Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1999-05-12
2000-10-31
Houtteman, Scott W.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
549510, C07H 2104
Patent
active
061404942
DESCRIPTION:
BRIEF SUMMARY
This invention concerns the use of a class of dyes in various biological applications. Some of the dyes are claimed as new compounds per se.
BACKGROUND TO THE INVENTION
The development of automated fluorescent methods has led to increased data generation in DNA sequencing projects. Smith et al U.S. Pat. No. 5,171,534 have described a fluorescent DNA sequencing system. Waggoner et al U.S. Pat. No. 5,268,486 have described the properties of some conjugates of cyanine dyes and Middendorf U.S. Pat. No. 5,230,781 and Patonay EP 670 374 have described the use of various cyanine dyes in DNA sequencing. Berger et al European patent 214 847 has described the use of other cyanine dyes some of which contain squarate groups in assays which involve a specific binding partner. Other squarate dyes are described by Pease et al in U.S. Pat. No. 4,830,786 and subsequent divisional patents, and by A J G Mank et al in Anal. Chem. 1995, 67, 1742-8. Cushman et al WO 93/09172 and Krutak et al WO94/19387 have described cyanine dyes containing squarate groups for use in thermoplastics and inks.
There is a need for methods of detecting biologically significant chemical species (hereafter biological molecules) at increased convenience and sensitivity in general and particularly for DNA sequencing and DNA mapping experiments.
SUMMARY OF PRESENT INVENTION
In one aspect the invention provides an adduct of any biological molecule with a squarate dye as defined below. Examples of biological molecules are peptides, proteins, antibodies, polysaccharides and drugs. Preferably the biological molecule is a nucleoside or nucleotide or analogue or oligonucleotide. An analogue of a nucleoside or nucleotide may be a nucleoside or nucleotide derivative or other sugar-heterocycle which inhibits or mimics biological activity of normal nucleosides or nucleotides towards nucleic acid modifying or polymerising enzymes. An example of a nucleotide analogue is a chain terminator, such as a dideoxynucleotide, as used in sequencing reactions. The adduct may have the formula Q--N--CO--Sq, where Q is a biological molecule such as a nucleoside or nucleotide or analogue or oligonucleotide residue, and Sq is a residue of a squarate dye, the two being joined by an amide linkage formed between an amine group of Q and a carboxylate group of Sq. Alternatively the linkage may be formed between a functional group such as carboxylic acid, or derivatives thereof, isothiocyanate, maleimide, iodoacetamide, or phosphoramidite, and a nucleophile such as an amine, thiol, hydroxy or other group, as known for other nucleotide or oligonucleotide adducts, whereby either the nucleophilic or electrophilic reactive grouping may be attached to the dye. Alternatively the dye containing O-alkyl or O-alkenyl or O-alkynyl on the central moiety but with no other reactive functional group on the rest of the molecule may react with an amine or thiol or alcohol group to form a covalent linkage to a biological molecule.
In a further aspect the invention provides for the labelling of species immobilised on solid supports. One example of this may be an immobilised labelled oligonucleotide monomer or a difunctional derivatised dye with one arm bound to a solid support which is used for automated DNA synthesis. In a later step the labelled oligonucleotide is cleaved from the support. Another application may be the labelling of a suitably derivatised solid support which is used in a heterogeneous luminescence based assay. In this instance the label may or may not be cleaved from the support.
In another aspect, the invention provides an improved fluorescent sequencing method, which comprises using an adduct as defined.
Squarate dyes are described in EP 214 847, the disclosure of which is incorporated herein by reference. A major aspect of the present invention is concerned with a family of squarate dyes that are particularly suitable for use in the adducts and the improved fluorescent sequencing methods defined above. According to this aspect, the invention provides a squarate dye of the for
REFERENCES:
patent: 4830786 (1989-05-01), Pease et al.
patent: 5227498 (1993-07-01), Lee et al.
patent: 5268486 (1993-12-01), Waggoner et al.
patent: 5486616 (1996-01-01), Waggoner et al.
patent: 5569587 (1996-10-01), Waggoner
patent: 5569766 (1996-10-01), Waggoner et al.
Mank et al., Anal. Chem., 67: 1742-1748 (1995).
Briggs Mark Samuel Jonathan
Bruce Ian Edward
Cummins William Jonathan
Hamilton Alan Lewis
West Richard Martin
Amersham Pharmacia Biotech UK Limited
Houtteman Scott W.
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