Methods and kits using extended rhodamine dyes

• US 8,628,975 B2
• Filed: 02/03/2012
• Issued: 01/14/2014
• Est. Priority Date: 06/03/1999
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Benson, Scott C., Menchen, Steven M.
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US13/365,554
• Publication Number:
  US 20120135885A1
• Time in Patent Office: 711 Days
• Field of Search: None
• US Class Current: 436/94
• 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 fragment analysis comprising the steps of:

forming one or more labeled polynucleotide fragments, each fragment covalently linked with a fluorescent extended rhodamine compound, wherein the extended rhodamine compound comprises the structure;

2. The method of claim 1 wherein the resolving step is an electrophoretic size-dependent separation process and the one or more labeled polynucleotide fragments are detected by fluorescence.

5. The method of claim 1, wherein the extended rhodamine dye is configured to be excited by an excitation light source having a wavelength greater than about 630 nm.

6. The method of claim 1, wherein the linkage is attached to R₈ of the extended rhodamine dye.

7. The method of claim 1, wherein the linkage connecting the extended rhodamine dye to the polynucleotide fragment is an aminohexyl linkage.

12. The method of claim 1, wherein the polynucleotide fragment label is introduced using a phosphoramidite labeled with the fluorescent extended rhodamine dye.

13. the method of claim 1, wherein the polynucleotide fragment is labeled at the 5′

terminus.

14. The method of claim 12, wherein the polynucleotide fragment label is introduced by using a N-hydroxysuccinimide ester of the fluorescent extended rhodamine dye.

15. The method of claim 1, wherein the linkage attaching the polynucleotide fragment to the fluorescent extended rhodamine dye is attached to R₈ of the extended rhodamine dye.

3. A method of fragment analysis comprising:

forming a labeled polynucleotide fragment, the fragment conjugated with a fluorescent extended rhodamine dye comprising the structures;

4. The method of claim 3, wherein the size-dependent separation process is electrophoresis and the detecting is by fluorescence.

8. The method of claim 3, wherein the extended rhodamine dye is configured to be excited by an excitation light source having a wavelength greater than about 630 nm.

9. The method of claim 3, wherein the linkage connecting the extended rhodamine dye to a nucleobase of the polynucleotide fragment is attached to R₈ of the extended rhodamine dye.

10. The method of claim 3, wherein the linkage connecting the extended rhodamine dye to the polynucleotide fragment is an alkynylamino-derivatized or alkenyl-derivatized linkage formed by reacting an activated N-hydroxysuccinimide (NHS) ester of the dye.

11. The method of claim 10, wherein the labeled polynucleotide fragment comprises a nucleotide having a stucture of one of the following formulae: