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Claims  |
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We claim:
1. A compound having the formula:
##STR11##
wherein: A' is hydrogen, fluoro, chloro, or a group that may be converted
to a linking functionality;
D' is fluoro, chloro, or an acidic anionic group;
X' is hydrogen, fluoro, or chloro;
Z.sub.3 and Z.sub.4 are separately hydrogen, halo, lower alkyl, lower
alkyloxy, or a group that may be converted to a linking functionality; and
wherein at least one of A', Z.sub.3, and Z.sub.4 is a group that may be
converted to a linking functionality.
2. The compound of claim 1 wherein A' is carboxyl, sulfonyl, or amino; B'
is carboxyl or sulfonyl; X' is hydrogen; Z.sub.3 and Z.sub.4 are
separately hydrogen, halo, methyl, methoxy, ethyl, ethoxy, carboxyl,
sulfonyl, or methylamino.
3. The compound of claim 2 wherein only one of A', Z.sub.3 and Z.sub.4 is
carboxyl, sulfonyl, methylamino, or amino.
4. The compound of claim 3 wherein A' and D' are carboxyl, Z.sub.3 is
hydrogen or chloro, and Z.sub.4 is hydrogen or chloro.
5. A method of distinguishing polynucleotides having different terminal
dideoxynucleotides in the chain termination method of DNA sequencing, the
method comprising the steps of:
forming a mixture of a first, a second, a third, and a fourth class of
polynucleotides,
each polynucleotide in the first class having a 3'-terminal
dideoxyadenosine and being labeled with a first dye selected from the
group consisting of 5- and 6-carboxyfluorescein, 5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and
6-carboxy-4,7-dichlorofluorescein, and
1',2',7',8'-dibenzo-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein,
each polynucleotide in the second class having a 3'-terminal
dideoxythymidine and being labeled with a second dye selected from the
group consisting of 5- and 6-carboxyfluorescein, 5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and
6-carboxy-4,7-dichlorofluorescein, and
1',2',7',8'-dibenzo-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein,
each polynucleotide in the third class having a 3'-terminal
dideoxyguanosine and being labeled with a third dye selected from the
group consisting of 5- and 6-carboxyfluorescein, 5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and
6-carboxy-4,7-dichlorofluorescein, and
1',2',7',8'-dibenzo-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein,
each polynucleotide in the fourth class having a b 3'-terminal
dideoxycytosine and being labeled with a fourth dye selected from the
group consisting of 5- and 6-carboxyfluorescein, 5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 1',2',7',8'-dibenzo-5- and
6-carboxy-4,7-dichlorofluorescein, and
1',2',7',8'-dibenzo-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein,
wherein the first, second, third, and fourth dyes are spectrally resolvable
from one another;
electrophoretically separating on a gel the polynucleotides in the mixture
so that bands of similarly sized polynucleotides are formed;
illuminating with an illumination beam the bands on the gel, the
illumination beam being capable of causing the dyes to fluoresce; and
identifying the class of the polynucleotides in the bands by the
fluorescence or absorption spectrum of the dyes.
6. The method of claim 5 wherein each polynucleotide of said first class is
labeled by attaching said first dye to said 3'-terminal dideoxyadenosine
by way of a linking group, each polynucleotide of said second class is
labeled by attaching said second dye to said 3'-terminal dideoxythymidine
by way of a linking group, each polynucleotide of said third class is
labeled by attaching said third dye to said 3'-terminal dideoxyguanosine
by way of a linking group, and each polynucleotide of said fourth class is
labeled by attaching said fourth dye to said 3'-terminal dideoxycytosine
by way of a linking group.
7. The method of claim 6 wherein said dideoxyadenosine is
2',3'-dideoxy-7-deazaadenosine, said dideoxycytidine is
2',3'-dideoxycytidine, said dideoxyguanosine is
2',3'-dideoxy-7-deazaguanosine or 2',3'-dideoxy-7-deazainosine, and said
dideoxythymidine is 2',3'-dideoxyuridine.
8. The method of claim 7 wherein said linking group links a 5 carbon of
said 2',3'-dideoxycytidine or 2',3'-dideoxyuridine to a 5 or 6 carbon of
said second dye or said fourth dye, respectively, and wherein said linking
group links a 7 carbon of said 2',3'-dideoxy-7-deazaadenosine or
2',3'-dideoxy-7-guanosine or 2',3'-dideoxy-7-deazainosine to a 5 or 6
carbon of said first dye or said third dye, respectively.
9. The method of claim 8 wherein said linking group is carboxyaminoalkynyl.
10. The method of claim 9 wherein said carboxyaminoalkynyl is
3-carboxyamino-1-propynyl.
11. The method of claim 5 wherein said dideoxyadenosine is
2',3'-dideoxy-7-deazaadenosine, said dideoxycytidine is
2',3'-dideoxycytidine, said dideoxyguanosine is
2',3'-dideoxy-7-deazaguanosine or 2',3'-dideoxy-7-deazainosine, and said
dideoxythymidine is 2',3'-dideoxyuridine.
