Use of gene specific universal primers for isolation of DNA sequences encoding laccase enzyme from a wild isolate of Schizophyllum commune

Abstract

Laccase enzymes plays a vital role in innumerable biotechnological applications and hence their large scale production has stimulated considerable research. In the present study, degenerate universal primer pairs were employed to isolate laccase gene from a wild isolate of laccase producing white rot fungi Schizophyllum commune. Primer pairs for this fungal isolate were also designed using laccase specific consensus sequences for fungi. The PCR product of 1000 bp 2 amplicon was visualized on agarose gel using the degenerate primer pair Cu1F/Cu3R. Matching of the sequenced gel purified DNA sequence resembled most of the putative phosphatases involved in cell cycle with 100% identity to S. commune. Here we report the false negative results obtained upon use of laccase specific degenerate primers as well as other primers specific for the genus. These failed to contribute towards isolation of laccase gene even after optimization of PCR conditions in terms of reaction volume, annealing temperature, number of cycles, touchdown PCR and gradient PCR. These findings constitute a practical guide for researchers addressing amplification of transcripts of this biotechnologically important enzyme from the not so well characterized genus of Schizophyllum.