U.S. patent application number 10/714407 was filed with the patent office on 2005-04-21 for 4,7-dichlorofluorescein dyes as molecular probes.
This patent application is currently assigned to Applera Corporation. Invention is credited to Chakerian, Vergine, Connell, Charles R., Fung, Steven, Hershey, N. Davis, Lee, Linda G., Menchen, Steven M., Woo, Sam L..
Application Number | 20050084870 10/714407 |
Document ID | / |
Family ID | 27030977 |
Filed Date | 2005-04-21 |
United States Patent
Application |
20050084870 |
Kind Code |
A1 |
Menchen, Steven M. ; et
al. |
April 21, 2005 |
4,7-dichlorofluorescein dyes as molecular probes
Abstract
Long wavelength, narrow emission bandwidth fluorescein dyes are
provided for detecting specially overlapping target substances. The
dyes comprise 4,7-dichlorofluorescein, and particularly 2',
4',5',7'-tetrachloro-4,7-di- chloro-5-(and 5-) carboxyfluoresceins.
Methods and kits for using the dyes in DNA analysis are
provided.
Inventors: |
Menchen, Steven M.;
(Fremont, CA) ; Lee, Linda G.; (Palo Alto, CA)
; Connell, Charles R.; (Redwood City, CA) ;
Hershey, N. Davis; (San Carlos, CA) ; Chakerian,
Vergine; (San Mateo, CA) ; Woo, Sam L.;
(Redwood City, CA) ; Fung, Steven; (Palo Alto,
CA) |
Correspondence
Address: |
MILA KASAN, PATENT DEPT.
APPLIED BIOSYSTEMS
850 LINCOLN CENTRE DRIVE
FOSTER CITY
CA
94404
US
|
Assignee: |
Applera Corporation
Foster City
CA
|
Family ID: |
27030977 |
Appl. No.: |
10/714407 |
Filed: |
November 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714407 |
Nov 14, 2003 |
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09949444 |
Sep 7, 2001 |
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6649598 |
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09949444 |
Sep 7, 2001 |
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09580754 |
May 30, 2000 |
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6403812 |
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09580754 |
May 30, 2000 |
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09273655 |
Mar 23, 1999 |
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6096723 |
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09273655 |
Mar 23, 1999 |
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08905855 |
Aug 4, 1997 |
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5885778 |
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08905855 |
Aug 4, 1997 |
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08400780 |
Mar 8, 1995 |
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5654442 |
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08400780 |
Mar 8, 1995 |
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07939813 |
Sep 3, 1992 |
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07939813 |
Sep 3, 1992 |
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07436455 |
Nov 14, 1989 |
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5188934 |
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Current U.S.
Class: |
435/6.12 ;
536/25.32 |
Current CPC
Class: |
Y10T 436/147777
20150115; Y10T 436/145555 20150115; C12Q 1/6869 20130101; A61K
48/00 20130101; C07D 311/82 20130101; C12Q 1/6816 20130101; Y10T
436/143333 20150115; Y10S 435/968 20130101; C12Q 2563/107 20130101;
C12Q 1/6869 20130101; C12Q 2563/107 20130101; C09B 11/08 20130101;
C12Q 1/6816 20130101; Y10S 436/80 20130101; C07H 21/00
20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
We claim:
1. In a method of detecting a plurality of electrophoretically
separated classes of DNA fragments, an improvement comprising
labelling DNA fragments of at least one class with a
4,7-dichlorofluorescein dye.
2. The method of claim 1 wherein said 4,7-dichlorofluorocein is
difined by the formula: 24wherein: A' is hydrogen, fluoro, chloro,
a linking functionality, or a group that may be converted to a
linking functionality; B' is fluoro, chloro, or an acidic anionic
group; X' is hydrogen, fluoro, or chloro; 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, a linking
functionality, or a group that may be converted to a linking
functionality, or when taken with Z.sub.1, benzo; Z.sub.3 and
Z.sub.4 are separately hydrogen, halo, lower alkyl, lower alkyloxy,
a linking functionality, or a group that may be converted to a
linking functionality; Z.sub.5, when taken alone, is hydrogen,
halo, lower alkyl, lower alkyloxy, a linking functionality, or a
group that may be converted to a linking functionality, or when
taken with Z.sub.6, benzo; Z.sub.6 is hydrogen or, when taken with
Z.sub.5, benzo; and wherein at least one of A, Z.sub.2, Z.sub.3,
Z.sub.4, and Z.sub.5 is a linking functionality or a group that may
be converted to a linking functionality.
