PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus

Abstract

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.