U.S. patent application number 09/902561 was filed with the patent office on 2002-05-16 for uv excitable fluorescent energy transfer dyes.
Invention is credited to Lee, Linda G..
Application Number | 20020058272 09/902561 |
Document ID | / |
Family ID | 23521062 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058272 |
Kind Code |
A1 |
Lee, Linda G. |
May 16, 2002 |
UV excitable fluorescent energy transfer dyes
Abstract
Novel energy transfer dyes which can be used with shorter
wavelength light sources are provided. These dyes include a donor
dye with an absorption maxima at a wavelength between about 250 to
450 nm and an acceptor dye which is capable of absorbing energy
emitted from the donor dye. One of the energy transfer dyes has a
donor dye which is a member of a class of dyes having a coumarin or
pyrene ring structure and an acceptor dye which is capable of
absorbing energy emitted from the donor dye, wherein the donor dye
has an absorption maxima between about 250 and 450 nm and the
acceptor dye has an emission maxima at a wavelength greater than
about 500 nm.
Inventors: |
Lee, Linda G.; (Palo Alto,
CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
943041050
|
Family ID: |
23521062 |
Appl. No.: |
09/902561 |
Filed: |
July 10, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09902561 |
Jul 10, 2001 |
|
|
|
09385352 |
Aug 27, 1999 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
536/24.3; 536/25.32 |
Current CPC
Class: |
C12Q 2563/107 20130101;
C12Q 1/6869 20130101; C12Q 1/6869 20130101; C07H 19/20 20130101;
C12Q 1/6818 20130101; C07H 21/04 20130101; C07H 21/00 20130101;
Y10T 436/143333 20150115; C09B 11/24 20130101; C07H 19/10
20130101 |
Class at
Publication: |
435/6 ; 536/24.3;
536/25.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. An energy transfer dye comprising: a donor dye having an
absorption maxima at a wavelength between about 250 to 450 nm; and
an acceptor dye capable of absorbing excitation energy emitted by
the donor dye and emitting light in response, the acceptor dye
having an emission maxima greater than about 500 nm.
2. The energy transfer dye according to claim 1 wherein the donor
dye has an absorption maxima at a wavelength between about 300 and
450 nm.
3. The energy transfer dye according to claim 1 wherein the donor
dye has an absorption maxima at a wavelength between about 350 and
400 nm.
4. The energy transfer dye according to claim 1 wherein the
acceptor dye has an emission maxima at a wavelength greater than
about 550 nm.
5. The energy transfer dye according to claim 1 wherein the
acceptor dye has an emission maxima at a wavelength between about
500 and 700 nm.
6. The energy transfer dye according to claim 1 wherein the
acceptor dye has an emission maxima at a wavelength at least about
150 nm greater than the absorption maxima of the donor dye.
7. The energy transfer dye according to claim 1 wherein the
acceptor dye is a member of a class of dyes selected from the group
consisting of fluorescein, rhodamine, asymmetric benzoxanthene,
xanthene, cyanine, phthalocyanine and squaraine dyes.
8. The energy transfer dye according to claim 1 wherein the
acceptor dye is selected from the group consisting of
4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes,
rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines,
N,N,N',N'-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G,
carboxy-X-rhodamines and Cy5.
9. The energy transfer dye according to claim 1 wherein the
acceptor dye is selected from the group consisting of R110, RG6,
TAMRA and ROX.
10. The energy transfer dye according to claim 1 further comprising
a linker attaching the donor dye to the acceptor dye.
11. The energy transfer dye according to claim 10 wherein the
linker linking the donor dye to the acceptor dye is such that the
acceptor dye absorbs substantially all of the excitation energy
emitted by the donor dye, the linker including a functional group
selected from the group consisting of an alkene, diene, alkyne, a
five and six membered ring having at least one unsaturated bond or
a fused ring structure.
12. The energy transfer dye according to claim 11 wherein the
linker functional group is a five or six membered ring selected
from the group consisting of cyclopentene, cyclohexene,
cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,
isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone,
benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,
indene, benzofuran, thionaphthene, indole and naphthalene.
13. An energy transfer dye comprising: a donor dye which is a
member of a class of dyes having a coumarin or pyrene ring
structure; and an acceptor dye capable of absorbing excitation
energy emitted by the donor dye and emitting light in response, the
acceptor dye having an emission maxima greater than about 500
nm.
14. The energy transfer dye according to claim 13 wherein the donor
dye has an absorption maxima at a wavelength between about 250 and
450 nm.
15. The energy transfer dye according to claim 13 wherein the donor
dye has an absorption maxima at a wavelength between about 300 and
450 nm.
16. The energy transfer dye according to claim 13 wherein the donor
dye has an absorption maxima at a wavelength between about 350 and
400 nm.
17. The energy transfer dye according to claim 13 wherein the
acceptor dye has an emission maxima at a wavelength greater than
about 550 nm.
18. The energy transfer dye according to claim 13 wherein the
acceptor dye has an emission maxima at a wavelength between about
500 and 700 nm.
19. The energy transfer dye according to claim 13 wherein the
acceptor dye has an emission maxima at a wavelength at least about
150 nm greater than the absorption maxima of the donor dye.
20. The energy transfer dye according to claim 13 wherein the donor
dye is selected from the group consisting of coumarin, pyrene and
pyrene sulfonate.
21. The energy transfer dye according to claim 13 wherein the
acceptor dye is a member of a class of dyes selected from the group
consisting of fluorescein, rhodamine, asymmetric benzoxanthene,
xanthene, cyanine, phthalocyanine and squaraine dyes.
22. The energy transfer dye according to claim 13 wherein the
acceptor dye is selected from the group consisting of
4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes,
rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines,
N,N,N',N'-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G,
carboxy-X-rhodamines and Cy5.
23. The energy transfer dye according to claim 13 wherein the
acceptor dye is selected from the group consisting of R110, RG6,
TAMRA and ROX.
24. The energy transfer dye according to claim 13 further
comprising a linker attaching the donor dye to the acceptor
dye.
25. The energy transfer dye according to claim 24 wherein the
linker linking the donor dye to the acceptor dye is such that the
acceptor dye absorbs substantially all of the excitation energy
emitted by the donor dye, the linker including a functional group
selected from the group consisting of an alkene, diene, alkyne, a
five and six membered ring having at least one unsaturated bond or
a fused ring structure.
26. The energy transfer dye according to claim 25 wherein the
linker functional group is a five or six membered ring selected
from the group consisting of cyclopentene, cyclohexene,
cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,
isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone,
benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,
indene, benzofuran, thionaphthene, indole and naphthalene.
27. An energy transfer dye selected from the group consisting of
DYE102, DYE104, DYE106, DYE108, DYE118 and DYE120.
28. A fluorescently labeled reagent comprising: a reagent selected
from the group consisting of a nucleoside, nucleoside
monophosphate, nucleoside diphosphate, nucleoside triphosphate,
oligonucleotide and oligonucleotide analog, modified to be linked
to an energy transfer fluorescent dye; and an energy transfer
fluorescent dye attached to the reagent, the energy transfer
fluorescent dye including a donor dye having an absorption maxima
at a wavelength between about 250 to 450 nm; and an acceptor dye
capable of absorbing excitation energy emitted by the donor dye and
emitting light in response, the acceptor dye having an emission
maxima greater than about 500 nm.
29. The energy transfer dye according to claim 28 wherein the donor
dye has an absorption maxima at a wavelength between about 300 and
450 nm.
30. The fluorescently labeled reagent according to claim 28 wherein
the donor dye has an absorption maxima at a wavelength between
about 350 and 400 nm.
31. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye has an emission maxima at a wavelength greater
than about 550 nm.
32. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye has an emission maxima at a wavelength between
about 500 and 700 nm.
33. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye has an emission maxima at a wavelength at least
about 150 nm greater than the absorption maxima of the donor
dye.
34. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye is is a member of a class of dyes selected from
the group consisting of fluorescein, rhodamine, asymmetric
benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine
dyes.
35. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye is selected from the group consisting of
4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes,
rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines,
N,N,N',N'-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G,
carboxy-X-rhodamines and Cy5.
36. The fluorescently labeled reagent according to claim 28 wherein
the acceptor dye is selected from the group consisting of R110,
RG6, TAMRA and ROX.
37. The fluorescently labeled reagent according to claim 28 further
comprising a linker attaching the donor dye to the acceptor
dye.
38. The fluorescently labeled reagent according to claim 37 wherein
the linker linking the donor dye to the acceptor dye is such that
the acceptor dye absorbs substantially all of the excitation energy
emitted by the donor dye, the linker including a functional group
selected from the group consisting of an alkene, diene, alkyne, a
five and six membered ring having at least one unsaturated bond or
a fused ring structure.
39. The fluorescently labeled reagent according to claim 38 wherein
the linker functional group is a five or six membered ring selected
from the group consisting of cyclopentene, cyclohexene,
cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,
isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone,
benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,
indene, benzofuran, thionaphthene, indole and naphthalene.
40. The fluorescently labeled reagent according to claim 28 wherein
the reagent is selected from the group consisting of
deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside
diphosphate and deoxynucleoside triphosphate.
41. The fluorescently labeled reagent according to claim 40 wherein
the deoxynucleotides are selected from the group consisting of
deoxycytosine, deoxyadenosine, deoxyguanosine, and
deoxythymidine.
42. The fluorescently labeled reagent according to claim 28 wherein
the reagent is selected from the group consisting of
dideoxynucleoside, dideoxynucleoside monophosphate,
dideoxynucleoside diphosphate and dideoxynucleoside
triphosphate.
43. The fluorescently labeled reagent according to claim 42 wherein
the dideoxynucleotides are selected from the group consisting of
deoxycytosine, deoxyadenosine, deoxyguanosine, and
deoxythymidine.
44. The fluorescently labeled reagent according to claim 28 wherein
the reagent is an oligonucleotide.
45. The fluorescently labeled reagent according to claim 44 wherein
the oligonucleotide has a 3' end which is extendable by using a
polymerase.
46. A fluorescently labeled reagent comprising: a reagent selected
from the group consisting of a nucleoside, nucleoside
monophosphate, nucleoside diphosphate, nucleoside triphosphate,
oligonucleotide and oligonucleotide analog, modified to be linked
to an energy transfer fluorescent dye; and an energy transfer
fluorescent dye attached to the reagent, the energy transfer
fluorescent dye including a donor dye which is a member of a class
of dyes having a coumarin or pyrene ring structure; and an acceptor
dye capable of absorbing excitation energy emitted by the donor dye
and emitting light in response, the acceptor dye having an emission
maxima greater than about 500 nm.
47. The fluorescently labeled reagent according to claim 46 wherein
the donor dye has an absorption maxima at a wavelength between
about 250 and 450 nm.
48. The fluorescently labeled reagent according to claim 46 wherein
the donor dye has an absorption maxima at a wavelength between
about 300 and 450 nm.
49. The fluorescently labeled reagent according to claim 46 wherein
the donor dye has an absorption maxima at a wavelength between
about 350 and 400 nm.
50. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye has an emission maxima at a wavelength greater
than about 550 nm.
51. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye has an emission maxima at a wavelength between
about 500 and 700 nm.
52. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye has an emission maxima at a wavelength at least
about 150 nm greater than the absorption maxima of the donor
dye.
53. The fluorescently labeled reagent according to claim 46 wherein
the donor dye is selected from the group consisting of coumarin,
pyrene and pyrene sulfonate.
54. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye is a member of a class of dyes selected from the
group consisting of fluorescein, rhodamine, asymmetric
benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine
dyes.
55. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye is selected from the group consisting of
4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes,
rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines,
N,N,N',N'-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G,
carboxy-X-rhodamines and Cy5.
56. The fluorescently labeled reagent according to claim 46 wherein
the acceptor dye is selected from the group consisting of R110,
RG6, TAMRA and ROX.
57. The fluorescently labeled reagent according to claim 46 further
comprising a linker attaching the donor dye to the acceptor
dye.
58. The fluorescently labeled reagent according to claim 57 wherein
the linker linking the donor dye to the acceptor dye is such that
the acceptor dye absorbs substantially all of the excitation energy
emitted by the donor dye, the linker including a functional group
selected from the group consisting of an alkene, diene, alkyne, a
five and six membered ring having at least one unsaturated bond or
a fused ring structure.
59. The fluorescently labeled reagent according to claim 58 wherein
the linker functional group is a five or six membered ring selected
from the group consisting of cyclopentene, cyclohexene,
cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole,
isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone,
benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine,
indene, benzofuran, thionaphthene, indole and naphthalene.
60. The fluorescently labeled reagent according to claim 46 wherein
the reagent is selected from the group consisting of
deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside
diphosphate and deoxynucleoside triphosphate.
61. The fluorescently labeled reagent according to claim 60 wherein
the deoxynucleotides are selected from the group consisting of
deoxycytosine, deoxyadenosine, deoxyguanosine, and
deoxythymidine.
62. The fluorescently labeled reagent according to claim 46 wherein
the reagent is selected from the group consisting of
dideoxynucleoside, dideoxynucleoside monophosphate,
dideoxynucleoside diphosphate and dideoxynucleoside
triphosphate.
63. The fluorescently labeled reagent according to claim 62 wherein
the dideoxynucleotides are selected from the group consisting of
deoxycytosine, deoxyadenosine, deoxyguanosine, and
deoxythymidine.
64. The fluorescently labeled reagent according to claim 46 wherein
the reagent is an oligonucleotide.
65. The fluorescently labeled reagent according to claim 64 wherein
the oligonucleotide has a 3' end which is extendable by using a
polymerase.
66. A fluorescently labeled reagent comprising: a reagent selected
from the group consisting of a nucleoside, nucleoside
monophosphate, nucleoside diphosphate, nucleoside triphosphate,
oligonucleotide and oligonucleotide analog, modified to be linked
to an energy transfer fluorescent dye; and an energy transfer
fluorescent dye attached to the reagent, the energy transfer
fluorescent dye being selected from the group consisting of DYE102,
DYE104, DYE106, DYE108, DYE118 and DYE120.
67. A method for sequencing a nucleic acid sequence comprising:
forming a mixture of extended labeled primers by hybridizing a
nucleic acid sequence with a fluorescently labeled oligonucleotide
primer in the presence of deoxynucleoside triphosphates, at least
one dideoxynucleoside triphosphate and a DNA polymerase, the DNA
polymerase extending the primer with the deoxynucleoside
triphosphates until a dideoxynucleoside triphosphate is
incorporated which terminates extension of the primer; separating
the mixture of extended primers; and determining the sequence of
the nucleic acid sequence by fluorescently measuring the mixture of
extended primers formed; the fluorescently labeled oligonucleotide
primer including an oligonucleotide sequence complementary to a
portion of the nucleic acid sequence being sequenced and having a
3' end extendable by a polymerase, and an energy transfer
fluorescent dye attached to the oligonucleotide, the energy
transfer fluorescent dye including a donor dye having an absorption
maxima at a wavelength between about 250 to 450 nm; and an acceptor
dye capable of absorbing excitation energy emitted by the donor dye
and emitting light in response, the acceptor dye having an emission
maxima greater than about 500 nm.
68. A method for sequencing a nucleic acid sequence comprising:
forming a mixture of extended labeled primers by hybridizing a
nucleic acid sequence with a fluorescently labeled oligonucleotide
primer in the presence of deoxynucleoside triphosphates, at least
one dideoxynucleoside triphosphate and a DNA polymerase, the DNA
polymerase extending the primer with the deoxynucleoside
triphosphates until a dideoxynucleoside triphosphate is
incorporated which terminates extension of the primer; separating
the mixture of extended primers; and determining the sequence of
the nucleic acid sequence by fluorescently measuring the mixture of
extended primers formed, the fluorescently labeled oligonucleotide
primer including an oligonucleotide sequence complementary to a
portion of the nucleic acid sequence being sequenced and having a
3' end extendable by a polymerase, and an energy transfer
fluorescent dye attached to the oligonucleotide, the energy
transfer fluorescent dye including a donor dye which is a member of
a class of dyes having a coumarin or pyrene ring structure; and an
acceptor dye capable of absorbing excitation energy emitted by the
donor dye and emitting light in response, the acceptor dye having
an emission maxima greater than about 500 nm.
69. A method for sequencing a nucleic acid sequence comprising:
forming a mixture of extended primers by hybridizing a nucleic acid
sequence with a primer in the presence of deoxynucleoside
triphosphates, at least one fluorescently labeled dideoxynucleoside
triphosphate and a DNA polymerase, the DNA polymerase extending the
primer with the deoxynucleoside triphosphates until a fluorescently
labeled dideoxynucleoside triphosphate is incorporated onto the
extended primer which terminates extension of the primer;
separating the mixture of extended primers; and determining the
sequence of the nucleic acid sequence by detecting the
fluorescently labeled dideoxynucleotide attached to the separated
mixture of extended primers; the fluorescently labeled
dideoxynucleoside triphosphate including a dideoxynucleoside
triphosphate, and an energy transfer fluorescent dye attached to
the dideoxynucleoside triphosphate, the energy transfer dye
including a donor dye having an absorption maxima at a wavelength
between about 250 to 450 nm; and an acceptor dye capable of
absorbing excitation energy emitted by the donor dye and emitting
light in response, the acceptor dye having an emission maxima
greater than about 500 nm.
70. A method for sequencing a nucleic acid sequence comprising:
forming a mixture of extended primers by hybridizing a nucleic acid
sequence with a primer in the presence of deoxynucleoside
triphosphates, at least one fluorescently labeled dideoxynucleoside
triphosphate and a DNA polymerase, the DNA polymerase extending the
primer with the deoxynucleoside triphosphates until a fluorescently
labeled dideoxynucleoside triphosphate is incorporated onto the
extended primer which terminates extension of the primer;
separating the mixture of extended primers; and determining the
sequence of the nucleic acid sequence by detecting the
fluorescently labeled dideoxynucleotide attached to the separated
mixture of extended primers; the fluorescently labeled
dideoxynucleoside triphosphate including a dideoxynucleoside
triphosphate, and an energy transfer fluorescent dye attached to
the dideoxynucleoside triphosphate, the energy transfer dye
including a donor dye which is a member of a class of dyes having a
coumarin or pyrene ring structure; and an acceptor dye capable of
absorbing excitation energy emitted by the donor dye and emitting
light in response, the acceptor dye having an emission maxima
greater than about 500 nm.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to fluorescent dyes and, more
specifically, energy transfer fluorescent dyes and their use.
[0003] 2. Description of Related Art
[0004] A variety of fluorescent dyes have been developed for
labeling and detecting components in a sample. In general,
fluorescent dyes preferably have a high quantum yield and a large
extinction coefficient so that the dye may be used to detect small
quantities of the component being detected. Fluorescent dyes also
preferably have a large Stokes' shift (i.e., the difference between
the wavelength at which the dye has maximum absorbance and the
wavelength at which the dye has maximum emission) so that the
fluorescent emission is readily distinguished from the light source
used to excite the dye.
[0005] One class of fluorescent dyes which has been developed is
energy transfer fluorescent dyes. In general, energy transfer
fluorescent dyes include a donor fluorophore and an acceptor
fluorophore. In these dyes, when the donor and acceptor
fluorophores are positioned in proximity with each other and with
the proper orientation relative to each other, the energy emission
from the donor fluorophore is absorbed by the acceptor fluorophore
and causes the acceptor fluorophore to fluoresce. It is therefore
important that the excited donor fluorophore be able to efficiently
absorb the excitation energy of the donor fluorophore and
efficiently transfer the energy to the acceptor fluorophore.
[0006] A variety of energy transfer fluorescent dyes have been
described in the literature. For example, U.S. Pat. No. 4,996,143
and WO 95/21266 describe energy transfer fluorescent dyes where the
donor and acceptor fluorophores are linked by an oligonucleotide
chain. Lee, et al., Nucleic Acids Research 20:10 2471-2483 (1992)
describes an energy transfer fluorescent dye which includes
5-carboxy rhodamine linked to 4'-aminomethyl-5-carboxy fluorescein
by the 4'-aminomethyl substituent on fluorescein. U.S Pat. No.
5,847,162 describes additional classes of energy transfer dyes.
[0007] Several diagnostic and analytical assays have been developed
which involve the detection of multiple components in a sample
using fluorescent dyes, e.g. flow cytometry (Lanier, et al., J.
Immunol. 132 151-156 (1984)); chromosome analysis (Gray, et al.,
Chromosoma 73 9-27 (1979)); and DNA sequencing. For these assays,
it is desirable to simultaneously employ a set of two or more
spectrally resolvable fluorescent dyes so that more than one target
substance can be detected in the sample at the same time.
Simultaneous detection of multiple components in a sample using
multiple dyes reduces the time required to serially detect
individual components in a sample. In the case of multi-loci DNA
probe assays, the use of multiple spectrally resolvable fluorescent
dyes reduces the number of reaction tubes that are needed, thereby
simplifying the experimental protocols and facilitating the
manufacturing of application-specific kits. In the case of
automated DNA sequencing, the use of multiple spectrally resolvable
fluorescent dyes allows for the analysis of all four bases in a
single lane thereby increasing throughput over single-color methods
and eliminating uncertainties associated with inter-lane
electrophoretic mobility variations. Connell, et al., Biotechniques
5 342-348 (1987); Prober, et al., Science 238 336-341 (1987),
Smith, et al., Nature 321 674-679 (1986); and Ansorge, et al.,
Nucleic Acids Research 15 4593-4602 (1989).
