To target single gene fragments from multiple organisms within a complex community of known and unknown organisms using PCR has required careful bioinformatics analyses and empirical testing of many primers. Ideal criteria for primers include: (i) primers should match genes of all known organisms within the group of interest, and should be able to target genes from unknown organisms in sub-taxonomic levels (i.e. domain-level primers targeting Bacteria should be conserved among all known bacteria, with the assumption that unknown bacteria will also contain these conserved regions), (ii) primer pairs should be balanced for melting temperature and produce robust amplification, and (iii) primers need to span one or more hyper-variable regions of the gene. Even in highly conserved regions of the ribosomal RNA (rRNA) genes, no primer set matches these criteria perfectly, although many excellent primer sets have been developed (e.g., [1,2–5]). To increase target range, pools of primers (degenerate primers) are used. Ribosomal RNA genes are the preferred targets for broad-spectrum analyses as the level of genetic diversity in conserved regions of rRNA genes is lower than that present in functional genes where amino acid sequences can be highly conserved even in the presence of substantial DNA-level changes due to the degeneracy of the genetic code. Therefore, degenerate primers used for PCR amplification of rRNA genes generally have lower levels of degeneracy than primers used for amplification of functional genes