation in the reaction.
[0025] A second signal primer which hybridizes to the second,
complementary strand of a double-stranded target sequence may
optionally be included in the reaction provided that the first and
second signal primers do not hybridize to each other. The second
signal primer hybridizes to the second strand of the target
sequence downstream of the second amplification primer and is
extended and displaced by extension of the second amplification
primer. The second signal primer extension product is rendered
double-stranded by hybridization and extension of the first
amplification primer. Generation of the double-stranded labeled
structure or sequence and separation of the dye pair proceed as for
the first strand of the target sequence. The second signal primer
preferably comprises the same 5' adapter sequence as the first
signal primer to allow detection of the products of amplification
of both target strands with a single reporter probe.
[0026] In addition, multiple signal primers per strand of target
may be employed if desired, each hybridizing to the target sequence
downstream of the other on the same strand, with all signal primers
being hybridized downstream of the amplification primer. In this
manner, each signal primer is displaced by extension of the
upstream detector nucleic acid and the most 5' signal primer is
displaced by the amplification primer. Use of multiple signal
primers has the advantage of increasing or amplifying the signal
generated per target, with an increase in sensitivity of the assay.
Again, it is preferable, but not necessary, that all of the signal
primers comprise the same 5' adapter sequence to allow detection of
all reaction products using a single reporter probe.
[0027] Multiple signal primers may also be used to simultaneously
detect a plurality of different target sequences. In this case, the
5' adapter sequences of the signal primers are preferably different
for each target to be detected. By labeling reporter probes
specific for the 5' adapter sequence of each target-specific signal
primer with donor/quencher dye pairs which are distinguishable, the
presence of each target may be determined by detecting changes in
the extent of fluorescence quenching in the reporter probe directed
to each target. This embodiment of the invention is particularly
useful for detection of single nucleotide sequence variations such
as are associated with certain disease states and conditions. The
target binding sequence of each signal primer may be selected to be
specific for a specific sequence variant of the target. Only those
signal primers which comprise the correct target binding sequence
for hybridization to the target will hybridize, be extended and
result in a complement of the adapter sequence being produced. The
reporter probe specific for that adapter sequence complement will
then produce a signal indicating which sequence variant(s) is/are
present by virtue of its distinguishing label.
[0028] Alternatively, for separate assay of multiple different
targets, the same 5' adapter sequence may be used in signal primers
directed to the multiple different target sequences. Specificity
for the different target sequences is conferred by varying the 3'
target binding sequence of the signal primer. This approach not
only simplifies the design and synthesis of signal primers, it
allows the same reporter probe to be used to detect any desired
target sequence. Commercially, this has the advantage that
production of only a single reporter probe is necessary to produce
assay systems for a variety of targets, thus lowering production
costs and simplifying the development of assays for new targets.
Further, synthesis of the various signal primers is simplified and
less expensive because they do not require labeling.
[0029] It will be apparent that, in addition to SDA, the signal
primers of the invention may be adapted for use in other primer
extension amplification methods (e.g., PCR, 3SR, TMA or NASBA). For
example, the methods may be adapted for use in PCR by substituting
PCR amplification primers and employing a strand displacing DNA
polymerase which lacks 5'.fwdarw.3' exonuclease activity (e.g.,
Sequencing Grade Taq from Promega or exo.sup.- Vent or exo.sup.-
Deep Vent from New England BioLabs) in the PCR. The signal primers
hybridize to the target downstream from the PCR amplification
primers. They are extended, displaced from the target and rendered
double-stranded essentially as described for SDA. The
single-stranded oligonucleotide comprising the complement of the
signal primer 5' adapter sequence is generated by denaturing the
double-stranded secondary amplification product, followed by
hybridization of the reporter probe and polymerase extension to
synthesize the complementary strand of the labeled reporter moiety
in the reporter probe. As in SDA systems, synthesis of the
complementary strand either directly or indirectly provides a
change in the proximity of donor and quencher dyes and changes the
degree of fluorescence quenching. An associated change in a
fluorescence parameter, such as intensity, serves as an indication
of target amplification.
[0030] For adaptation of the inventive methods to 3SR, TMA or
NASBA, a 5'.fwdarw.3' exonuclease deficient reverse transcriptase
with strand displacing activity is employed, with hybridization of
the signal primer to the RNA target downstream of an amplification
primer. In a reaction scheme similar to that previously described,
the hybridized signal primer is 1) extended, and 2) displaced by
extension of the upstream amplification primer. The displaced
signal primer extension product is then made entirely
double-stranded by hybridization and extension of the second
amplification primer which contains an RNA polymerase promoter. The
promoter sequence, which is located on the 5' tail of the second
amplification primer, is made double-stranded by extension of the
3' end of the signal primer extension product. From the
double-stranded promoter, RNA polymerase generates RNA copies
complementary to the signal primer extension product. The 3' end of
each RNA copy contains a sequence complementary to the adapter
sequence of the signal primer. This sequence then hybridizes to a
complementary region of the reporter probe. If the reporter probe
is extendible, reverse transcriptase will extend the 3' end of the
probe upon the RNA template to produce a reporter probe extension
product. RNase H will then degrade the RNA strand of this
heteroduplex, freeing the reporter probe extension product to
hybridize with the second amplification primer containing the
promoter sequence. Conversion of the promoter sequence to the
double-stranded form will initiate a new round of RNA synthesis,
yielding products that are complementary to the reporter probe
extension product, including the full reporter moiety sequence.
Hybridization of reporter probes to these RNA targets will cause
the reporter moiety to unfold, producing signal as donor and
quencher dyes are separated and quenching is reduced. In addition,
the reporter probes will be extended upon the RNA target as
described above and the cycle will be repeated.
[0031] If the reporter probes are not extendible (capped) the
adapter sequence of the signal primer must be selected to contain
sequences such that the complement of the adapter sequence will
hybridize to the reporter moiety of the reporter probe. The
reaction will proceed as described above, except that the capped
reporter probes will not be extended and the RNA complements of the
signal primer extension product will hybridize to the capped
reporter probe (including the reporter moiety). Signal will be
produced as the reporter moiety unfolds and quenching of donor
fluorescence is relieved during hybridization.
[0032] For reduced background, it is preferred that the signal
primers of the invention be used as described above, with the
signal primer extension product being separated from the target
sequence by displacement due to extension of the upstream
amplification primer. However, it will be apparent that the
amplification primers known for use in the various nucleic acid
amplification reactions may themselves be used for hybridization of
the reporter probe if the primers contain appropriate adapter
sequences. In this embodiment, the adapter sequence of an SDA
primer is located between the nickable restriction endonuclease
site that drives SDA and the target binding sequence. SDA with this
primer will produce an amplified product that contains at its 3'
end a sequence complementary to the reporter probe. Binding of the
reporter probe to this complementary sequence will produce signal
as described above. For PCR and NASBA the amplification primers are
modified by addition of a noncomplementary 5' tail as described
above for the signal primer. In the case of NASBA, the primer
lacking the RNA polymerase promoter is the primer modified with the
5' adapter sequence. During PCR and NASBA, complements of the
adapter-containing primer extension products are produced as
described above for the signal primers. These complementary
sequences are made single-stranded either by heat denaturation
(PCR) or enzymatic digestion of RNA template (RNase H in NASBA),
and the single-stranded complement then binds to reporter probe as
described above for signal primers. The use of amplification
primers as signal primers eliminates the need for the additional
signal primer in the reaction, but because background may be higher
in this embodiment the sensitivity of the assay may be
decreased.
[0033] In other alternative embodiments, the signal primers of the
invention may be used in non-amplification based assay formats to
detect target sequences. In a first non-target amplification
embodiment, the 3' single-stranded target binding sequence of the
signal primer hybridizes to the 3' end of the target sequence such
that the 5' adapter sequence forms a 5' overhang. The target
sequence functions as a primer for synthesis of a strand
complementary to the signal primer using a polymerase to extend the
target sequence using the 5' overhang as a template. If the target
binding sequence of the signal primer hybridizes to only a portion
of the target sequence, the target sequence also forms a 5'
overhang and the signal primer may be similarly extended using the
5' overhang of the target as a template. Alternatively, the signal
primer may be non-extendible as synthesis of a copy of the target
sequence is not required in this embodiment of the invention. In
either case, the complement of the adapter sequence of the signal
primer is synthesized. Upon separation of the two strands, the
complement of the signal primer adapter sequence in the target will
hybridize to the 3' end of the reporter probe, rendering the
labeled reporter moiety double-stranded upon polymerase extension
of the recessed 3' end of the adapter sequence complement. An
advantage of this embodiment over the reaction described in U.S.
