Optimal Conditions for Chromosomal Dna Isolation and Pcr Amplification of the Internal Transcribe Spacer Rdna Region of Four Riau Penicillium Isolates

Abstract

The genus Penicillium has currently 549 described species. Many members of this genus are economically important, both in positive and negative ways. Correct molecular identification of isolated species is important for their utilization. Penicillium LBKURCC37.1, LBKURCC37.2, LBKURCC38 and LBKURCC39 are four strains isolated from Giam Siak Kecil-Bukit Batu Biosphere Reserve natural forest located in Riau. Although already identified morphology as members of Penicillium, their species identity still needs to be determined by molecular methods. The aim of this study was to determine optimal conditions for chromosomal DNA isolation and amplification of the Internal Transcribe Spacer (ITS) ribosomal DNA (rDNA) region of Penicillium LBKURCC37.1, LBKURCC37.2, LBKURCC38 and LBKURCC39, for subsequent use in molecular and phylogenetic analysis. Our results show that chromosomal DNA could be isolated from two to three days old cultures, depending on the strain. Various annealing temperatures were explored for Polymerase Chain Reaction (PCR) amplification of the ITS rDNA regions of the fungal chromosomal DNA. All strains required an annealing temperature of 42°C before producing PCR bands representing the ITS rDNA region with sizes ranging from 590 bp to 732 bp.