29] Identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning

Abstract

Publisher Summary This chapter discusses the identification of archaea in objects of art by denaturing gradient gel electrophoresis analysis and shotgun cloning. Archaea are regarded as occurring in extreme habitats, such as hypersaline, anaerobic, alkaline, acidic, and hypenhermophilic environments. Further, they have not been previously regarded as important in global ecology. In this “extreme” environment, the formation of biofilms may enable archaea to survive exposure to suboptimal conditions. This chapter describes the development and application of a strategy combining denaturing gradient gel electrophoresis (DGGE) analysis and shotgun cloning for the analysis of archaea in biodeteriorated materials of cultural heritage. The combination of DGGE analysis with the construction of clone libraries to study archaeal communities has already been described here, but instead of DGGE, restriction fragment length polymorphism (RFLP) was used for the screening of clones. RFLP analysis of 16S rDNA has been used for several years as a method for comparing rDNAs. PCR products obtained with universal primers are digested with restriction enzymes. This method has been used for the study of communities, where the potentially large number of fragments can be resolved by using polyacrylamide gels to produce a communityspecific pattern. The combination of the methods described offers advantages over other methods that have been previously described: the parallel application of DGGE analysis and clone libraries combines the potential of both methods and overcomes their limitations; and the screening of the clones by DGGE analysis makes the procedure easier and faster than other molecular methods such as RFLP analysis.