Homology cloning of cDNA or genomic DNA

The principle of cloning utilizes hybridization of single-stranded DNA probes to denatured DNA fixed on membranes to detect the identical DNA molecules present in a large population of diverse DNAs. In homology or low-stringency cloning, hybridization of oligonucleotide, cDNA, or genomic DNA probes to denatured DNA from plasmid or bacteriophage libraries is carried out under low-stringency conditions that promote hybridization in the presence of sequence mismatch (i.e., mispaired nucleotides). The procedures used are identical to those for screening libraries at high stringency, with the exception that hybridization and washing conditions are altered to permit hybridization with mismatched sequence; salt concentration is increased and the hybridization and wash temperatures are decreased. This unit describes conditions that have been used to clone and identify novel genes and cDNA clones using low-stringency hybridization of known probes to membranes that contain libraries of bacterial or bacteriophage DNA.