Methods of labelling polynucleotides with dibenzorhodamine dyes

• US 6,919,445 B2
• Filed: 05/20/2003
• Issued: 07/19/2005
• Est. Priority Date: 11/25/1997
• Status: Expired due to Fees

• Current Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., Benson, Scott C., Menchen, Steven Michael
• Primary Examiner(s): NORTHINGTON DAVIS, ZINNA
• Application Number:
  US10/441,950
• Publication Number:
  US 20040072209A1
• Time in Patent Office: 791 Days
• Field of Search: 435/6, 435/91.5, 536/22.1, 536/25.3, 536/26.6, 549/382
• US Class Current: 536/26.6
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 311/78 Ring systems having three or more relevant rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Dibenzorhodamine compounds having the structure
are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure
including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure
including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH₂OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Claims

1. A method of polynucleotide sequencing comprising the steps of:

forming a mixture of a first, a second, a third, and a fourth class of polynucleotides such that;

each polynucleotide in the first class includes a 3′

-terminal dideoxyadenosine and is labeled with a first dye;

each polynucleotide in the second class includes a 3′

-terminal dideoxycytidine and is labeled with a second dye;

each polynucleotide in the third class includes a 3′

-terminal dideoxyguanosine and is labeled with a third dye; and

each polynucleotide in the fourth class includes a 3′

-terminal dideoxythymidine and is labeled with a fourth dye;

wherein at least one of the first, second, third, or fourth dyes is a dibenzorhodamine compound comprising the structure

including nitrogen- and aryl-substituted forms thereof;

the other of the dyes being spectrally resolvable from the dibenzorhodamine dye(s) and from each other;

electrophoretically separating the polynucleotides thereby forming bands of similarly sized polynucleotides;

illuminating the bands with an illumination beam capable of causing the dyes to fluoresce; and

identifying the classes of the polynucleotides in the bands by the fluorescence spectrum of the dyes.

2. The method of claim 1, wherein the step of electrophoretically separating is carried out in an electrophoretic matrix is uncrosslinked polyacrylamide or crosslinked polyacrylamide.

3. The method of claim 1, wherein the uncrosslinked polyacrylamide or crosslinked polyacrylamide has a concentration of from 2-20 weight percent.

4. The method of claim 1, wherein the uncrosslinked polyacrylamide or crosslinked polyacrylamide concentration is from 4-8 weight percent.

5. The method of claim 1, wherein the electrophoretic matrix comprises a denaturing agent selected from urea and formamide.