[0008] There are several difficulties associated with obtaining a
set of fluorescent dyes for simultaneously detecting multiple
target substances in a sample, particularly for analyses requiring
an electrophoretic separation and treatment with enzymes, e.g., DNA
sequencing. For example, each dye in the set must be spectrally
resolvable from the other dyes. It is difficult to find a
collection of dyes whose emission spectra are spectrally resolved,
since the typical emission band half-width for organic fluorescent
dyes is about 40-80 nanometers (nm) and the width of the available
spectrum is limited by the excitation light source. As used herein
the term "spectral resolution" in reference to a set of dyes means
that the fluorescent emission bands of the dyes are sufficiently
distinct, i.e., sufficiently non-overlapping, that reagents to
which the respective dyes are attached, e.g. polynucleotides, can
be distinguished on the basis of the fluorescent signal generated
by the respective dyes using standard photodetection systems, e.g.
employing a system of band pass filters and photomultiplier tubes,
charged-coupled devices and spectrographs, or the like, as
exemplified by the systems described in U.S. Pat. Nos. 4,230,558,
4,811,218, or in Wheeless et al, pgs. 21-76, in Flow Cytometry:
Instrumentation and Data Analysis (Academic Press, New York,
1985).
[0009] The fluorescent signal of each of the dyes must also be
sufficiently strong so that each component can be detected with
sufficient sensitivity. For example, in the case of DNA sequencing,
increased sample loading can not compensate for low fluorescence
efficiencies, Pringle et al., DNA Core Facilities Newsletter, 1
15-21 (1988). The fluorescent signal generated by a dye is
generally greatest when the dye is excited at its absorbance
maximum. It is therefore preferred that each dye be excited at
about its absorbance maximum.
[0010] A further difficulty associated with the use of a set of
dyes is that the dyes generally do not have the same absorbance
maximum. When a set of dyes are used which do not have the same
absorbance maximum, a trade off is created between the higher cost
associated with providing multiple light sources to excite each dye
at its absorbance maximum, and the lower sensitivity arising from
each dye not being excited at its absorbance maximum.
[0011] In addition to the above difficulties, the charge, molecular
size, and conformation of the dyes must not adversely affect the
electrophoretic mobilities of the fragments. The fluorescent dyes
must also be compatible with the chemistry used to create or
manipulate the fragments, e.g., DNA synthesis solvents and
reagents, buffers, polymerase enzymes, ligase enzymes, and the
like.
[0012] Because of the multiple constraints on developing a set of
dyes for multicolor applications, particularly in the area of four
color DNA sequencing, only a few sets of fluorescent dyes have been
developed. Connell, et al., Biotechniques 5 342-348 (1987); Prober,
et al., Science 238 336-341 (1987); and Smith, et al., Nature 321
674-679 (1986); and U.S. Pat. No. 5,847,162.
[0013] Energy transfer fluorescent dyes possess several features
which make them attractive for use in the simultaneous detection of
multiple target substances in a sample, such as in DNA sequencing.
For example, a single donor fluorophore can be used in a set of
energy transfer fluorescent dyes so that each dye has strong
absorption at a common wavelength. Then, by varying the acceptor
fluorophore in the energy transfer dye, a series of energy transfer
dyes having spectrally resolvable fluorescence emissions can be
generated.
[0014] Energy transfer fluorescent dyes also provide a larger
effective Stokes' shift than non-energy transfer fluorescent dyes.
This is because the Stokes' shift for an energy transfer
fluorescent dye is based on the difference between the wavelength
at which the donor fluorophore maximally absorbs light and the
wavelength at which the acceptor fluorophore maximally emits light.
In general, a need exists for fluorescent dyes having larger
Stokes' shifts.
[0015] The sensitivity of any assay using a fluorescent dye is
dependent on the strength of the fluorescent signal generated by
the fluorescent dye. A need therefore exists for fluorescent dyes
which have a strong fluorescence signal. With regard to energy
transfer fluorescent dyes, the fluorescence signal strength of
these dyes is dependent on how efficiently the acceptor fluorophore
absorbs the energy emission of the donor fluorophore.
SUMMARY OF THE INVENTION
[0016] The present invention relates to energy transfer dyes which
can be used with shorter wavelength light sources. The present
invention also relates to reagents which include the energy
transfer dyes of the present invention. The present invention also
relates to methods which use dyes and reagents adapted to shorter
wavelength light sources. Kits are also provided which include the
dyes and reagents.
[0017] Energy transfer dyes are provided which include a donor dye
with an absorption maxima at a wavelength between about 250 to 450
nm and an acceptor dye which is capable of absorbing energy from
the donor dye.
[0018] It is noted that energy transfer may occur by a variety of
mechanisms. For example, the emission of the donor dye does not
need to overlap with the absorbance of the acceptor dye for many of
the dyes of the present invention.
[0019] In one variation, the donor dye has an absorption maxima
between about 300 and 450 nm, more preferably between about 350 and
400 nm.
[0020] The acceptor dye preferably has an emission maxima greater
than about 500 nm. In one variation, the acceptor dye has an
emission maxima at a wavelength greater than about 550 nm. The
acceptor dye may also have an emission maxima at a wavelength
between about 500 and 700 nm. The acceptor dye may also be selected
relative to the donor dye such that the acceptor dye has an
emission maxima at a wavelength at least about 150 nm greater than
the absorption maxima of the donor dye.
[0021] In another embodiment of the present invention, the energy
transfer dye has a donor dye which is a member of a class of dyes
having a coumarin or pyrene ring structure and an acceptor dye
which is capable of absorbing energy from the donor dye.
[0022] In one variation of this embodiment, the donor dye has an
absorption maxima between about 250 and 450 nm, preferably between
about 300 and 450 nm, and more preferably between about 350 and 400
nm.
[0023] In another variation of this embodiment, the acceptor dye
has an emission maxima at a wavelength greater than about 500 nm,
and optionally more than 550 nm. The acceptor dye may also have an
emission maxima at a wavelength between about 500 and 700 nm. The
acceptor dye may also be selected relative to the donor dye such
that the acceptor dye has an emission maxima at a wavelength at
least about 150 nm greater than the absorption maxima of the donor
dye.
[0024] An energy transfer dye according to the present invention
may also have the structure of "antennae" dyes or dendrimers in
which large numbers of donor dyes are coupled to one acceptor dye
where the donor dye either has an absorption maxima between 250 and
450 nm or has a coumarin or pyrene ring structure.
[0025] The present invention also relates to fluorescent reagents
containing any of the energy transfer dyes of the present
invention. In general, these reagents include any molecule or
material to which the energy transfer dyes of the invention can be
attached. The presence of the reagent is detected by the
fluorescence of the energy transfer dye. One use of the reagents of
the present invention is in nucleic acid sequencing.
[0026] Examples of classes of the fluorescent reagents include
deoxynucleosides and mono-, di- or triphosphates of a
deoxynucleoside labeled with an energy transfer dye. Examples of
deoxynucleotides include deoxycytosine, deoxyadenosine,
deoxyguanosine or deoxythymidine, and analogs and derivatives
thereof.
[0027] Other classes of the reagents include analogs and
derivatives of deoxynucleotides which are not extended at the 3'
position by a polymerase. A variety of analogs and derivatives have
been developed which include a moiety at the 3' position to prevent
extension including halides, acetyl, benzyl and azide groups.
Dideoxynucleosides and dideoxynucleoside mono-, di- or
triphosphates which cannot be extended have also been developed.
Examples of dideoxynucleotides include dideoxycytosine,
dideoxyadenosine, dideoxyguanosine or dideoxythymidine, and analogs
and derivatives thereof.
[0028] The fluorescently labeled reagent may also be an
oligonucleotide. The oligonucleotide may have a 3' end which is
extendable by using a nucleotide polymerase. Such a labeled
oligonucleotide may be used, for example, as a dye-labeled primer
in nucleic acid sequencing.
[0029] The present invention also relates to methods which use the
energy transfer dyes and reagents of the present invention. In one
embodiment, the method includes forming a series of different sized
oligonucleotides labeled with an energy transfer dye of the present
invention, separating the series of labeled oligonucleotides based
on size and detecting the separated labeled oligonucleotides based
on the fluorescence of the energy transfer dye.
[0030] In another embodiment, the method includes forming a mixture
of extended labeled primers by hybridizing a nucleic acid with an
oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one dideoxynucleoside triphosphate and a
DNA polymerase, the DNA polymerase extending the primer with the
deoxynucleoside triphosphates until a dideoxynucleoside
triphosphate is incorporated which terminates extension of the
primer. Once terminated, the mixture of extended primers are
separated and the separated extended primers detected by detecting
an energy transfer dye of the present invention that was
incorporated onto either the oligonucleotide primer, a
deoxynucleotide triphosphate, or a dideoxynuceotide
triphosphate.
[0031] The present invention also relates to methods for sequencing
a nucleic acid using the energy transfer dyes of the present
invention. In one embodiment, the method includes forming a mixture
of extended labeled primers by hybridizing a nucleic acid sequence
with an oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one dideoxynucleoside triphosphate and a
DNA polymerase. The oligonucleotide primer and/or the
dideoxynucleotide is labeled with an energy transfer dye of the
present invention. The DNA polymerase is used to extend the primer
with the deoxynucleoside triphosphates until a dideoxynucleoside
triphosphate is incorporated which terminates extension of the
primer. The mixture of extended primers are then separated and the
sequence of the nucleic acid determined by detecting the energy
transfer dye on the extended primer.
[0032] The present invention also relates to methods for detecting
oligonucleotides and reagents labeled with energy transfer dyes
using shorter wavelength light sources. The light sources used in
these methods preferably provide energy at a wavelength less than
450 nm. In one variation, the light source provides energy at a
wavelength between about 250 and 450 nm, preferably between about
300 and 450 nm, and most preferably between about 350 and 450 nm.
In one particular embodiment, the light source used provides energy
at about 400 nm.
[0033] In one embodiment, the method includes forming a series of
different sized oligonucleotides labeled with an energy transfer
dye, separating the series of labeled oligonucleotides based on
size and detecting the separated labeled oligonucleotides based on
the fluorescence of the energy transfer dye upon exposure to a
shorter wavelength light source.
[0034] In another embodiment, the method includes forming a mixture
of extended labeled primers by hybridizing a nucleic acid with an
oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one dideoxynucleoside triphosphate and a
DNA polymerase, the DNA polymerase extending the primer with the
deoxynucleoside triphosphates until a dideoxynucleoside
triphosphate is incorporated which terminates extension of the
primer. Once terminated, the mixture of extended primers are
separated. The separated extended primers are detected by exposing
the extended primer to light having a wavelength between about 250
and 450 nm and measuring light emitted by an energy transfer dye at
a wavelength greater than about 500 nm. The energy transfer dye is
incorporated onto either the oligonucleotide primer, a
deoxynucleotide triphosphate, or a dideoxynuceotide
triphosphate.
[0035] The present invention also relates to methods for sequencing
a nucleic acid using a shorter wavelength light source. In one
embodiment, the method includes forming a mixture of extended
labeled primers by hybridizing a nucleic acid sequence with an
oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one dideoxynucleoside triphosphate and a
DNA polymerase. The oligonucleotide primer and/or the
dideoxynucleotide is labeled with an energy transfer dye adapted
for use with a shorter wavelength light source. The DNA polymerase
is used to extend the primer with the deoxynucleoside triphosphates
until a dideoxynucleoside triphosphate is incorporated which
terminates extension of the primer. The mixture of extended primers
are then separated and the sequence of the nucleic acid determined
by exposing the extended primer to light having a wavelength
between about 250 and 450 nm and measuring light emitted by the
energy transfer dye at a wavelength greater than about 500 nm.
