DNA complexes with dyes designed for energy transfer as fluorescent markers

• US 5,646,264 A
• Filed: 01/23/1995
• Issued: 07/08/1997
• Est. Priority Date: 03/14/1990
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Benson, Scott C., Glazer, Alexander N.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/340,749
• Time in Patent Office: 897 Days
• Field of Search: 536/25.32, 536/26.6, 549/223, 546/107, 546/108, 546/176
• US Class Current: 536/25.32
• CPC Class Codes: C07D 221/12 Phenanthridines
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/06 linked by a carbon chain containing only aliphatic carbon atoms
• CPC Class Codes: C07D 417/14 containing three or more hetero rings
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44747 Composition of gel or of carrier mixture

Abstract

Heteromultimeric fluorophores are provided for binding to DNA, which allow for the detection of DNA in electrical separations and preparation of probes having high-fluorescent efficiencies and large Stokes shifts. In addition, by appropriate choice of fluorescent molecules, one can use a single narrow wavelength band excitation light source, while obtaining fluorescent emissions having sufficient separation to be readily discriminated.

Claims

1. A kit comprising at least two fluorescent multimers, each multimer having at least two fluorophores, at least one fluorophore comprising benzthiazole, at least one of said multimers characterized by:

a) comprising at least two different fluorophores, which fluorophores absorb and emit at different wavelengths when bound to dsDNA;

b) having a Stokes shift of at least about 25 nm;

c) capable of intercalating dsDNA at a ratio of at least one dye per 200 bp;

d) having from 2 to 4 fluorophores;

wherein at least two of said fluorescent multimers can be excited within the same narrow light bandwidth range of not more than 30 nm when bound to dsDNA, and have emission maxima at least 25 nm apart, wherein said multimer has a fluorophore comprising benzthiazole.

2. A kit comprising at least two fluorescent multimers wherein said multimers are selected from the group consisting of thiazole orange dimer of formula 8 of FIG. 2, thiazole orange-thiazole blue dimer of formula 7 of FIG. 2, and thiazole orange-ethidium dimer of formula 11 of FIG. 3.

3. A kit comprising at least two fluorescent multimers, wherein at least one of said multimers comprises phenanthridine and is selected from the group consisting of fluorescein-ethidium dimer of formula 13 of FIG. 4 and ethidium dimer and the other multimers characterized by:

(a) comprising at least two different fluorophores, which fluorophores absorb and emit at different wavelengths when bound to dsDNA;

(b) has a stokes shift of at least about 25 nm;

(c) capable of intercalating dsDNA at a ratio of at least one dye per 200 bp;

(d) having from two to four fluorophores;

wherein said fluorescent multimer can be excited within the same narrow light band width range when bound to dsDNA of said phenanthridine comprising multimer and has an emission maxima at least 25 nm apart from said phenanthridine comprising multimer.