Rapid and easy method to extract and preserve DNA from Cryptococcus neoformans and other pathogenic yeasts

Abstract

The mucopolysaccharide capsule of Cryptococcus neoformans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time-saving technique to obtain high yields of good-quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months.

MfLIP1, a gene encoding an extracellular lipase of the lipid-dependent fungus Malassezia furfur

“…Low stringency conditions were used for Southern blots (hybridization at room temperature instead of 42 uC and wash at 40 uC instead of 68 uC) to identify genes similar to MfLIP1 in the genome of M. furfur. For the construction of the genomic library, DNA was extracted with a urea lysis method as described by Gupta et al (2000) and Sansinforiano et al (1998) to obtain sufficiently large fragments. For all sequencing reactions, the BigDye Cycle Sequencing Kit (Applied Biosystems) was used.…”

Obtaining, Storing and Archiving Specimens and Tissue Samples for Use in Molecular Studies

“…For example, Sansinforiano et al [71] used lysis buffer with a high urea concentration for preserving the impermeable mucopolysaccharide capsules of Cryptococcus neoformans and other pathogenic yeasts, and acquired high molecular weight DNA after storage at ambient temperature for up to 6 months. Details of these methods are listed in Protocol 2.…”

Draft Genome Sequence of the Oleaginous Yeast Cryptococcus curvatus ATCC 20509

“…Cells were lysed during mid-log-phase growth by treatment with a buffer containing 8 M urea, 0.5 M NaCl, 20 mM Tris, 20 mM EDTA, and 2% SDS, and the DNA was isolated by phenol-chloroform–isoamyl alcohol extraction as previously described (6). Following isolation, RNA was removed via RNase treatment, and the DNA was ethanol precipitated.…”