Methods and kits using extended rhodamine dyes

• US 7,776,568 B2
• Filed: 08/24/2004
• Issued: 08/17/2010
• Est. Priority Date: 06/03/1999
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Menchen, Steven M., Benson, Scott C.
• Primary Examiner(s): LIU, SUE XU
• Application Number:
  US10/925,411
• Publication Number:
  US 20050027130A1
• Time in Patent Office: 2,184 Days
• Field of Search: 435/91.2, 435/968, 435/975, 436/800, 536/22.1, 549/382, 549/384
• US Class Current: 435/91.2
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/08 Aza-anthracenes
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 471/06 Peri-condensed systems
• CPC Class Codes: C07D 487/06 Peri-condensed systems
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Extended rhodamine compounds exhibiting favorable fluorescence characteristics having the structure

are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having the structure

In addition, methods of making and using the dyes as fluorescent labels are disclosed.

Claims

1. A method of PCR enzymatic synthesis comprising amplifying a template DNA with nucleotide triphosphates, a polymerase, and two or more primers wherein the primers are complementary to the template DNA sequence and at least one of the primers is a labelled nucleic acid comprising the formula:

NUC-L-D

wherein

NUC is a polynucleotide and analogs thereof;

L is a linkage;

wherein L is attached to the polynucleotide at a position selected from the 5′

terminus, the phosphodiester backbone, a nucleobase, and the 3′

terminus; and

D is an extended rhodamine dye comprising the structure;

2. A kit for PCR enzymatic synthesis comprising one or more nucleotide triphosphates, a polymerase, and two or more primers wherein one or more of the nucleotide triphosphates is a labelled nucleic acid compound comprising the structure:

3. A kit for PCR enzymatic synthesis comprising one or more nucleotide triphosphates, polymerase, and two or more primers wherein at least one of the primers is a labelled nucleic acid comprising the formula:

NUC-L-D

wherein

NUC is a polynucleotide and analogs thereof

L is a linkage;

wherein L is attached to the polynucleotide at a position selected from the 5′

terminus, the phosphodiester backbone, a nucleobase, and the 3′

terminus; and

D is an extended rhodamine dye comprising the structure;