Isolation of Adrenergic Receptor Genes

In order to isolate a single gene, phage or cosmid libraries can be screened by the conventional technique of hybridization as described by Sambrook et al. (1 ) using end-labeled oligonucleotide probes or gene-specific probes. The probes are labeled either by nick translation, end labeling, or random priming using radioactive or nonradioactive techniques. Newer methods that use polymerase chain reaction (PCR) to screen phage libraries have been described by Yu and Bloem (2 ). Below, we describe a method for screening a cosmid library using PCR rather than a conventional colony hybridization technique. The methodology is based on a report by Takumi and Lodish (3 ). The cosmid libraries produce transformed bacterial colonies containing large cosmid vectors that behave as plasmids. These plasmids can be extracted from the bacteria by standard techniques for plasmid isolation once the appropriate clone is selected.