Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-05-30
2002-06-11
Krass, Frederick (Department: 1614)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C549S224000, C549S225000, C549S382000, C549S388000
Reexamination Certificate
active
06403812
ABSTRACT:
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 labelled with distinguishable fluorescent tages, e.g. Lanier et al, J. Immunol., Vol. 132, pgs. 151-156 (1984)(flow cytometry); Gray et al, Chromosoma, Vol. 73, pgs. 9-27 (1979)(flow system karyotyping); Fung et al, U.S. Pat. No. 4,855,225 (DNA sequencing); and Mayrand et al, Applied and Theoretical Electrophoresis, Vol. 3, pgs. 1-11 (1992)(analysis of electrophoretically separated polymerase chain reaction (PCR) products). 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.
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); 35Smith 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 that 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 interfer with the activity of the polymerase or reverse transcriptase employed.
Because of the 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 fluorescent dyes (1) which are physiochemically similar to readily available dyes, (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, and (4) which are amenable for use with a wide range of preparative and manipulative techniques.
SUMMARY OF THE INVENTION
The invention is directed to a method of concurrently detecting spacially 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.
wherein:
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;
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;
Z
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
3
is hydrogen or chloro;
Z
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
4
is hydrogen or chloro;
B′ 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
3
, and Z
4
is a linking functionality or a group that may be converted to a linking functionality. Preferably, only one of A′, Z
3
, and Z
4
is a linking functionality or a group that may be converted to a linking functionality.
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
Chakerian Vergine
Connell Charles R.
Fung Steven
Hershey N. Davis
Lee Linda G.
Krass Frederick
Powers Vincent M.
The Perkin-Elmer Corporation
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