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Description  |
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TECHNICAL FIELD
The field of this invention is fluorescent tags and their use.
BACKGROUND
There is an increasing demand to be able to identify and quantify
components of mixtures. The greater the complexity of the mixture, the
greater the interest in being able to simultaneously detect a plurality of
the components present. As illustrative of this situation is DNA
sequencing, where it is desirable to efficiently excite from one to four
fluorescently tagged components with a laser source at a single
wavelength, while providing for fluorescent signal emission at a plurality
of distinctive wavelengths. In this situation, the different labels should
not adversely affect the electrophoretic mobility of the sequences to
which they are attached.
Currently, there are four methods used for automated DNA sequencing: (1)
the DNA fragments are labeled with one fluorophore and then the fragments
run in adjacent sequencing lanes (Ansorge et al., Nucleic Acids Res. 15,
4593-4602 (1987); (2) the DNA fragments are labeled with four different
fluorophores and all the fragments are electrophoretically separated and
detected in a single lane (Smith et al.; Nature 321, 674-679 (1986); (3)
each of the dideoxynucleosides in the termination reaction is labeled with
a different fluorophore and the four sets of fragments are run in the same
lane (Prober et al., Science 238, 336-341 (1987); or (4) the sets of DNA
fragments are labeled with two different fluorophores and the DNA
sequences coded with the dye ratios (Huang et at., Anal. Chem. 64,
2149-2154 (1992).
All of these techniques have significant deficiencies. Method 1 has the
potential problems of lane-to-lane variations in mobility, as well as a
low throughput. Methods 2 and 3 require that the four dyes be well excited
by one laser source and that they have distinctly different emission
spectra. In practice, it is very difficult to find two or more dyes that
can be efficiently excited with a single laser and that emit well
separated fluorescent signals.
As one selects dyes with distinctive red-shifted emission spectra, their
absorption maxima will also move to the red and all the dyes can no longer
be efficiently excited by the same laser source. Also, as more different
dyes are selected, it becomes more difficult to select all the dyes such
that they cause the same mobility shift of the labeled molecules.
It is therefore of substantial interest that improved methods be provided
which allow for multiplexing of samples, so that a plurality of components
can be determined in the same system and in a single run. It is also
desirable for each label to have strong absorption at a common wavelength,
to have a high quantum yield for fluorescence, to have a large Stokes
shift of the emission, that the various emissions be distinctive, and that
the labels introduce the same mobility shift. It is difficult to
accomplish these conflicting goals by simply labeling the molecules with a
single dye.
SUMMARY OF THE INVENTION
The subject invention provides compositions and methods for analyzing a
mixture using a plurality of fluorescent labels. To generate the labels,
pairs or families of fluorophores are bound to a backbone, particularly a
nucleic acid backbone, where one of the members of the families is excited
at about the same wavelength. By exploiting the phenomenon of energy
transfer, the other members of each of the families emit at detectably
different wavelengths. The range of distances between donor and acceptor
chromophores is chosen to ensure efficient energy transfer. Furthermore,
labels used conjointly are selected to have approximately the same
mobility in a separation system. This is achieved by changing the mobility
of the labeled entity by varying the distance between the two or more
members of the family of fluorophores and choosing labels with the same
mobility. The subject invention finds particular application in
sequencing, where the fluorophores may be attached to universal or other
primers and different fluorophore combinations used for the different
dideoxynucleosides. Kits of combinations of labels are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color.
Copies of this patent with color drawings will be provided by the Patent
and Trademark Office upon request and payment of the necessary fee.
