Improved methods for the detection of unique sequences in Southern blots of mammalian DNA by non-radioactive biotinylated DNA hybridization probes.
Biotinylated DNA hybridization probes offers a stable, cheap and non-radioactive alternative to probes labelled with 32P. Insufficient sensitivity has, however, up till now, been prohibitive for the use of such probes in detecting unique sequences in Southern blots of human DNA. By optimizing the steps in the procedure we have improved the sensitivity enough for such use. We have showed (1) that long probes (greater than 500 nucleotides) perform unproportionally better than short probes; (2) that a simple affinity labelling with avidin alkaline phosphatase conjugate performs better than laborious immunochemical systems; (3) that use of 3% BSA as blocking agent at 37 degrees C and the presence of 0.5 mol/l NaCl together with 1% BSA during the affinity labelling nearly eliminate background staining; (4) that a dramatic gain in sensitivity is gained by affinity labelling at pH 9.0 instead of 7.5; (5) that biotin-labelling can be highly reproducibly performed on a preparative scale with cheap and easily synthesized bio-11-dUTP in a two step nick-translation and (6) that biotinylated probes and hybridization mixtures can be stored for months and reused. The study has resulted in the presentation of a fast procedure, which is generally applicable to routine DNA diagnostic work, also in parts of the world where it is difficult to get a regular supply of 32P.
University Department of Clinical Chemistry, Aarhus Kommunehospital, Denmark.
N
N
Gregersen
J
J
Koch
S
S
Kølvraa
K B
KB
Petersen
L
L
Bolund
D001483:Base Sequence; D001710:Biotin; D003101:Collodion; D004247:DNA / Q000032:analysis* / Q000276:immunology; D006801:Humans; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D014544:Uridine Triphosphate / Q000032:analysis
D010761:Phosphorus Radioisotopes; D001710:Biotin; D003101:Collodion; D004247:DNA; D014544:Uridine Triphosphate