U.S. patent application number 11/196263 was filed with the patent office on 2006-09-07 for cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods.
This patent application is currently assigned to Carnegie Mellon University. Invention is credited to Alan S. Waggoner.
Application Number | 20060199949 11/196263 |
Document ID | / |
Family ID | 27505617 |
Filed Date | 2006-09-07 |
United States Patent
Application |
20060199949 |
Kind Code |
A1 |
Waggoner; Alan S. |
September 7, 2006 |
Cyanine dyes as labeling reagents for detection of biological and
other materials by luminescence methods
Abstract
The present invention pertains to luminescent dyes and methods
for covalently attaching the dyes to a component or mixture of
components so that the components may be detected and/or quantified
by luminescence detection methods. The dyes are cyanine and
cyanine-type dyes that contain or are derivatized to contain a
reactive group. The reactive group is covalently reactive with
amine, hydroxy and/or sulfhydryl groups on the component so that
the dye can be covalently bound to the component. In addition, the
dyes are preferably soluble in aqueous or other medium in which the
component is contained. The components to be labeled can be either
biological materials, such as antibodies, antigens, peptides,
nucleotides, hormones, drugs, or non-biological materials, such as
polymers, glass, or other surfaces. Any luminescent or light
absorbing detecting step can be employed in the method of the
invention.
Inventors: |
Waggoner; Alan S.;
(Pittsburgh, PA) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
Carnegie Mellon University
Pittsburgh
PA
|
Family ID: |
27505617 |
Appl. No.: |
11/196263 |
Filed: |
August 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10103119 |
Mar 22, 2002 |
6989275 |
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11196263 |
Aug 4, 2005 |
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09740486 |
Dec 19, 2000 |
6956032 |
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10103119 |
Mar 22, 2002 |
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08745712 |
Nov 12, 1996 |
6225050 |
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09740486 |
Dec 19, 2000 |
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07831759 |
Sep 22, 1992 |
5627027 |
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08745712 |
Nov 12, 1996 |
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06854347 |
Apr 18, 1986 |
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07831759 |
Sep 22, 1992 |
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Current U.S.
Class: |
530/409 ;
536/25.32; 536/55.3; 544/209; 546/269.7; 548/113; 548/156; 548/217;
548/455 |
Current CPC
Class: |
C09B 23/02 20130101;
C07H 5/06 20130101; C07H 21/00 20130101; C07D 209/14 20130101; C07D
209/60 20130101; Y10T 436/173845 20150115; C07D 403/12
20130101 |
Class at
Publication: |
530/409 ;
536/025.32; 536/055.3; 548/455; 548/156; 548/217; 548/113; 544/209;
546/269.7 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07H 21/00 20060101 C07H021/00; C07H 5/06 20060101
C07H005/06; C07D 417/02 20060101 C07D417/02; C07D 413/02 20060101
C07D413/02; C07D 403/02 20060101 C07D403/02 |
Goverment Interests
[0001] This invention was made in part under National Institutes of
Health contract number 5R01NS19353-02. The United States government
may have rights in this invention.
Claims
1. A method for imparting luminescence to a component which
contains or is derivatized to contain an amine, sulhydryl or
hydroxy group in a liquid, said method comprising the steps of:
adding to a liquid containing said component, wherein said
component is selected from the group consisting of proteins, cells,
nucleic acids, sugars, carbohydrates and combinations thereof a
luminescent cyanine dye having the structure ##STR18## wherein: the
dotted lines each represent carbon atoms necessary for the
formation of said cyanine dye, X and Y are selected from the group
consisting of O, S and CH.sub.3--C--CH.sub.3, m is an integer
selected from the group consisting of 1, 2, 3 and 4, and at least
one of R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.7 contain a
reactive group covalently reactive with amine or hydroxy groups;
and reacting said cyanine dye with said component under reaction
conditions sufficient for forming a covalent bond between said
reactive group and said amine or hydroxy group so that said dye
labels said component.
2. The method recited in claim 1 wherein said reactive group is
selected from the group consisting of isothiocyanate, isocyanate,
phosphoramidite, monochlorotriazine, dichlorotriazine, mono- or di-
halogen substituted pyridine, mono- or di- halogen substituted
diazine, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido
ester, glyoxal and aldehyde.
3. The method recited in claim 2 wherein when any of said R.sub.3,
R.sub.4 and R.sub.7 is not a reactive group, said remaining
R.sub.3, R.sub.4 and R.sub.7 are selected from the group consisting
of hydrogen, C.sub.1-C.sub.4 alkyl or the group E-F, a polar group
wherein E is a spacer group having the structure
--(CH.sub.2).sub.n-- and n of said spacer group is 1, 2, 3, 4 or 5,
and F is hydroxy, protected hydroxy, sulfonate, sulfate, carboxyl,
or lower alkyl substituted amino; and when any of R.sub.1 and
R.sub.2 is not said reactive group, said remaining R.sub.1 or
R.sub.2 are a C.sub.1-C.sub.4 alkyl group or said E-F group and n
of said spacer group is 1, 2, 3, 4 or 5.
4. A luminescent cyanine dye comprising a cyanine dye having the
structure ##STR19## wherein: the dotted lines each represent carbon
atoms necessary for the formulation of said cyanine; X and Y are
independently selected from the group consisting of O, S and
CH.sub.3--C--CH.sub.3; m is an integer from 1-4; at least one of
the groups R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.7 is a
reactive group, reactive with amino, sulfhydryl or hydroxy
nucleophiles; and when any of said R.sub.3, R.sub.4 and R.sub.7 is
not a reactive group, said remaining R.sub.3, R.sub.4 and R.sub.7
are selected from the group consisting of hydrogen, C.sub.1-C.sub.4
alkyl or the group E-F, a polar group wherein E is a spacer group
having the structure --(CH.sub.2).sub.n-- and n of said spacer
group is 1, 2, 3, 4 or 5, and F is hydroxy, protected hydroxy,
sulfonate, sulfate, carboxyl, or lower alkyl substituted amino; and
when any of R.sub.1 and R.sub.2 is not said reactive group, said
remaining R.sub.1 or R.sub.2 are a C.sub.1-C.sub.4 alkyl group or
said E-F group and n of said spacer group is 1, 2, 3, 4 or 5.
5. The cyanine dye recited in claim 4 wherein said reactive group
is selected from the group consisting of isothiocyanate,
isocyanate, phosphoramidite, monochlorotriazine, dichlorotriazine,
mono- or di-halogen substituted pyridine, mono- or di-halogen
substituted diazine, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido
ester, glyoxal and aldehyde.
6. A luminescent labeled component comprising: a component selected
from the group consisting of proteins, nucleic acids, cells,
carbohydrates, sugars, and combinations thereof having amino,
sulfhydryl or hydroxy groups covalently bound to a luminescent
cyanine dye having the structure ##STR20## wherein: the dotted
lines each represent carbon atoms necessary for the formulation of
said cyanine; X and Y are independently selected from the group
consisting of O, S and CH.sub.3--C--CH.sub.3; m is an integer from
1-4; at least one of the groups R.sub.1, R.sub.2, R.sub.3, R.sub.4
and R.sub.7 is a reactive group, reactive with amino, sulfhydryl or
hydroxy nucleophiles.
7. The component recited in claim 6 wherein at least one of said
reactive groups is a phosphoramidite.
8. The component recited in claim 6 wherein said reactive group is
selected from the group consisting of isothiocyanate, isocyanate,
phosphoramidite, monochlorotriazine, dichlorotriazine, mono- or
di-halogen substituted pyridine, mono- or di-halogen substituted
diazine, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxy sulfosuccinimide ester, imido
ester, glyoxal and aldehyde; and when any of said R.sub.3, R.sub.4
and R.sub.7 is not a reactive group, said remaining R.sub.3,
R.sub.4 and R.sub.7 are selected from the group consisting of
hydrogen, C.sub.1-C.sub.4 alkyl or the group E-F, a polar group
wherein E is a spacer group having the structure
--(CH.sub.2).sub.n-- and n of said spacer group is 1, 2, 3, 4 or 5,
and F is hydroxy, protected hydroxy, sulfonate, sulfate, carboxyl,
or lower alkyl substituted amino; and when any of R.sub.1 and
R.sub.2 is not said reactive group, said remaining R.sub.1 or
R.sub.2 are a C.sub.1-C.sub.4 alkyl group or said E-F group and n
of said spacer group is 1, 2, 3, 4 or 5.
Description
BACKGROUND OF THE INVENTION
[0002] Cyanine and related polymethine dyes having light absorbing
properties have been employed in photographic films. Although such
dyes require light absorbing properties, they do not require
luminescence (fluorescent or phosphorescent) properties. Cyanine
dyes having luminescence properties heretofore have had very
limited utilization. One such utilization involved specifically
labeling the sulfhydryl group of proteins. In one report, Salama,
G., Waggoner, A. S., and Abramson, J. have reported under the title
Sulfhydryl Reagent Dyes Trigger the Rapid Release of Ca.sup.2+ from
Sarcoplasmic Reticulum Vesicles (SR), Biophysical Journal, 47, 456a
(1985) that cyanine chromophores having an iodoacetyl group was
used to form covalent bonds with sulfhydryl groups on the
Sarcoplasmic Reticulum protein at Ph 6.7 to trigger Ca.sup.2+
release. The report also stated that fluorescent dyes were used to
label and isolate those proteins.
