PCR optimization and troubleshooting, with special reference to the amplification of ribosomal DNA in lichenized fungi

Factors affecting the outcome of a PCR is discussed. Nested PCR is described and a protocol that has been shown to work well for the amplification of ribosomal genes in ascomycetes is described. It is claimed that DNA extracts of lichenized fungi, contrary to many other organism groups, are: often contaminated by other fungi: This poses a problem due to the preferential amplification of short intron-lacking sequences over longer, intron-containing ones, particularly when extension time is limited. A simple protocol for optimizing and troubleshooting PCRs is presented. (C) 1999 The British Lichen Society.