Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects.
A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.
Möhlenhoff
P
P
Geomicrobiology, University of Oldenburg, D-26111 Oldenburg, Germany.
Müller
L
L
Gorbushina
A A
AA
Petersen
K
K
D018441:Biofilms; D004271:DNA, Fungal / Q000032:analysis*; D004275:DNA, Ribosomal / Q000032:analysis*; D004586:Electrophoresis / Q000379:methods*; D005658:Fungi / Q000254:growth & development / Q000302:isolation & purification* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010151:Paintings; D016133:Polymerase Chain Reaction; D012339:RNA, Ribosomal, 28S / Q000235:genetics
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012339:RNA, Ribosomal, 28S