[0003] In a report of A. S. Waggoner, P. L. Jenkins and J. P.
Carpenter entitled The Kinetics of Conformational Changes in a
Region of the Rhodopsin Molecule Away From the Retinylidene Binding
Site, Biophysical Journal, 33, 292a (1981), the authors state that
the sulfhydryl group on the F1 region of cattle rhodopsin has been
covalently labeled with a cyanine dye having absorbance at 660 nm.
Again, this report used cyanine dyes for labeling specifically the
sulfhydryl group of a protein, but does not disclose that
fluorescent dyes were used.
[0004] An article entitled International workshop on the
application of fluorescence photobleaching techniques to problems
in cell biology, Jacobson K., Elson E., Koppel D., Webb W. Fed.
Proc. 42:72-79 (1983), reports on a paper delivered by A. Waggoner
relating to cyanine-type fluorescent probes which can be conjugated
to proteins and can be excited in the deeper red region of the
spectrum.
[0005] The only cyanine probes mentioned in any of the above three
reports are those which covalently attach specifically to the
sulfhydryl group of a protein. The only specific cyanine compound
mentioned is one having an iodoacetyl group, which group causes the
cyanine dye to be covalently reactive with a sulfhydryl group. None
of the articles listed above discloses the covalent reaction of a
cyanine dye with any material other than a protein or with any
group on a protein other than a sulfhydryl group.
[0006] However, many non-protein materials do not have sulfhydryl
groups and many proteins do not have a sufficient number of
sulfhydryl groups to make these groups useful for purposes of
fluorescence probing. Furthermore, sulfhydryl groups (--SHSH--) are
easily oxidized to disulfides (--S--S--) in the presence of air and
thereby become unavailable for covalent attachment to a
fluorescence probe.
SUMMARY OF THE INVENTION
[0007] In accordance with the present invention, cyanine and
related polymethine dyes have been developed having substituent
groups which are covalently reactive under suitable reaction
conditions with amine (--NH.sub.2) and hydroxy (--OH) groups on
proteins, nucleic acids, cells, sugars, carbohydrates and other
materials for purposes of fluorescence and phosphorescence
detection of those materials. The present invention offers
considerable advantages over the use of the iodoacetyl cyanine dye
of the prior art and its specific reactivity with sulfhydryl
groups. Amine and hydroxy groups are more prevalent in proteins and
other materials than are sulfhydryl groups and are more stable.
Thereby, when fluorescent cyanine dyes are used for detecting the
presence of certain proteins, a stronger fluorescent or
phosphorescent light intensity signal will be given off because a
larger number of dye molecules can be attached to the protein which
is being probed. Furthermore, amine and hydroxy groups are more
easily added to components which it is desired to label, such as
polymer particles, which do not naturally contain either
sulfhydryl, amine or hydroxy groups.
[0008] According to the present invention, the iodoacetyl cyanine
dye which was used in the prior art at a pH of 6.7 to react with
sulfhydryl groups can be sometimes used in a process under
appropriate temperature and reaction time conditions at pH's above
7 at which they will covalently react with neutral amine and
hydroxy groups.
[0009] This invention also relates to a method wherein luminescent
cyanine dyes which contain a group which is covalently reactive
with amine or hydroxy groups are used to label proteins, nucleic
acids, cells, sugars, carbohydrates or other materials having an
amine or hydroxy group in a mixture so that the presence and amount
of labeled protein or other material can be detected after the
labeled components have been separated by chromatographic methods.
According to the above cited references, apparently the sulfhydryl
group was selected for covalent reaction specifically because there
are so few of these groups on a protein molecule and because in
some cases the sulfhydryl group plays a significant role in the
function of the protein. Therefore, it was possible for the authors
to attempt to ascertain the specific location of a sulfhydryl group
on a protein structure. Also, in those references the
sulfhydryl-specific dye was used as a probe to detect or to produce
structural changes in a specific protein. Then, in order to
interpret a change in light absorption by the dye or the calcium
ion released by dye binding, it was necessary to know where the
probe is bound.
[0010] Because there are so few sulfhydryl groups on most protein
molecules, those groups may not be sufficiently numerous to provide
adequate total luminescence for detection studies. In contrast,
amine and hydroxy groups are significantly more numerous and are
widely dispersed on a protein molecule enabling a fluorescent probe
to be attached to multiple sites on the molecule, thereby
precluding interpretation of light absorption or fluorescence
changes, by facilitating the detection of the protein. Therefore,
the method of the present invention is clearly in contrast to the
method of the references cited above.
[0011] The present invention relates to the labeling with
luminescent polymethine cyanine and related polymethine dyes, such
as merocyanine, styryl and oxonol, of proteins, sugars,
carbohydrates and other materials, including nucleic acids, DNA,
drugs, toxins, blood cells, microbial materials, particles, etc.,
at an amine or hydroxy site on those materials. The dyes are
advantageously soluble in aqueous or other medium in which the
labeled material is contained. The present invention relates to a
two-step labeling process in addition to a single step labeling
process. In the two-step labeling process, a primary component,
such as an antibody, can be labeled at sites thereon, including
amine, hydroxy or sulfhydryl sites, and the labeled component is
used as the probe for a secondary component, such as an antigen for
which the antibody is specific.
[0012] In the prior art discussed above, specificity of site of
attachment by a cyanine probe was achieved by using a probe which
is covalently reactive with a sulfhydryl group. According to the
two-step method of the present invention, cyanine and related
probes can be reacted in a first step with amine, sulfhydryl or
hydroxy groups on a first component, such as an antibody, and then
the antibody can achieve the desired specificity in a second
component, such as an antigen, in a second or staining step, the
specificity being determined by the antigen site of attachment to
the antibody.
[0013] The present invention is directed also to the luminescent
polymethine cyanine and related. compounds which contain groups
enabling them to be covalently attached to amine or hydroxy groups
on a target molecule. It is directed to monoclonal antibodies and
other components labeled with these luminescent cyanine compounds
which are capable of being probes for antigens. When the target is
a type of cell, the present invention can be employed to measure
the amount of labeled antibodies which are attached to that type of
cell. The measurement can be made by determining the relative
brightness or dimness of the luminescence of the cells.
[0014] The present invention can be employed to determine the
concentration of a particular protein or other component in a
system. If the number of reactive groups on a protein which can
react with a probe is known, the fluorescence per molecule can be
known and the concentration of these molecules in the system can be
determined by the total luminescence intensity of the system.
[0015] The method can be employed to quantify a variety of proteins
or other materials in a system by labeling all of a mixture of
proteins in the system and then. separating the labeled proteins by
any means, such as chromatographic means. The amount of separated
proteins that are luminescent can then be determined. In
chromatographic detection systems, the location of the dye on the
labeled material can be ascertained.
[0016] This invention can also be employed to determine the number
of different cells which are tagged by an antibody. This
determination can be made by tagging a plurality of types of cells
in a system, and then separating the tagged cells outside of the
system. Also, tagged cells can be separated from non-tagged cells
outside of the system.
[0017] Another embodiment of the present invention comprises a
multiparameter method employing a plurality of luminescent cyanine
or related dyes attached respectively to a plurality of different
primary components, such as antibodies, each specific for a
different secondary component, such as an antigen, in order to
identify each of a plurality of said antigens in a mixture of
antigens. According to this embodiment, each of said antibodies is
separately labeled with a dye having different light absorption and
luminescence wavelength characteristics than the dye used for
labeling the other probes. Then, the labeled antibodies are all
added to a biological preparation being analyzed containing
secondary components, such as antigens, which can be respectively
stained by particular labeled antibodies. Any unreacted dye
materials may be removed from the preparation as by washing, if
they interfere with the analysis. The biological preparation is
then subjected to a variety of excitation wavelengths, each
excitation wavelength used being the excitation wavelength of a
particular conjugated dye. A luminescence microscope or other
luminescence detection system, such as a flow cytometer or
fluorescence spectrophotometer, having filters or monochrometers to
select the rays of the excitation wavelength and to select the
wavelengths of luminescence is employed to determine the intensity
of rays of the emission wavelength corresponding to the excitation
wavelength. The intensity of luminescence at wavelengths
corresponding to the emission wavelength of a particular conjugated
dye indicates the quantity of antigen which has been bound the
antibody to which the dye is attached. If desired, a light
absorption detection method can be employed. The two-step method of
the invention can be applied to any system in which a primary
material conjugated with a dye is used in a luminescence or light
absorption detection system to detect the presence of another
material to which the primary material-dye conjugate is directed.
For example, the dye can be conjugated to a fragment of DNA or RNA
to form a dye conjugated DNA or RNA fragment which is then directed
to a main strand of DNA or RNA to which the piece is complementary.
The same test method can be employed to detect the presence of any
complementary main strand of DNA.