Pat. No. 5,866,336 is that use of the overhang allows synthesis of
the complement of the adapter sequence in a single extension step
rather than two. That is, the complement of the adapter sequence is
appended directly to the original target, thus allowing target
detection without requiring amplification. In a second preferred
non-target amplification embodiment of the invention the signal
primer is hybridized to an internal sequence of the target with an
additional primer hybridized upstream to displace it (commonly
referred to as a "bumper" primer). The signal primer and bumper
primer are extended such that the signal primer extension product
is displaced from the target sequence. A second pair of primers are
hybridized to the extension product and extended such that the
downstream primer extension product contains the complement of the
adapter sequence and is displaced from the signal primer extension
product by extension of its bumper primer. The reporter probe
hybridizes to the complement of the adapter sequence and the
adapter sequence is extended as described herein to synthesize the
complement of the reporter moiety. Because this is an isothermal
reaction which depends on strand displacement to separate
complementary strands, extension of the first bumper primer renders
the target double-stranded and unable to participate in any further
reaction steps. Although a copy is generated and displaced, this is
not considered target amplification because the copy represents a
subsequence of the original target which is detected as an
indication of the presence of the target and only one copy of the
subsequence is generated per original target sequence.
[0034] The foregoing disclosure primarily relates to preferred
embodiments in which the reporter moiety is labeled with a
fluorescent donor/quencher dye pair and synthesis of the complement
of the reporter moiety is detected by an increase in fluorescence.
This label system allows synthesis of the complement to be detected
in real-time and/or in a homogeneous assay (i.e., without
separation of the label prior to detection). However, other labels
useful in the invention will be apparent to those skilled in the
art. For example, a single fluorescent label may be employed on the
reporter moiety with detection of a change in fluorescence
polarization in the presence of the complement of the reporter
moiety (see U.S. Pat. No. 5,593,867). Non-fluorescent labels are
also useful. For example, the reporter moiety may be labeled with a
lipophilic dye and contain a restriction site which is cleaved in
the presence of the complement of the reporter moiety (see U.S.
Pat. No. 5,550,025). Alternatively, the reporter probe may be
radiolabeled and the products resulting from synthesis of the
complement of the reporter moiety may be resolved by
electrophoresis and visualized by autoradiography. Immunological
labels may also be employed. A reporter probe labeled with a hapten
can be detected after synthesis of the complement of the reporter
moiety by first removing unreacted reporter probe (for example by
adapter-specific capture on a solid phase) and then detecting the
hapten label on the reacted reporter probe using standard
chemiluminescent or colorimetric ELISAs. A biotin label may be
substituted for the hapten and detected using methods known in the
art.
[0035] The label indicating the presence of the complement of the
reporter moiety may be detected at a selected endpoint in the
reaction. However, because oligonucleotides with increased distance
between the donor and the quencher are produced concurrently with
hybridization and primer extension, the label may also be monitored
as the reaction is occurring, i.e., in "real-time". This
homogeneous, real-time assay format can be used to provide
semi-quantitative or quantitative information about the initial
amount of target present. For example, the rate at which the label
(e.g., fluorescence intensity) changes during the reaction (either
as part of target amplification or in non-amplification detection
methods) is an indication of initial target levels. As a result,
when more initial copies of the target sequence are present, the
label more rapidly reaches a selected threshold value (i.e.,
shorter time to positivity). In addition, the rate of change in the
label during the course of the reaction is more rapid in samples
containing higher initial amounts of target than in samples
containing lower initial amounts of target. These or other
measurements as are known in the art may be made as an indication
of the presence of target or as an indication of target
amplification. The initial amount of target is typically determined
by comparison of the experimental results to results for known
amounts of target.
