Enzymatic addition of fluorescein- or biotin-riboUTP to oligonucleotides results in primers suitable for DNA sequencing and PCR.
An enzymatic, post-synthetic, non-isotopic modification of oligonucleotides giving primers that are substrates for chain elongation by DNA polymerases is described. It is shown that terminal deoxynucleotidyl transferase incorporates preferentially and almost exclusively a single fluorescein- or biotin-riboUTP at the 3' terminus of oligonucleotides. This one-step procedure, using readily available materials, permits an economical enzymatic labeling of oligonucleotides designed for fluorescence-based or solid-state DNA sequencing or PCR and offers a convenient alternative to chemical modification.
Igloi
G L
GL
Institut für Biologie III, University of Freiburg, FRG.
Schiefermayr
E
E
D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism; D017931:DNA Primers; D019793:Fluorescein; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014544:Uridine Triphosphate / Q000378:metabolism*
D017931:DNA Primers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D001710:Biotin; D004253:DNA Nucleotidylexotransferase; D019793:Fluorescein; D014544:Uridine Triphosphate