An advanced molecular strategy to identify bacterial communities on art objects

The application of culture-independent techniques based on molecular biolog ical methods, especially on the PCR amplification of 16S rRNA genes, attemp ts to overcome same shortcomings of conventional cultivation methods and re veals far more complex bacterial communities on art objects than can be sho wn by cultivation methods. One of the major challenges of investigating mic robial growth on art objects by molecular means is the extraction of DNA, d ue to small sample amounts and PCR inhibitors. In the present study, we int roduce a DNA extraction protocol, which allowed the extraction of PCR-ampli fiable DNA from samples derived from lime wall paintings and loamy soil und erground. The DNA extracts were used to amplify 16S ribosomal fragments, wh ich were subsequently analyzed by denaturing gradient gel electrophoresis ( DGGE). In parallel with the DGGE analysis, clone libraries containing PCR f ragments of the ribosomal gene were constructed and clones were screened by DGGE. Clone libraries allow the inclusion of the entire 16S rDNA sequence in the phylogenetic analyses of microorganisms, providing a more reliable p hylogenetic identification of microorganisms than is obtained from sequence analyses of excised and directly sequenced DGGE bands. (C) 2001 Elsevier S cience B.V. All rights reserved.