Alternative dye-labeled primers for automated DNA sequencing

• US 5,614,386 A
• Filed: 06/23/1995
• Issued: 03/25/1997
• Est. Priority Date: 06/23/1995
• Status: Expired due to Fees

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Metzker, Michael L., Gibbs, Richard A.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/494,216
• Time in Patent Office: 641 Days
• Field of Search: 435/91.1, 435/6, 435/5, 435/91.2, 536/26.6, 204/182.8, 548/405
• US Class Current: 435/91.1
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

Methods for the use of a class of dyes for improved DNA sequencing are provided. A new class of dyes, BODIPY® fluorophore, has been described recently. The parent heterocyclic molecule of the BODIPY® fluorophore is a dipyrrometheneboron difluoride compound which is modified to create a broad class of spectrally-discriminating fluorophores. The present invention provides methods for the use of BODIPY® fluorophore-labeled DNA. BODIPY® fluorophore have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. BODIPY® fluorophore have narrower band width, insensitivity to solvent or pH, and improved photostability, thus, BODIPY® fluorophores lead to improved DNA sequencing and/or detection in any method where electrophoresis and detection of DNA is required. Additionally, the spectral properties of the BODIPY® fluorophores are sufficiently similar in wavelength and intensity to be used with conventional equipment known in the art.

Claims

1. A method for distinguishing polynucleotide sequences in a hybridization method of DNA sequencing, said method comprising the steps of:

synthesizing a first, a second, a third and a fourth class of oligonucleotides, wherein all of said classes of oligonucleotides have a same length, said first, second, third and fourth classes of oligonucleotides differ from the oligonucleotides of each other class by one nucleotide base at a 3'"'"', a 5'"'"' or an internal position, and each oligonucleotide of the first class has a dideoxyadenosine at said position and is labeled with a first fluorophore;

each oligonucleotide in the second class has a dideoxycytidine at said position and is labeled with a second fluorophore;

each oligonucleotide in the third class having a dideoxyguanosine at said position and is labeled with a third fluorophore; and

each oligonucleotide in the fourth class has a dideoxythymidine at said position and is labeled with a fourth fluorophore;

wherein at least one of said fluorophore is a 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

) fluorophore;

hybridizing said oligonucleotides to a single-stranded DNA target immobilized to a solid support, wherein said solid support is in a grid format, to form a hybridized product;

washing said hybridized product to remove any unhybridized oligonucleotide or target;

illuminating with an illumination beam the solid support, said illumination beam being capable of causing said BODIPY®

fluorophores to fluoresce; and

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

2. The method of claim 1 wherein said at least one 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

) fluorophore has an adsorption maxima of about 450 to 700, and an emission maxima of about 450 to 700.

3. The method of claim 2, wherein said at least one 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

) fluorophore has adsorption maxima of about 480 to 650, and an emission maxima of about 480 to 650.