FIG. 1 is a graph of the absorption and emission spectra of FAM-3-TAM in
1.times.TBE;
FIG. 2 is a CE electropherogram of FAM-3-TAM. The sample was analyzed by
typical capillary electrophoresis DNA sequencing conditions with 488 nm
excitation. The green trace is the fluorescence signal detected in the
green channel (525 nm), and the red trace is the fluorescence signal
detected in the red channel (590 nm). Both channels are detected
simultaneously;
FIG. 3 is a graph of the absorption and emission spectra of FAM-4-ROX in
1.times.TBE;
FIG. 4 is a CE electropherogram of FAM-4-ROX. The sample was analyzed by
typical capillary electrophoresis DNA sequencing conditions with 488 nm
excitation. The green trace is the fluorescence signal detected in the
green channel (525 nm), and the red trace is the fluorescence signal
detected in the red channel (590 nm). Both channels are detected
simultaneously;
FIG. 5 is a CE electropherogram of FAM-4-ROX and ROX primer. The two
primers at the same concentration were mixed together in 80% formamide and
injected into the capillary. The fluorescence signals were detected in the
green and red channels simultaneously with 476 nm excitation;
FIG. 6 is a CE electropherogram of a FAM-3-ROX, FAM-4-ROX and FAM-10-ROX
mixture, showing the dependence of the mobility on the distance between
the donor and acceptor. The sample was analyzed by typical capillary
electrophoresis DNA sequencing conditions with 488 nm excitation; and
FIG. 7 is a comparison of the mobility shift of different dye primers on
M13 mp 18 A fragment DNA samples.
DESCRIPTION OF SPECIFIC EMBODIMENTS
Novel fluorescent labels, combinations of fluorescent labels, and their use
in separation systems involving the separation of a plurality of
components are provided. Particularly, the fluorescent labels comprise
pairs of fluorophores, which with one exception where the fluorophores are
the same, involve different fluorophores having overlapping spectra, where
the donor emission overlaps the acceptor absorption, so that there is
energy transfer from the excited fluorophore to the other member of the
pair. It is not essential that the excited fluorophore actually fluoresce,
it being sufficient that the excited fluorophore be able to efficiently
absorb the excitation energy and efficiently transfer it to the emitting
fluorophore.
The donor fluorophores in the different families of fluorophores may be the
same or different, but will be able to be excited efficiently by a single
light source of narrow bandwidth, particularly a laser source. The donor
fluorophores will have significant absorption, usually at least about 10%,
preferably at least about 20% of the absorption maxima within 20 nm of
each other, usually within 10 nm, more usually within 5 nm, of each other.
The emitting or accepting fluorophores will be selected to be able to
receive the energy from donor fluorophores and emit light, which will be
distinctive and detectably different. Therefore, one will be able to
distinguish between the components of the mixture to which the different
labels have been bound. Usually the labels will emit at emission maxima
separated by at least 10 nm, preferably at least 15 nm, and more
preferably at least 20 nm.
Usually the donor fluorophores will absorb in the range of about 350-800
nm, more usually in the range of about 350-600 nm or 500-750 nm, while the
acceptor fluorophores will emit light in the range of about 450-1000 nm,
usually in the range of about 450-800 nm. As will be discussed
subsequently, one may have more than a pair of absorbing molecules, so
that one may have 3 or more molecules, where energy is transferred from
one molecule to the next at higher wavelengths, to greatly increase the
difference in wavelength between absorption and observed emission.
The two fluorophores will be joined by a backbone or chain, usually a
polymeric chain, where the distance between the two fluorophores may be
varied. The physics behind the design of the labels is that the transfer
of the optical excitation from the donor to the acceptor depends on
1/R.sup.6, where R is the distance between the two fluorophores. Thus, the
distance must be chosen to provide efficient energy transfer from the
donor to the acceptor through the well-known Foerster mechanism. Thus, the
distance between the two fluorophores as determined by the number of atoms
in the chain separating the two fluorophores can be varied in accordance
with the nature of the chain. Various chains or backbones may be employed,
such as nucleic acids, both DNA and RNA, modified nucleic acids, e.g.
where oxygens may be substituted by sulfur, carbon, or nitrogen,
phosphates substituted by sulfate or carboxylate, etc., polypeptides,
polysaccharides, various groups which may be added stepwise, such as
di-functional groups, e.g. haloamines, or the like. The fluorophores may
be substituted as appropriate by appropriate functionalization of the
various building blocks, where the fluorophore may be present on the
building block during the formation of the label, or may be added
subsequently, as appropriate. Various conventional chemistries may be
employed to ensure that the appropriate spacing between the two
fluorophores is obtained.
The molecular weights of the labels (fluorophores plus the backbone to
which they are linked) will generally be at least about 250 Dal and not
more than about 5,000 Dal, usually not more than about 2,000 Dal. The
molecular weight of the fluorophore will generally be in the range of
about 250 to 1,000 Dal, where the molecular weights of the acceptor-donor
pairs on different labels to be used together will usually not differ by
more than about 20%. The fluorophores may be bound internal to the chain,
at the termini, or one at one terminus and another at an internal site.