3. The method of claim 2 wherein: A' is carboxyl, sulfonyl,
isothlocyanato, succinimidyl carboxytate, phosphoramidite, or
amino; B' is carboxyl or sulfonyl; X' is hydrogen or chloro;
Z.sub.2, when taken alone, is hydrogen, methyl, ethyl, methoxy,
ethoxy, or chloro; Z.sub.3, and Z.sub.4 are separately hydrogen,
methyl, ethyl, methoxy, ethoxy, chloro, carboxyl, sulfonyl,
isothiocyanate, succinimidyl carboxylate, phospboamidite, or
methylamino: Z.sub.5, when taken alone, is hydrogen, methyl, ethyl,
methoxy, ethoxy or chloro; and wherein only one of A', Z.sub.3, and
Z.sub.4 is carboxyl, sulfonyl, metylamino, isothiocyanate,
succinimidyl carboxylate, phosphoramidite, or amino.
4. The method of claim 3 wherein Z.sub.3, and Z.sub.4 are
separately hydrogen, methyl, ethyl, methoxy, ethoxy, or chloro.
5. The method of claim 4 wherein Z.sub.2, when taken alone, is
hydrogen, methoxy, ethoxy, or chloro; Z.sub.3, and Z.sub.4 are
separately hydrogen, methoxy, ethoxy, chloro; and Z.sub.5 when
taken alone, is hydrogen, methoxy, ethoxy, or chloro.
6. The method of claim 5 wherein B' is carboxy and A' is carboxy,
succinimidyl, carboxylate, or phosphoramidite.
7. A compound having the formula: 25wherein A' is hydrogen, fluoro,
chloro, a linking functionality or a group that may be converted to
a linking functionality; B' 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, a linking functionality, 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 linking functionality or a
group that may be converted to a linking functionality.
8. The compound of claim 7 wherein A' is carboxyl, sulfonyl,
isothiocyanate, succinimidyl carboxylate, phosphoramidite, or
amino: B' is carboxyl or sulfonyl: X' is hydrogen; Z.sub.3 and
Z.sub.4 are separately hydrogen, halo, carboxyl, sulfonyl, or
methylamino.
9. The compound of claim 8 wherein only one of A', Z.sub.3, and
Z.sub.4 is carboxyl, sulfonyl, methylamino, or amino.
10. The compound of claim 9 wherein A' and B' are carboxyl, Z.sub.3
is hydrogen or chloro, and Z.sub.4 is hydrogen or chloro.
11. A kit for detecting a plurality of electrophoretically
separated classes of DNA fragments comprising an oligonucleotide
labelled with a 4,7-dichlorofluorescein dye.
12. The kit of claim 11 further compounding: an enzyme selected
from the group consisting of acid polymerase and nucleic acid
ligase; and a reaction buffer.
13. The kit of claim 12 wherein said enzyme is a nucleic acid
polymerase and wherein said kit further includes a nucleoside
triphosphate mix.
14. The kit of claim 12 wherein said enzyme is a nucleic acid
ligase.
15. A kit for sequencing DNA comprising: an oligonucleotide with a
4,7-dichlorofluorescein dye; a nucleic acid polymerase; a reaction
buffer; and a nucleoside triphosphate mix.
16. The kit of claim 15 wherein said 4,7-dichlorofluorescein dye is
defined by the formula: 26wherein: A' is hydrogen, fluoro, chloro,
or a group that may be converted to a linking functionality; B' is
fluoro, chloro, or an acidic anionic group; X' hydrogen, fluoro, or
chloro; 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 that may be converted to a linking
functionality, or when taken with Z.sub.1, 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.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; Z.sub.6 hydrogen
or, when taken with Z.sub.5, benzo; 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 a linking functionality.
17. The kit of claim 16 wherein: A' and B' are carboxy; X' hydrogen
or chloro; Z.sub.2, when taken alone, is hydrogen, methyl, ethyl,
methoxy, ethoxy, or chloro Z.sub.3, and Z.sub.4 are separately
hydrogen, methyl, ethyl, methoxy, ethoxy, chloro; and Z.sub.5, when
taken alone, is hydrogen, methyl, ethyl, methoxy, ethoxy, or
chloro.
18. A kit for sequencing DNA comprising: a dye-terminator mix
wherein at least one dye-terminator is labelled with a
4,7-dichlorofluorescein dye; a nucleic acid polymerase; a
nucleoside triphosphate mix; and a reaction buffer.