[0036] In a preferred variation of the embodiment, the extended
primer is exposed to light having a wavelength between about 300
and 450 nm. The extended primer may also be exposed to light having
a wavelength between about 350 and 400 nm. In another preferred
variation of the embodiment, the light emitted by the energy
transfer dye has a wavelength greater than about 550 nm. The light
emitted by the energy transfer dye may also have a wavelength
between about 500 and 700 nm. In another embodiment, the light
emitted by the energy transfer dye has a wavelength at least about
150 nm greater than the wavelength of the light to which the
extended primer is exposed.
[0037] The present invention also relates to kits containing the
dyes and reagents for performing DNA sequencing using the dyes and
reagents of the present invention. A kit may include a set of 2, 3,
4 or more energy transfer dyes or reagents of the present
invention. Optionally the kits may further include a nucleotide
polymerase, additional nucleotides and/or reagents useful for
performing nucleic acid sequencing.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1 illustrates examples of energy transfer dyes
according to the present invention.
[0039] FIG. 2 illustrates examples of donor dyes which include a
pyrene ring structure.
[0040] FIG. 3 illustrates examples of donor dyes which include a
coumarin ring structure.
[0041] FIG. 4 illustrates the structure of a dendrimer
energy-transfer dye.
[0042] FIG. 5 illustrates classes of acceptor dyes including
xanthene dyes, cyanine dyes, phthalocyanine dyes and squaraine
dyes.
[0043] FIG. 6 illustrates the general structure of xanthene dyes
and classes of xanthene dyes like fluorescein, rhodamine and
asymmetric benzoxanthene.
[0044] FIG. 7 illustrates structures of acceptor dyes which may be
used in the dyes of the present invention.
[0045] FIG. 8 illustrates examples of --C(O)R.sub.22-- subunits of
linkers which may be used in the present invention.
[0046] FIG. 9 illustrates the synthesis scheme of energy transfer
dye DYE104.
[0047] FIG. 10 illustrates the synthesis scheme of energy transfer
dye DYE106.
[0048] FIG. 11 illustrates the synthesis scheme of energy transfer
dye DYE108.
[0049] FIG. 12 shows the fluorescence emission spectra of energy
transfer dyes according to the present invention.
[0050] FIG. 13 illustrates the synthesis scheme of energy transfer
dye DYE120.
[0051] FIG. 14 shows the fluorescence emission spectra of energy
transfer dye DYE120 according to the present invention.
DETAILED DESCRIPTION
[0052] The present invention relates to energy transfer dyes which
may be used with shorter wavelength light sources. For example, the
energy transfer dyes are preferably adapted to be excited at
wavelengths between about 250 and 450 nm. The present invention
also relates to reagents which include the energy transfer dyes of
the present invention. The present invention further relates to
methods which use the dyes and reagents. Kits are also provided
which include the dyes and reagents.
[0053] I. Energy Transfer Dyes
[0054] The energy transfer dyes of the present invention include a
donor dye and an acceptor dye which is capable of emitting energy
in response to absorbing energy from the donor dye.
[0055] In one embodiment, the energy transfer dyes may be excited
at wavelengths between about 250 and 450 nm. According to this
embodiment, the donor dye preferably has an absorption maxima at a
wavelength between about 250 to 450 nm, more preferably between
about 300 and 450 nm, and most preferably between about 350 and 450
nm.
[0056] In another embodiment, the energy transfer dyes include an
donor dye having a coumarin or pyrene ring structure.
[0057] The acceptor dye may be any dye which is capable of
absorbing energy from the donor dye. In one embodiment, the
acceptor dye has an emission maxima greater than about 500 nm, more
preferably greater than 550 nm. In another embodiment, the acceptor
dye has an emission maxima between about 500 and 700 nm. In another
embodiment, the acceptor dye is selected such that it has an
emission maxima at a wavelength at least about 150 nm greater than
the absorption maxima of the donor dye.
[0058] The energy transfer dyes may also include a linker which
couples the donor dye to the acceptor dye. The linker preferably
couples the donor dye to the acceptor dye such that the acceptor
dye is able to absorb substantially all of the energy by the donor
dye.
[0059] Particular examples of energy transfer dyes of the present
invention are illustrated in FIG. 1. In these examples
5-carboxyfluorescein, which has an emission maxima of 523 nm, is
used as the acceptor dye. Coumarin-based donor dyes DYE116, which
has an absorption maxima at 376 nm, DYE114 (absorption maximum=328
nm), and DYE112 (absorption maximum=362 nm) or pyrene-based donor
dye DYE110 (absorption maximum=396 nm) are conjugated to a
5-carboxyfluorescein acceptor derivatized with a
4-aminomethylbenzoic linker (5CF-B). The structures of the 5CF-B
conjugates, DYE102, DYE104, DYE106, and DYE108, are shown in FIG.
1.
[0060] A. DonorDye
[0061] In one embodiment, the donor dye has an absorption maxima at
a wavelength between about 250 to 450 nm, more preferably between
about 300 and 450 nm, most preferably between about 350 and 400
nm.
[0062] In another embodiment, the donor dye has a pyrene ring
structure. As used herein, pyrene dyes include all molecules
including the general structure 1
[0063] The present invention is intended to encompass all pyrene
dyes since all may be used in the present invention. Particular
examples of pyrene dyes, DYE110, DYE122, DYE124 and DYE126, are
illustrated in FIG. 2. In the figure, X is a functional group which
may be used to attach substituents, such as the acceptor dye, to
the donor dye.
[0064] In another embodiment, the donor dye has a coumarin ring
structure. As used herein, coumarin dyes include all molecules
including the general structure. 2
[0065] The present invention is intended to encompass all coumarin
dyes since all may be used in the present invention. Particular
examples of coumarin dyes are illustrated in FIG. 3. In the figure,
X is a functional group which may be used to attach substituents,
such as the acceptor dye, to the donor dye.
[0066] The present invention also relates to energy transfer dyes
where multiple donor dyes are coupled to an acceptor dye. Coumarin
dyes are water-soluble and coumarin conjugates show much better
quantum yields than larger dyes, for which the quantum yields in
water are about 1/3 that of free acceptor dyes. The present
invention utilizes the small size and solubility of the coumarins
to synthesize "antennae" dyes or dendrimers in which large numbers
of donor dyes are coupled to one acceptor dye. An example of a
dendrimer energy transfer dye (DYE118) is shown in FIG. 4.
[0067] B. Acceptor Dye
[0068] The acceptor dye may be any dye which is capable of
absorbing energy from the donor dye. In one embodiment, the
acceptor dye has an emission maxima greater than about 500 nm, more
preferably greater than 550 nm. In another embodiment, the acceptor
dye has an emission maxima between about 500 and 700 nm. In another
embodiment, the acceptor dye is selected such that it has an
emission maxima at a wavelength at least about 150 nm greater than
the absorption maxima of the donor dye.
[0069] Examples of classes of acceptor dyes which may be used in
the energy transfer fluorescent dye of this embodiment include, but
are not limited to, xanthene dyes, cyanine dyes, phthalocyanine
dyes and squaraine dyes. The general structures of these dyes are
illustrated in FIG. 5. The substituents illustrated on these dyes
may be selected from the wide variety of substituents which may be
incorporated onto these different classes of dyes since all dyes
having the general xanthene, fluorescein, rhodamine, asymmetric
benzoxanthene, cyanine, phthalocyanine and squaraine ring
structures are intended to fall within the scope of this
invention.
[0070] One particular class of acceptor dyes which may be used in
the energy transfer dyes of the present invention are xanthene
dyes. As used herein, xanthene dyes include all molecules having
the general structure illustrated in FIG. 6 where Y.sub.1 and
Y.sub.2 taken separately are either hydroxyl, oxygen, iminium or
amine, the iminium and amine preferably being a tertiary iminium or
amine. Examples of classes of xanthene dyes are fluorescein,
rhodamine and asymmetric benzoxanthene classes of dyes which are
also illustrated in FIG. 6. The substituents illustrated on these
dyes may be selected from the wide variety of substituents which
may be incorporated onto these different classes of dyes since all
dyes having the general xanthene, fluorescein, rhodamine, and
asymmetric benzoxanthene ring structures are intended to fall
within the scope of this invention. Fluorescein and rhodamine dyes
may be linked to a substituent, such as an acceptor dye, a
nucleoside, or an oligonucleotide, in a variety of locations.
Illustrated with an asterik "*" in FIG. 6 are preferred locations
for substitutions.
[0071] Fluorescein and rhodamine classes of dyes are members of a
particular subclass of xanthene dyes where R.sub.17 is a phenyl or
substituted phenyl having the general formula 3
[0072] Substituents X.sub.1-X.sub.5 on the phenyl ring can include
hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl,
alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy,
where adjacent substituents are taken together to form a ring, and
combinations thereof. As illustrated in FIG. 5, dyes where Y.sub.1
is hydroxyl and Y.sub.2 is carboxyl are fluorescein dyes and where
Y.sub.1 is amine and Y.sub.2 is iminium are rhodamine dyes.
[0073] R.sub.11-R.sub.17 may be any substituent which is compatible
with the energy transfer dyes of the present invention, it being
noted that the R.sub.11-R.sub.17 may be widely varied in order to
alter the spectral and mobility properties of the dyes. Examples of
R.sub.11-R.sub.17 substituents include, but not limited to
hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl,
alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy,
phenyl, substituted phenyl, where adjacent substituents are taken
together to form a ring, and combinations thereof.
[0074] In one embodiment, R.sub.15 and R.sub.16 are taken together
to form a substituted or unsubstituted benzene ring. This class of
xanthene dyes are referred to herein as asymmetric benzoxanthene
dyes and are described in U.S. Pat. No. 5,840,999, entitled
Asymmetric Benzoxanthene Dyes, by Scott C. Benson, et al. which is
incorporated herein by reference.
[0075] In one particular embodiment, the acceptor dye is a member
of the class of dyes where Y.sub.1 is amine, Y.sub.2 is iminium,
and X.sub.2 and X.sub.5 are chlorine, referred to herein as
4,7-dichlororhodamine dyes. Dyes falling within the
4,7-dichlororhodamine class of dyes and their synthesis are
described in U.S. Pat. No. 5,847,162, entitled:
"4,7-Dichlororhodamine Dyes" which is incorporated herein by
reference.
[0076] R.sub.11-R.sub.17 and X.sub.1-X.sub.5 may also each
independently be a linking moiety which may be used to attach the
energy transfer dye to a reagent, such as a nucleotide, nucleoside
or oligonucleotide. Examples of linking moieties include
isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinylamine,
succinimidyl ester, or other active carboxylate whenever the
complementary functionality is amine. Preferably the linking group
is maleimide, halo acetyl, or iodoacetamide whenever the
complementary functionality is sulfhydryl. See R. Haugland,
Molecular Probes Handbook of Fluorescent Probes and Research
Chemicals, Molecular probes, Inc. (1992). In a particularly
preferred embodiment, the linking group is an activated NHS ester
formed from a carboxyl group on either the donor or acceptor dye
which can be reacted with an aminohexyl-oligomer to form a dye
labeled oligonucleotide primer.