12. The method of claim 11 wherein said first dye is
2',7'-dimethoxy-5-carboxy-4,7-dichlorofluorescein, said fourth dye is
5-carboxy-4,7-dichlorofluorescein, said third dye is
2',7'-dimethoxy-4',5'-dichloro-6-carboxy-4,7-dichlorofluorescein or
1',2',7',8'-dibenzo-4',5'-dichloro-5-carboxy-4,7-dichlorofluorescein, and
said second dye is 1',2',7',8'-dibenzo-5-carboxy-4,7-dichlorofluorescein.
13. In a chain termination method of DNA sequencing, the method of the type
wherein four classes of DNA fragments are formed such that DNA fragments
of the same class have the same terminating base and are labelled with the
same fluorescent dye, an improvement comprising:
labelling DNA fragments of at least one class with a
4,7-dichlorofluorescein dye selected from the group defined by the
formula:
##STR12##
wherein: A' is hydrogen, fluoro, chloro, or a group that may be converted
to a linking functionality;
D' is fluoro, chloro, or an acidic anionic group;
X' is hydrogen, fluoro, or chloro;
Z' is hydrogen or, when taken with Z.sub.2, benzo;
Z.sub.2, when taken along, is hydrogen, halo, lower alkyl, lower alkyloxy,
or a group that may be converted to a linking functionality, or when taken
with Z.sub.1, Z.sub.2 is benzo;
Z.sub.3 and Z.sub.4 are separately hydrogen, halo, lower alkyl, lower
alkyloxy, or a group that may be converted to a linking functionality;
Z.sub.6 is hydrogen or, when taken with Z.sub.5, benzo;
Z.sub.5, when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy,
or a group that may be converted to a linking functionality, or when taken
with Z.sub.6, benzo; and wherein only one of A', A.sub.2, Z.sub.3,
Z.sub.4, and Z.sub.5 is a group that may be converted to a linking
functionality.
14. The method of claim 13 wherein A' and D' are carboxyl; X' is hydrogen;
Z.sub.2, when taken alone, is hydrogen, chloro, methyl, ethyl, ethoxy, or
methoxy; Z.sub.3 and Z.sub.4 are separately hydrogen, chloro, methoyl,
ethyl, methoxy, or ethyoxy; and Z.sub.5, when taken alone, is hydrogen,
chloro, methyl, ethyl, ethoxy, or methoxy.
15. The method of claim 14 wherein Z.sub.1, Z.sub.3, Z.sub.4, and Z.sub.6
are hydrogen, and Z.sub.2 and Z.sub.5 are methoxy.
16. The method of claim 14 wherein Z.sub.1 and Z.sub.6 are hydrogen,
Z.sub.3 and Z.sub.4 are chloro, and Z.sub.2 and Z.sub.5 are methoxy, or
wherein Z.sub.1 and Z.sub.2 are benzo, Z.sub.5 and Z.sub.6 are benzo, and
Z.sub.3 and Z.sub.4 are chloro.
17. The method of claim 14 wherein Z.sub.1 and Z.sub.2 taken together are
benzo, Z.sub.5 and Z.sub.6 taken together are benzo, and Z.sub.3 and
Z.sub.4 are hydrogen.
18. The method of claim 14 wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, and R.sub.6 are hydrogen. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The invention relates generally to fluorescent labelling techniques, and
more particularly, to the use of 4,7-dichlorofluoresceins for detecting
multiple target substances in the same sample.
BACKGROUND
Many diagnostic and analytical techniques require that multiple target
substances in the same sample be labeled with distinguishable fluorescent
tags, e.g. in flow cytometry as exemplified by Lanier et al, J. Immunol.,
Vol. 132, Pgs. 151-156 (1984); and chromosome analysis as exemplified by
Gray et al, Chromosoma, Vol 73, pgs. 9-27 (1979). This requirement is
particularly difficult to satisfy in DNA sequence analysis where at least
four spectrally resolvable dyes are needed in most automated procedures.
Presently there are two basic approaches to DNA sequence determination: the
dideoxy chain termination method, e.g. Sanger et al, Proc. Natl. Acad.
Sci., Vol. 74, pgs. 5463-5467 (1977); and the chemical degradation method,
e.g. Maxam et al, Proc. Natl. Acad. Sci., Vol. 74, pgs.560-564 (1977). The
chain termination method has been improved in several ways, and serves as
the basis for all currently available automated DNA sequencing machines,
e.g. Sanger et al, J. Mol. Biol., Vol. 143, pgs. 161-178 (1980); Schreier
et al, J. Mol. Biol., Vol. 129, pgs. 169-172 (1979); Smith et al, Nucleic
Acids Research, Vol. 13, pgs. 2399-2412 (1985); Smith et al, Nature, Vol.
321, pgs. 674-679 (1987); Prober et al, Science, Vol. 238, pgs. 336-341
(1987), Section II, Meth. Enzymol., Vol. 155, pgs. 51-334 (1987); Church
et al, Science, Vol 240, pgs. 185-188 (1988); and Connell et al,
Biotechniques, Vol. 5, pgs. 342-348 (1987).
