Probe production for in situ hybridization by PCR and subsequent covalent labeling with fluorescent dyes.
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.
Dirsch
Olaf
O
Institute of Pathology, University of Cologne, Germany.
Ji
Yuan
Y
Bohr
Julia
J
Shen
Kai
K
Levison
Derek
D
Dahmen
Uta
U
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
C524080:5-(aminoallyl)-2'-deoxyuridine 5'-triphosphate; D000498:Allyl Compounds; D015342:DNA Probes; D005456:Fluorescent Dyes; D019793:Fluorescein; D014544:Uridine Triphosphate