19. The kit of claim 18 wherein said dye-terminator mix comprise
dideoxynudeotide triphosphates selected from the group consisting
of dideoxyadenosine triphosphate, dideoxycytidine triphosphate,
dideoxyguanosine triphosphate, and dideoxythymidine triphosphate
wherein each of said dideoxynudeotide triphosphates is separately
labelled with a dye 3elected from the group consisting of 5- and
carboxyfluorescein. 5- and carboxyl-4,7-dichlorofluorescein,
2',7'-dimethoxy-5- and carboxy4,7-dichlorofluorocein,
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,
1',2',7',8'-dibenzo-4',5'-dichloro-5- and
6-carboxy-4,7dichlorofluorescein, 2',7'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, and 2',4',5',7'-tetrachloro-5-
and 6-carboxy-4,7-dichlorofluorescein.
20. The kit of claim 19 wherein said dideoxythymidine triphosphate
is labelled with 6-carboxyfluorescein, said dideoxycytidine
triphosphate is labelled with
2',4',5',7'-tetrachloro-5-carboxyfluorescein, said dideoxyadenosine
triphosphate is labelled with 2',4',5',7'-tetrachloro-4,-
7-dichloro-5-carboxyfluorescein, and said dideoxyguanosine
triphosphate is labelled with
1',2',7',8'-dibenzo-4,7-dichloro-5-carboxyfluorescein.
21. The kit of claim 20 wherein said nucleic acid polymerase is
Sequenase.TM..
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
09/949,444 filed Sep. 7, 2001, which is a continuation of
application Ser. No. 09/580,754 filed May 30, 2000, now Pat. No.
6,403,812 which is a continuation of application Ser. No.
09/273,655 filed Mar. 23, 1999, now Pat. No. 6,096,723 which is a
continuation of application Ser. No. 08/905,855 filed Aug. 4, 1997,
now Pat. No. 5,885,778, which is a continuation of application Ser.
No. 08/400,780 filed Mar. 8, 1995, now Pat. No. 5,654,442 which is
a continuation of application Ser. No. 07/939,813 filed Sep. 3,
1992, abandoned, which is a continuation-in-part of application
Ser. No. 07/436,455 filed Nov. 14, 1989, now Pat. No. 5,188,934,
which are all incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to fluorescent labelling
techniques, and more particularly, to the use of
4,7-dichlorofluosceins for detecting multiple target substances in
the same sample.
BACKGROUND
[0003] Many diagnostic and analytical techniques require that
multiple target substances in the same sample be labelled with
distinguishable fluorescent tages, e.g. Lanler at al. J. Immunol.,
Vol. 132, pgs. 151-158 (196)(flow cyometry): Gray et al.
Chromosoma, Vol. 73. pgs. 9-27 (1979) (flow system kayrotyping);
Fung at al. U.S. Pat. No. 4,055,255 (DNA sequencing); and Mayrand
et al, Applied and Theoretical Electrophoresis, Vol. 3, pgs. 1-11
(1992) (analysis of electrophoretically separated polymerase chain
reaction (PCR) product). This requirement is particularly difficult
to satisfy in DNA sequence analysis where at least four spectrally
resolvable dyes are needed in most automated sequencing
approaches.
[0004] 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.569-564 (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. Singer
et al. J. Mol. Biol. Vol. 143. pgs. 161-178 (1980); Schmier et al.
J. Mol. Biol. Vol. 129, pp. 169-172 (1979); Smith et al. Nucleic
Acids Research Vol. 13, pgs. 2399-2412 (1985): 35Smith et al,
Nature, Vol. 321. pgs. 674-479 (1987); Prober et al, Science, Vol.
238, pgs. 338-341 (1987) Section II, Meth. Enzyml., 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).
[0005] 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 sots 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 at al (1987,
cited above), or to the base of a terminal dideoxynucleotide, e.g.
Prober at 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.
[0006] 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 width
for organic fluorescent dyes is about 4080 nanometers (nm) and the
width of the visible strum is only about 350400 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.
[0007] 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).
[0008] in view of the above, many analytical and diagnostic
techniques such s DNA sequencing, would be significantly advanced
by the availability of new fluorescent dyes (1) which are
physiochemically similar to readily available dyes, (2) which
permit detection of specially 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, and (4) which are
amenable for use with a wide range of preparative and manipulative
techniques.