[0018] The cyanine and related dyes of this invention are
especially well adapted for the analysis of a mixture of components
wherein dyes of a variety of excitation and emission wavelengths
are required because specific cyanine and related dyes can be
synthesized having a wide range of excitation and emission
wavelengths. Specific cyanine and related dyes having specific
excitation and emission wavelengths can be synthesized by varying
the number of methine groups or by modifying the cyanine ring
structures. In this manner, it is possible to synthesize dyes
having particular excitation wavelengths to correspond to a
particular excitation light source, such as a laser, e.g., a HeNe
laser or a diode laser.
[0019] This invention relates to the covalent reaction of highly
luminescent and highly light absorbing cyanine and related dye
molecules under reaction conditions to amine and hydroxy groups on
proteins, peptides, sugars, carbohydrates, nucleic acids,
derivatized nucleic acids, lipids, certain other biological
molecules, biological cells, as well as to non-biological
materials, such as soluble polymers, polymeric particles, and other
particles and surfaces. Because luminescence involves highly
sensitive optical techniques, the presence of these dye "labels"
can be detected and quantified even when the label is present in
very low amounts. Thus the dye labeling reagents can be used to
measure the quantity of a material that has been labeled. The most
useful dyes are highly light absorbing (.epsilon.=100,000 to
250,000 liters per mole centimeter, or higher) and very luminescent
and they have quantum yields of at least 0.1 to 0.8, or more. The
qualities apply to the dyes themselves and to the dyes conjugated
to a labeled material.
[0020] An important application for these color labeling reagents
is the production of luminescent monoclonal antibodies. Monoclonal
antibodies are protein molecules that bind very tightly and very
specifically to certain chemical sites or "markers" on cell
surfaces or within cells. These antibodies, therefore, have an
enormous research and clinical use for identifying certain cell
types (e.g., HLA classification, T-cell subsets, bacterial and
viral classification, etc.) and diseased cells. In the past, the
amount of antibody bound to a cell has been quantified by tagging
the antibody in various ways. Tagging has been accomplished with a
radioactive label (radio immune assay), an enzyme (ELISA
techniques), or a fluorescent dye (usually fluorescein, rhodamine,
Texas Red or phycoerythrin). Most manufacturers and users of
clinical antibody reagents would like to get away from the problems
involved in the use of radioactive tracers so luminescence is
considered one of the most promising alternatives. In fact, many
companies now market fluorescein, Texas Red, rhodamine and
phycoerythrin labeled monoclonal antibodies.
[0021] In recent years, optical/electronic instrumentation for
detecting fluorescent antibodies on cells has become more
sophisticated. For example, flow cytometry can be used to measure
the amount of fluorescent antibody on individual cells at a rate up
to 5,000 cells per second. Microscopy and solution fluorescence
techniques have also advanced. These instruments can excite
fluorescence at many wavelengths of the UV, visible, and near IR
regions of the spectrum. Yet most of the useful fluorescent
labeling reagents available today can. be excited only in the
400-580 nm region of the spectrum. The exceptions are some of the
phycobiliprotein-type pigments isolated from marine organisms which
can be covalently attached to proteins and which can be excited at
somewhat longer wavelengths. Therefore, there is a large spectral
window ranging from 580 to roughly 900 nm where new labeling
reagents need to become available for labeling biological and
non-biological materials for analysis with now available
instrumentation. New reagents excitable in this spectral region
would make it possible to perform color luminescence analyses of
markers on cells because antibodies with different specificities
could each be tagged with a different colored fluorescent dye. Thus
the presence of several markers could be determined simultaneously
for each cell analyzed.
[0022] This invention also relates to the luminescent (fluorescent
or phosphorescent) cyanine, merocyanine, styryl and oxonol dyes
themselves that can be covalently linked to biological and
non-biological materials. Merocyanine, styryl and oxonol dyes are
considered to be related to cyanine dyes for purposes of this
invention. The present invention also relates to conjugate
complexes comprised of components selected from the group
consisting of nucleic acid molecules (DNA and RNA), proteins,
cells, sugars and carbohydrates that are labelled with the
luminescent cyanine dyes described herein. The new labeling
reagents themselves, but more particularly when conjugated to a
labeled component, can be excited by light of first defined
wavelengths, e.g. by light in wavelength regions of the spectrum
ranging from 450 nm to 900 nm. Background fluorescence of cells
generally occurs at a lower wavelength. Therefore, the labeling
reagents will distinguish over background fluorescence.
Particularly of interest are the derivatives that absorb light at
633 nm since they can be excited by inexpensive, intense, stable,
long-life, HeNe laser sources. Light of second defined wavelengths
that is fluoresced or phosphoresced by the labeled component can
then be detected. The fluoresced or phosphoresced light generally
has a greater wavelength than the excitation light. The detection
step can employ a luminescence microscope having a filter for
absorption of scattered light of the excitation wavelength and for
passing the wavelength that corresponds to the luminescence
corresponding to the particular dye label used with the specimen.
Such an optical microscope is described in Ser. No. 711,065, filed
Mar. 12, 1985.
[0023] Not all cyanine and related dyes are luminescent. However,
the dyes of this invention include those of the cyanine and related
dyes which are luminescent. They are relatively photostable and
many are soluble in the reaction solution, preferably a-water
solution. The conjugated dyes themselves, but more particularly
when conjugated to a labeled component, have molar extinction
coefficients (.epsilon.) of at least 50,000, and preferably at
least 120,000 liters per mole centimeter. The extinction
coefficient is a measure of the capability of the molecules to
absorb light. The conjugated dyes of this invention have quantum
yields of at least 5 percent and preferably at least 20 percent. In
addition, the conjugated dyes of this invention absorb and emit
light in the 400 to 900nm spectral range, and preferably in the 600
to 900 nm spectral range.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The present invention can be better understood by reference
to the figures in which:
[0025] FIGS. 1(a) and (b) are absorbance scans of oligonucleotide
synthesis products after deprotection and ethanol precipitation of
the DNA but before HPLC purification;
[0026] FIG. 2 is an HPLC trace showing separation of the labelled
oligonucleotide from unlabelled DNA and free dye of Example 7;
[0027] FIGS. 3(a) and (b) are the absorbance scans of the dye
primers of Examples 7 and 8 after HPC purification; and
[0028] FIGS. 4(a) and (b) are DNA sequence traces for the dye
primers of Examples 7 and 8.
BACKGROUND PROCEDURES
[0029] Luminescent probes are valuable reagents for the analysis
and separation of molecules and cells and for the detection and
quantification of other material. A very small number of
luminescent molecules can be detected under optimal circumstances.
Barak and Webb visualized fewer than 50 fluorescent lipid analogs
associated with the LDL reception of cells using a SIT camera, J.
Cell Biol. 90:595-604 (1981). Flow cytometry can be used to detect
fewer than 10,000 fluorescein molecules associated with particles
or certain cells (Muirhead, Horan and Poste, Bio/Technology
3:337-356 (1985)). Some specific examples of the application of
fluorescent probes are (1) identification and separation of
subpopulations of cells in a mixture of cells by the techniques of
fluorescence flow cytometry, fluorescence-activated cell sorting
and fluorescence microscopy; (2) determination of the concentration
of a substance that binds to a second species (e.g.,
antigen-antibody reactions) in the technique of fluorescence
immunoassay; (3) localization of substances in gels and other
insoluble supports by the techniques of fluorescence staining.
These techniques are described by Herzenberg et al., "Cellular
Immunology," .3rd ed., chapt. 22; Blackwell Scientific
Publications, 1978 (fluorescence-activated cell sorting); and by
Goldman, "Fluorescence Antibody Methods," Academic Press, New York,
(1968) (fluorescence microscopy and fluorescence staining); and in
Applications of Fluorescence in the Biomedical Sciences, ed. Taylor
et al., Alan Liss Inc., (1986).
[0030] When employing fluorescers for the above purposes, there are
many constraints on the choice of the fluorescer. One constraint is
the absorption and emission characteristics of the fluorescer,
since many ligands, receptors, and materials in the sample under
test, e.g. blood, urine, cerebrospinal fluid, will fluoresce and
interfere with an accurate determination of the fluorescence of the
fluorescent label. This phenomenon is called autofluorescence or
background fluorescence. Another consideration is the ability to
conjugate the fluorescer to ligands and receptors and other
biological and non-biological materials and the effect of such
conjugation on the fluorescer. In many situations, conjugation to
another molecule may result in a substantial change in the
fluorescent characteristics of the fluorescer and, in some cases,
substantially destroy or reduce the quantum efficiency of the
fluorescer. It is also possible that conjugation with the
fluorescer will inactivate the function of the molecule that is
labeled. A third consideration is the quantum efficiency of the
fluorescer which should be high for sensitive detection. A fourth
consideration is the light absorbing capability, or extinction
coefficient, of the fluorescers, which should also be as large as
possible. Also of concern is whether the fluorescent molecules will
interact with each other when in close proximity, resulting in
self-quenching. An additional concern is whether there is
non-specific binding of the fluorescer to other compounds or
container walls, either by themselves or in conjunction with the
compound to which the fluorescer is conjugated.
