Multichromophore fluorescent probes using DNA intercalation complexes

• US 6,428,667 B1
• Filed: 10/10/2000
• Issued: 08/06/2002
• Est. Priority Date: 03/14/1990
• Status: Expired due to Fees

• Current Assignee: University of California Berkeley (University of California)
• Original Assignee: University of California Berkeley (University of California)
• Inventors: Mathies, Richard A., Glazer, Alexander N., Peck, Konan
• Primary Examiner(s): Whisenant, Ethan C.
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/686,147
• Time in Patent Office: 665 Days
• Field of Search: 435/6, 435/91.1, 435/183, 436/94, 336/23.1, 336/24.3, 336/24.33, 336/25.32, 204/450, 204/456
• US Class Current: 204/456
• 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 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• 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
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Novel fluorescent labeling techniques and fluorescent labels are provided, employing high affinity non-covalently binding and intercalating fluorescent dyes and dsDNA. The dyes find application to provide highly sensitive labeling of nucleic acids in electrophoretic gels and as pre-prepared labels for binding to a wide variety of specific binding pair members. The DNA-dye fluorescer complex can be used for labels in diagnostic assays, detection of specific nucleic acid sequences, and the like.

Claims

1. In a method for performing a separation of a composition comprising employing an electrical field for directing movement of components in said composition in a differential manner, the improvement which comprises:

having present during said separation in said electrical field a moiety comprising (a) a probe having a double stranded DNA of at least about 20 base pairs and (b) non-covalently bound intercalated in said double stranded DNA at least one fluorescent molecule characterized by having at least two positive charges, having at least one fluorescent monomeric unit and having a binding affinity to double stranded DNA of at least about 5×

10⁶ M<sup>−

1</sup> and up to about 1 fluorescent unit per 4 nucleotides in a nucleic acid strand, wherein said fluorescent unit is characterized by having at least one aromatic ring per monomeric unit.

2. A method according to claim 1, wherein said fluorescent molecule is a dimer and said monomeric units are phenanthridinium and/or acridinium.

3. A method according to claim 1, wherein said separation is gel electrophoresis and said method further comprises combining a probe having a double stranded DNA containing sample with said dimer prior to said separation.

4. A method according to claim 3, wherein said dimer has a plurality of positive charges.

5. A method according to claim 4, wherein said dimer is an ethidium dimer.

6. A method according to claim 2, wherein said moiety is joined to other than double stranded DNA.

7. A method according to claim 6, wherein said moiety comprises a polypeptide or saccharide.

8. The method according to claim 1, wherein said separating is by gel electrophoresis.

9. The method according to claim 8, wherein the individual bands “

generated by gel electrophoresis”

are detected.

10. The method according to claim 11, wherein said probe further comprises single stranded nucleic acid.