SUMMARY OF THE INVENTION
[0009] The invention is directed to a method of concurrently
detecting specially overlapping target substances using
4,7-dichlorofluorescein dyes, and in particular, methods of DNA
sequence determination employing 4,7-dichlorofluorescein dyes. The
invention also includes 2',7'-dichloro-5 (and 6-)
carboxy-4,7-dichlorofluorescein defined by Formula I. 1
[0010] wherein:
[0011] A' is hydrogen, fluoro, chloro, a linking functionality,
such as isothiocyanate, succinimidyl carboxylate, or
phosphoramidite, or a group, such as carboxyl, sulfonyl, or amino,
that may be converted to a linking functionality: preferably A' is
a linking functionality or a group that may be converted to a
linking functionality;
[0012] X' is 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;
[0013] Z.sub.3 is hydrogen, fluoro, chloro, a linking
functionality, such as isothiocyanate, succinimidyl carboxylate, or
phosphoramidite, 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;
[0014] Z.sub.4 is hydrogen, fluoro, chloro, a linking
functionality, such as isothiocyanate, succinimidyl carboxylate, or
phosphoramidite, 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;
[0015] B' is fluoro, chloro, or an acidic anionic group;
preferably, B' is carboxyl or sulfonyl, and most preferably B' is
carboxyl;
[0016] and wherein at least one of A', Z.sub.3, and Z.sub.4 is a
linking functionality or a group that may be converted to a linking
functionality. Preferably, only one of A', Z.sub.3 and Z.sub.4 is a
linking functionality or a group that may be converted to a linking
functionality.
[0017] The invention also includes kits for carrying out the method
of the invention. Generally, kits are provided for detecting a
plurality of electrophoretically separated classes of DNA
fragments. In particular, kits are included for carrying out DNA
sequencing wherein at beat one class of primer extension product is
fluorescently labelled with a 4,7-dichlorofluorescein dye. Such DNA
sequencing kits include kits with dye-labelled primers and, as an
alternative embodiment, kits with dye-labelled terminators.
[0018] 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.
[0019] 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
high r 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
[0020] 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.
[0021] As used herein the term imperially resolvable in reference
to a set of dyes means that the fluorescent emission bonds of the
dyes are sufficiently distinct, i.e. sufficiently non-overlapping,
that target substances to which the selective 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 Cyometry:.
instrumentation and Data Analysis (Academic Pros, Now York,
1985)
[0022] 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, and the
like. More preferably, the term "lower alkyl" denotes an alkyl
having from 1 to 3 carbon atoms.
[0023] 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.
[0024] Preferably, the 4,7-dichloro-5- (and 6-) carboxyfluorescein
dyes of the invention include those defined by Formula II. 2
[0025] wherein:
[0026] A', B' and X' are defined as above;
[0027] Z.sub.1 hydrogen or, when taken with Z.sub.2, benzo;
[0028] 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
functionally, or when taken with Z.sub.1, Z.sub.2 is methylamino,
that may be convened to an active linking functionally, 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.
[0029] 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, and
Z.sub.4, and Z.sub.5 is a group that may be converted to an active
linking functionality.
[0030] Many dyes for use in 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. 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. Sulfonylfluorosceins, i.e. A or B is
sulfonyl, are synthesized following the methods disclosed by Lee et
al. Cytometry. Vol. 10, pgs. 151-16 (1989), modified by
substituting appropriate reactants to give 5- or 6-carboxyl or
sulfonylfluoescein products. Preferably, when labelling
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 used. The 5 and 6 lissome of the dyes are
readily separated by reverse phase HPLC, e.g. Edmundson et al. Mol.
Immunol., Vol. 21, pg. 561 (1194). Generally, it is believed that
the flit eluting peak is the 6-isomer and the second eluting peak
is the 5-isomer.
[0031] 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 functionally that can be reacted with a complementary
functionality on a target substance to form a linking group. The
following table lists linking functionalities that can be formed
whenever A is carboxyl, sulfonyl or amino, suitable complementary
function and the resulting linking groups suitable for use with the
invention.
1 Com- Linking plementary Linking Functionality Functionality Group
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
[0032] Preferably the linking functionality k isothiocyanate,
sulfonyl chloride, 4,6-dichlorotriazinylamine, or succinimidyl
carboxylate whenever the complementary functionality is amine. And
preferably the lining 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. 4,318,848, and Kasai et al,
Anal. Chem., Vol. 47, pgs. 3437 (1975). Accordingly, these
references are incorporated by reference. Dye phosphoramidites are
formed as taught by Stein et al. Gene, Vol. 72, pgs. 333-341
(1988); Fung at of, U.S. Pat. No. 4,757,141; European patent
application 89118946.8 filed 13 Sep. 1989; and European patent
application 88307934.5 filed 26 Aug. 1988. Substitutes R.sub.1,
R.sub.2, and R.sub.3 can take a variety of forms, e.g. as taught by
Beaucage et al. Tetrahedron, Vol. 48, pgs. 2223-2311 (192;
Canthers, pgs. 47-94 in Norang, editor, Synthesis and Applications
of DNA and RNA (cademic Press, New York, 1987); and the like.