Both the chain termination and chemical degradation methods require the
generation of one or more sets of labeled DNA fragments, each having a
common origin and each terminating with a known base. The set or sets of
fragments must then be separated by size to obtain sequence information.
In both methods, the DNA fragments are separated by high resolution gel
electrophoresis. In most automated DNA sequencing machines, fragments
having different terminating bases are labeled with different fluorescent
dyes, which are attached either to a primer, e.g. Smith et al (1987, cited
above), or to the base of a terminal dideoxynucleotide, e.g. Prober et al
(cited above). The labeled fragments are combined and loaded onto the same
gel column for electrophoretic separation. Base sequence is determined by
analyzing the fluorescent signals emitted by the fragments as they pass a
stationary detector during the separation process.
Obtaining a set of dyes to label the different fragments is a major
difficulty in such DNA sequencing systems. First, it is difficult to find
three or more dyes that do not have significantly overlapping emission
bands, since the typical emission band halfwidth for organic fluorescent
dyes is about 40-80 nanometers (nm) and the width of the visible spectrum
is only about 350-400 nm. Second, even when dyes with non-overlapping
emission bands are found, the set may still be unsuitable for DNA
sequencing if the respective fluorescent efficiencies are too low. For
example, Pringle et al, DNA Core Facilities Newsletter, Vol. 1, pgs. 15-21
(1988), present data indicating that increased gel loading cannot
compensate low fluorescent efficiencies. Third, when several fluorescent
dyes are used concurrently, excitation becomes difficult because the
absorption bands of the dyes are often widely separated. The most
efficient excitation occurs when each dye is illuminated at the wavelength
corresponding to its absorption band maximum. When several dyes are used
one is often forced to make a trade off between the sensitivity of the
detection system and the increased cost of providing separate excitation
sources for each dye. Fourth, when the number of differently sized
fragments in a single column of a gel is greater than a few hundred, the
physiochemical properties of the dyes and the means by which they are
linked to the fragments become critically important. The charge, molecular
weight, and conformation of the dyes and linkers must not adversely affect
the electrophoretic mobilities of closely sized fragments so that
extensive band broadening occurs or so that band positions on the gel
become reversed, thereby destroying the correspondence between the order
of bands and the order of the bases in the nucleic acid whose sequence is
to be determined. Finally, the fluorescent dyes must be compatible with
the chemistry used to create or manipulate the fragments. For example, in
the chain termination method, the dyes used to label primers and/or the
dideoxy chain terminators must not interfere with the activity of the
polymerase or reverse transcriptase employed.
Because of these severe constraints only a few sets of fluorescent dyes
have been found that can be used in automated DNA sequencing and in other
diagnostic and analytical techniques, e.g. Smith et al (1985, cited
above); Prober et al (cited above); Hood et al, European patent
application 8500960; and Connell et al (cited above).
In view of the above, many analytical and diagnostic techniques, such as
DNA sequencing, would be significantly advanced by the availability of new
sets of fluorescent dyes (1) which are physiochemically similar, (2) which
permit detection of spacially overlapping target substances, such as
closely spaced bands of DNA on a gel, (3) which extend the number of bases
that can be determined on a single gel column by current methods of
automated DNA sequencing, (4) which are amenable for use with a wide range
of preparative and manipulative techniques, and (5) which otherwise
satisfy the numerous requirements listed above.
SUMMARY OF THE INVENTION
The invention is directed to a method of concurrently detecting spacially
overlapping target substances using 4,7-dichlorofluorescein dyes. The
invention also includes methods of DNA sequence determination employing
4,7-dichlorofluorescein dyes, and compounds consisting of the
1',2',7',8'-dibenzo-5 (and 6-)carboxy-4,7,-dichlorofluoresceins defined by
Formula I.
##STR1##
wherein: A' is hydrogen, fluoro, chloro, or a group, such as carboxyl,
sulfonyl, or amino, that may be converted to a linking functionality;
preferably A is a group that may be converted to a linking functionality;
X' is a hydrogen, fluoro or chloro, such that whenever A' is a substituent
of the 6 carbon atom X' is a substituent of the 5 carbon atom, and
whenever A' is a substituent of the 5 carbon atom X' is a substituent of
the 6 carbon atom; preferably, X' is hydrogen;
Z.sub.3 is hydrogen, fluoro, chloro, or a group, such as carboxyl,
sulfonyl, or methylamino, that that may be converted to a linking
functionality; preferably, Z.sub.3 is hydrogen or chloro;
Z.sub.4 is hydrogen, fluoro, chloro, or a group, such as carboxyl,
sulfonyl, or methylamino, that may be converted to a linking
functionality; preferably, Z.sub.4 is hydrogen or chloro; group;
D' is fluoro, chloro, or an acidic anionic group; preferably, B' is
carboxyl or sulfonyl, and most preferably B' is carboxyl; and
wherein at least one of A', Z.sub.3, and Z.sub.4 is a group that may be
converted to a linking functionality. Preferably, only one of A', Z.sub.3,
and Z.sub.4 is a group that may be converted to a linking functionality.