[0036] Many donor/quencher dye pairs known in the art are useful in
preferred embodiments of the present invention. These include, for
example, fluorescein isothiocyanate (FITC)/tetramethylrhodamine
isothiocyanate (TRITC), FITC/Texas Red.TM. (Molecular Probes),
FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB), FITC/eosin
isothiocyanate (EITC), N-hydroxysuccinimidyl 1-pyrenesulfonate
(PYS)/FITC, FITC/Rhodamine X, FITC/tetramethylrhodamine (TAMRA),
and others. The selection of a particular donor/quencher pair is
not critical. For energy transfer quenching mechanisms it is only
necessary that the emission wavelengths of the donor fluorophore
overlap the excitation wavelengths of the quencher, i.e., there
must be sufficient spectral overlap between the two dyes to allow
efficient energy transfer, charge transfer or fluorescence
quenching. P-(dimethyl aminophenylazo) benzoic acid (DABCYL) is a
non-fluorescent quencher dye which effectively quenches
fluorescence from an adjacent fluorophore, e.g., fluorescein or
5-(2'-aminoethyl) aminonaphthalene (EDANS). Certain donor/quencher
pairs are exemplified above and in the following Examples, however,
others will be apparent to those skilled in the art and are also
useful in the invention. Any dye pair which produces fluorescence
quenching in the reporter probes of the invention are suitable for
use in the methods of the invention, regardless of the mechanism by
which quenching occurs. Terminal and internal labeling methods are
also known in the art and may be routinely used to link the donor
and quencher dyes at their respective sites in the reporter
probe.
EXAMPLE 1
[0037] Strand Displacement Amplification reactions containing
signal primers according to the invention were run essentially as
described in U.S. Pat. No. 5,547,861 for detection of a synthetic
target sequence. A first reaction contained 10.sup.6 copies of the
target sequence, SDA amplification primers appropriate for
amplification of the synthetic target sequence, 100 nm of a signal
primer according to the invention comprising a target binding
sequence specific for the target and a 5' tail sequence identical
to the 3' sequence of a reporter probe, and 200 nm of the reporter
probe. The sequence of the reporter probe contained an RERS in the
5' region flanked by fluorescein and Rhodamine X (Rox) such that
fluorescence of fluorescein was quenched when the RERS was intact.
The sequences of the signal primer and reporter probe (shown in the
5' to 3' direction) are shown below. The target binding sequence is
shown in italics, the 5' adapter sequence of the signal primer and
the identical 3' sequence of the reporter probe are underlined and
the RERS of the reporter probe is bolded.
1 Signal Primer (SEQ ID NO:1): CCAAAATGACAGCTTCTGATGGAATGAC-
TCACTGAGTTGGAACGT Reporter Probe (SEQ ID NO:2):
(fluorescein)TACCTCGAGT(rox)GCAGCCAAAAGACAGCTTCTGATGGAA
[0038] A second reaction contained no target and the same signal
primer as in the first reaction. A third reaction was a control
reaction which contained only 10.sup.6 copies of target and the
reporter probe (i.e., no signal primer). Fluorescein fluorescence
was detected in real-time during the amplification reactions. As
shown in FIG. 2, donor fluorescence remained low and constant in
the absence of target, indicating quenching of fluorescence
throughout the reaction due failure of the RERS of the reporter
probe to be converted to double-stranded form and cleaved. In the
absence of signal primer donor fluorescence also remained quenched
throughout the amplification reaction. In the presence of target,
signal primer and reporter probe, however, donor fluorescence was
initially low but increased during the time course of the
amplification reaction as the RERS of the reporter probe was
converted to double-stranded form and cleaved to reduce the extent
of fluorescence quenching. These results demonstrate that the
signal primers and reporter probes of the invention can be used to
detect a nucleic acid target sequence by monitoring changes in the
extent of fluorescence quenching.
[0039] In a similar experiment, 0 and 250 copies of cloned HIV
target DNA were detected using a variety of signal primers in
combination with one of two reporter probes, each having the same
sequence but labeled with different donor/quencher dye pairs. The
sequences of the signal primers and reporter probes are shown in
the 5' to 3' direction below. The target binding sequence is shown
in italics, the 5' adapter sequence of the signal primer and the
identical 3' sequence of the reporter probe are underlined and the
RERS of the reporter probe is bolded.
