Simple Method for Preparation of Fluor/Hapten-Labeled dUTP

Abstract

Many projects, such as multiplex-fluorescence in situ hybridization (M-FISH) karyotyping, require the use of relatively large amounts of multiple fluor- or hapten-labeled nucleotides for the preparation of DNA probes. Such a requirement makes these experimental approaches prohibitively expensive for many researchers. The cost of such nucleotides can be reduced approximately 99% by purchasing the chemical precursors, fluor or hapten succinimidyl esters and 5-(3-aminoallyl)-2′-deoxyuridine 5′ triphosphate (AA-dUTP), and performing the simple coupling/purification described here. It is possible to finish four to ten different fluor/hapten dUTP preparations of 2.5 μM scale within a 24 h period. The reagent cost for each preparation ranges from $33–$237 per μM, depending on the fluor/hapten. This laboratory uses such nucleotide preparations to prepare FISH probes by nick translation or PCR amplification.