ALTERNATIVE DYE-LABELED PRIMERS, RIBONUCLEOTIDES, DEOXYRIBONUCLEOTIDES, AND DIDEOXYRIBONUCLEOTIDES FOR AUTOMATED DNA ANALYSIS AND HOMOGENEOUS AMPLIFICATION/DETECTION ASSAYS

摘要

Methods for the use of a class of dyes for improved DNA sequencing are provided. A new class of dyes, BODIPY® fluorophores, has been described recently. The parent heterocyclic molecule of the BODIPY® fluorophores 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 for dye-primer sequencing in which the BODIPY®s are attached to the 5' end of sequencing primers, methods for DNA sequencing by the chain termination method of DNA sequencing and for internal labelling of polynucleotides by enzymatic incorporation of fluorescently-labeled ribonucleotides or deoxyribonucleotides, and provides oligonucleotides labelled with substituted 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®fluorophore) compounds for perfoming the Taqman assay. BODIPY® fluorophores have improved spectral characteristics compared to conventional fluorescein and rhodamine dyes. BODIPY® fluorophores 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.