The fluorophores may be selected so as to be from a similar chemical
family, such as cyanine dyes, xanthenes or the like. Thus, one could have
the donors from the same chemical family, each donor-acceptor pair from
the same chemical family or each acceptor from the same family.
The subject labels find particular application in various separation
techniques, such as electrophoresis, chromatography, or the like, where
one wishes to have optimized spectroscopic properties, high sensitivity
and comparable influence of the labels on the migratory aptitude of the
components being analyzed. Of particular interest is electrophoresis, such
as gel, capillary, etc. Among chromatographic techniques are HPLC,
affinity chromatography, thin layer chromatography, paper chromatography,
and the like.
It is found that the spacing between the two fluorophores will affect the
mobility of the label. Therefore, one can use different dye pairs and by
varying the distance between the different dye pairs, within a range which
still permits good energy transfer, provide for substantially constant
mobility for the labels. The mobility is not related to the specific
spacing, so that one will empirically determine the effect of the spacing
on the mobility of a particular label. However, because of the flexibility
in the spacing of the fluorophores in the labels, by synthesizing a few
different labels with different spacings and different dye pairs, one can
now provide for a family of fluorescent labels, which share a common
excitation, that have strong and distinctive emission and a substantially
common mobility. Usually, the mobility will differ by not more than about
20% of each other, preferably not more than about 10% of each other, and
more preferably within about 5% of each other, when used in a particular
separation. The mobility may usually be determined by carrying out the
separation of the labels by themselves or the labels bound to a common
molecule which is relevant to the particular separation, e.g. a nucleic
acid molecule of the appropriate size, where one is interested in
sequencing.
A wide variety of fluorescent dyes may find application. These dyes will
fall into various classes, where combinations of dyes may be used within
the same class or between different classes. Included among the classes
are dyes, such as the xanthene dyes, e.g. fluoresceins and rhedamines,
coumafins, e.g. umbelliferone, benzimide dyes, e.g. Hoechst 33258,
phenanthridine dyes, e.g. Texas Red, and ethidium dyes, acridine dyes,
cyanine dyes, such as thiazole orange, thiazole blue, Cy 5, and Cyfr,
carbazole dyes, phenoxazine dyes, porphyrin dyes, quinoline dyes, or the
like. Thus, the dyes may absorb in the ultraviolet, visible or infra-red
ranges. For the most part, the fluorescent molecules will have a molecular
weight of less than about 2 kDal, generally less than about 1.5 kDal.
The energy donor should have strong molar absorbance coefficient at the
desired excitation wavelength, desirably greater than about 10.sup.4,
preferably greater than about 10.sup.5 cm.sup.-1 M.sup.-1. The excitation
maximum of the donor and the emission maximum of the acceptor (fluorester)
will be separated by at least 15 nm or greater. The spectral overlap
integral between the emission spectrum of the donor chromophore and the
absorption spectrum of the acceptor chromophore and the distance between
the chromophores will be such that the efficiency of energy transfer from
donor to acceptor will range from 20% to 100%.
Separation of the donor and acceptor based on number of atoms in the chain
will vary depending on the nature of the backbone, whether rigid or
flexible, involving ring structures or non-cyclic structures or the like.
Generally the number of atoms in the chain (the atoms in the ring
structures will be counted as the lowest number of atoms around one side
of the ring for inclusion in the chain) will be below about 200, usually
below about 150 atoms, preferably below about 100, where the nature of the
backbone will influence the efficiency of energy transfer between donor
and acceptor.
While for the most part, pairs of fluorophores will be used, there can be
situations where up to four different, usually not more than three
different, fluorophores bound to the same backbone may find use. By using
more fluorophores, one may greatly extend the Stokes shift, so that one
may excite in the visible wavelength range and emit in the infra-red
wavelength range, usually below about 1000 nm, more usually below about
900 nm. Detecting light in the infra-red wavelength range has many
advantages, since it will not be subject to interference from Raman and
Rayleigh light resulting from the excitation light. In order to maintain
the mobility constant, one may use the same number of fluorophores on the
labels, having a multiplicity of the same fluorophore to match the number
of fluorophores on labels having different fluorophores for the large
Stokes shift.