Preferably, R.sub.1 and R.sub.2, taken separately, are methyl,
ethyl, or isopropyl, and R.sub.1 and R.sub.2, taken together with
the nitrogen to which they are attached, is a heterocycle having
from four to eight carbon atoms and one to two heteroatoms selected
from the group consisting of nitrogen, oxygen, and sulfur. More
preferably. R.sub.1 and R.sub.2, taken together with the nitrogen
to which they am attached is morpholino. Preferably, R.sub.3 is
selected from the group consisting of methyl, chlorophenyl,
.beta.-cyanoethyl, methylsulfonylethyl, and nitrophenylethyl.
Preferably, the phosphoramidite-derived linking group is oxidized
to form a phosphorus(V) linkage, e.g. as taught by Beaucage et al
(cited above); Stec et al, PCT application PCTIUS91/01010; Beaucage
et al, U.S. Pat. No. 5,003,097; or the like.
[0033] When dyes of the invention are used to label
dideoxynucleotides for DNA sequencing, preferably they are linked
to the 5 carbon of pyrimidin 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. 8455647
(1987); Gebeyehu et al. Nucleic Acids Research. Vol. 15. pgs.
4513-4535 (1987); Haralambidis et al, Nucleic Acids Research, Vol.
15, pgs. 48584876 (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 dideoxynudeotide. Preferably,
the linking group is 3carboxyamino-1-propynyl. The synthesis of
such alkynylamino-derivatized dideoxynudeotide is taught by Hobbs
et al in European patent application number 87305844.0 and U.S.
Pat. No. 5,047,519, which are incorporated herein by reference.
Briefly, the alkynylamino-derivatized dideoxynucleotides are formed
by placing the appropriate halodideoxynucleoside (usually
5-iodopyrimidine and 7-Iodo-7-deazapurine dideoxynudeosides 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, triethylamine 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 hydroxyhalide
generated in the coupling reaction. When a protected alkynylamine
is used, methano/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
methoxymethyl, 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.
[0034] 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 a few bases in length to several thousand bases
in length. e.g. from 6 to a few tens to several hundreds or to
several thousands of bases in length (of single stranded), or in
the size range of a few basepairs in length to several thousand
basepairs in length, e.g. from 8 to a few tens to several hundred
or to several thousand basepairs in length (of double
stranded).
[0035] 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. 4,805,735; 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).
[0036] 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 special
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 electrophoresis,
particularly gel electrophoresis.
[0037] Classes of polynucleotides can arise in a variety of
contexts. For example, they can arise as products of restriction
enzyme digests, or as extension products in polymerase or "gas
reactions. Preferably, classes identified in accordance with the
invention are defined in term: 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 United, London, 1981); or Osterman, Methods of
Protein and Nucleic Acid Research, Vol. 1 (Springer-Verlag, Beriln,
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 Enzymoloay, Vol. 65, pgs. 299-305
(1980); Maniatis et 91., "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, polynucleotides having sizes in the range of been 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 ascytamid to bacyamide, formed in a
Triborate 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.
[0038] 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 is
simultaneously at these lines, e.g. Cyonics, Ltd. (Sunnyvale,
Calif.) Model 2001. or the like.
[0039] 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, e.g. following the teachings of Ward et al, U.S. Pat. No.
4,711,955; directly to the 5'-hydroxyl via a phosphoramidite
linking functionality: or on the base of a dideoxynudeotide, e.g.
via the alkynylamino linking groups disclosed by Hobbs et al,
European patent application number 87305844.0 which is incorporated
herein by reference.
[0040] Kits of the invention can take a variety of forms, but
usually pride the means for the fluorescent detection of multiple
DNAs separated by size. Kits may be used for detecting amplified
nucleic acids separated by size (e.g. by electrophoresis), for DNA
sequencing, and the like. Generally, the kits will include either
an oligonucleotide labelled with a 4,7-dichlorofluorescein dye, or
in an embodiment of the DNA sequencing kit a dye-terminator mix
wherein at least one of the dye-terminators is labelled with a
4,7-dichlorofluorescein dye. Usually, the dye-terminator is a
dideoxynucloside triphosphate, as d scribed above, labelled with a
fluorescent dye.
[0041] Kits for detecting amplified nucleic acids comprise at least
one oligonucleotide labelled with a 4,7-dichlorofluorescein dye, an
enzyme selected from the group consisting of nucleotide polymerase
and nucleic acid ligase, and a reaction buffer. Whenever the kit
includes a DNA polymerase, it further includes a nucleoside
triphosphate mix, e.g. a 50 mM aqueous solution of EDTA containing
the appropriate concentration of nudeoside triphosphates for a
particular application, e.g. amplification, sequencing, or the
like. When the kit provides a nucleoside triphosphate mix for DNA
sequencing it is understood that such triphosphates include
analogs, such as nucleoside-5'-O-(1-thiotriphosphates), e.g. as
taught by Lee et al, Nucleic Acids Research, Vol. 20, pgs.