[0031] The applicability and value of the methods indicated above
are closely tied to the availability of suitable fluorescent
compounds. In particular, there is a need for fluorescent
substances that emit in the longer wavelength visible region
(yellow to near infrared), since excitation of these chromophores
produces less autofluorescence and also multiple chromophores
fluorescing at different wavelengths can be analyzed simultaneously
if the full visible and near infrared regions of the spectrum can
be utilized. Fluorescein, a widely used fluorescent compound, is a
useful emitter in the green region although in certain immunoassays
and cell analysis systems background autofluorescence generated by
excitation at fluorescein absorption wavelengths limits the
detection sensitivity. However, the conventional red fluorescent
label rhodamine has proved to be less effective than fluorescein.
Texas Red is a useful labeling reagent that can be excited at 578
nm and fluoresces maximally at 610 nm.
[0032] Phycobiliproteins have made an important contribution
because of their high extinction coefficient and high quantum
yield. These chromophore-containing proteins can be covalently
linked to many proteins and are used in fluorescence antibody
assays in microscopy and flow cytometry. The phycobiliproteins have
the disadvantages that (1) the protein labeling procedure is
relatively complex; (2) the protein labeling efficiency is not
usually high (typically an average of 0.5 phycobiliprotein
molecules per protein); (3) the phycobiliprotein is a natural
product and its preparation and purification is complex; (4) the
phycobiliproteins are expensive; (5) there are at present no
phycobiliproteins available as labeling reagents that fluoresce
further to the red region of the spectrum than allophycocyanine,
which fluoresces maximally at 680 nm; (6) the phycobiliproteins are
large proteins with molecular weights ranging from 33,000 to
240,000 and are larger than many materials that it is desirable to
label, such as metabolites, drugs, hormones, derivatized
nucleotides, and many proteins including antibodies. The latter
disadvantage is of particular importance because antibodies,
avidin, DNA-hybridization probes, hormones, and small molecules
labeled with the large phycobiliproteins may not be able to bind to
their targets because of steric limitations imposed by the size of
the conjugated complex.
[0033] Other techniques involving histology, cytology, immunoassays
would also enjoy substantial benefits from the use of a fluorescer
with a high quantum efficiency, absorption and emission
characteristics at longer wavelengths, having simple means for
conjugation and being substantially free of nonspecific
interference.
OUTLINE OF INVENTION
[0034] This invention employs reactive fluorescent cyanine and
related dyes having relatively large extinction coefficients and
high quantum yields for the purpose of detection and quantification
of labeled components. Fluorescent cyanine and related dyes can
preferably be used to label biological materials such as nucleic
acids, proteins, carbohydrates, sugars, cells and combinations
thereof. More specifically, the cyanine type dyes of the invention
can label antibodies, antigens, avidin, peptides, derivatized
nucleotides, bacteria, viruses, blood cells, tissue cells,
hormones, lymphokines, trace biological molecules, toxins and
drugs. Fluorescent dyes can also be used to label non-biological
materials, such as soluble polymers and polymeric particles, glass,
monomers, drugs and other surfaces and particles which contain or
are derivatized to contain functionalities capable of binding
covalently to the amino, hydroxy or sulfhydryl reactive
nucleophiles of the cyanine dye molecule. The component being
labeled can be in a mixture including other materials. The mixture,
in which the labeling reaction occurs, can be a liquid mixture,
particularly a water mixture. The detection step can occur with the
mixture in a liquid or dry condition, such as a microscope
slide.
[0035] This invention requires cyanine dyes to be modified by the
incorporation into the cyanine molecule of a reactive group that
will covalently attach to a target molecule, preferably at an amine
or hydroxy site, and in some instances at a sulfhydryl site. This
invention also employs modification or use of cyanine and related
dye structures to enhance their solubility in the test liquid to
help prevent aggregation of the dye on the surface of proteins that
are being labeled and to help prevent nonspecific binding of
labeled materials to biological materials and to surfaces and assay
apparatus.
[0036] The cyanine and related dyes offer an important advantage
over existing fluorescent labeling reagents. First, cyanine and
related dyes have been synthesized that absorb and emit in a region
of the spectrum ranging from 400 to nearly 1100 nm. Thus reactive
derivatives of these dyes can be made for assays that require
simultaneous measurement of a number of labeled materials.
Multicolor (or multiparameter) analysis of this sort may be
desirable for the sake of simplicity, cost effectiveness, or for
determining ratios of different labeled species on each particle in
a complex mixture of particles (e.g., ratios of antigen markers on
individual blood cells in a complex mixture by multiparameter flow
cytometry or fluorescence microscopy). Second, many cyanine and
related dyes strongly absorb and fluoresce light. Third, many
cyanine and related dyes are relatively photostable and do not
rapidly bleach under the fluorescence microscope. Fourth, cyanine
and related dye derivatives can be made which are simple and
effective coupling reagents. Fifth, many structures and synthetic
procedures are available and the class of dyes is versatile.
Therefore, many structural modifications can be made to make the
reagents more or less water soluble. Their charge can be changed so
they will not perturb the molecule to which they are attached and
so nonspecific binding can be reduced. Sixth, unlike the
phycobiliproteins the cyanine type dyes are relatively small
(molecular weight=1,000) so they don't sterically interfere
appreciably with the ability of the labeled molecule to reach its
binding sight or carry out its function.
[0037] Thus cyanine type dye labeling agents offer many potential
advantages. These dyes can be used to selectively label one or more
components in a liquid, especially an aqueous liquid. The labeled
component can then be detected by optical or luminescence methods.
Alternately, the labeled component can then be used to stain a
second component for which it has a strong affinity, and the
presence of the second component is then detected by optical or
luminescence methods. In this case, the dye is reacted with an
amine, hydroxy or sulfhydryl group on the labeled component. For
example, the labeled component can be an antibody and the stained
component for which it has a strong affinity can be a biological
cell, an antigen or a hapten, or a biological cell or particle
containing said antigen or hapten. In another example, the labeled
component is avidin and the stained component can be biotinylated
materials. Also, lectins conjugated with polymethine cyanine type
dyes can be used to detect and quantify specific carbohydrate
groups. In addition, luminescent cyanine and related dyes can be
attached to fragments of DNA or RNA. The labeled fragments of
DNA.or RNA can be used as fluorescent hybridization probes to
identify the presence and quantity of specific complementary
nucleotide sequences in samples of DNA or RNA. Also, the dye can be
attached to a hormone or ligand (such as a hormone, protein,
peptide, lymphokine, metabolite) which in turn can be attached to a
receptor.
Reactive Cyanine Dyes are Described in Patents for Other Uses
[0038] Miraha et al. (U.S. Pat. No. 4,337,063), and Masuda et al.
(U.S. Pat. No. 4,404,289 and U.S. Pat. No. 4,405,711) have
synthesized a variety of cyanine dyes possessing
N-hydroxysuccinimide active ester groups. These patents show that
these reagents can be used as photographic sensitizers. The
possible fluorescence properties of these reagents are not
mentioned in the patents and, indeed, fluorescence is not required
for their process. Most of the dyes mentioned in those patents are
only weakly fluorescent, they are not especially photostable, and
their solubility properties are not optimal for many uses that
would involve fluorescence detection of labeled materials.
[0039] Exekiel et al. (British Patent 1,529,202) have presented
numerous cyanine dye derivatives that can be used as covalently
reacting molecules. The reactive group used in these reagents are
azine groups to which the mono- and dichloro-triazine groups
belong. The British patent relates to the development and use of
these reagents as photographic film sensitizers. Fluorescence is
not required for the process and most of the reagents described are
not fluorescent. The British patent does not relate to the
development and use of reactive cyanine dyes for the purpose of
detecting and quantifying labeled materials.
Description of the Preferred Embodiments
[0040] The present invention pertains to methods for covalently
attaching luminescent cyanine and cyanine-type dyes to biological
materials, non-biological molecules and macromolecules, and
particles in order to make the material that has been labeled
luminescent so that the labeled material can be detected and/or
quantified by luminescence detection methods.
[0041] This invention relates to a method for the detection of a
component in a liquid comprising adding to said liquid a dye
selected from the group consisting of cyanine, merocyanine, oxonol
and styryl dyes which is soluble in the liquid and contains a
substituent to make it covalently reactive with amine and hydroxy
groups, and possibly to sulfhydryl groups, on said component so
that it labels said component. The labeled component is then
detected and/or quantified by luminescence or light absorption
methods. If the labeled component is an antibody, DNA fragment,
hormone, lymphokine, or drug, the labeled component can be used to
identify the presence of a second component to which it binds, and
then the second component can be detected and/or quantified.
[0042] Any available luminescence or light absorbing detecting step
can be employed. For example, the detecting step can be an optical
detecting step wherein the liquid is illuminated with light of
first defined wavelengths. Light at second defined wavelengths that
is fluoresced or phosphoresced by the labeled component is then
detected. The detection also can be by optical light absorption.
For example, the detecting step can comprise passing light of first
defined wavelengths through the liquid and then ascertaining the
wavelength of the light that is transmitted by the liquid.
[0043] If desired, the detecting step can comprise chemical
analysis to chemically detect attachment of the cyanine or related
chromophore to the component.
[0044] The basic structures of cyanine, merocyanine, styryl and
oxonol dyes that can be modified to create covalent labeling
reagents are shown below wherein the dotted lines each represent
the carbon atoms necessary for the formulation of the dye.