Throughout, the Colour Index (Association of Textile Chemists, 2nd Ed.,
1971) carbon numbering scheme is used, i.e. primed numbers refer to
carbons in the xanthene structure and unprimed numbers refer to carbons in
the 9'-phenyl.
The invention is based in part on the discovery that the fluorescent
properties of 4,7-chloro-5- (and 6-)carboxyfluorescein and related dyes
are highly favorable for use as molecular probes. Their emission band
widths are generally 20-30 percent narrower than analogs lacking the
4,7-dichloro derivatives, their emission and absorption maxima are at
wavelengths generally about 10-30 nm higher than analogs lacking the
4,7-dichloro derivatives, and their fluorescent efficiencies are high, in
some cases being nearly triple those of analogs lacking the 4,7-dichloro
derivatives.
DETAILED DESCRIPTION OF THE INVENTION
As mentioned above, the invention is based in part on the discovery of a
class of fluorescein dyes that have absorption and emission maxima at
unusually long wavelengths, narrow emission band widths and other
favorable fluorescent properties. In addition, the invention includes the
novel fluorescein analogs defined by Formula I as members of this class of
dyes. These dyes permit the assembly of novel sets of spectrally
resolvable, physiochemically similar dyes particularly useful in automated
DNA sequence analysis.
As used herein the term "spectrally resolvable" in reference to a set of
dyes means that the fluorescent emission bands of the dyes are
sufficiently distinct, i.e. sufficiently non-overlapping, that target
substances to which the respective dyes are attached, e.g.
polynucleotides, can be distinguished on the basis of the fluorescent
signal generated by the respective dyes by standard photodetection
systems, e.g. employing a system of band pass filters and photomultiplier
tubes, or the like, as exemplified by the systems described in U.S. Pat.
Nos. 4,230,558, 4,811,218, or the like, or in Wheeless et al, pgs. 21-76,
in Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New
York, 1985).
The term "lower alkyl" as used herein directly or in connection with ethers
denotes straight-chain and/or branched chain alkyl groups containing from
1-6 carbon atoms, e.g. the term includes methyl, ethyl, propyl, isopropyl,
tert-butyl, isobutyl, and the like.
The term "halo" as used herein denotes the halogen atoms fluorine,
chlorine, bromine, and iodine; more preferably, the term denotes fluorine
or chlorine; and most preferably, the term denotes chlorine.
Preferably, the 4,7-chloro-5- (and 6-) carboxyfluorescein dyes of the
invention include those defined by Formula II.
##STR2##
wherein: A', D' and X' are defined as above;
Z.sub.1 is hydrogen or, when taken with Z.sub.2, benzo;
Z.sub.2, when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy,
or a group, such as carboxyl, sulfonyl, or methylamino, that may be
converted to an active linking functionality, or when taken with Z.sub.1,
Z.sub.2 is benzo; preferably, when taken alone, Z.sub.2 is hydrogen,
methyl, ethyl, fluoro, chloro, methoxy, or ethoxy;
Z.sub.3 and Z.sub.4 are hydrogen, halo, lower alkyl, lower alkyloxy, or a
group, such as carboxyl, sulfonyl, or methylamino, that may be converted
to a linking functionality; more preferably, Z.sub.3 and Z.sub.4 are
hydrogen, fluoro, chloro, methyl, ethyl, methoxy, or ethoxy;
Z.sub.5 is hydrogen or, when taken with Z.sub.6, benzo; and
Z.sub.6, when taken alone, is hydrogen, halo, lower alkyl, lower alkyloxy,
or a group, such as carboxyl, sulfonyl, or methylamino, that may be
converted to an active linking functionality, or when taken with Z.sub.5,
Z.sub.6 is benzo; preferably, when taken alone, Z.sub.6 is hydrogen,
methyl, ethyl, fluoro, chloro, methoxy, or ethoxy;
and
wherein at least one of A, Z.sub.2, Z.sub.3, Z.sub.4, and Z.sub.5 is a
group that may be converted to an linking functionality. Preferably, only
one of A, Z.sub.2, Z.sub.3, Z.sub.4, and Z.sub.5 is a group that may be
converted to an active linking functionality.
Many dyes of the invention are commercially available or can be synthesized
by techniques known in the art, e.g. Ghatak et al, J. Ind. Chem. Soc.,
Vol. 6, pgs. 465-471 (1929); and Khanna et al, U.S. Pat. No. 4,439,356.