2471-2483 (1992). Nucleic acid polymerases include DNA polymerases,
RNA polymerases, and reverse transcriptases, and the like.
Preferably, whenever the kit is for PCR amplification, the nucleic
acid polymerase is Taq polymerase, e.g. as disclosed by G U.S. Pat.
No. 4,889,818. Guidance for selecting a PCR reaction buffers and
nucleoside triphosphate mixes for particular embodiments can be
found in innis et al, Editors, PCR Protocois: A Guide to Methods
and Applications (Academic Press, New York, 1990). A typical
10.times.PCR reaction buffer comprises 15 mM MgCl.sub.2, 500 mM
KCl, and TrHCl, pH 8.3.
[0042] Preferably, whenever the kit permits a ligase based
amplification reaction, e.g. as disclosed by Landegren et al, U.S.
Pat. No. 4,988,617 or the like, the nucleic acid ligase is a
thermostable ligase, such as disclosed by Barany, Proc. Natl. Acad.
Sci., Vol. 88, pgs. 189-193 (1991). Guidance for selecting a
ligase-based reaction buffer can be found in Landegren et al (cited
above), Wu et al, Genomics, Vol. 4, pos. 5W-569 (1989): Barany
(cited above), and Nickerson et al, Proc. Natl. Acad. Sci., Vol.
87, pgs. 8923-8927 (1990). A typical ligation reaction buffer
comprises 20 mM TrisHCl, pH 7.6; 50 mM KCl; 10 mM MgCl.sub.2; 1 mM
EDTA; 10 mM NAD.sup.+, and 10 mM dithiothreitol.
[0043] The dye-labelled oligonudeotides of the kit can have a wide
range of lengths, but preferably their length are in the range of 6
to 60 nucleotides. More preferably, the oligonucleotides for
ligation kits are in the range of 6 to 30 nudeotides in length, and
most preferably, the oligonudeotides for ligation kits are in the
range of 16 to 25 nucleotides in length. The particular nucieotide
sequence of the oligonucleotides are, of course, dictated bythe
target sequences sought to be amplified. In embodiments for PCR
amplification, selection of oligonudeotides for use as PCR primers
is well known in the art, e.g. Innis et al (cited above), Hiller
and Green, PCR Methods and Applications, Vol. 1, pgs. 124-128
(1991), and the like.
[0044] Preferably, in kits for DNA sequencing wherein
dye-terminators are provided, each dideoxynucdeoside triphosphate
is separately labelled with a dye selected from the set comprising
5- and 6-carboxyfluorescein, 5- and
Scarboxy-4.7-dlchlorofluorescein, 2',7'-dimethoxy-5- and
6-carboxy-4,7-dichlorofluorescein,
2',7'-dimethoxy-4',5'-dichloro-4',5'- and 6-carboxyfluorescein,
2',7'-dimethoxy-4',5'-dichloro-5- and
6-carboxy-4,7-dichloofluorescein, 1',2',7',8'-dibenzo-5- and
6-carboxy-4,7dichlorofluorescein,
1',2',7',8'-benzo-4',5'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein, 2',7'-dichloro-5- and
6-carboxy-4,7-dichlorofluorescein. and 2',4',5',7'-tetrachloro-5-
and 6-carboxy-4,7-dichlorofluorescein. More preferably.
dideoxythymidine triphosphate is labelled with 6-carboxyfluorescein
("6-FAM"), dideoxycytidine triphosphate is labelled with
2',4',5',7'-tetrachloro-5cr- boxyfluocein ("5-ZOE"),
dideoxyadenosine triphosphate is labelled with
2',4',5',7'-tetrachloro-4,7dichloro-5-carboxyfluorescein ("5-HEX"),
and dideoxyguanosine triphosphate is labelled with
1',2',7',8'-dibenzo-4,7-di- chloro-7-carboxyfluoressein ("5-NAN").
it is understood that dideoxyadenosine includes
2',3'-dideoxy-7deazaadenosine and dideoxyguanosine includes
1',3'-dideoxy-7-deazaguanosine and 2'3'-dideoxy-7-deazainosins, and
dideoxythymidine includes 2',3'-dideoxyuridine. Usually, the
dideoxynudeoside triphosphates are labelled by way of a linking
group. Preferably, the linking group links a 5 carbon of the
2',3'-dideoxycytidine or 2',3'-dideoxyuridine to a 5 or 8 carbon of
a dye, and the linking group links a 7 carbon of the
2',3'-dideoxy-7-deazaadenosine or 2',3'-dideoxy-7-guanosine
or2',3'-dideoxy-7-deazainosine to a 5 or 6 carbon of a dye.
