Some situations may call for the use of degenerate primers. These are mixtures of primers that are similar, but not identical. These may be convenient when amplifying the same gene from different organisms, as the sequences are probably similar but not identical. This technique is useful because the genetic code itself is degenerate, meaning several different codons can code for the same amino acid. This allows different organisms to have a significantly different genetic sequence that code for a highly similar protein. For this reason, degenerate primers are also used when primer design is based on protein sequence, as the specific sequence of codons are not known. Therefore, primer sequence corresponding to the amino acid isoleucine might be "ATH", where A stands for adenine, T for thymine, and H for adenine, thymine, or cytosine, according to the genetic code for each codon, using the IUPAC symbols for degenerate bases. Degenerate primers may not perfectly hybridize with a target sequence, which can greatly reduce the specificity of the PCR amplification.
Degenerate primers are widely used and extremely useful in the field of microbial ecology. They allow for the amplification of genes from thus far uncultivated microorganisms or allow the recovery of genes from organisms where genomic information is not available. Usually, degenerate primers are designed by aligning gene sequencing found in GenBank. Differences among sequences are accounted for by using IUPAC degeneracies for individual bases. PCR primers are then synthesized as a mixture of primers corresponding to all permutations of the codon sequence.