The subject invention finds particular application with nucleic acid
chains, where the nucleic acid chains find use as primers in sequencing,
the polymerase chain reaction, particularly for sizing, or other system
where primers are employed for nucleic acid extension and one wishes to
distinguish between various components of the mixture as related to the
particular labels. For example, in sequencing, universal primers may be
employed, where a different pair of fluorophores are used for each of the
different dideoxynucleosides used for the extension during sequencing.
A large number of nucleosides are available, which are functionalized, and
may be used in the synthesis of a polynucleotide. By synthesizing the
subject nucleic acid labels, one can define the specific sites at which
the fluorophores are present. Commercially available synthesizers may be
employed in accordance with conventional ways, so that any sequence can be
achieved, with the pair of fluorophores having the appropriate spacing.
Where different primers have been used in PCR, each of the primers may be
labeled in accordance with the subject invention, so that one can readily
detect the presence of the target sequence complementary to each of the
different primers. Other applications which may find use include
identifying isozymes, using specific antibodies, identifying lectins using
different polysaccharides, and the like.
As already indicated, the subject labels find particular use in sequencing.
For example, universal primers may be prepared, where the primer may be
any one of the universal primers, having been modified by bonding of the
two fluorophores to the primer. Thus, various commercial primers are
available, such as primers from pUC/M13, .lambda.gt10, .lambda.gt11, and
the like. See, Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd ed., CSHL, 1989, Section 13. DNA sequences are cloned in an
appropriate vector having a primer sequence joined to the sequence to be
sequenced. Different 2', 3' ddNTPs are employed, so that termination
occurs at different sites, depending upon the particular ddNTP which is
present in the chain extension. By employing the subject palmers, each
ddNTP will be associated with a particular label. After extension with the
Klenow fragment, the resulting fragments may then be separated in a single
lane by electrophoresis or in a single capillary by electrophoresis, where
one can detect the terminating nucleotide by virtue of the fluorescence of
the label.
One may also use the subject labels with immune complexes, where the
ligands or receptors, e.g. antibodies, may be labeled to detect the
different complexes or members of the complexes. Where the ligands may
have the same migratory aptitude in the method separation, to determine
the presence of one or more of such ligands, the different antibodies
could be labeled with the different labels fluorescing at different
wavelengths, so as to be detectable, even where there is overlap of the
compositions in the separation.
Kits are provided having combinations of labels, usually at least 2. Each
of the labels will have the acceptor-donor pair, usually with comparable
backbones, where the labels will be separated along the backbone to give
comparable mobility in the separation method to be used. Each of the
labels in a group to be used together will absorb at about the same
wavelength and emit at different wavelengths. Each of the labels in the
group will have about the same effect on mobility in the separation
method, as a result of the variation in placement of the different
fluorophores along the backbone.
The kits will generally have up to about 6, usually about up to about 4
different labels which are matching, but may have 2 or more sets of
matching labels, having 2-6 different labels.
Of particular interest are labels comprising a nucleic acid backbone, where
the labels will generally have at least about 10 nucleotides and not more
than about 50 nucleotides, usually not more than about 30 nucleotides. The
labels may be present on the nucleotides which hybridize to the
complementary sequence or may be separated from those nucleotides. The
fluorophores will usually be joined to the nucleotide by a convenient
linking arm of from about 2 to 20, usually 4 to 16 atoms in the chain. The
chain may have a plurality of functionalities, particularly
non-oxo-carbonyl, more particularly ester and amide, amino, oxy, and the
like. The chain may be aliphatic, alicyclic, aromatic, heterocyclic, or
combinations thereof, usually comprising carbon, nitrogen, oxygen, sulfur,
or the like in the chain.
The entire nucleic acid sequence may be complementary to the 5' primer
sequence or may be complementary only to the 3' portion of the sequence.
Usually, there will be at least about 4 nucleotides, more usually at least
about 5 nucleotides which are complementary to the sequence to be copied.
The primers are combined with the sequence to be copied in the appropriate
plasmid having the primer sequence at the 3' end of the strand to be
copied and dNTPs added with a small amount of the appropriate ddntp. After
extension, the DNA may be isolated and transferred to a gel or capillary
for separation.
The kits which are employed will have at least two of the subject labels,
which will be matched by having substantially the same absorption for the
donor molecule, distinct emission spectra and substantially the same
mobility. Generally for single stranded nucleic acids, the separation will
be from about 1-15, more usually 1-12, preferably about 2-10 nucleosides
between fluorophores.