Preferably, the linking group is carboxyaminoalkynyl, and most
preferably, the linking group is 3carboxyamino-1propynyl.
[0045] Preferably, in kits for DNA sequencing wherein
dye-temoinators are provided, the nucleic acid polymerase is
Sequenase.TM..
EXAMPLE 1
4,7-dichloro-5-(and 6-)carboxyfluorescein ("ALF")
[0046] 0.58 g of 3,6-dichlonttrimellitic acid, 0.72 g of
resorcinol, 0.5 ml concentrated suifuti acid, and 3 ml of propionic
acid were refluxed 12 hours under argon. The reaction mixture w8
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 mbiture was taken into 100 ml ethyl acetate, washed
with 1 N 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:methano/silica gel) were evaporated to dryness. This
resldue was dissolved in 10 ml methanol and then 1 ml of 4 N 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 2
4,7-dichloro-5-(and 6-) carboxvfluomscein N-hvdroxvsuccinimide
(NHS) ester
[0047] 13.7 ng of fluorescein from Example 1, 3.3 mg of 30 NHS, 6,4
mg DCC and 1 ml ethyl acate were stired 0.5 hours. The solid was
filtered, and the supematnt was washed three times with 1:1
bine:water, dried with sodium sulfate, and evaporated to dryness
yielding 15 mg of NHS ester.
EXAMPLE 3
Conjugation of 4,7-dichloro-5-(and 6-)carboxyfluorescein with
aminoalkyloligonucleotides
[0048] 5 mg of NHS ester from Example II were dissolved 5 in 20 ul
of DMSO: 3 ul of this solution were added to a solution consisting
of 20 ul of 1.0 mM 5'-aminophosphate oligonucleotide (an 18-mer) in
water and 10 ul of 1 M sodium bkarbond/sodium carbonate buffer. pH
9.0. After one hour in the dark, the solution was passed through a
10 ml Sophedox G 25 (medium) column with 0.1 M triethylammonium
acetate buffer, pH 7.0. The band of colored material eluting in the
exclusion volume was collected. Reverse phase HPLC showed two major
fluent peaks, corresponding to the 5- and 6-isomers of the dye
conjugated onto the DNA. The peaks were collected, and the
fluorescence speera in 50% urea at pH 8.0 showed full width at half
m of 34 nm with the emission maxima at 528 nm.
EXAMPLE 4
2',7'-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein
("BUB")
[0049] The procedure of Example I was followed except that the
following metals and quantities were substituted: 1.47 g
4-methylresorcinol, 0.60 g of 3,8-dichlorimellitic ad., 0.2 ml
concentrated sulfuric acid, and 4 ml propionic acid. The procedure
yielded 0.180 g of 4,7-dichloro-2',7'-dimet- hoxy-5-(and
6-)carboxyfluorescein.
EXAMPLE 5
2',7'-dimethoxy-5-(and 6-)carboxy 4,7-dichlorofluorescein NHS
ester
[0050] 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, 8.4 mg DCC, and 2
ml ethyl acetate.
EXAMPLE 6
Conjugation of 4,7-dichloro-2',7'-dimethoxy 5-(and
6-)carboxyfluorescein with amino-alkyloligonucleotide
[0051] The procedure of Example III was followed using the dye NHS
ester of ample V. The fluorescence aspect 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 82.
EXAMPLE 7
2'7'-dimethoxy-4',5'-dichloro-5-(and
6-)carboxy-4,7-dichlorofluorescein ("LOU")
[0052] This dye was prepared from the dye of Example IV and sodium
hypochie in aqueous sodium hydride.
EXAMPLE 8
4,7-dichloro-2',7'-dimethoxy-4',5'-dichloro-5-(and
6-)carboxyfluorescein NHS ester
[0053] 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 ma DCC, and 0.2 ml ethyl
acetate as in Example II.
EXAMPLE 9
Conjugation of 4,7-dichloro-2',7'-dimethoxy 4',5'-dichloro-5-(and
6-)carboxyfluorescein with aminoalkyloligonucleotides
[0054] The dye nucleotide conjugate of this example was prepared as
in Example III using tho dye NHS ester from Example VIII. The
fluorescence spectra of the two peaks collected during revere phase
HPLC showed full widths at half max of 38 nm with emulsion maxima
at 558 nm in 50% urea at pH 8.2.
EXAMPLE 10
1',2',7',8'-dibenzo-5-(and 6-)carboxy-4,7-dichlorofluorescein
("NAN")
[0055] First 3,6-dichlorotrimellitic acid trichloride was prepared:
A mixture of 0.5 g of 3,6-dichloromellitic 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 trichloro was
used without further purification. NAN was prepared as follows: A
mixture of 2,7 g of 1,3-dihydroxynaphthalene, 2.84 g of
3,6dichlorotrimellitic acid trichloride, and 8 ml of propionic add
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 1 M NaHCO.sub.3. The aqueous
solution was heated to boiling and acidified with concentrated HCl.