Alternatively, fluorescein analogs, i.e. A=B=carboxyl, can be synthesized
by reacting substituted resorcinol with substituted benzophenone or with
substituted trimellitic acid in the presence of propionic acid, as
illustrated in the examples. Sulfonylfluoresceins, i.e. A or B is
sulfonyl, are synthesized following the methods disclosed by Lee et al,
Cytometry, Vol. 10, pgs. 151-164 (1989), modified by substituting
appropriate reactants to give 5- or 6-carboxyl- or or sulfonylfluorescein
products. Preferably, when labeling polynucleotides in DNA sequencing the
5- and 6- isomers of the dyes are used separately because they typically
have slightly different electrophoretic mobilities that can lead to band
broadening if mixtures of the isomers are used. The 5- and 6- isomers of
the dyes are readily separated by reverse phase HPLC, e.g. Edmundson et
al, Mol. Immunol., Vol. 21, pg. 561 (1984). Generally, it is believed that
the first eluting peak is the 6- isomer and the second eluting peak is the
5- isomer.
Dyes of the invention can be attached to target substances by a variety of
means well known in the art. For example, Haugland, Handbook of
Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene,
1989) provides guidance and examples of means for linking dyes to target
substances. Substituent A is converted to a linking functionality that can
be reacted with a complementary functionality on a target substance to
form a linking group. The following table lists illustrative linking
functionalities that can be formed whenever A is carboxyl sulfonyl or
amine, suitable complementary functionalities, and the resulting linking
groups suitable for use with the invention.
______________________________________
Linking Complementary
Linking
Functionality
Functionality
Group
______________________________________
NCS NH.sub.2 NHCSNH
##STR3## NH.sub.2
##STR4##
SO.sub.2 X NH.sub.2 SO.sub.2 NH
##STR5## NH.sub.2
##STR6##
##STR7## SH
##STR8##
##STR9## SH
##STR10##
______________________________________
Preferably the linking functionality is isothiocyanate, sulfonyl chloride,
4,6-dichlorotriazinylamine, or succinimidyl carboxylate whenever the
complementary functionality is amine. And preferably the linking
functionality is maleimide, or iodoacetamide whenever the complementary
functionality is sulfhydryl. Succinimidyl carboxylates can be formed by
condensing the 5- and/or 6-carboxyls of the above dyes with
N-hydroxysuccinimide using dicyclohexylcarbodiimide (DCC), e.g. as
illustrated in examples 6 and 8 of Khanna et al, U.S. Pat. No. 4,318,846,
and Kasai et al, Anal. Chem., Vol. 47, pgs. 34-37 (1975). Accordingly,
these references are incorporated by reference.
When dyes of the invention are used to label dideoxynucleotides for DNA
sequencing, preferably they are linked to the 5 carbon of pyrimidine bases
and to the 7 carbon of 7-deazapurine bases. For example, several suitable
base labeling procedures have been reported that can be used with the
invention, e.g. Gibson et al, Nucleic Acids Research, Vol. 15, pgs.
6455-6467 (1987); Gebeyehu et al, Nucleic Acids Research, Vol. 15, pgs.
4513-4535 (1987); Haralambidis et al, Nucleic Acids Research, Vol. 15,
pgs. 4856-4876 (1987); and the like. Preferably, the linking group between
the dye and a base is formed by reacting an N-hydroxysuccinimide (NHS)
ester of a dye of the invention with an alkynylamino-derivatized base of a
dideoxynucleotide. Preferably, the linking group is
3-carboxyamino-1-propynyl. The synthesis of such alkynylamino-derivatized
dideoxynucleotides is taught by Hobbs et al in European patent application
number 87305844.0, which is incorporated herein by reference. Briefly, the
alkynylamino-derivatized dideoxynucleotides are formed by placing the
appropriate halodideoxynucleoside (usually 5-iodopyrimidine and
7-iodo-7-deazapurine dideoxynucleosides as taught by Hobbs et al (cited
above)) and Cu(I) in a flask, flushing with Ar to remove air, adding dry
DMF, followed by addition of an alkynylamine, triethyl-amine and Pd(0).
The reaction mixture can be stirred for several hours, or until thin layer
chromatography indicates consumption of the halodideoxynucleoside. When an
unprotected alkynylamine is used, the alkynylamino-nucleoside can be
isolated by concentrating the reaction mixture and chromatographing on
silica gel using an eluting solvent which contains ammonium hydroxide to
neutralize the hydrohalide generated in the coupling reaction. When a
protected alkynylamine is used, methanol/methylene chloride can be added
to the reaction mixture, followed by the bicarbonate form of a strongly
basic anion exchange resin. The slurry can then be stirred for about 45
minutes, filtered, and the resin rinsed with additional methanol/methylene
chloride. The combined filtrates can be concentrated and purified by
flash-chromatography on silica gel using a methanol-methylene chloride
gradient. The triphosphates are obtained by standard techniques.
Target substances of the invention can be virtually anything that the dyes
of the invention can be attached to. Preferably the dyes are covalently
attached to the target substances. Target substances include proteins,
polypeptides, peptides, polysaccharides, polynucleotides, lipids, and
combinations and assemblages thereof, such as chromosomes, nuclei, living
cells, such as bacteria, other microorganisms, and mammalian cells,
tissues, and the like. As used herein the term "polynucleotide" means a
single stranded or double stranded chain of DNA or RNA in the size range
of about 10-1000 bases in length (if single stranded), or in the size
range of about 10-1000 basepairs in length (if double stranded).