2 Signal Primers: GAAAGACGTTAGCCACCATACGGATACCCCT-
TTTCTTTTAAAATTGTG (SEQ ID NO:3, UA1)
GAAAGACGTTAGCCACCATACGGATACCCCTTTTCTTTTAAAATTGTGGATG (SEQ ID NO:4,
UA2) GAAAGACGTTAGCCACCATACGGATACCCCTTTTCTTTTAAAATT (SEQ ID NO:5,
UA3) GAAAGACGTTAGCCACCATACGGATACCCCTTTTCTTTTAAAATTG (SEQ ID NO:6,
UA3.1) ACGTTAGCCACCATACGGATACCCCTTTTCTTTTAAAATTG- TG (SEQ ID NO:7,
UA4) ACGTTAGCCACCATACGGATACCCCTTTTCTTTTAA- AATTGTGGATG (SEQ ID
NO:8, UA5) ACGTTAGCCACCATACGGATACCCCTT- TTCTTTTAAAATT (SEQ ID NO:9,
UA6) ACGTTAGCCACCATACGGATACCCC- TTTTCTTTTAAAATTG (SEQ ID NO:10,
UA6.1) AGCCACCATACGGATACCCCTTTTCTTTTAAAATTGTG (SEQ ID NO:11, UA7)
AGCCACCATACGGATACCCCTTTTCTTTTAAAATTGTGGATG (SEQ ID NO:12, UA8)
AGCCACCATACGGATACCCCTTTTCTTTTAAAATT (SEQ ID NO:13, UA9)
AGCCACCATACGGATACCCCTTTTCTTTTAAAATTG (SEQ ID NO:14, UA9.1) Reporter
Probes (SEQ ID NO:15)
(fluorescein)TGCCCGAGT(dabcyl)GAAAGACGTTAGCCACCATACGGAT
(fluorescein)TGCCCGAGT(rox)GAAAGACGTTAGCCACCATACGGAT
[0040] The signal primers differed in length and T.sub.m of the
target binding sequence and of the reporter binding sequence.
Fluorescein fluorescence was monitored during amplification. To
compare the reporter probe/signal primer combinations, results were
expressed as the area under the fluorescence curve or "MOTA". The
more area under the curve, the more fluorescence generated by a
particular reporter probe/signal primer combination and the more
efficient the detection of amplified products. Both reporter probes
worked well in combination with all signal primers for detection of
the HIV target, although performance was generally not as good as
for reporter probes containing hairpin reporter moieties. However,
linear reporter probes such as these are shorter than reporter
probes containing secondary structures and are therefore easier to
synthesize with higher yield. Higher MOTA values were obtained
using the fluorescein-dabcyl reporter probe, suggesting that this
dye pair may have a higher quenching efficiency.
EXAMPLE 2
[0041] SDA reactions were prepared to contain the different signal
primers shown in Example 1, either 0 or 5,000 copies of the cloned
HIV target, and a reporter probe. The sequence of the reporter
probe was as follows:
3 (dabcyl)TAGTGCCCGAGCACT(rox)GAAAGACGTTAGCCACCATACGGAT (SEQ ID
NO:16, TBD9)
[0042] SEQ ID NO:16 contains a BsoBI RERS in the single-stranded
loop of a hairpin structure at the 5' end. The SDA reactions
contained 500 nm SDA amplification primers, 50 nM bumper primers,
and 200 nM each signal primers and reporter probes. Rhodamine
fluorescence was monitored during amplification. For each signal
primer/reporter probe combination rhodamine fluorescence increased
in the presence of target during the amplification reaction. In the
absence of target rhodamine fluorescence remained low throughout
the reaction. The results of one of the reactions are shown in FIG.
3A, for signal primer SEQ ID NO:3, with the multiple curves
representing replicate samples. Results indicated that the length
and T.sub.m of the adapter sequence did not significantly affect
assay performance. However, the T.sub.m of the target binding
sequence of the signal primer influenced signal generation, with
signal primers comprising longer target binding sequences
performing better than those with shorter target binding
sequences.
[0043] The experiment was repeated using three different reporter
probes, including SEQ ID NO:16.
[0044] The additional reporter probes were as follows:
4 (fluorescein)TAGTGCCCGAGCACT(dabcyl)ACGTTAGCCACCATACGGAT (SEQ ID
NO:17, TBD10) (fluorescein)TAGTGCCCGAGCACT(dabcyl)AGCCAC- CATACGGAT
(SEQ ID NO:18, TBD11)
[0045] In this experiment the concentration of the upstream
amplification primer was reduced to 100 nM. Amplification was
performed in the presence of either 0 or 250 copies of target DNA.
Reactions containing target showed a rapid increase in fluorescein
fluorescence after as little as 5 min. of incubation. In contrast,
reactions without target exhibited low fluorescein fluorescence
throughout the reaction period. Results for a reaction containing
SEQ ID NO:8 abd SEQ ID NO:17 are shown in FIG. 3B, with the
multiple curves representing replicate samples. The reporter
probe/signal primer combinations SEQ ID NO:16/SEQ ID NO:4 and SEQ
ID NO:17/SEQ ID NO:8 produced similar MOTA values (62,147 and
66,051 respectively), whereas the SEQ ID NO:18/SEQ ID NO:12
combination was less efficient (MOTA=49,879) suggesting less
efficient hybridization and conversion due to the shorter probe and
primer length.