[0077] As used here, alkyl denotes straight-chain and branched
hydrocarbon moieties, i.e., methyl, ethyl, propyl, isopropyl,
tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, and the
like. Substituted alkyl denotes an alkyl moiety substituted with
any one of a variety of substituents, including, but not limited to
hydroxy, amino, thio, cyano, nitro, sulfo, and the like. Haloalkyl
denotes a substituted alkyl with one or more halogen atom
substituents, usually fluoro, chloro, bromo, or iodo. Alkene
denotes a hydocarbon wherein one or more of the carbon-carbon bonds
are double bonds, and the non-double bonded carbons are alkyl or
substituted alkyl. Alkyne denotes a hydocarbon where one or more of
the carbons are bonded with a triple bond and where the non-triple
bonded carbons are alkyl or substituted alkyl moieties. Sulfonate
refers to moieties including a sulfur atom bonded to 3 oxygen
atoms, including mono- and di-salts thereof, e.g., sodium
sulfonate, potassium sulfonate, disodium sulfonate, and the like.
Amino refers to moieties including a nitrogen atom bonded to 2
hydrogen atoms, alkyl moieties, or any combination thereof. Amido
refers to moieties including a carbon atom double bonded to an
oxygen atom and single bonded to an amino moiety. Nitrile refers to
moieties including a carbon atom triple bonded to a nitrogen atom.
Alkoxy refers to a moiety including an alkyl moiety single bonded
to an oxygen atom. Aryl refers to single or multiple phenyl or
substituted phenyl, e.g., benzene, naphthalene, anthracene,
biphenyl, and the like.
[0078] In another embodiment, the acceptor dye is selected such
that the acceptor dye has an emission maximum that is greater than
about 500 nm and an emission maximum that is at least about 150 nm
greater than the absorption maxima of the donor dye. This class of
dyes of the present invention exhibit unusually large Stokes'
shifts, as measured by the difference between the absorbance of the
donor and the emission of the acceptor. In addition, these dyes
exhibit efficient energy transfer in that minimal donor
fluorescence is observed. Interestingly, energy is transferred from
the donor to the acceptor in some of the dyes belonging to this
class even though the absorbance spectrum of the acceptor dye does
not overlap with the emission spectrum of the donor dye.
[0079] Particular examples of acceptor dyes which may be used in
the dyes of the present invention include, but are not limited to
isomers of carboxyfluorescein (e.g., 5 and 6 carboxy),
4,7-dichlorofluoresceins, 4,7-dichlororhodamines, fluoresceins,
asymmetric benzoxanthene dyes, isomers of carboxy-HEX (e.g., 5 and
6 carboxy), NAN, CI-FLAN, TET, JOE, ZOE, rhodamine, isomers of
carboxyrhodamine (e.g., 5 and 6 carboxy), isomers of carboxy R110
(e.g., 5 and 6 carboxy), isomers of carboxy R6G (e.g., 5 and 6
carboxy), 4,7-dichlorofluoresceins (See U.S. Pat. No. 5,188,934),
4,7-dichlororhodamines (See U.S. Pat. No. 5,847,162), asymmetric
benzoxanthene dyes (See U.S. Pat. No. 5,840,999), isomers of
N,N,N',N'-tetramethyl carboxyrhodamine (TAMRA) (e.g., 5 and 6
carboxy), isomers of carboxy-X-rhodamine (ROX) (e.g., 5 and 6
carboxy) and Cy5. Illustrated in FIG. 7 are the structures of these
dyes.
[0080] C. Linkers
[0081] The donor dye may be joined with the acceptor dye using a
wide variety of linkers which have been developed, all of which are
intended to fall within the scope of the present invention. The
energy transfer dyes which include a linker may generally be
illustrated as
DONOR ---- LINKER ---- ACCEPTOR
[0082] In a preferred embodiment, the linker joins the donor dye to
the acceptor dye such that the acceptor dye absorbs substantially
all of the energy by the donor dye. While not being bound by
theory, it is believed that the efficiency of energy transmission
from the donor dye to the acceptor dye is dependent upon the
separation between the dyes and relative orientation of the dyes.
Described in U.S. Pat. No. 5,800,996 are linkers which have been
found to be effective for providing a very high level of energy
transfer between the donor and acceptor dye. U.S. Pat. No.
5,800,996 also describes methods for synthesizing dyes
incorporating these linkers. U.S. Pat. No. 5,800,996 is
incorporated herein by reference in its entirety.
[0083] In one particular embodiment, the linker used in the energy
transfer dyes of the present invention is such that the acceptor
dye absorbs substantially all of the excitation energy by the donor
dye. Such linkers may include a functional group which provides
structural rigidity to the linker. Examples of such functional
groups include an alkene, diene, alkyne, a five and six membered
ring having at least one unsaturated bond and/or having a fused
ring structure.
[0084] Examples of functional groups with a five or six membered
ring with at least one unsaturatd bond and/or a fused ring
structure include cyclopentene, cyclohexene, cyclopentadiene,
cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole,
pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine,
pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran,
thionaphthene, indole and naphthalene.
[0085] One linker according to the present invention for linking a
donor dye to an acceptor dye in an energy transfer dye includes the
subunit structure --C(O)R.sub.22--, where R.sub.22 includes a
functional group such as the ones described above which provides
structural rigidity. FIG. 8 illustrates examples of
--C(O)R.sub.22-- subunits of linkers which may be used in the
linkers of the present invention.
[0086] One embodiment of this linker has the general structure
--R.sub.21Z.sub.1C(O)R.sub.22R.sub.28--, where R.sub.21 is a
C.sub.1-5 alkyl attached to the donor dye, C(O) is a carbonyl
group, Z.sub.1 is either NH, sulfur or oxygen, R.sub.22 is a
substituent which includes an alkene, diene, alkyne, a five and six
membered ring having at least one unsaturated bond or a fused ring
structure which is attached to the carbonyl carbon, and R.sub.28
includes a functional group which attaches the linker to the
acceptor dye.
[0087] In one embodiment of this linker, the linker has the general
structure --R.sub.21Z.sub.1C(O)R.sub.22R.sub.29Z.sub.2C(O)-- where
R.sub.2, and R.sub.22 are as detailed above, Z.sub.1 and Z.sub.2
are each independently either NH, sulfur or oxygen, R.sub.29 is a
C.sub.1-5 alkyl, and the terminal carbonyl group is attached to the
ring structure of the acceptor dye. In the variation where Z.sub.2
is nitrogen, the --C(O)R.sub.22R.sub.29Z.sub.2-- subunit forms an
amino acid subunit.
[0088] A preferred embodiment of this linker is where R.sub.21 and
R.sub.29 are methylene, Z.sub.1 and Z.sub.2 are NH, and R.sub.22 is
benzene.
[0089] In yet another variation, the linker has the general formula
R.sub.25Z.sub.3C(O) or R.sub.25Z.sub.3C(O)R.sub.26Z.sub.4C(O) where
R.sub.25 is attached to the donor dye, C(O) is a carbonyl group and
the terminal carbonyl group is attached to the acceptor dye,
R.sub.25 and R.sub.26 are each selected from the group of C.sub.1-4
alkyl and Z.sub.3 and Z.sub.4 are each independently either NH, O
or S.
[0090] In another variation of this embodiment, the linker includes
a R.sub.27Z.sub.5C(O) group where R.sub.27 is a C.sub.1-5 alkyl
attached to the donor dye, Z.sub.5 is either NH, sulfur or oxygen,
and C(O) is a carbonyl group attached to the acceptor dye.
[0091] II. Reagents Including Energy Transfer Dyes of the Present
Invention
[0092] The present invention also relates to reagents which
incorporate an energy transfer dye according to the present
invention. As described in greater detail in Section III, these
reagents may be used in a wide variety of methods for detecting the
presence of a component in a sample.
[0093] The reagents of the present invention include any molecule
or material to which the energy transfer dyes of the invention can
be attached and used to detect the presence of the reagent based on
the fluorescence of the energy transfer dye. Types of molecules and
materials to which the dyes of the present invention may be
attached to form a reagent include, but are not limited to
proteins, polypeptides, polysaccharides, nucleotides, nucleosides,
oligonucleotides, oligonucleotide analogs (such as a peptide
nucleic acid), lipids, solid supports, organic and inorganic
polymers, and combinations and assemblages thereof, such as
chromosomes, nuclei, living cells, such as bacteria, other
microorganisms, mammalian cells, and tissues.
[0094] Preferred classes of reagents of the present invention are
nucleotides, nucleosides, oligonucleotides and oligonucleotide
analogs which have been modified to include an energy transfer dye
of the invention. Examples of uses for nucleotide and nucleoside
reagents include, but are not limited to, labeling oligonucleotides
formed by enzymatic synthesis, e.g., nucleoside triphosphates used
in the context of PCR amplification, Sanger-type nucleotide
sequencing, and nick-translation reactions. Examples of uses for
oligonucleotide reagents include, but are not limited to, as DNA
sequencing primers, PCR primers, oligonucleotide hybridization
probes, and the like.
[0095] One particular embodiment of the reagents are labeled
nucleosides, such as cytosine, adenosine, guanosine, and thymidine,
labeled with an energy transfer fluorescent dye of the present
invention. These reagents may be used in a wide variety of methods
involving oligonucleotide synthesis. Another related embodiment are
labeled nucleotides (NTP), e.g., mono-, di- and triphosphate
nucleoside phosphate esters. These reagents include, in particular,
deoxynucleoside triphosphates (dNTP), such as deoxycytosine
triphosphate, deoxyadenosine triphosphate, deoxyguanosine
triphosphate, and deoxythymidine triphosphate, labeled with an
energy transfer fluorescent dye of the present invention. These
reagents may be used, for example, as polymerase substrates in the
preparation of dye labeled oligonucleotides. These reagents also
include labeled dideoxynucleoside triphosphates (ddNTP), such as
dideoxycytosine triphosphate, dideoxyadenosine triphosphate,
dideoxyguanosine triphosphate, and dideoxythymidine triphosphate,
labeled with an energy transfer fluorescent dye of the present
invention. These reagents may be used, for example, in dye
termination sequencing.
[0096] Another embodiment of reagents are oligonucleotides which
includes an energy transfer dye of the present invention. These
reagents may be used, for example, in dye primer sequencing.
[0097] As used herein, "nucleoside" refers to a compound consisting
of a purine, deazapurine, or pyrimidine nucleoside base, e.g.,
adenine, guanine, cytosine, uracil, thymine, deazaadenine,
deazaguanosine, and the like, linked to a pentose at the 1'
position, including 2'-deoxy and 2'-hydroxyl forms, e.g. as
described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,
San Francisco, 1992). The term "nucleotide" as used herein refers
to a phosphate ester of a nucleoside, e.g., mono, di and
triphosphate esters, wherein the most common site of esterification
is the hydroxyl group attached to the C-5 position of the pentose.
"Analogs" in reference to nucleosides include synthetic nucleosides
having modified base moieties and/or modified sugar moieties, e.g.
described generally by Scheit, Nucleotide Analogs (John Wiley, New
York, 1980). The terms "labeled nucleoside" and "labeled
nucleotide" refer to nucleosides and nucleotides which are
covalently attached to an energy transfer dye through a
linkage.
