A method for recovering strand-specific probes from nick-translated DNA fragments

• Published in: Analytical biochemistry Vol. 140; no. 1; pp. 121 - 128
• Main Authors: Dutton, F.Lee, Chovnick, Arthur
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.01.1984; Elsevier
• Subjects: Bacteriophages - genetics; Base Sequence; Biological and medical sciences; Cell physiology; Chromatography, Agarose; Cloning, Molecular; DNA - isolation & purification; DNA, Recombinant; DNA, Viral; Drosophila melanogaster - genetics; Fundamental and applied biological sciences. Psychology; Hormonal regulation; M13 clones; Molecular and cellular biology; nick translation; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Peptide Fragments - analysis; phage M13; Protein Biosynthesis; rosy; strand-specific probes; xanthine dehydrogenase; Xanthine Dehydrogenase - genetics; xanthine dehydrogenase; nucleic acid hybridization; rosy; M13 clones; strand-specific probes; nick translation
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(84)90142-8

A method of preparing strand-specific probes for DNA·DNA or DNA·RNA hybridizations is described. Double-stranded DNA fragments are first isolated from any recombinant DNA clone containing the desired sequence, and then labeled in vitro by nick-translation ( T. Maniatis, A. Jeffrey, and D. G. Kleid (1975) Proc. Natl. Acad. Sci. USA 72, 1184–1188; P. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg (1977) J. Mol. Biol. 113, 237–251). Sequences homologous to the desired strand are captured by annealing the denatured nick-translate to viral strands of an appropriate M13 clone, and recovered by elution of the resulting hybrids from a column of agarose A50M (Bio-Rad). By this method, separate probes with specificity to either strand, as well as the double-stranded probe, may conveniently be prepared from a single nick-translation reaction. Probes may be obtained which are homologous either to the full length of the cloned region or to selected portions thereof by selecting appropriate M13 clones for annealing. The probe is recovered as a population of fragments several hundred bases or less in length, which have been found ideal for saturating liquid hybridizations, and should be similarly well suited for in situ hybridizations to cytological preparations.