Solution hybridization using cloned, 5'-32P-labeled, double-stranded DNA to distinguish among closely related nucleic acids.
A rapid, sensitive, and specific solution-hybridization method is described that utilizes cloned, double-stranded DNA. The DNA is treated with restriction enzyme(s), and fragments are 32P-labeled at their 5' termini with polynucleotide kinase. A single fragment is partially purified by gel electrophoresis, denatured, and annealed with unlabeled RNA or DNA. The reaction mix is treated with S1 nuclease, precipitated with TCA, and the [32P]DNA counted. Hybridization is recognized only when the 32P label is associated with duplexes that are TCA-precipitable. The specificity of the method was analyzed by annealing experiments with cloned, human globin DNAs. Restriction fragments labeled in the 3' untranslated region of human beta globin clones formed S1-resistant, TCA-precipitable duplexes with beta globin DNA, but not with delta or gamma globin DNA. Thus, a major advantage of the method is that it can distinguish among homologous nucleic acids whose uniformly labeled cDNAs cross hybridize under moderately stringent conditions. This assay is as sensitive as the [3H]cDNA hybridization method, and it circumvents the requirement for purified mRNA as a template for [3H]cDNA synthesis. It also avoids a gel electrophoresis step, it is rapid and quantitative, and many samples can be simultaneously analyzed.
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005914:Globins / Q000235:genetics; D006801:Humans; D008722:Methods; D008836:Micrococcal Nuclease; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D010957:Plasmids
D004274:DNA, Recombinant; D010761:Phosphorus Radioisotopes; D005914:Globins; D004247:DNA; D004262:DNA Restriction Enzymes; D008836:Micrococcal Nuclease