[0098] As used herein, the term "oligonucleotide" refers to linear
polymers of natural or modified nucleoside monomers, including
double and single stranded deoxyribonucleosides, ribonucleosides,
.alpha.-anomeric forms thereof, and the like. Usually the
nucleoside monomers are linked by phosphodiester linkages, where as
used herein, the term "phosphodiester linkage" refers to
phosphodiester bonds or analogs thereof including phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like, including associated counterions, e.g., H, NH.sub.4, Na, and
the like if such counterions are present. The oligonucleotides
range in size form a few monomeric units, e.g. 8-40, to several
thousands of monomeric units. Whenever an oligonucleotide is
represented by a sequence of letters, such as "ATGCCTG," it will be
understood that the nucleotides are in 5'.fwdarw.3' order from left
to right and that "A" denotes deoxyadenosine, "C" denotes
deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes
thymidine, unless otherwise noted.
[0099] Nucleoside labeling can be accomplished using any of a large
number of known nucleoside labeling techniques using known
linkages, linking groups, and associated complementary
functionalities. The linkage linking the dye and nucleoside should
(i) be stable to oligonucleotide synthesis conditions, (ii) not
interfere with oligonucleotide-target hybridization, (iii) be
compatible with relevant enzymes, e.g., polymerases, ligases, and
the like, and (iv) not quench the fluorescence of the dye.
[0100] Preferably, the dyes are covalently linked to the 5-carbon
of pyrimidine bases and to the 7-carbon of 7-deazapurine bases.
Several suitable base labeling procedures have been reported that
can be used with the invention, e.g. Gibson et al, Nucleic Acids
Research, 15 6455-6467 (1987); Gebeyehu et al, Nucleic Acids
Research, 15 4513-4535 (1987); Haralambidis et al, Nucleic Acids
Research, 15 4856-4876 (1987); Nelson et al., Nucleosides and
Nucleotides, 5(3) 233-241 (1986); Bergstrom, et al., JACS, 111
374-375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and 5,449,767,
each of which is incorporated herein by reference.
[0101] Preferably, the linkages are acetylenic amido or alkenic
amido linkages, the linkage between the dye and the nucleotide base
being formed by reacting an activated N-hydroxysuccinimide (NHS)
ester of the dye with an alkynylamino-, alkynylethoxyamino- or
alkenylamino-derivatize- d base of a nucleotide. More preferably,
the resulting linkage is proargyl-1-ethoxyamido
(3-(amino)ethoxy-1-propynyl), 3-(carboxy)amino-1-propynyl or
3-amino-1-propyn-1-yl.
[0102] Several preferred linkages for linking the dyes of the
invention to a nucleoside base are shown below. 4
[0103] where R.sub.1 and R.sub.2 taken separately are H, alkyl, a
protecting group or a fluorescent dye.
[0104] The synthesis of alkynylamino-derivatized nucleosides is
taught by Hobbs et al. in European Patent Application No.
87305844.0, and Hobbs et al., J. Org. Chem., 54 3420 (1989), which
is incorporated herein by reference. Briefly, the
alkynylamino-derivatized nucleotides are formed by placing the
appropriate halodideoxynucleoside (usually 5-iodopyrimidine and
7-iodo-7-deazapurine dideoxynucleosides as taught by Hobbs et al.
(cited above)) and Cu(I) in a flask, flushing with argon to remove
air, adding dry DMF, followed by addition of an alkynylamine,
triethyl-amine and Pd(0). The reaction mixture can be stirred for
several hours, or until thin layer chromatography indicates
consumption of the halodideoxynucleoside. When an unprotected
alkynylamine is used, the alkynylamino-nucleoside can be isolated
by concentrating the reaction mixture and chromatographing on
silica gel using an eluting solvent which contains ammonium
hydroxide to neutralize the hydrohalide generated in the coupling
reaction. When a protected alkynylamine is used, methanol/methylene
chloride can be added to the reaction mixture, followed by the
bicarbonate form of a strongly basic anion exchange resin. The
slurry can then be stirred for about 45 minutes, filtered, and the
resin rinsed with additional methanol/methylene chloride. The
combined filtrates can be concentrated and purified by
flash-chromatography on silica gel using a methanol-methylene
chloride gradient. The triphosphates are obtained by standard
techniques.
[0105] The synthesis of oligonucleotides labeled with an energy
transfer dye of the present invention can be accomplished using any
of a large number of known oligonucleotide labeling techniques
using known linkages, linking groups, and associated complementary
functionalities. For example, labeled oligonucleotides may be
synthesized enzymatically, e.g., using a DNA polymerase or ligase,
e.g., Stryer, Biochemistry, Chapter 24, W. H. Freeman and Company
(1981), or by chemical synthesis, e.g., by a phosphoramidite
method, a phosphite-triester method, and the like, e.g., Gait,
Oligonucleotide Synthesis, IRL Press (1990). Labels may be
introduced during enzymatic synthesis utilizing labeled nucleoside
triphosphate monomers, or introduced during chemical synthesis
using labeled non-nucleotide or nucleotide phosphoramidites, or may
be introduced subsequent to synthesis.
[0106] Generally, if the labeled oligonucleotide is made using
enzymatic synthesis, the following procedure may be used. A
template DNA is denatured and an oligonucleotide primer is annealed
to the template DNA. A mixture of deoxynucleoside triphosphates is
added to the reaction including dGTP, dATP, dCTP, and dTTP where at
least a fraction of one of the deoxynucleotides is labeled with a
dye compound of the invention as described above. Next, a
polymerase enzyme is added under conditions where the polymerase
enzyme is active. A labeled polynucleotide is formed by the
incorporation of the labeled deoxynucleotides during polymerase
strand synthesis. In an alternative enzymatic synthesis method, two
primers are used instead of one, one primer complementary to the
+strand and the other complementary to the--strand of the target,
the polymerase is a thermostable polymerase, and the reaction
temperature is cycled between a denaturation temperature and an
extension temperature, thereby exponentially synthesizing a labeled
complement to the target sequence by PCR, e.g., PCR Protocols,
Innis et al. eds., Academic Press (1990).
[0107] Generally, if the labeled oligonucleotide is made using a
chemical synthesis, it is preferred that a phosphoramidite method
be used. Phosphoramidite compounds and the phosphoramidite method
of polynucleotide synthesis are preferred in synthesizing
oligonucleotides because of the efficient and rapid coupling and
the stability of the starting materials. The synthesis is performed
with the growing oligonucleotide chain attached to a solid support,
so that excess reagents, which are in the liquid phase, can be
easily removed by filtration, thereby eliminating the need for
purification steps between cycles.
[0108] In view of the utility of phosphoramidite reagents in
labeling nucleosides and oligonucleotides, the present invention
also relates to phosphoramidite compounds which include an energy
transfer dye of the present invention.
[0109] Nucleoside labeling with the dyes can be accomplished using
any of a large number of known nucleoside labeling techniques using
known linkages, linking groups, and associated complementary
functionalities. Preferably, the dyes are covalently linked to the
5-carbon of pyrimidine bases and to the 7-carbon of 7-deazapurine
bases. Several suitable base labeling procedures have been reported
that can be used with the invention, e.g. Gibson et al. Nucleic
Acid Res. 15 6455-6467 (1987); Gebeyehu et al. Nucleic Acid Res. 15
4513-4535 (1987); Haralambidis et al. Nucleic Acid Res. 15
4856-4876; Nelson et al. Nucleosides and Nucleotides 5 233-241
(1986); Bergstrom et al. J. Am. Chem. Soc. 111:374-375 (1989); U.S.
Pat. Nos. 4,855,225, 5,231,191, and 5,449,767, each of which is
incorporated herein by reference.
[0110] Preferably, the linkages are acetylenic amido or alkenic
amido linkages, the linkage between the dye and nucleotide base
being formed by reacting an activated N-hydroxysuccinimide (NHS)
ester of the dye with an alkynylamino, alkynylethoxyamino, or
alkenylamino-derivatized base of a nucelotide. More preferably, the
resulting linkage is proargyl-1-ethoxyamido
(3-(amino)ethoxy-1-propynyl), 3-(carboxy)amino-1-propynyl or
3-amino-1-propyn-1-yl.
[0111] The synthesis of alkynylamino-derivatized nucleosides is
taught by Hobbs et al. J. Org. Chem. 54:3420 (1989), which is
incorporated herein by reference. Briefly, the
alkynylamino-derivatized nucleosides are formed by placing the
appropriate halodideoxynucleoside (usually 5-iodopryrimidine and
7-iodo-deazapurine dideoxynucleosides as taught by Hobbs et al. as
cited above) and Cu(I) in a flask, flushing with argon to remove
air, adding dry DMF. followed by addition of an alkynylamine,
triethyl-amine and Pd(0). The reaction mixture can be stirred for
several hours, or until thin layer chromatography indicates
consumption of the halodideoxynucleoside. When an unprotected
alkynylamine is used, the alkynylaminonucleoside can be isolated by
concentrating the reaction mixture and chromatographing on silica
gel using an eluting solvent which contains ammonium hydroxide to
neutralize the hydrohalide generated in the coupling reaction. When
a protected alkynylamine is used, methanol/methylene chloride can
be added to the reaction mixture, followed by the bicarbonate form
of a strongly basic anion exchange resin. The slurry can then be
stirred for about 45 minutes, filtered, and the resin rinsed with
additional methanol/methylene chloride. The combined filtrate can
be concentrated and purified by flash-chromatography on silica gel
using a methanol-methylene chloride gradient. The triphosphates are
obtained by standard techniques.
[0112] Detailed descriptions of the chemistry used to form
oligonucleotides by the phosphoramidite method are provided in
Caruthers et al., U.S. Pat. No. 4,458,066; Caruthers et al., U.S.
Pat. No. 4,415,732; Caruthers et al., Genetic Engineering, 4 1-17
(1982); Users Manual Model 392 and 394 Polynucleotide Synthesizers,
pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991),
each of which are incorporated by reference in their entirety.
[0113] The following briefly describes the steps of a typical
oligonucleotide synthesis cycle using the phosphoramidite method.
First, a solid support including a protected nucleotide monomer is
treated with acid, e.g., trichloroacetic acid, to remove a
5'-hydroxyl protecting group, freeing the hydroxyl for a subsequent
coupling reaction. An activated intermediate is then formed by
simultaneously adding a protected phosphoramidite nucleoside
monomer and a weak acid, e.g., tetrazole, to the reaction. The weak
acid protonates the nitrogen of the phosphoramidite forming a
reactive intermediate. Nucleoside addition is complete within 30 s.
Next, a capping step is performed which terminates any
polynucleotide chains that did not undergo nucleoside addition.
Capping is preferably done with acetic anhydride and
1-methylimidazole. The internucleotide linkage is then converted
from the phosphite to the more stable phosphotriester by oxidation
using iodine as the preferred oxidizing agent and water as the
oxygen donor. After oxidation, the hydroxyl protecting group is
removed with a protic acid, e.g., trichloroacetic acid or
dichloroacetic acid, and the cycle is repeated until chain
elongation is complete. After synthesis, the polynucleotide chain
is cleaved from the support using a base, e.g., ammonium hydroxide
or t-butyl amine. The cleavage reaction also removes any phosphate
protecting groups, e.g., cyanoethyl. Finally, the protecting groups
on the exocyclic amines of the bases and the hydroxyl protecting
groups on the dyes are removed by treating the polynucleotide
solution in base at an elevated temperature, e.g., 55.degree.
