title: Probe production for in situ hybridization by PCR and subsequent covalent labeling with fluorescent dyes.
abstract: A simple procedure for fluorescent labeling of probes just before in situ hybridization is provided. Aminoallyl-dUTP is introduced during probe production by polymerase chain reaction (PCR). The aminoallyl-dUTP functions as a reactive site for subsequent labeling of the probe. Activated fluorescent dyes such as fluorescein are covalently attached to the probe through the formation of a stable amide bond. Labeled probes are purified by size-exclusion gel chromatography to remove unincorporated dye. Target genes used to demonstrate the efficacy of this technique with in situ hybridization are rat Y-chromosome and rat granulocyte colony-stimulating factor receptor. PCR amplicons containing aminoallyl-dUTP were produced in high yield. Probes obtained after labeling with activated fluorophores demonstrated high intrinsic activity within in situ hybridizations. The introduction of aminoallyl-dUTP into the PCR reaction enables the production of "unlabeled" probes by PCR having a shelf life, which is not limited by the storage and stability challenges of fluorophore-labeled probes. Subsequent labeling of the probes with activated fluorescent dyes just before use allows one step in situ hybridization with high activity and minimal background staining.
authors.affiliation: Institute of Pathology, University of Cologne, Germany.
authors.forename: Olaf
authors.initials: O
authors.lastname: Dirsch
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ji
authors.forename: Julia
authors.initials: J
authors.lastname: Bohr
authors.forename: Kai
authors.initials: K
authors.lastname: Shen
authors.forename: Derek
authors.initials: D
authors.lastname: Levison
authors.forename: Uta
authors.initials: U
authors.lastname: Dahmen
mesh_terms: D000498:Allyl Compounds / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015342:DNA Probes / Q000096:biosynthesis* / Q000737:chemistry; D005260:Female; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D011917:Rats, Inbred Lew; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C524080:5-(aminoallyl)-2'-deoxyuridine 5'-triphosphate; D000498:Allyl Compounds; D015342:DNA Probes; D005456:Fluorescent Dyes; D019793:Fluorescein; D014544:Uridine Triphosphate