Biotinylation of DNA on membrane supports: a procedure for preparation and easy control of labeling of nonradioactive single-stranded nucleic acid probes.
We have used M13 single-stranded DNA bound by uv to small pieces of nylon membrane for the synthesis of biotinylated single-stranded DNA probes. The labeling method requires a large fragment of DNA polymerase I and random hexanucleotides. There is no need for previous linearization of the template. The clean probe is removed from the membrane by a single wash step. The synthesized probe is completely free of unincorporated precursors. This makes possible the easy control of the reaction of incorporation of biotinylated analogues into the probe by simple staining on the filter, thus allowing evaluation of the efficiency of labeling. The DNA membrane can be stored for reuse. With the procedure described it is possible to biotinylate many DNA fragments in parallel, simultaneously controlling the efficiency of labeling in a time- and cost-saving manner.
Didenko
V V
VV
Department of Immunology, Institute of Transplantology and Artificial Organs, Moscow, Russia.
D001710:Biotin / Q000737:chemistry*; D015342:DNA Probes / Q000138:chemical synthesis* / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000138:chemical synthesis* / Q000737:chemistry; D005374:Filtration; D015151:Immunoblotting; D007553:Isotope Labeling / Q000379:methods; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009757:Nylons; D013698:Templates, Genetic
D015342:DNA Probes; D004277:DNA, Single-Stranded; D008567:Membranes, Artificial; D009757:Nylons; D001710:Biotin