C.
[0114] Any of the phosphoramidite nucleoside monomers may be
dye-labeled phosphoramidites. If the 5'-terminal position of the
nucleotide is labeled, a labeled non-nucleotidic phosphoramidite of
the invention may be used during the final condensation step. If an
internal position of the oligonucleotide is to be labeled, a
labeled nucleotidic phosphoramidite of the invention may be used
during any of the condensation steps. Subsequent to their
synthesis, oligonucleotides may be labeled at a number of positions
including the 5'-terminus. See Oligonucleotides and Analogs,
Eckstein ed., Chapter 8, IRL Press (1991) and Orgel et al., Nucleic
Acids Research 11(18) 6513 (1983); U.S. Pat. No. 5,118,800, each of
which are incorporated by reference
[0115] Oligonucleotides may also be labeled on their phosphodiester
backbone (Oligonucleotides and Analogs, Eckstein ed., Chapter 9) or
at the 3'-terminus (Nelson, Nucleic Acids Research 20(23)
6253-6259, and U.S. Pat. Nos. 5,401,837 and 5,141,813, both patents
hereby incorporated by reference. For a review of oligonucleotide
labeling procedures see R. Haugland in Excited States of
Biopolymers, Steiner ed., Plenum Press, NY (1983).
[0116] In one preferred post-synthesis chemical labeling method an
oligonucleotide is labeled as follows. A dye including a carboxy
linking group is converted to the n-hydroxysuccinimide ester by
reacting with approximately 1 equivalent of
1,3-dicyclohexylcarbodiimide and approximately 3 equivalents of
n-hydroxysuccinimide in dry ethyl acetate for 3 hours at room
temperature. The reaction mixture is washed with 5% HCl, dried over
magnesium sulfate, filtered, and concentrated to a solid which is
resuspended in DMSO. The DMSO dye stock is then added in excess
(10-20.times.) to an aminohexyl derivatized oligonucleotide in 0.25
M bicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6
hours, e.g., U.S. Pat. No. 4,757,141. The dye labeled
oligonucleotide is separated from unreacted dye by passage through
a size-exclusion chromatography column eluting with buffer, e.g.,
0.1 molar triethylamine acetate (TEM). The fraction containing the
crude labeled oligonucleotide is further purified by reverse phase
HPLC employing gradient elution.
[0117] III. Methods Employing Dyes and Reagents of the Present
Invention
[0118] The energy transfer dyes and reagents of the present
invention may be used in a wide variety of methods for detecting
the presence of a component in a sample by labeling the component
in the sample with a reagent containing the dye. In particular, the
energy transfer dyes and reagents of the present invention are well
suited for use in methods which combine separation and fluorescent
detection techniques, particularly methods requiring the
simultaneous detection of multiple spatially-overlapping analytes.
For example, the dyes and reagents are particularly well suited for
identifying classes of oligonucleotides that have been subjected to
a biochemical separation procedure, such as electrophoresis, where
a series of bands or spots of target substances having similar
physiochemical properties, e.g. size, conformation, charge,
hydrophobicity, or the like, are present in a linear or planar
arrangement. As used herein, the term "bands" includes any spatial
grouping or aggregation of analytes on the basis of similar or
identical physiochemical properties. Usually bands arise in the
separation of dye-oligonucleotide conjugates by
electrophoresis.
[0119] Classes of oligonucleotides can arise in a variety of
contexts. In a preferred category of methods referred to herein as
"fragment analysis" or "genetic analysis" methods, labeled
oligonucleotide fragments are generated through template-directed
enzymatic synthesis using labeled primers or nucleotides, e.g., by
ligation or polymerase-directed primer extension; the fragments are
subjected to a size-dependent separation process, e.g.,
electrophoresis or chromatography; and, the separated fragments are
detected subsequent to the separation, e.g., by laser-induced
fluorescence. In a particularly preferred embodiment, multiple
classes of oligonucleotides are separated simultaneously and the
different classes are distinguished by spectrally resolvable
labels.
[0120] One such fragment analysis method is amplified fragment
length polymorphisim detection (AmpFLP) and is based on amplified
fragment length polymorphisms, i.e., restriction fragment length
polymorphisms that are amplified by PCR. These amplified fragments
of varying size serve as linked markers for following mutant genes
through families. The closer the amplified fragment is to the
mutant gene on the chromosome, the higher the linkage correlation.
Because genes for many genetic disorders have not been identified,
these linkage markers serve to help evaluate disease risk or
paternity. In the AmpFLPs technique, the polynucleotides may be
labeled by using a labeled oligonucleotide PCR primer, or by
utilizing labeled nucleotide triphosphates in the PCR.
[0121] Another fragment analysis method is nick translation. Nick
translation involves a reaction to replace unlabeled nucleotide
triphosphates in a double-stranded DNA molecule with labeled ones.
Free 3'-hydroxyl groups are created within the unlabeled DNA by
"nicks" caused by deoxyribonuclease I (DNAase I) treatment. DNA
polymerase I then catalyzes the addition of a labeled nucleotide to
the 3'-hydroxyl terminus of the nick. At the same time, the 5' to
3'-exonuclease activity of this enzyme eliminates the nucleotide
unit from the 5'-phosphoryl terminus of the nick. A new nucleotide
with a free 3'-OH group is incorporated at the position of the
original excised nucleotide, and the nick is shifted along by one
nucleotide unit in the 3' direction. This 3' shift will result in
the sequential addition of new labeled nucleotides to the DNA with
the removal of existing unlabeled nucleotides. The nick-translated
polynucleotide is then analyzed using a separation process, e.g.,
electrophoresis.
[0122] Another exemplary fragment analysis method is based on
variable number of tandem repeats, or VNTRs. VNTRs are regions of
double-stranded DNA that contain adjacent multiple copies of a
particular sequence, with the number of repeating units being
variable. Examples of VNTR loci are pYNZ22, pMCT118, and Apo B. A
subset of VNTR methods are those methods based on the detection of
microsatellite repeats, or short tandem repeats (STRs), i.e.,
tandem repeats of DNA characterized by a short (2-4 bases) repeated
sequence. One of the most abundant interspersed repetitive DNA
families in humans is the (dC-dA)n--(dG-dT)n dinucleotide repeat
family (also called the (CA)n dinucleotide repeat family). There
are thought to be as many as 50,000 to 100,000 (CA)n repeat regions
in the human genome, typically with 15-30 repeats per block. Many
of these repeat regions are polymorphic in length and can therefore
serve as useful genetic markers. Preferably, in VNTR or STR
methods, label is introduced into the polynucleotide fragments by
using a dye-labeled PCR primer.
[0123] Another exemplary fragment analysis method is DNA
sequencing. In general, DNA sequencing involves an extension I
termination reaction of an oligonucleotide primer. Included in the
reaction mixture are deoxynucleoside triphosphates (dNTPs) which
are used to extend the primer. Also included in the reaction
mixture is at least one dideoxynucleoside triphosphate (ddNTP)
which when incorporated onto the extended primer prevents the
further extension of the primer. After the extension reaction has
been terminated, the different termination products that are formed
are separated and analyzed in order to determine the positioning of
the different nucleosides.
[0124] Fluorescent DNA sequencing may generally be divided into two
categories, "dye primer sequencing" and "dye terminator
sequencing". In dye primer sequencing, a fluorescent dye is
incorporated onto the primer being extended. Four separate
extension/termination reactions are then run in parallel, each
extension reaction containing a different dideoxynucleoside
triphosphate (ddNTP) to terminate the extension reaction. After
termination, the reaction products are separated by gel
electrophoresis and analyzed. See, for example, Ansorge et al.,
Nucleic Acids Res. 15 4593-4602 (1987).
[0125] In one variation of dye primer sequencing, different primers
are used in the four separate extension/termination reactions, each
primer containing a different spectrally resolvable dye. After
termination, the reaction products from the four
extension/termination reactions are pooled, electrophoretically
separated, and detected in a single lane. See, for example, Smith
et al., Nature 321 674-679 (1986). Thus, in this variation of dye
primer sequencing, by using primers containing a set of spectrally
resolvable dyes, products from more than one extension/termination
reactions can be simultaneously detected.
[0126] In dye terminator sequencing, a fluorescent dye is attached
to each of the dideoxynucleoside triphosphates. An
extension/termination reaction is then conducted where a primer is
extended using deoxynucleoside triphosphates until the labeled
dideoxynucleoside triphosphate is incorporated into the extended
primer to prevent further extension of the primer. Once terminated,
the reaction products for each dideoxynucleoside triphosphate are
separated and detected. In one embodiment, separate
extension/termination reactions are conducted for each of the four
dideoxynucleoside triphosphates. In another embodiment, a single
extension/termination reaction is conducted which contains the four
dideoxynucleoside triphosphates, each labeled with a different,
spectrally resolvable fluorescent dye.
[0127] Thus according to one aspect of the invention, a method is
provided for conducting dye primer sequencing using one or more
oligonucleotide reagents of the present invention. According to
this method, a mixture of extended labeled primers are formed by
hybridizing a nucleic acid sequence with a fluorescently labeled
oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one dideoxynucleoside triphosphate and a
DNA polymerase. The fluorescently labeled oligonucleotide primer
includes an oligonucleotide sequence complementary to a portion of
the nucleic acid sequence being sequenced, and an energy transfer
fluorescent dye attached to the oligonucleotide.
[0128] According to the method, the DNA polymerase extends the
primer with the deoxynucleoside triphosphates until a
dideoxynucleoside triphosphate is incorporated which terminates
extension of the primer. After termination, the mixture of extended
primers are separated. The sequence of the nucleic acid sequence is
then determined by fluorescently detecting the mixture of extended
primers formed.
[0129] In a further embodiment of this method, four dye primer
sequencing reactions are run, each primer sequencing reaction
including a different fluorescently labeled oligonucleotide primer
and a different dideoxynucleoside triphosphate (ddATP, ddCTP, ddGTP
and ddTTP). After the four dye primer sequencing reactions are run,
the resulting mixtures of extended primers may be pooled. The
mixture of extended primers may then be separated, for example by
electrophoresis and the fluorescent signal from each of the four
different fluorescently labeled oligonucleotide primers detected in
order to determine the sequence of the nucleic acid sequence.
[0130] According to a further aspect of the invention, a method is
provided for conducting dye terminator sequencing using one or more
dideoxynucleoside triphosphates labeled with an energy transfer dye
of the present invention. According to this method, a mixture of
extended primers are formed by hybridizing a nucleic acid sequence
with an oligonucleotide primer in the presence of deoxynucleoside
triphosphates, at least one fluorescently labeled dideoxynucleotide
triphosphate and a DNA polymerase. The fluorescently labeled
dideoxynucleotide triphosphate includes a dideoxynucleoside
triphosphate labeled with an energy transfer fluorescent dye of the
present invention.