##STR1##
[0045] The following are more specific examples of polymethine
cyanine type dyes: ##STR2##
[0046] In these structures [0047] X and Y are selected from the
group consisting of O, S and CH.sub.3--C--CH.sub.3; [0048] Z is
selected from the group consisting of O and S; [0049] m is an
integer selected from the group consisting of 1, 2, 3 and 4 and,
preferably an integer from 1-3.
[0050] In the above formulas, the. number of methine groups
determines in part the excitation color. The cyclic azine
structures can also determine in part the excitation color. Often,
higher values of m contribute to increased luminescence and
absorbance. At values of m above 4, the compound becomes unstable.
Thereupon, further luminescence can be imparted by modifications at
the ring structures. When m=2, the excitation wavelength is about
650 nm and the compound is very fluorescent. Maximum emission
wavelengths are generally 15-100 nm greater than maximum excitation
wavelengths.
[0051] At least one, preferably only one, and possibly two or more
of said R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 groups in each molecule is or contains a reactive group
covalently reactive with amine, protected or unprotected hydroxy or
sulfhydryl nucleophiles for attaching the dye to the labeled
component. For certain reagents, at least one of said R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 groups on
each molecule may also be a group that increases the solubility of
the chromophore, or affects the selectivity of labeling of the
labeled component or affects the position of labeling of the
labeled component by the dye. Reactive groups that may be attached
directly or indirectly to the chromophore to form R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 groups may include
reactive moieties such as groups containing isothiocyanate,
isocyanate, monochlorotriazine, dichlorotriazine, mono- or
di-halogen substituted pyridine, mono- or di-halogen substituted
diazine, phosphoramidite, maleimide, aziridine, sulfonyl halide,
acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide
ester, imido ester, hydrazine, axidonitrophenyl, azide,
3-(2-pyridyl dithio)-proprionamide, glyoxal and aldehyde.
[0052] Specific examples of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 groups that are especially useful for
labeling components with available amino-, hydroxy-, and sulfhydryl
groups include: ##STR3## where at least one of Q or W is a leaving
group such as I, Br or Cl, ##STR4##
[0053] Specific examples of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 groups that are especially useful for
labeling components with available sulfhydryls which can be used
for labeling antibodies in a two-step process: ##STR5## where Q is
a leaving group such as I or Br, ##STR6## where n is 0 or an
integer.
[0054] Specific examples of R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6 and R.sub.7 groups that are especially useful for
labeling components by light-activated cross linking include:
##STR7##
[0055] For the purpose of increasing water solubility or reducing
unwanted nonspecific binding of the labeled component to
inappropriate components in the sample or to reduce the
interactions between two or more reactive chromophores on the
labeled component.which might lead to quenching of fluorescence,
the R.sub.1, R.sub.2, R.sub.31 R.sub.4, R.sub.5, R.sub.6 and
R.sub.7 groups can be selected from the well known polar and
electrically charged chemical groups. When any of the R.sub.3,
R.sub.4 or R.sub.7 groups are not a reactive group they may be
hydrogen, C.sub.1-C.sub.4 alkyl, or the group -E-F, a polar group,
wherein F is hydroxy, protected hydroxy, sulfonate, sulfate,
carboxylate, lower alkyl substituted amino or quaternary amino and
wherein E is a spacer group such as --(CH.sub.2)-- where n is 0, 1,
2, 3, 4 or 5. If R.sub.1 or R.sub.4 is not a reactive group it can
be the E-F group, wherein n of the spacer is 1, 2, 3, 4 or 5, or
C.sub.1-C.sub.4 alkyl. Useful examples include alkyl sulfonate;
--(CH.sub.2).sub.3--SO.sub.3.sup.-; and
--(CH.sub.2).sub.4--SO.sub.3.sup.-.
[0056] The polymethine chain of the luminescent dyes of this
invention may also contain one or more cyclic chemical groups that
form bridges between two or more of the carbon atoms of the
polymethine chain. These bridges might serve to increase the
chemical or photostability of the dye and might be used to alter
the absorption and emission wavelength of the dye or change its
extinction coefficient or quantum yield. Improved solubility
properties may be obtained by this modification.
[0057] In accordance with this invention the labeled component can
be antibodies, proteins, peptides, enzyme substrates, hormones,
lymphokines, metabolites, receptors, antigens, haptens, lectins,
toxins, carbohydrates, sugars, oligosaccharides, polysaccharides,
nucleic acids, deoxy nucleic acids, derivatized nucleic acids,
derivatized deoxy nucleic acids, DNA fragments, RNA fragments,
derivatized DNA fragments, derivatized RNA fragments, natural
drugs, virus particles, bacterial particles, virus components,
yeast components, blood cells, blood cell components, biological
cells, noncellular blood components, bacteria, bacterial
components, natural and synthetic lipid vesicles, synthetic drugs,
poisons, environmental pollutants, polymers, polymer particles,
glass particles, glass surfaces, plastic particles and plastic
surfaces.
[0058] A cyanine or related chromophore can be prepared which when
reacted with a component can absorb light at 633 nm and the
detecting step can employ a helium neon laser that emits light at
this wavelength of the spectrum. Also, a cyanine or related dye can
be prepared which when reacted with a component can absorb light
maximally between 700 nm and 900 nm and the detecting step can
employ a laser diode that emits light in this region of the
spectrum.
Selectivity
[0059] The reactive groups listed above are relatively specific
Ifor labeling particular functional groups on proteins and other
biological or non-biological molecules, macromolecules, surfaces or
particles provided that appropriate reaction conditions are used,
including appropriate pH conditions.
Properties of the Reactive Cyanine and Merocyanine Dyes and Their
Products
[0060] The spectral properties of the dyes of this invention are
not appreciably altered by the functionalization described in this
specification. The spectral properties of labeled proteins and
other compounds are also not very different from the basic dye
molecule that has not been conjugated to a protein or other
material. The dyes described in this invention alone or conjugated
to a labeled material generally have large extinction coefficients
(.epsilon.=100,000 to 250,000), have quantum yields as high as 0.4
in certain cases, and absorb and emit light in the spectral range
of 400 to 900 nm. Thus, they are especially valuable as labeling
reagents for luminescence detection.
Optical Detection Methods
[0061] Any method can be employed for detecting a labeled or
stained component. The detecting method can employ a light source
that illuminates the mixture containing the labeled material with
light of first defined wavelengths. Known devices are employed that
detect light at second wavelengths that is transmitted by the
mixture or is fluoresced or luminesced by the mixture. Such
detection devices include fluorescence spectrometers, absorption
spectrophotometers, fluorescence microscopes, transmission light
microscopes and flow cytometers.
[0062] The method of this invention can also employ chemical
analysis methods to detect attachment of the dye to the labeled
component or components. Chemical analysis methods can include
infrared spectrometry, NMR spectrometry, absorption spectrometry,
fluorescence spectrometry, mass spectrometry and chromatographic
methods.
EXAMPLES
Example 1
Reactive dye I:
Anhydro-1-methyl-1'-sulfobutyl-5-N-(3,5-dichloro-2,4,6-triazinyl)-aminome-
thyl-indodicarbocyanine hydroxide
[0063] ##STR8##
[0064] 5-aminomethyl-1,3,3 trimethyl-2-methylene-indolenine (A) was
synthesized according to Gale et al., Austr. J. Chem. 30:689-694
(1977). To a solution of 1.0 g cynuric chloride in dry methylene
dichloride (40ml) cooled to -12.degree. C. under nitrogen was added
dropwise with stirring a solution of 1.01 g of the
aminomethyl-indolenine and 0.7 ml triethylamine in 60 ml methylene
dichloride. The resulting brown solution was stirred for 1.5 hr. at
0.degree. C., diluted with 100 ml methylene dichloride and washed
with 80 ml water. The light red organic layer was washed with sat.
salt solution, dried over sodium sulfate and concentrated to give
1.07 g of a reddish oil, (B).
[0065] 2,3,3-trimethyl-(3H)-indole (0.48 g) and butane-1,4-sultone
(0.42 g) were heated for three hours in a stoppered flask at
120.degree. C. The viscous semi-solid red mass was cooled, washed
with ether (2.times.5 ml) and dried under vacuum. The product was
dissolved in 10 ml acetic anhydride, and malonaldehyde dianil
hydrochloride (0.77 g) was added. Heating at 125.degree. C. for 30
minutes produced a solution of the anil intermediate which was
cooled to room temperature. To this mixture was added a solution of
(B) in 6 ml acetic anhydride, and it was again heated to
120.degree. C. for 30 minutes. The resulting deep blue solution was
cooled in ice and diluted with ether (40 ml). The precipitate was
collected, washed with ether (10 ml), suspended in 8 ml ethanol and
reprecipitated with ether (40 ml). The resulting semi-solid mass
was chromatographed over 200 g of silica gel. Elution with
chloroform-ethanol (4:1) gave 0.74 g of the pure dye I. [0066]
.lamda. Abs=648 nm (Ethanol), .epsilon..sub.648=198,000 (Ethanol),
Max [0067] .lamda. Fluor=670 nm (Ethanol) Max
Example 2
Reactive dye II:
1-(.gamma.)-isothiocyanatopropyl-1'-methyl-indocarbocyanine
bromide,
[0068] ##STR9##
[0069] 2,3,3-trimethyl-(3H)-indole and
N-(3-bromo-propyl)-phthalimide (2.7 g) were heated at 110.degree.
