SECTION 6-3 - Hybridization with Synthetic 32P End-Labeled Probe

Abstract

This chapter discusses the method of hybridization with synthetic 32P end-labeled probe, which identifies homologous DNA or RNA species bound to a filter by hybridization with a labeled synthetic oligonucleotide probe. The method may be used on circular filters to visualize plaques or colonies that contain complementary nucleotide sequences or on filters blotted from gels to visualize DNA or RNA species. The base pairing between synthetic probe and DNA or RNA is governed by the complementary hydrogen bonding of nucleic acids. The hydrogen bonding among the pairs involves association and dissociation kinetics. Increasing lengths of complementary nucleic acids provide increased stability and thus favor association. The use of oligonucleotide probes to identify species with homologous complementary sequences requires an awareness of these empirically derived factors. There should be sufficient sensitivity to detect specific nucleotide sequences without interference from nonspecific hybridization to background. The equipment required to carry out the procedure is a bag-sealing apparatus and an X-ray film-developing system.