[0131] According to this method, the DNA polymerase extends the
primer with the deoxynucleoside triphosphates until a fluorescently
labeled dideoxynucleoside triphosphate is incorporated into the
extended primer. After termination, the mixture of extended primers
are separated. The sequence of the nucleic acid sequence is then
determined by detecting the fluorescently labeled dideoxynucleoside
attached to the extended primer.
[0132] In a further embodiment of this method, the step of forming
a mixture of extended primers includes hybridizing the nucleic acid
sequence with four different fluorescently labeled
dideoxynucleoside triphosphates, i.e., a fluorescently labeled
dideoxycytosine triphosphate, a fluorescently labeled
dideoxyadenosine triphosphate, a fluorescently labeled
dideoxyguanosine triphosphate, and a fluorescently labeled
dideoxythymidine triphosphate.
[0133] In each of the above-described fragment analysis methods,
the labeled oligonucleotides are preferably separated by
electrophoretic procedures, e.g. Gould and Matthews, cited above;
Rickwood and Hames, Eds., Gel Electrophoresis of Nucleic Acids: A
Practical Approach, (IRL Press Limited, London, 1981); or Osterman,
Methods of Protein and Nucleic Acid Research, Vol. 1
Springer-Verlag, Berlin, 1984). Preferably the type of
electrophoretic matrix is crosslinked or uncrosslinked
polyacrylamide having a concentration (weight to volume) of between
about 2-20 weight percent. More preferably, the polyacrylamide
concentration is between about 4-8 percent. Preferably in the
context of DNA sequencing in particular, the electrophoresis matrix
includes a strand separating, or denaturing, agent, e.g., urea,
formamide, and the like. Detailed procedures for constructing such
matrices are given by Maniatis et al., "Fractionation of Low
Molecular Weight DNA and RNA in Polyacrylamide Gels Containing 98%
Formamide or 7M Urea," in Methods in Enzymology, 65 299-305 (1980);
Maniatis et al., "Chain Length Determination of Small Double- and
Single-Stranded DNA Molecules by Polyacrylamide Gel
Electrophoresis," Biochemistry, 14 3787-3794 (1975); Maniatis et
al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, New York, 1982), pgs. 179-185; and ABI PRISM.TM. 377
DNA Sequencer User's Manual, Rev. A, January 1995, Chapter 2 (p/n
903433, The Perkin-Elmer Corporation, Foster City, Calif.), each of
which are incorporated by reference. The optimal polymer
concentration, pH, temperature, concentration of denaturing agent,
etc. employed in a particular separation depends on many factors,
including the size range of the nucleic acids to be separated,
their base compositions, whether they are single stranded or double
stranded, and the nature of the classes for which information is
sought by electrophoresis. Accordingly application of the invention
may require standard preliminary testing to optimize conditions for
particular separations. By way of example, oligonucleotides having
sizes in the range of between about 20-300 bases have been
separated and detected in accordance with the invention in the
following matrix: 6 percent polyacrylamide made from 19 parts to 1
part acrylamide to bis-acrylamide, formed in a Tris-borate EDTA
buffer at pH 8.3.
[0134] After electrophoretic separation, the dye-oligonucleotide
conjugates are detected by measuring the fluorescence emission from
the dye labeled polynucleotides. To perform such detection, the
labeled polynucleotides are illuminated by standard light sources,
e.g. high intensity mercury vapor lamps, lasers, or the like.
Previously, fluorescein and rhodamine-based dyes and
fluorescein-linked energy transfer dyes have been used which are
excited at a wavelength between 488 and 550 nm. However, the donor
dyes used in the energy transfer dyes of the present invention
typically have absorption maxima below 450 nm and thus may be
excited at shorter wavelengths, preferably between 250 and 450
nm.
[0135] IV. Detection Methods Using Shorter Wavelength Light
Sources
[0136] The present invention also relates to detection methods,
such as the detection methods described above in Section III, in
which a shorter wavelength light source is used, preferably a light
source emitting light between 250 and 450 nm. As noted above,
several of the energy transfer dyes of the present invention have
the feature of having a donor dye with an emission maxima between
about 250 and 450 nm and an acceptor dye which has an emission
maxima at a wavelength greater than about 500 nm. As a result,
these dyes enable these shorter wavelength light sources to be
used. Accordingly, the present invention relates to methods for
using these shorter wavelength light sources. It is noted that the
use of these shorter wavelength light sources in detection methods,
such as the ones described in Section III, is not intended to be
limited to the energy transfer dyes of the present invention but
rather are intended to encompass the use of any energy transfer dye
which can be excited using light having a wavelength between 250
and 450 nm
[0137] V. Kits Incorporating the Energy Transfer Dyes
[0138] The present invention also relates to kits having
combinations of energy transfer dyes and/or reagents. In one
embodiment, the kit includes at least two spectrally resolvable
energy transfer dyes according to the present invention. In this
kit, the energy transfer dyes preferably include the same donor dye
so that a single light source is needed to excite the dyes.
[0139] In another embodiment, the kit includes dideoxycytosine
triphosphate, dideoxyadenosine triphosphate, dideoxyguanosine
triphosphate, and dideoxythymidine triphosphate, each
dideoxynucleotide triphosphate labeled with an energy transfer dye
according to the present invention. In one embodiment, each energy
transfer dye is spectrally resolvable from the other energy
transfer dyes attached to the other dideoxynucleotide
triphosphates. In this kit. the energy transfer dyes preferably
include the same first xanthene dye.
[0140] In yet another embodiment, the kit includes at least two
oligonucleotides, each oligonucleotide including an energy transfer
dye according to the present invention. In one embodiment, each
oligonucleotide contains an energy transfer dye which is spectrally
resolvable from the energy transfer dyes attached to the other
oligonucleotides. In another embodiment, the kit includes at least
four oligonucleotides which each contain a spectrally resolvable
energy transfer dye.
[0141] The energy transfer dyes and their use in DNA sequencing is
illustrated by the following examples. Further objectives and
advantages other than those set forth above become apparent from
the examples.
EXAMPLES
[0142] 1. Method of Synthesis of DYE104
[0143] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide
(DMF), 20 .mu.L) was added to a solution of the succimidyl ester of
coumarin DYE114 (20 .mu.L of a 5 mg/200 .mu.L DMF solution).
Diisopropylethylamine (5 .mu.L) was added. After 5 min, 200 .mu.L
of 5% HCl was added. The mixture was centrifuged. The solid was
dissolved in bicarbonate solution and purified by reverse-phase
HPLC. The synthesis scheme of DYE104 is illustrated in FIG. 9.
[0144] 2. Method of Synthesis of DYE106
[0145] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide
(DMF), 20 .mu.L) was added to a solution of the succimidyl ester of
coumarin DYE116 (20 .mu.L of a 5 mg/200 .mu.L DMF solution).
Diisopropylethylamine (5 .mu.L) was added. After 5 min, 200 .mu.L
of 5% HCl was added. The mixture was centrifuged. The solid was
dissolved in bicarbonate solution and purified by reverse-phase
HPLC. The synthesis scheme of DYE106 is illustrated in FIG. 10.
[0146] 2. Method of Synthesis of DYE108
[0147] A solution of 5CF-B (8 mg in 0.45 mL dimethylformamide
(DMF), 20 .mu.L) was added to a solution (20 .mu.L of a 5 mg/200
.mu.L DMF solution) of DYE110, trisulfopyrene acetyl azide (or
Cascade Blue acetyl azide, Molecular Probes). Diisopropylethylamine
(5 .mu.L) was added. After 5 min, 200 .mu.L of 5% HCl was added.
The mixture was centrifuged. The solid was dissolved in bicarbonate
solution and purified by reverse-phase HPLC. The synthesis scheme
of DYE108 is illustrated in FIG. 11.
[0148] 3. Comparison of Fluorescence Emission Spectra of
5CF-B-conjugates
[0149] The following example compares the fluorescence emission
spectra of a series of energy transfer dyes according to the
present invention. Dye solutions of 5CF-B, DYE102, DYE104, DYE106,
and DYE108 were measured in Tris-EDTA.
[0150] The structures of these dyes are illustrated in FIG. 1. FIG.
12 provides a graph of the relative fluorescence emission of each
of these dyes when excited at 365 nm. FIG. 12 also show the
emission maxima of the individual dye components. As shown in FIG.
12, the emissions of the donor dyes do not overlap with the
absorbance of the acceptor dye. The best conjugate, DYE108, is more
than 10-fold brighter than 5CF-B alone.
[0151] Table 1 shows the relative spectral data and relative
quantum yields of 5CF-B conjugates. As can be seen from Table 1,
the quantum yields are high and the energy transfer is practically
quantitative, as observed by the lack of emission of the donor
dyes. Coumarin based dyes DYE104 and DYE102 and pyrene based dye
DYE108 display high quantum yields indicating that the acceptor is
able to absorb substantially all of the energy emitted by the donor
dye. In contrast, the DYE106 (coumarin) displays poor quantum yield
and inefficient energy transfer.
1TABLE 1 EX/EM Maxima of Quantum Yield of Conjugate 5CF-B conjugate
Individual Dyes (nm) Relative to 5CF-B 5CF-B 495/523 1.00 DYE106
376/468 0.17 DYE104 328/386 0.93 DYE108 396/410 0.91 DYE102 362/459
0.87
[0152] 4. Method of Synthesis of Pyrenetrisulfonate-Rhodamine Dye
(DYE120)
[0153] D-Rox succinimidyl ester (3 mg), 1,4-cyclohexanediamine (7
mg), DMF (100 .mu.L) and diisopropylethylamine (10 .mu.L) were
combined. After 5 min ethyl ether was added. The mixture was
centrifuged and decanted. The residue was dissolved in methanol and
an aliquot was purified by reverse-phase HPLC to separate the
d-Rox-acid from the the d-Rox-cyclohexanediamine adduct. The
purified adduct was concentrated to dryness and dissolved in 10
.mu.L DMF.
[0154] A solution of DYE110, Cascade Blue acetyl azide, was made
(Molecular Probes, 8 mg/100 .mu.L DMF). To 5 .mu.L of the Dye 110
solution was added the d-Rox-cyclohexanediamine adduct and 2 .mu.L
diisopropylamine. The mixture was purified by reverse-phase HPLC.
The synthesis scheme of pyrenetrisulfonate-d-Rox dye (DYE120) is
illustrated in FIG. 13.
[0155] Normalized excitation and emission spectra of the
pyrenetrisulfonate-d-Rox adduct (DYE120) are shown in FIG. 14. Very
little pyrenetrisulfonate emission (410 nm) was observed. The
excitation spectra showed a peak at 400 nm that was 50% of the
maximum peak at 600 nm.
[0156] While the present invention is disclosed by reference to the
preferred embodiments and examples detailed above, it is to be
understood that these examples are intended in an illustrative
rather than limiting sense, as it is contemplated that
modifications will readily occur to those skilled in the art, which
modifications will be within the spirit of the invention and the
scope of the appended claims. With regard to all of the molecular
structures provided herein, it is intended that these molecular
structures encompass not only the exact electronic structure
presented, but also include all resonant structures and protonation
states thereof.
* * * * *