C. in a sealed tube for four hours. The resulting red viscous mass
solidified on cooling was washed with ether (2.times.20 ml) and
then dried. The 1-(3-phthalimido propyl)-2,3,3-trimethyl indolinium
bromide intermediate (C) was crystallized from ethanol-ether.
[0070] To produce the second intermediate (D), 2,3,3-trimethyl
(3H)-indole (1.6 g) and methyl toluenesulfonate (1.9 g) were heated
at 120.degree. C. for 4 hours then cooled to room temperature. To
form the cyanine dye intermediate, (E), N,N'-diphenyl formamidine
(2 g) and acetic anhydride (25 ml) were added to the solution
containing (D) and the mixture was heated with stirring for ten
minutes at 110.degree. C. then cooled. To this solution was added
an ethanol solution containing intermediate (C). The mixture was
heated to complete dissolution, triethylamine (1.5 ml) was added
and the reaction mixture was again heated for 20 minutes at
125.degree. C. An intense red solution resulted which was cooled,
diluted with ether (30 ml) and filtered. The residue was washed
thoroughly with ether, dried and then refluxed for four hours in
40% aqueous hydrobromic acid (50 ml). The product, (E), was
precipitated by neutralization with sodium bicarbonate and
collected by filtration, yielding 1.6 g of a semi-solid.
[0071] To an ice-cold solution of thiophosgene (0.45 ml) in 50 ml
chloroform, was added drop-wise during thirty minutes a solution of
the aminopropyl dye (E) (0.8 g) in chloroform (60 ml). The reaction
mixture was stirred at 0-5.degree. C. for one hour, allowed to warm
to room temperature and stirred overnight. Solvent was removed by
rotary evaporation, and the residue was chromatographed over silica
gel (80 g). Elution with 5% ethanol in chloroform gave 0.15 g of
pure final product. m.p. 109-112. [0072] .lamda. Abs=55 nm
(Ethanol), .epsilon..sub.551=107,000 (Ethanol), Max [0073] .lamda.
Fluor=565 nm (Ethanol) Max
Example 3
Reactive dye III:
Anhydro-1-methyl-1'-(3-sulfopropyl)-5-iodoacetamido-indocarbocyanine
hydroxide
[0074] ##STR10##
[0075] The first intermediate, 5-amino-1,3,3
trimethyl-2-methylene-indolenine (F) was prepared according to Gale
and Wilshire (1974). The second intermediate,
5-chloroacetamido-1,3,3-trimethyl-2-methylene indolenine (G) was
prepared by adding chloroacetylchloride (3.2 ml) in 180 ml dry
benzene dropwise with stirring to 1.88 g of (F) in 180 ml dry
benzene at 0-5.degree. C. After stirring for 2 hr. more, 100 ml
cold water was added. The aqueous layer was separated, made
alkaline with ammonium hydroxide and extracted with ether. The
ether extract was dried over anhydrous magnesium sulfate and
concentrated. Recrystallization of the residue from
dichloromethane-hexane (3-10) gave 2 g of yellow crystals, (G),
m.p. 128-131.degree. C.
[0076] The third intermediate, (H), was formed by the following
procedure. A 1.7 g sample of 2,3,3-trimethyl-(3H)-indole and 1.3 g
propane-1,3-sultone were heated in a sealed tube for 3 hr. at
100.degree. C. The mixture solidified on cooling and was washed
with ether (2.times.5 ml) and dried in vacuo. To this solid was
added 2.0 g of N,N'-diphenyl formamidine and 12 ml acetic anhydride
and the mixture was heated at 120.degree. C. for 30 minutes. After
cooling the solution was diluted with ether (30 ml), then filtered.
The residue was crystallized from ethanol-ether to give 1.5 g of
the anil, (H), m.p. 308-310.degree. C.
[0077] A 1.3 g sample of anil (H) and 0.8 g of the
chloroacetamido-indolenine, (G), were dissolved in 15 ml acetic
anhydride, heated for 10 min. at 120.degree. C. and cooled to room
temperature. The solution was diluted with ether (40 ml). The red
solid was collected and dried yielding 1.6 g of almost pure dye,
(J). The chloroacetamido dye (J) (1.4 g) was refluxed for 2.5 hr.
in methanol (150 ml) containing 1.4 g sodium iodide. After
concentration of the reaction mixture to 10 ml, the product was
precipitated by the addition of 200 ml acetone. The solid was
collected, washed with acetone and ether to give 1.7 g of crude
product. The dye was purified by chromatography over silica gel (80
g) eluting with 30% ethanol in chloroform which yielded 0.83 g of
pure dye. m.p. 245-250. [0078] .lamda. Abs=565 nm (Ethanol),
.epsilon..sub.565=116,000 (Ethanol), Max [0079] .lamda. Fluor=590
nm (Ethanol) Max
Example 4
Reactive dye IV:
Anhydro-1-(2-carboxyethyl)-1'-(4-sulfobutyl)-indotricarbocyanine
hydroxide N-hydroxysuccinimide ester
[0080] ##STR11##
[0081] The intermediate (K),
N-(2-carboxyethyl)-2,3,3-trimethyl-indolinium bromide, was prepared
as follows. A sample of 5.8 g 2,3,3-trimethyl-(3H)-indole,
3-bromo-proprionic acid (5.1 g) and sodium iodide (5.0 g) were
dissolved in 2-propanone (15 ml) and heated at reflux for 18 hours.
The reaction mixture was cooled on ice, then diluted with 60 ml
diethyl ether. After 2 hours on ice, the precipitate was collected
and recrystallized from isopropanol. The product, (K), was a
mauve-colored powder (4.5 g) with melting point of 166-167.degree.
C. The cyanine dye intermediate (L),
1-(2-carboxyethyl)-1'-(4-sulfobutyl)-indotricarbocyanine, was
prepared as follows.
Anhydro-N-sulfobutyl-2,3,3-trimethyl-indolinium hydroxide (0.50 g)
and glutaconaldehyde dianil hydrochloride (0.55 g) were dissolved
in 10 ml of acetic anhydride, were refluxed for 20 minutes, cooled
on ice and diluted with diethyl ether (40 ml). The resulting
supernatant was decanted. The sticky residue was dissolved in
acetic acid (5 ml) and reprecipitated with 25 ml ether. The
supernatant was decanted. To the residue was added a solution of
the above (K), N-carboxyethyl indolenene (0.5 g) in 15 ml methanol
and then 0.25 ml triethylamine. This mixture was warmed on a hot
plate for 45 minutes, cooled and diluted with 50 ml ether. The
precipitate was collected, dissolved in a small volume of acetic
acid and reprecipitated with ether. The crude product (0.8 g) was
purified by chromatography over silica gel (3 cm.times.400 cm
column) with methanol-chloroform (4:6) elution. The material
collected (130 mg) was pure dye intermediate, (L), according to
TLC.
[0082] The reactive N-hydroxysuccinimide ester of the
indotricarbocyanine was prepared as follows. The carboxyl-dye
intermediate (L) (30 mg) and 30 mg of N-hydroxysuccinimide (5
equivalents) were dissolved in a mixture of acetonitrile (6 ml) and
dimethylformamide (1 ml). Dicyclohexylcarbodiimide (15 mg, 1.5
equivalents) was added and the mixture was stirred at 22.degree. C.
for 20 hours. The urea by-product was removed by filtration and
washed with acetonitrile (3.times.2 ml). The combined filtrates
were concentrated by rotary evaporation and the dye was
precipitated from the remaining DMF by the addition of 20 ml ether.
The resulting solid was collected on a sintered glass filter and
dried under vacuum. A dark blue powder (30 mg) was obtained. The
active ester showed, by TLC, single products when reacted with
either methylamine in methanol or taurine in methanol/water. [0083]
.lamda. Abs=750 nm (water), E.sub.750=150,000 (water), Max [0084]
.lamda. Fluor=775 nm (water) Max
Example 5
Labeling Antibody with a Reactive Cyanine dye IV
[0085] To 1 mg of sheep anti-mouse-IgG antibody in 250 ul of 0.1M
sodium carbonate/bicarbonate (pH 9.2) was added 10 ul of dye
solution (4.42 mg/ml DMSO), giving a molar ratio of dye:protein of
10:1. The reaction mixture was stirred at 22.degree. C. for 2
hours, then passed over a Sephadex G-15 desalting column (2.Sml bed
volume), preconditioned with phosphate buffered saline. The
dye-conjugated protein obtained from the column had an apparent
ratio of about 5 dyes/protein. Other reactive cyanine dyes bearing
N-hydroxysuccinimide esters, isothiocyanate, or dichlorotriazine
groups were attached to antibody molecules by essentially the same
procedure.