A number of complementary functionalities can be attached to the 5' or 3'
ends of synthetic oligonucleotides and polynucleotides, e.g. amino groups,
Fung et al, U.S. Pat. No. 4,757,141 and Miyoshi et al, U.S. Pat. No.
4,605,735; or sulfhydryl groups, Connolly, Nucleic Acids Research, Vol.
13, pgs. 4485-4502 (1985), and Spoat et al, Nucleic Acids Research, Vol.
15, pgs. 4837-4848 (1987).
Dyes of the invention are particularly well suited for identifying classes
of polynucleotides that have been subjected to a biochemical separation
procedure, such as gel electrophoresis, where a series of bands or spots
of target substances having similar physiochemical properties, e.g. size,
conformation, charge, hydrophobicity, or the like, are present in a linear
or planar arrangement. As used herein, the term "bands" includes any
spacial grouping or aggregation of target substance on the basis of
similar or identical physiochemical properties. Usually bands arise in the
separation of dye-polynucleotide conjugates by gel electrophoresis.
Classes of polynucleotides can arise in a variety of contexts. For example,
they can arise as products of restriction enzyme digests. Preferably,
classes identified in accordance with the invention are defined in terms
of terminal nucleotides so that a correspondence is established between
the four possible terminal bases and the members of a set of spectrally
resolvable dyes. Such sets are readily assembled from the dyes of the
invention by measuring emission and absorption bandwidths with
commercially available spectrophotometers. More preferably, the classes
arise in the context of the chemical or chain termination methods of DNA
sequencing, and most preferably the classes arise in the context of the
chain termination method. In either method dye-polynucleotide conjugates
are separated by standard gel electrophoretic procedures, e.g. Gould and
Matthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis of
Nucleic Acids: A Practical Approach, (IRL Press Limited, London, 1981); or
Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1
(Springer-Verlag, Berlin, 1984). Preferably the type of gel is
polyacrylamide having a concentration (weight to volume) of between about
2-20 percent. More preferably, the polyacrylamide gel concentration is
between about 4-8 percent. Preferably the gel includes a strand
separating, or denaturing, agent. Detailed procedures for constructing
such gels are given by Maniatis et al., "Fractionation of Low Molecular
Weight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7 M
Urea," in Methods in Enzymology, Vol. 65, pgs. 299-305 (1980); Maniatis et
al., "Chain Length Determination of Small Double- and Single-Stranded DNA
Molecules by Polyacrylamide Gel Electrophoresis," Biochemistry, Vol. 14,
pgs. 3787-3794, (1975); and Maniatis et al., Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Laboratory, New York, 1982), pgs.
179-185. Accordingly these references are incorporated by reference. The
optimal gel concentration, pH, temperature, concentration of denaturing
agent, etc. employed in a particular separation depends on many factors,
including the size range of the nucleic acids to be separated, their base
compositions, whether they are single stranded or double stranded, and the
nature of the classes for which information is sought by electrophoresis.
Accordingly application of the invention may require standard preliminary
testing to optimize conditions for particular separations. By way of
example, oligonucleotides having sizes in the range of between about
20-300 bases have been separated and detected in accordance with the
invention in the following gel: 6 percent polyacrylamide made from 19
parts to 1 part acrylamide to bis-acrylamide, formed in a Tris-borate EDTA
buffer at pH 8.3 (measured at 25.degree. C.) with 48 percent
(weight/volume) urea. The gel was run at 50.degree. C.
The dye-polynucleotide conjugates on the gel are illuminated by standard
means, e.g. high intensity mercury vapor lamps, lasers, or the like.
Preferably, the dye-polynucleotides on the gel are illuminated by laser
light generated by a argon ion laser, particularly the 488 and 514 nm
emission lines of an argon ion laser. Several argon ion lasers are
available commercially which lase simultaneously at these lines, e.g.
Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like.
In the chain termination method, dyes of the invention can be attached to
either primers or dideoxynucleotides. Dyes can be linked to a
complementary functionality on the 5' end of the primer, e.g following the
teaching in Fung et al, U.S. Pat. No. 4,757,141 which is incorporated
herein by reference; on the base of a primer; or on the base of a
dideoxynucleotide, e.g. via the alkynylamino linking groups disclosed by
Hobbs et al, European patent application number 87305844.0 which is
incorporated herein by reference.