The resulting red solid (0.2 0) was filtered and dried.
EXAMPLE 11
1',2',7',8'-dibenzo-4',5'-dichloro-5-(and
6-)carboxy-4,7-dichlorofluoresce- in ("DEB")
[0056] 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% reacion. The solution
was acidified with HCI. extracted with 20 ml of ethyl acetate. dred
(Na.sub.2SO.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 800:60:18 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 ng of DEB.
EXAMPLE 12
Formation of 1',2',7',8'-dibenzo-5-(and
6-)carboxy-4,7-dichlorofluorescein NHS ester
[0057] 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 sold appeared.
[0058] Thin layer Schromatography on a silica gel using 800:80:16
methylene chloride:methanol:acetic acid showed complete conversion
to the NHS ester. The ethyl acetate solution was wished with dilute
HCl, dried (NaSO.sub.4) and concentrated to a red solid (15
mg).
EXAMPLE 13
Using ALF-, BUB-, LOU-, and NAN-oligonucleotide conjugates as
dye-labeled primers in DNA sequence analysis
[0059] 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 manufacturers 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 ester 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-dichlorofluoresce- in 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-4,7-dich- lorofluosceins 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 14
Using ALF-, BUB-, DEB and NAN-oligonucleotide conjugates as
dye-labeled primers in DNA sequence analysis
[0060] The same procedure was followed as described for Example
XIII, except for the following: (i)
1',2',7',8'-dibenzo-4',5'-dichlorocarboxy-4- ,7-dichlorofluoroscein
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 (ii) 5 nm
bandpass filters wore centered at 540, 580, 580, and 610 nm.
EXAMPLE 15
2',7'-dichloro-5-(and 6)-carboxy-4,7-dichlorofluoresein ("5-(and
6-)TET")
[0061] A mixture of 4-chlororesorcinol (10 g),
4,7-dichlorotrimellitic acid (10 g), and methanesulfonic acid (30
mL) were combined and heated to 140.degree. C.-150.degree. C. for
two hours. The red mixture was poured into water (100 mL) and
extracted with ethyl acetate (100 mL). The organic phase was washed
twice with dilute aqueous HCl and concentrated to go brown solid
(19 g). Pyridin (40 mL) and acetic anhydride (10 mL) were added to
the solid and the mixture refluxed for 0.5 hours. The solution was
allowed to cool for 1 hour at 4.degree. C.
[0062] Crystals were separated by function to yield a white solid
(5.4 ). Hydrolysis of a small portion (by addition of 0.02 mL of
0.1 N NsCl and 0.02 mL of ethanol to 2 ng solid) followed by
analysis on reverse phase HPLC showed that the solid contained a
92:8 ratio of isomers (6-carboxy TET:5carboxy TET ). A second
recrystallization provided nearly isometrically pure dye as the
diacetate (99:1 ratio). 5-TET can be recovered from the filtrate by
hydrolysis of the discelate form of 5-TET followed by
recrystallization from acetonitrile.
[0063] Sodium hydroxide (3 g) and water (10 mL) were added to 8TET
diacetate (8.8 g)(obtained as the first of two peaks off the HPLC
column). Additional water (50 mL) was added until the solution
became homogeneous. To the dark red solution was added concentrated
HCl (15 mL). A yellow precipitated formed. The mixture was
extracted with ethyl acetate (100 mL). The organic layer was
concentrated to pale yellow, nearly colorless solid (7.4 g of
6-TET).
EXAMPLE 16
2',4',5',7'-tetrachloro-5-(and 6-)carboxy-4,7-dichlorofluorescein
("5- and 6-HEX")
[0064] To a 1-liter Erienmeyer flask equipped with a magnetic
stirring bar was added 5- or 8-TET (6.3 ) and 1 M bonate buffer at
pH 9.4 (60 mL). Household bleach (sodium hypochlorite, 50 ml) was
added dropwise over 20 minutes. The progress of the reaction was
monitored by reverse phase HPLC. A total of 67 mL of bleach was
added. The solution was acidified with concentrated HCl (15 mL) and
extracted with ethyl acetate (100 mL). The organic phase was
concentrated to a bright yellow solid (7.3 g). .sup.1H NMR
(DMSO-d.sub.6) .delta.8.1(1H); 7.4 (2H).
Sequence CWU 1
1
1 1 18 DNA Artificial Sequence m13 universal primer 1 tcccagtcac
gacgttgt 18
* * * * *