The following examples are offered by way of illustration and not by way of
limitation.
Experimental
Design and Synthesis of Energy Transfer Fluorescent Dye Tagged
Oligonucleotide Labels for Genetic Analysis.
Deoxyoligonucleotides (12-base long) with the sequence 5'-GTTTTCCCAGTC-3',
selected from the M13 universal primer, were synthesized with
donor-acceptor fluorophore pairs separated by different distances.
Specifically, the 12-mer contains a modified base introduced by the use of
5'dimethoxytrityl-5-›N-(trifluoroacetylaminohexy)-3-acrylimido!-2'-deoxyUr
idine, 3'-›(2-cyanoethyl)-(N,N-diisopropyl)!-phosphoramidite
(Amino-Modifier C6 dT) (Structure 1), which has a primary amine linker arm
at the C-5 position.
##STR1##
The donor dye was attached to the 5' side of the oligomer, and the
acceptor dye was attached to the primary amine group on the modified T.
The distances between the donor and acceptor were changed by varying the
position of the modified T on the oligomer. The primers are denoted as
D-N-A, where D is the donor, A is the acceptor and N is the number of
bases between D and A. In all the primers prepared, D is Applied
Biosystems Inc. ("ABI") dye FAM, a fluorescein derivative, A is ABI dyes
TAM or ROX which are both rhodamine derivatives. As a representative
example, the structure of FAM-3-TAM is shown below (Structure 2).
##STR2##
Structure 2. FAM-3-TAM
The advantages of the energy transfer approach described here are (1) that
a large Stokes shift and much stronger fluorescence signals can be
generated when exciting at 488 nm and (2) that the mobility of the primers
can be tuned by varying the distances between the donor and acceptor to
achieve the same mobility. The visible spectrum of FAM-3-TAM has both the
absorption of FAM (495 nm) and TAM (560 nm); however with excitation at
488 nm nearly all of the emission comes out from T with a maximum at 579
nm (FIG. 1). This demonstrates efficient fluorescence energy transfer from
FAM to TAM. This can also be seen by running the primer down a capillary
electrophoresis (CE) column and detecting in red and green channels. With
a FAM- and TAM-labeled primer, nearly all the emission is seen in the red
channel (590 nm) (FIG. 2), indicating that the energy from donor FAM was
transferred almost completely to the acceptor TAM, producing a Stokes
shift of 91 nm. The observation of a single peak indicates the primer is
pure. The same outcome is seen for FAM-4-ROX, which gives even a larger
Stokes shift of 114 nm (FIGS. 3 and 4). Enhancement of the fluorescence
signals of the energy transfer primers compared to single dye labeled
primer is seen, where an ABI ROX primer at the same concentration as that
of FAM-4-ROX (measured by UV) was injected in the same capillary. The
resulting fluorescence signal of FAM-4-ROX is seen to be more than ten
times higher than that of the ROX primer (FIG. 5).
For the successful application of donor-acceptor fluorophore labeled
primers to DNA sequencing, it is essential that the primers produce the
same mobility shifts of the DNA fragments and display distinct
fluorescence signals. It was found that the mobility of the primers
depends on the distance between the donor and acceptor (FIG. 6). FAM4-ROX,
FAM-3-ROX and FAM-10-ROX were separated on a capillary and detected in red
and green channels. For FAM-10-ROX the increased distance between the dyes
reduces the amount of energy transfer, resulting in almost equal signals
in the two channels. As the separation distance is reduced, the amount of
energy transfer increases as evidenced by the reduced relative green
signal. FAM-3-ROX and FAM-4-ROX both exhibit excellent energy transfer,
but their mobilities are distinctly different, which offers the potential
of tuning the mobility shift by varying the distance. To get an exact
match of the mobility of two primers that have distinctly different
emission spectra, FAM-3-FAM, FAM-4-FAM and FAM-10-FAM were also prepared.
Among a library of primers prepared (FAM-N-FAM, FAM-N-TAM, FAM-N-ROX), it
was found that sequencing fragments terminating in A, generated with
FAM-10-FAM and FAM-3-ROX using Sequenase 2, have very similar mobility
shifts (FIG. 7), demonstrating the potential for DNA sequence analysis.
