Screening cDNA Libraries by Hybridization with Double-Stranded DNA Probes and Oligonucleotides

Probably the most commonly used method to screen a cDNA library is hybridization to a labeled DNA probe. This probe may be a single-stranded oligonucleotide or a double-stranded cDNA or polymerase chain reaction (PCR) product. The DNA may be either radioactively or nonradioactively labeled. The sequence of an oligonucleotide probe may be derived from a number of sources, e.g., degenerate probes may be obtained by back translating a peptide sequence of an unknown protein, or they may be a short conserved region of sequence within a cDNA from another member of a multigene family or from a cognate cDNA from another species (see Note 1 ). Double-stranded DNA probes may be a partial cDNA obtained by screening another library or a PCR product or a cDNA from another member of a gene family or from another species. This chapter concentrates on the methods for labeling DNA probes and hybridization to filter-bound library DNA.