A rapid microwave method for isolation of genomic DNA and identification of white rot fungi

Abstract

White rot fungi (WRF) produce lignolytic enzymes comprised by laccases and peroxidases responsible for mineralization of recalcitrant lignin. Because of the so-called lignin modifying enzymes(LME’s), these fungi have potential applications in biodegradation and bioremediation processes. Increased demand for lignolytic enzymes to exploit their various applications has sparked interest in identifying and characterizing new novel strains of WRF. Despite this undisputed biotechnological significance, molecular identification of WRF, remains a daunting task for researchers as genomic DNA isolation is a tedious process, unsuccessful many a times because of their rigid and resistant cell walls. A rapid, effective and efficient method to identify the innumerable fungal strains within no time is the need of the hour. The fungal mycelia of various unknown as well as know isolates of WRF, after alternative washing with TE buffer and sterile water were suspended in TE buffer. Fungi in solution were then exposed to microwave. The crude extract contained genomic DNA which was extracted and amplified using ITS primers for further identification. Based on sequencing results the identity of known cultures was confirmed, while the unknown cultures were identified as Clitopilus scyphoides (AGUM004, BankIt2098576 MH172163); Ganoderma rasinaceum (AGUM007, BankIt2098576 MH172163); Schizophyllum sp (KONA001 BankIt2098576 MH172164; AGUM011 BankIt2098576 MH172165and AGUM021 BankIt2098576 MH172166respectively), Coprinellus disseminatus (BANG001, BankIt2098576 MH172167) and Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167). The microwave method described for isolating quality DNA of WRF without further purification steps proved a novel method requiring less than ten minutes and minimized the chances of the presence of PCR inhibitors.