EXAMPLE 3
[0046] In this experiment a reporter probe comprising a hairpin and
a nickable rather than cleavable BsoBI RERS was tested in SDA. The
reporter probe had the following sequence (SEQ ID NO:19,
TBD13.1):
5 (fluorescein)TAGTGCTCGGGCACT(dabcyl)GAAAGACGTTAGCCACCATACGGAT
[0047] This reporter probe was used with SEQ ID NO:4 as the signal
primer in the amplification reaction. A mean MOTA value of 48,000
was obtained in the presence of 250 copies of HIV target DNA,
compared with a score of less than 150 from negative controls. The
lower MOTA score observed as compared to reporter probe SEQ ID
NO:16, which has the same 3' tail sequence may be due to
inefficient priming of the polymerase off the short oligonucleotide
that is left after nicking of the BsoBI site. Performance of the
reaction may be enhanced by increasing the length of the hairpin to
stabilize this oligonucleotide and provide a larger region for
binding of the polymerase.
EXAMPLE 4
[0048] In this experiment SDA was performed using a reporter probe
containing a G-quartet structure and an RERS as the reporter
moiety. This reporter probe had the following sequence (SEQ ID
NO:20, TBD14):
6
(fluorescein)GGTTGGCTCGAGGTTGGT(dabcyl)GAAAGACGTTAGCCACCATACGGAT
[0049] An increase in fluorescein fluorescence was observed during
the course of amplification of 250 copies of HIV target DNA. No
such increase in fluorescence was observed in the absence of
target.
Sequence CWU 1
1
20 1 45 DNA Artificial Sequence Description of Artificial
SequenceSynthetic sequence for experimental model 1 ccaaaatgac
agcttctgat ggaatgactc actgagttgg aacgt 45 2 37 DNA Artificial
Sequence Description of Artificial SequenceSynthetic sequence for
experimental model 2 tacctcgagt gcagccaaaa gacagcttct gatggaa 37 3
48 DNA Human immunodeficiency virus 3 gaaagacgtt agccaccata
cggatacccc ttttctttta aaattgtg 48 4 52 DNA Human immunodeficiency
virus 4 gaaagacgtt agccaccata cggatacccc ttttctttta aaattgtgga tg
52 5 45 DNA Human immunodeficiency virus 5 gaaagacgtt agccaccata
cggatacccc ttttctttta aaatt 45 6 46 DNA Human immunodeficiency
virus 6 gaaagacgtt agccaccata cggatacccc ttttctttta aaattg 46 7 43
DNA Human immunodeficiency virus 7 acgttagcca ccatacggat accccttttc
ttttaaaatt gtg 43 8 47 DNA Human immunodeficiency virus 8
acgttagcca ccatacggat accccttttc ttttaaaatt gtggatg 47 9 40 DNA
Human immunodeficiency virus 9 acgttagcca ccatacggat accccttttc
ttttaaaatt 40 10 41 DNA Human immunodeficiency virus 10 acgttagcca
ccatacggat accccttttc ttttaaaatt g 41 11 38 DNA Human
immunodeficiency virus 11 agccaccata cggatacccc ttttctttta aaattgtg
38 12 42 DNA Human immunodeficiency virus 12 agccaccata cggatacccc
ttttctttta aaattgtgga tg 42 13 35 DNA Human immunodeficiency virus
13 agccaccata cggatacccc ttttctttta aaatt 35 14 36 DNA Human
immunodeficiency virus 14 agccaccata cggatacccc ttttctttta aaattg
36 15 34 DNA Human immunodeficiency virus 15 tgcccgagtg aaagacgtta
gccaccatac ggat 34 16 40 DNA Human immunodeficiency virus 16
tagtgcccga gcactgaaag acgttagcca ccatacggat 40 17 35 DNA Human
immunodeficiency virus 17 tagtgcccga gcactacgtt agccaccata cggat 35
18 30 DNA Human immunodeficiency virus 18 tagtgcccga gcactagcca
ccatacggat 30 19 40 DNA Human immunodeficiency virus 19 tagtgctcgg
gcactgaaag acgttagcca ccatacggat 40 20 43 DNA Human
immunodeficiency virus 20 ggttggctcg aggttggtga aagacgttag
ccaccatacg gat 43
* * * * *