EXAMPLES
Example I. 4,7-dichloro-5-(and 6-)carboxyfluorescein ("ALF")
0.58 g of 3,6-dichlorotrimellitic acid, 0.72 g of resorcinol, 0.5 ml
concentrated sulfuric acid, and 3 ml of propionic acid were refluxed 12
hours under argon. The reaction mixture was poured into 150 ml water; the
precipitate was dried, taken into 3 ml pyridine and acetylated with 2 ml
acetic anhydride for 1 hour. The acetylation mixture was taken into 100 ml
ethyl acetate, washed with 1N hydrochloric acid, water, and evaporated to
dryness. The residue was placed on 15 grams of silica gel and eluted with
50 ml ethyl acetate, then 4:1 ethyl acetate:methanol. Fractions containing
UV active material with R.sub.f of about 0.2 (4:1 ethyl
acetate:methanol/silica gel) were evaporated to dryness. This residue was
dissolved in 10 ml methanol and then 1 ml of 4N sodium hydroxide was
added. After 10 minutes, the reaction mixture was diluted to 200 ml with
water and then 0.5 ml of concentrated hydrochloric acid was added. The
total mixture was extracted with 200 ml of ethyl acetate, after which the
ethyl acetate was dried with sodium sulfate and evaporated to dryness
yielding 102 mg of yellow-green solid.
Example II. 4,7-dichloro-5-(and 6-) carboxyfluorescein N-hydroxysuccinimide
(NHS) ester
13.7 mg of fluorescein from Example I, 3,3 mg of NHS, 6,4 mg DCC, and 1 ml
ethyl acetate were stirred 0.5 hours. The solid was filtered, and the
supernatant was washed three times with 1:1 brine:water, dried with sodium
sulfate, and evaporated to dryness yielding 15 mg of NHS ester.
Example III. Conjugation of 4,7-dichloro-5-(and 6-) carboxyfluorescein with
aminoalkyloligonucleotides
5 mg of NHS ester from Example II were dissolved in 20 ul of DMSO; 3 ul of
this solution were added to a solution consisting of 20 ul of 1.0 mM
5'-aminohexylphosphate oligonucleotide (an 18-mer) in water and 10 ul of
1M sodium bicarbonate/sodium carbonate buffer, pH 9.0. After one hour in
the dark, the solution was passed through a 10 ml Sephadex G-25 (medium)
column with 0.1M triethylammonium acetate buffer, pH 7.0. The band of
colored material eluting in the exclusion volume was collected. Reverse
phase HPLC showed two major fluorescent peaks, corresponding to the 5- and
6- isomers of the dye conjugated onto the DNA. The peaks were collected,
and the fluorescence spectra in 50% urea at pH 8.0 showed full width at
half max of 34 nm with the emission maxima at 528 nm.
Example IV. 2',7'-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein
("BUB")
The procedure of Example I was followed except that the following materials
and quantities were substituted: 1,47 g 4-methoxyresorcinol, 0.60 g of
3,6-dichlorotrimellitic acid, 0.2 ml concentrated sulfuric acid, and 4 ml
propionic acid. The procedure yielded 0.180 g of
4,7-dichloro-2',7'-dimethoxy-5-(and 6-)carboxyfluorescein.
Example V. 2',7'-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein NHS
ester
18 mg of this dye NHS ester were prepared as in Example II using 18 mg of
dye from Example IV, 3.5 mg NHS, 6.4 mg DCC, and 2 ml ethyl acetate.
Example VI. Conjugation of 4,7-dichloro-2',7'-dimethoxy -5-(and
6-)carboxyfluorescein with aminoalkyloligonucleotide
The procedure of Example III was followed using the dye NHS ester of
Example V. The fluorescence spectra of the two peaks collected during
reverse phase HPLC showed full widths at half max of 37 nm with emission
maxima at 544 nm in 50% urea at pH 8.2.
Example VII. 2',7'-dimethoxy-4',5'-dichloro-5-(and
6-)carboxy-4,7-dichlorofluorescein ("LOU")
This dye was prepared from the dye of Example IV and sodium hypochlorite in
aqueous sodium hydroxide.
Example VIII. 4,7-dichloro-2',7'-dimethoxy-4',5'-dichloro-5-(and
6-)carboxyfluorescein NHS ester
1.1 mg of this dye NHS ester was prepared from 0.7 mg of the dye from
Example VII, 0.45 mg of NHS, 0.7 mg DCC, and 0.2 ml ethyl acetate as in
Example II.
Example IX. Conjugation of 4,7-dichloro-2',7'-dimethoxy
-4',5'-dichloro-5-(and 6-)carboxyfluorescein with
aminoalkyloligonucleotides
The dye oligonucleotide conjugate of this example was prepared as in
Example III using the dye NHS ester from Example VIII. The fluorescence
spectra of the two peaks collected during reverse phase HPLC showed full
widths at half max of 38 nm with emission maxima at 558 nm in 50% urea at
pH 8.2.
Example X. 1',2',7',8'-dibenzo-5-(and 6-)carboxy-4,7-dichlorofluorescein
("NAN")
First, 3,6-dichlorotrimellitic acid trichloride was prepared: A mixture of
0.5 g of 3,6-dichlorotrimellitic acid and 1.3 g of phosphorous
pentachloride was heated at 130.degree. C. for 40 minutes. The mixture was
cooled to room temperature and poured into ice. The mixture was then
extracted with 40 ml ether, the organic fraction was washed twice with 15
ml water, dried with MgSO.sub.4, and concentrated to a clear oil (0.7 g).
