Method, system and reagents for DNA sequencing

• US 5,608,063 A
• Filed: 03/28/1995
• Issued: 03/04/1997
• Est. Priority Date: 07/02/1986
• Status: Expired due to Fees

• Current Assignee: PerkinElmer Life Sciences, Inc.
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Trainor, George L., Hobbs, Frank W. Jr.
• Primary Examiner(s): BERNHARDT, EMILY A
• Application Number:
  US08/412,409
• Time in Patent Office: 707 Days
• Field of Search: 544/243, 544/244
• US Class Current: 544/244
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 471/04 Ortho-condensed systems
• CPC Class Codes: C07F 9/6512 having the nitrogen atoms in positions 1 and 3
• CPC Class Codes: C07F 9/65586 at least one of the hetero rings does not contain nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

A DNA sequencing system and method are described to detect the presence of radiant energy emitted from different excited reporter dye-labeled species (DNA fragments) following separation in time and/or space, and the identity of the species which emit radiant energy closely spaced in wavelength. Functions of the emitted energy are obtained which vary over the wavelengths of the closely spaced spectra in different senses and the functions ratioed, whereby the ratio is indicative of the identity of the DNA fragments.

The emitting portion of the reporter-labeled DNA fragment is preferably one of a family of fluorescent dyes based on 9-carboxyethyl-6-hydroxy-3 -oxo-3H-xanthene. These xanthene dyes are covalently attached to the DNA fragments through the carboxylic acid functionality, preferably via an amide linkage. The dyes may be protected by including an alkoxy group at the 9-position. A spacer may be inserted between the dye and the amine. The fluorescent dye preferably is attached to the DNA chain terminators and provides many advantages. Thus only DNA sequencing fragments resulting from bona fide termination events will carry a reporter. The DNA sequencing may also be labeled using the xanthene dyes which have general utility as fluorescent labels. Also acyclonucleoside triphosphates are described as being useful as chain terminators in DNA sequencing using a modification of the Sanger method.

Claims

1. A fluorescent-labeled triethylammonium salt having the structure:

##STR22## wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine, or 7-deazahypoxanthine; and

A is a fluorophore having the structure;

##STR23## wherein n=2 or 3, and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano; and

R₃ is a straight-chained C₁ -C₄ alkylene moiety, provided that if B is a pyrimidine, B is N₁ -linked to the acyclic sugar moiety and C₅ -linked to one end of the triple bond (pyrimidine numbering), and if B is a 7-deazapurine, B is N₉ -linked to the acyclic sugar moiety and C₇ -linked to one end of the triple bond (purine numbering).

2. A fluorescent-labeled triethylammonium salt having the structure:

##STR24## wherein B is uracil, cytosine, 7-deazaadenine, 7-deazaguanine, or 7-deazahypoxanthine; and

A is a fluorophore having the structure;

##STR25## wherein n=2 or 3, and R₁ and R₂ are H, lower alkyl, halo, lower alkoxy, and cyano; and

R₃ is a straight-chained C₁ -C₄ alkylene moiety

provided that if B is a pyrimidine, B is N₁ -linked to the acyclic sugar moiety and C₅ -linked to one end of the triple bond (pyrimidine numbering), and if B is a 7-deazapurine, B is N₉ -linked to the acyclic sugar moiety and C₇ -linked to one end of the triple bond.

3. The fluorescent-labeled triethylammonium salt of claim 2 wherein R₃ is CH₂.