Example 6
Staining and Microscopic Visualization of Human Lymphocytes with
Cyanine Dye IV Conjugated to Sheep Antimouse IgG Antibody
[0086] Freshly isolated peripheral lymphocytes were treated at zero
degrees for 30 minutes with mouse anti-Beta2-microglobulin (0.25
.mu.g/10**6 cells). The cells were washed twice with DMEM buffer
and were then treated with dye IV-labeled sheep anti-mouse-IgG
antibody (1 .mu.g per 10**6 cells). After a 30 minute incubation at
0.degree. C., the excess antibody was removed and the cells were
again washed twice with DMEM buffer. Aliquots of the cells were
fixed on slides for analysis by fluorescence microscopy. Under the
microscope the stained lymphocytes on the slide were excited with
light at 705-740 nm and the fluorescence at 750-805 nm was detected
with a COHU red sensitive intensified television camera attached to
an image digitizer and television monitor. The cells stained by
this method showed fluorescence under the microscope. In a control
experiment, use of the primary mouse anti-B.sub.2-microglobulin
antibody was omitted but the staining and analysis was otherwise
carried out as described above. The control sample showed no
fluorescence under the microscope indicating that dye IV-labeled
sheep anti-mouse antibody does not give significant nonspecific
binding to lymphocytes.
Experimental for the Preparation of Cyanine Dye Phosphoramidites
for Use as 5' Labelling Reagents
Example 7
Preparation of
1-Ethyl-2-{5-[1-(5-(2-cyanoethyl-N,N-diisopropylphosphoramidite)pentyl)-3-
,3-dimethyl-2
3-dihydro-1H-benzo[b]azol-2-yliden]-1,3-pentadienyl}-3,3,-dimethyl-3H-ben-
zo[b]azolium-5-sulfonate
1) 2,3,3-Trimethyl-1-(5-methylcarbonyloxypentyl)-3H-benzo[b]azolium
iodide
[0087] A mixture of 2,3,3-trimethyl-3H-benzo[b]azole (2.39 g, 15
mmol) and 5-iodopentyl acetate (3.84 g, 15 mmol) in
1,2-dichlorobenzene (15 ml) were heated at 120.degree. C. for 6 h
and then cooled to ambient temperature. The mixture was then
dripped into Et.sub.2O which precipitated the title compound as a
red gum which was collected and used without any further
purification in subsequent reactions.
ii)
1-Ethyl-2-{5-hydroxypentyl)-3,3-dimethyl-2,3-dihydro-1H-benzo[b]azol-2-
-yliden-1,3-pentadienyl}-3,3,-dimethyl-3H-benzo[b]azolium-5-sulfonate
[0088] A mixture of
1-ethyl-2,3,3-trimethyl-3H-benzo[b]azolium-5-sulfonate (267 mg, 1
mmol) and malonaldehyde bis(phenylimine) monohydrochloride (293 mg,
1.1 mmol) in AcOH (2.5 ml) and Ac.sub.2O (2.5 ml) were heated at
ca. 110.degree. C. for 1.5 h. The reaction mixture was then colled
to ambient temperature and concentrated in vacua. To the residue
was added
2,3,3-trimethyl-1-1-(5-methylcarbonyloxypentyl)-3H-benzo[b]azolium
iodide (ca 500 mg) as a solution in pyridine (2.5 ml) followed by
Ac.sub.2) (2.5 ml). the resultant mixture was left stirring at
ambient temperature and monitored by UV. The absorption at 450nm
decreased to virtually zero with a corresponding increase in the
absorption at 644 nm. The mixture was then concentrated in vacuo,
redissolved in dichloromethane, washed with 1M HCl, saturated
NaHCO.sub.3, brine, dried over MgSO.sub.4 and concentrated in
vacuo. The residue was redissolved in MeOH (50 ml) and
K.sub.2CO.sub.3 (200 mg) was added. After stirring at ambient
temperature for 45 min the reaction mixture was concentrated in
vacuo to ca.5 ml, diluted with dichloromethane, washed with 1M HCl,
brine, dried over MgSO.sub.4 and concentrated in vacuo. The residue
was then purified by flash column chromatography, SiO.sub.2
dichloromethane-25% MeOH:dichloromethane gradient, to yield the
title compound as a dark blue solid (350 mg, 64%) (The product dye
was assumed to be the internal salt) .lamda..sub.max 644 (MeOH) 644
nm; .delta..sub.H (270 MHz; CDCl.sub.3/CD.sub.3OD) 1.31 (3H, t,
CH.sub.2CH.sub.3), 1.38-1.85 (18H, m), 3.53 (2H, t, CH.sub.2OH),
3.87-4.04 (4H, m, CH.sub.2N and CH.sub.2N), 6.03 (2H, m,
.alpha.-vinyl H), 6.45 (1H, t, .gamma.-vinyl H), 6.98-7.11 (2H, m,
ArH), 7.17-7.40 (3H, m, ArH) and 7.73-7.95 (4H, m, ArH and 2
.beta.-vinyl H).
iii)
1-Ethyl020{5-[1-(5-(2-cyanoethyl-N,N-diisopropylphosphoromnidite)pent-
yl)-3,3-dimethyl-2,3-dihydro-1H-benzo[b]azol-2-yliden]1,3-pentadienyl-3,3,-
-dimethyl-3H-benzo[b]azolium-5-sulfonate (I)
[0089]
1-Ethyl-2-{5-(1-(5-hydroxypentyl)-3,3-dimethyl-2,3-dihydro-1H-benz-
o[b]azol-2-yliden]-1,3-pentadienyl}-3,3,-dimethyl-3H-benzo[b]azolium-5-sul-
fonate (100 mg) was dried azeotropically with MeCN and then under
high vacuum. MeCN (5 ml) was added to the dried dye followed by a
solution of 2-cyanothyl-N,N,N',N'-tetraisopropylphosphorodiamidite
(ca. 250 mg in MeCN (2 ml)) and then tetrazole (50 .mu.l of a 10
mg/ml solution in MeCN). After a few minutes DMF (1 ml) was added
followed by a further aliquot of tetrazole solution (100 .mu.l)
after ca. 1 hr. After ca. a further 1 hr the MeCN was removed in
vacuo and the crude solution of phosphoramidite was used stored at
-70.degree. C. under nitrogen. No purification was attempted and
the crude reaction mixture was used in subsequent reactions.
Example 8
Potassium
3-ethyl-2-{5-[3-(5-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-
pentyl)-1,1-dimethyl-2,3-dihydro-1H-naptho[2,1-bazol-2-yliden]-1,3-pentadi-
enyl}-1,1,-dimethyl-1H-naptho[2,1b]azolium-6,8-disulfonate
1)
1,1,2-Trimethyl-3-(5-methylcarbonyloxypentyl)-1H-naptho[2,1b]azolium
iodide
[0090] This prepared is a similar manner to
2,3,3-trimethyl-1-(5-methylcarbonyloxypentyl)-3H-benzo[b]azolium
iodide but starting from 1,1,2-trimethyl-1H-naptho[2,1-b]azole and
5-iodopentyl acetate.
ii) Potassium
3-ethyl-2-{5-[3-(5-hydroxypentyl)-1,1-dimethyl-2,3-dihydro-1H-naptho[2,1--
bazol-2-yliden]-1,3-pentadienyl}-1,1,-dimethyl-1H-naptho[2,1b]azolium-6,8--
disulfonate
[0091] A mixture of potassium
3-ethyl-1,1,2-trimethyl-1H-naptho[2,1-b]azolium-6,8-disulfonate
(1.08 g) and malonaldehyde bis(phenylimine) monohydrochloride 512
mg) in AcOH(20 ml) was heated at 120.degree. C. for 8 h and then
cooled to ambient temperature.
1,1,2-Trimethyl-3-(5-methylcarbonyloxypentyl)-1H-naptho[2,1-b]azolium
iodide (835 mg) was added followed by pyridine (20 ml) and
Ac.sub.2O (2ml) and the mixture was allowed to stand at ambient
temperature for 16 h before being concentrated in vacuo. The
residue was then purified by reversed phase HPLC to afford the
title compound as a dark solid,
iii) Potassium
3-ethyl-2-{5-[3-(5-(2-cyanoethyl-N,N-diisopropylphosphoramidite)pentyl)-1-
,1-dimethyl-2,3-dihydro-1H-naptho[2,1-bazol-2-yliden]-1,3-peritadienyl}-1,-
1,-dimethyl-1H-naptho[2,1b]azolium-6,8-disulfonate (II)
[0092] To a stirred solution of potassium
3-ethyl-2-{5-[3-(5-hydroxypentyl)-1,1-dimethyl-2,3-dihydro-1H-naptho[2,1--
b]azol-2-yliden]-1,3-pentadienyl}-1,1-dimethyl-1H-naptho[2,1]b]azolium-6,8-
-disulfonate (65 mg) in DMF (2.5 ml) at ambient temperature was
added diisopropylethylamine (148 .mu.l, 0.85 mmol) followed by
2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (100 .mu.l).
After 90 min further aliquots of diisopropylethylamine (148 .mu.l)
and 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (100 .mu.l)
were added to the reaction mixture. Stirring was continued for a
while and then the reaction vessel was sealed under nitrogen and
stored in a -70.degree. C. freezer until use. No purification was
attempted and the crude reaction mixture was used in subsequent
reactions. ##STR12##
1-Ethyl-2-{5-[1-(5-hydroxypentyl)-3,3-dimethyl-2,3-dihydro-1H-benzo[b]azol-
-2-yliden]-1,3-pentadienyl}-3,3,-dimethyl-3H-benzo[b]azolium-5-sulfonate
[0093] ##STR13##
1-Ethyl-2-{5-[1-(5-(2-cyanoethyl-N,N-diisopropylphosphoramidite)pentyl)-3,-
3-dimethyl-2,3-dihydro-1H-benzo[b]azol-2-yliden]-1,3-pentadienyl}-3,3,-dim-
ethyl-3H-benzo[b]azolium-5-sulfonate (Example I)
[0094] ##STR14##
2,3,3-Trimethyl-1-(5-methylcarbonyloxypentyl)-3H-benzo[b]azolium
iodide
[0095] ##STR15##
Potassium
3-ethyl-2-{5-[3-(5-hydroxypentyl)-1,1-dimethyl-2,3-dihydro-1H-na-
ptho[2,1-b]azol-2-yliden]-1,3-pentadienyl}-1,1-dimethyl-1H-naptho[2,1-b]az-
olium-6,8-disulfonate
[0096] ##STR16##
Potassium
3-ethyl-2-{5-[3-(5-(2-cyanoethyl-N,N-diisopropylphosphoramidite)-
pentyl)-1,1-dimethyl-2,3-dihydro-1H-naptho[2,1-b]azol-2-yliden]-1,3-pentad-
ienyl}-1,1-dimethyl-1H-naptho[2,1-b]azolium-6,8-disulfonate
(Example II)
[0097] ##STR17##
1,1,2-Trimethyl-3-(5-methylcarbonyloxypentyl)-1H-naptho[2,1-b]azolium
iodide
Synthesis of oligonucleotide primers labelled at the 5' terminus
with Example 7 or Example 8
Materials
[0098] Example 7, 100 mg/ml in dimethylformamide.
[0099] Example 8, 65 mg/ml in dimethylformamide.
[0100] ABI394 reagent kit for .beta.-cyanoethyl phosphoramidite
chemistry, P/N 400852X.
[0101] ABI cpg T synthesis columns, 0.2 .mu.mol.
[0102] Glen Research iPr-Pac-dG-CE phosphoramidite.
[0103] Glen Research dT-CE phosphoramidite.
[0104] Glen Research Ac-dC-CE phosphoramidite.
[0105] Glen Research Pac-dA-CE phosphoramidite.
[0106] 50 mM Potassium carbonate in methanol.
[0107] Amersham International cyanine reference dyes referred to as
Cy5 and Cy5.5.
DNA Synthesis.
[0108] An ABI394 instrument was programmed to synthesize the
sequence [0109] 5'-5tgtaaaacgacggccagt-3'
[0110] The sequence is an 18 base universal [-21] M13 forward
primer. The 0.2 .mu.mol CE standard cycle was used. Dye
phosphoramidites were located at bottle position #5. The end cycle
was set to Trityl-On and Manual cleavage/deprotection.
[0111] An interrupt was programmed to halt synthesis at the start
of the dye coupling cycle to allow manual control of reagent
delivery. Three consecutive deliveries of
tetrazole+dye-phosphoramidite were used, each with a coupling time
of 200 seconds. This was followed by the standard capping and
iodine oxidation reactions. The final acetonitrile wash was
extended to 60 seconds to flush residual dye from the column.
Cleavage from the Column and Base Deprotection.
[0112] The column was opened and the resin put into a 1.5 ml
Sarstedt tube. 1 ml of deprotection reagent [50 mM potassium
carbonate in methanol] was added. The tube was capped, wrapped in
foil to exclude light and gently mixed for 2 hours at ambient
temperature [-20.degree. C.].
[0113] The tube was uncapped and centrifuged under vacuum in a
SpeedVac for 15 minutes until the volume reduced to about 0.5 ml.
The DNA solution was removed to a fresh tube leaving the CPG
support resin behind. The volume was measured and DNA precipitated
by addition of 1M MgCl.sub.2 to 20 mM final concentration and 2.5
aqueous volumes of absolute ethanol. After 10 minutes on ice in the
dark the DNA was pelleted by centrifugation at 13000.times.g for 5
minutes.
[0114] The DNA pellet was re-dissolved in 0.1 ml TE pH8.0 and
re-precipitated as above.
[0115] Finally the DNA was taken up in 0.2 ml TE pH8.0 for
determining the absorption spectrum and for HPLC purification.
OD Spectrum
[0116] 0.1 ml of a 1:200 dilution of the crude dye-primer was
scanned between 215-750 nm in a Philips PUV8700 UV/Visible
spectrophotometer. DNA concentration and approximate % labelling
were calculated prior to HPLC purification.
[0117] Example 7 gave a OD260/647 ratio of 1.04, equivalent to
.about.75% labelled DNA based upon an .epsilon..sub.m of 220000
M.sup.-1 cm.sup.-1 for Cy5.
[0118] Example 8 gave a OD260/681 ratio of 6.87, equivalent to
.about.13% labelled DNA using an .epsilon..sub.m of 190000 M.sup.-1
cm.sup.-1 fr Cy5. FIGS. 1(a) and (b) show absorbance scans of the
oligonucleotide synthesis products after deprotection and ethanol
precipitation of the DNA but before HPLC purification.
HPLC Purification
[0119] Dye-primers were separated on a 24.times.0.4 cm Spherisorb
ODS-2 C.sub.18 column using a Varian Vista 5500 system. The program
used was as follows: TABLE-US-00001 Time % Buffer A % Buffer B Flow
rate 0 95 5 1 ml/min 5 95 5 '' 50 90 50 '' 60 30 70 '' 70 30 70 ''
80 95 5 '' 90 95 5 '' 91 0 Buffer A = 0.1M ammonium acetate Buffer
B = acetonitrile Column eluate was monitored at 260 nm and either
650 nm or 680 nm
[0120] Dye primers eluted at approximately 20% acetonitrile. The
HPLC trace for Example 7 is shown in FIG. 2. The two lines record
dye absorbance at 648 nm [tallest peak] and DNA at 260 nm.
Increasing acetonitrile concentration is indicated by the diagonal
line. There is a small amount of unlabelled DNA eluting around 10%
acetonitrile and some free dye eluting late in the run [far left].
The major product is a single peak of dye-primer exhibiting
absorbance at both wavelengths.
[0121] Dye-Primer containing HPLC fractions were evaporated to
dryness in a Speedvac, re-dissolved in 0.1 ml TE buffer and ethanol
precipitated [as described earlier] to remove salt. A second OD
spectrum was taken to determine the primer concentration prior to
DNA sequencing. See FIG. 3.
DNA Sequencing Reactions
[0122] Primers were diluted in TE buffer to 2 pmol/.mu.l and used
to sequence bacteriophage M13 mp8 single stranded DNA using a cycle
sequencing kit RPN2444 provided by Amersham International's
ThermoSequenase. Briefly, 2 pmol of fluorescent primer was mixed
with 0.4 pmol of DNA template and made up to 20 .mu.l with sterile
deionised water. The mixture was split into 4 equal aliquots
designated A,C,G and T. Next 2 .mu.l of A,C,G or T reagent
[containing ThermoSequenase DNA polymerase, reaction buffer and
nucleotides] was added to the appropriate tube which was then
capped and transferred to a Perkin-Elmer 9600 thermal cycler. The
thermocycler program consisted of denaturation at 95.degree. C. for
30 seconds followed by annealing and polymerisation at 60.degree.
C. for 30 seconds and was repeated 25 times. After cycling, 3 .mu.l
of formamide loading buffer was added and the samples were
evaporated under vacuum in a SpeedVac for 10 minutes to reduce the
volume to .about.3 .mu.l. The samples were then loaded on to a 6%
HydroLink Long Ranger gel containing 6M urea and 1.2.times. TBE
buffer and electrophoresed at 1500V for 10 hours. Two different
automated fluorescent DNA sequences were used. For Example 7, a
modified Vistra DNA Sequencer 725 with a 10 mW 633 nm Helium-Neon
laser and 650 nm interference filter set and for Example 8, a
prototype Hitachi SQ-Y1 instrument with a 3 mW 650 nm laser diode
and 680 nm color glass filter. Examples of the analysed data are
shown in FIG. 4.
[0123] FIG. 4(a) Sequence data for the dye-primer of Example 7 was
prepared from from a red-laser version of the Vistra DNA Sequencer
725 instrument. The first 400 bases are shown from a total of 660
bases called by V2.01 analysis software.
[0124] FIG. (4b) Sequence data for the dye-primer of Example 8 was
prepared on a Hitachi SQ-Y1 automated fluorescent DNA sequencer.
The first 400 bases are shown out of 1072 bases called by V1-0
analysis software.
[0125] The detection limit of both dye primers in fluorescent
sequencing applications was in the attomole range per DNA band,
comparable that of Texas Red primers normally used on the Vistra
DNA sequencer 725.
[0126] Band resolution and sequence accuracy were good, typically
>98.0% accuracy for 600 bases.
* * * * *