Identification of Homologous Gene Sequences by PCR with Degenerate Primers

Degenerate primers are mixtures of similar oligonucleotides that are used in a PCR, so-called degenerate PCR, to amplify unknown DNA sequences, typically coding sequences of genes. Degenerate primers are designed based on sequence data of related and already sequenced gene homologs. This method is useful for identifying new members of a gene family or orthologous genes from different organisms where genomic information is not available. We describe here how to design degenerate primers, set up the PCR (with genomic DNA or cDNA as a template), clone the resulting PCR fragments, and sequence them. Since this method only yields partial coding sequences, complete gene sequences must then be achieved by other approaches such as inverse PCR (see Chapter 16), 5′ RACE, 3′ RACE, or circular RACE (see Chapter 15).