The acid trichloride was used without further purification. NAN was
prepared as follows: A mixture of 2.7 g of 1,3-dihydroxynaphthalene, 2.84
g of 3,6-dichlorotrimellitic acid trichloride, and 8 ml of propionic acid
was refluxed for 2 hours. Water (50 ml) and ethyl acetate (50 ml) were
added. The layers were separated and the organic layer was extracted three
times with 50 ml of 1M NaHCO.sub.3. The aqueous solution was heated to
boiling and acidified with concentrated HCl. The resulting red solid (0.2
g) was filtered and dried.
Example XI. 1',2',7',8'-dibenzo-4',5'-dichloro-5-(and
6-)carboxy-4,7-dichlorofluorescein ("DEB")
20 mg of NAN, sodium hydroxide (34 ul of a 15% solution), water (1 ml), and
sodium hypochlorite (170 ul of a 5% solution) were combined. Reverse phase
HPLC showed 92% reaction. The solution was acidified with HCl, extracted
with 20 ml of ethyl acetate, dried (Na.sub.2 SO.sub.4), and concentrated
to 20 mg. The solid was purified by chromatography on a silica gel column
(1" diameter.times.2" height), eluting with 600:60:16 methylene
chloride:methanol:acetic acid. The dye solution was concentrated, and
dilute HCl and ethyl acetate added. The organic phase was dried
(MgSO.sub.4) and concentrated to 20 mg of DEB.
Example XII. Formation of 1',2',7',8'-dibenzo-5-(and
6-)carboxy-4,7-dichlorofluorescein NHS ester
NAN (10 mg) was dissolved in 2 ml of ethyl acetate, and NHS (10 mg) and DCC
(5 mg) was added. After 20 minutes, the solution was dark red in color and
a crystalline solid appeared. Thin layer chromatography on a silica gel
using 600:60:16 methylene chloride:methanol:acetic acid showed complete
conversion to the NHS ester. The ethyl acetate solution was washed with
dilute HCl, dried (NaSO.sub.4) and concentrated to a red solid (15 mg).
Example XIII.Using ALF-, BUB-, LOU-, and NAN-oligonucleotide conjugates as
dye-labeled primers in DNA sequence analysis
An all-fluorescein set of dyes was used to label DNA fragments in the chain
termination approach employing the Applied Biosystems (Foster City,
Calif.) Model 370A automated DNA sequencer. The manufacturer's protocol
(User Bulletin DNA Sequencer Model 370, Issue No. 2, Aug. 12, 1987), which
is incorporated by reference) was followed for amplification of the
unknown DNA in M13 and preparation of separately labeled DNA fragments for
gel electrophoretic separation. Dye-labeled primers were prepared as
described in the examples above. That is, NHS esters of the respective
dyes were prepared and reacted with the 5'-aminohexyl-derivatized M13
universal primer (5'-TCCCAGTCACGACGTTGT-3') to form the dye-labeled
primers for the four separate dideoxy reaction mixtures. The following
modifications were made to the standard protocol:
5-carboxy-4,7-dichlorofluorescein labeled the primer in the
dideoxycytidine reaction,
2',7'-dimethoxy-5-carboxy-4,7-dichlorofluorescein labeled the primer in
the dideoxyadenosine reaction,
2',7'-dimethoxy-4',5'-dichloro-6-carboxy-4,7-dichlorofluorescein labeled
the primer in the dideoxyguanosine reaction,
1',2',7',8'-dibenzo-5-carboxy-4,7 -dichlorofluorescein labeled the primer
in the dideoxythymidine reaction, labeled DNA fragments from the
respective reactions were combined in the following molar ratios for
loading onto the gel: 1:1:4:2 ddC reaction:ddA reaction:ddG reaction:ddT
reaction, and detection was accomplished with a modified filter wheel
using 10-nm bandpass filters centered at 535, 550, 565, and 580 nm.
Example XIV. Using ALF-, BUB-, DEB-, and NAN-oligonucleotide conjugates as
dye-labeled primers in DNA sequence analysis
The same procedure was followed as described for Example XIII, except for
the following: (i)
1',2',7',8'-dibenzo-4',5'-dichloro-5-carboxy-4,7-dichlorofluorescein
labeled the primer in the dideoxyguanosine reaction, (ii) labeled DNA
fragments from the respective reactions were combined in the following
molar ratios for loading on the gel: 1:1:2:15 ddC reaction:ddA
reaction:ddG reaction:ddT reaction, and (iii) 5 nm bandpass filters were
centered at 540, 560, 580, and 610 nm.
The foregoing disclosure of preferred embodiments of the invention has been
presented for purposes of illustration and description. It is not intended
to be exhaustive or to limit the invention to the precise form disclosed,
and obviously many modifications and variations are possible in light of
the above teaching. The embodiments were chosen and described in order to
best explain the principles of the invention and its practical
application, to thereby enable others skilled in the art to best utilize
the invention in various embodiments and with various modifications as are
suited to the particular use contemplated. It is intended that the scope
of the invention be defined by the claims appended hereto.
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