The emission of FAM-10-FAM and FAM-3-ROX are at 525 nm and 605 nm
respectively. The water Raman signals are trivial at these two
wavelengths. Thus, the signal to noise ratio is increased dramatically.
I. Preparation of 12-mer Oligonucleotides Containing a Modified T and a FAM
Label at the 5' Position.
The following three primers were prepared on an ABI Model 394 DNA
synthesizer in a 0.2 .mu.mol scale:
##STR3##
The modified base T* containing an amino linker arm was introduced to the
defined position by using Amino-Modifier C6 dT phosphoramidite (Glen
Research) and FAM was introduced by using 6-FAM amidite (ABI) in the last
step of the synthesis. After the base sequences were completed, the
oligonucleotides were cleaved from the solid support (CPG) with 1 ml
concentrated NH.sub.4 OH. The amino protecting groups on the bases (A, G,
C and T*) were removed by heating the NH.sub.4 OH solution for 4 hours at
55.degree. C. Capillary electrophoresis analysis indicated that the
oligomers were .about.80% pure, and they were used directly in the next
dye-coupling step.
II. Attachment of the Second Fluorescent Dye to the Amino Linker Arm of the
Oligomers 1, 2 and 3.
As a representative example, the reaction scheme to couple the second dye
(TAM) to the oligomer 1 is shown below:
##STR4##
FAM-3-TAM
The FAM-labeled oligonucleotides (1, 2 and 3) in 40 .mu.L 0.5M Na.sub.2
CO.sub.3 /NaHCO.sub.3 buffer were incubated overnight at room temperature
with approximately 150 fold excess of either TAM-NHS ester, ROX-NHS ester
or FAM-NHS ester in 12 .mu.L DMSO. Unreacted dye was removed by size
exclusion chromatography on a Sephadex G-25 column. The two dye labeled
oligonucleotides were then purified by 6M urea-TBE, 20% acrylamide gel
electrophoresis (40 cm.times.0.8 cm). The pure primers were recovered from
the gel and desalted with Oligonucleotide Purification Cartridge. The
purity of the primers was shown to be >99% by capillary gel
electrophoresis.
III. Preparation of DNA Sequencing Fragments with FAM-3-ROX and FAM-10-FAM.
M13mp18 DNA sequencing fragments terminated in A were produced using
Sequenase 2.0 (USB). Two annealing solutions were prepared in 600 .mu.L
vials: (1) 10 .mu.L of reaction buffer, 40 .mu.L of M13mp18
single-stranded DNA, and 6 .mu.L of FAM-3-ROX; (2) 6 .mu.L of reaction
buffer, 20 .mu.L of M13mp18 single-stranded DNA and 3 .mu.L FAM-10-FAM.
Each vial was heated to 65.degree. C. for 5 min and then allowed to cool
to room temperature for 30 min, and then placed on ice for 20 min to
ensure that the shorter primers had completely hybridized to the template.
3 .mu.L DTT, 20 .mu.L of ddA termination mixture and 12 .mu.L diluted
Sequenase 2.0 were added to each vial on ice. The reaction mixtures were
incubated initially at 20.degree. C. for 20 min and then at 37.degree. C.
for another 20 min. Reactions were stopped by adding 10 .mu.L 50 mM EDTA,
40 .mu.L 4M NH.sub.4 OH and 300 .mu.L 95% EtOH. The solutions were mixed
well and then placed on ice for 20 min. The fragments were desalted twice
with 75% cold EtOH, dried under vacuum and dissolved in 4 .mu.L of 95%
(v/v) formamide and 50 mM EDTA. The sample was heated for 3 min to
denature the DNA and then placed on ice until sample injection on the
capillary electrophoresis instrument. Electrokinetic injection was
performed at 10 kV for 30 s.
It is evident from the above results, that one can tune related
compositions, e.g. polynucleotides functionalized with 2 fluorophores to
provide for different emission wavelengths and high emission quantum
yields, while having substantially the same excitation-light absorbance
and mobility. In this way, mixtures of compositions may be independently
analyzed, where the different components may be differentially labeled
with labels having differing fluorescence emission bands. Furthermore, the
compositions can be readily prepared, can be used in a wide variety of
contexts, and have good stability and enhanced fluorescent properties.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to be
incorporated by reference.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in the art in light of
the teachings of this invention that certain changes and modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
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