This chapter deals with both classical and modern molecular methods that can be useful for the identification of microorganisms, elucidation and comparison of microbial communities, and investigation of their diversity and functions. The most important and critical steps necessary for all molecular methods is DNA isolation from microbial communities and environmental samples; these are discussed in the first part. The second part provides an overview over DNA polymerase chain reaction (PCR) amplification and DNA sequencing methods. Protocols and analysis software as well as potential pitfalls associated with application of these methods are discussed. Community fingerprinting analyses that can be used to compare multiple microbial communities are discussed in the third part. This part focuses on Denaturing Gradient Gel Electrophoresis (DGGE), Terminal Restriction Fragment Length Polymorphism (T-RFLP) and Automated rRNA Intergenic Spacer Analysis (ARISA) methods. In addition, classical and next-generation metagenomics methods are presented. These are limited to bacterial artificial chromosome and Fosmid libraries and Sanger and next-generation 454 sequencing, as these methods are currently the most frequently used in research. Isolation of nucleic acids: This chapter discusses, the most important and critical steps necessary for all molecular methods is DNA isolation from microbial communities and environmental samples. Nucleic acid isolation methods generally include three steps: cell lysis, removal of unwanted substances, and a final step of DNA purification and recovery. The first critical step is the cell lysis, which can be achieved by enzymatic or mechanical procedures. Removal of proteins, polysaccharides and other unwanted substances is likewise important to avoid their interference in subsequent analyses. Phenol-chloroform-isoamyl alcohol is commonly used to recover DNA, since it separates nucleic acids into an aqueous phase and precipitates proteins and other cell components into the organic phase. The last step is the purification of nucleic acids, which may dramatically reduce the efficiency of the recovery. The method used should, therefore, result in a compromise between yield and purity. PCR and DNA sequencing: Application of DNA polymerase chain reaction (PCR) amplification and DNA sequencing in order to amplify and determine the DNA of microbial marker genes, allows the understanding of microbial diversity with an ever-increasing resolution and accuracy. This chapter provides an overview over DNA PCR amplification and DNA sequencing methods. It discusses protocols and analysis software as well as potential pitfalls associated with application of these methods. While PCR is a molecular technique to amplify virtually unlimited amounts of a particular DNA sequence from only a few DNA copies of input material, DNA sequencing refers to the actual determination of the sequence of nucleotides of a strand of DNA (or RNA). In phylogeny-based analyses, taxa are operationally defined by relatedness to another sequence in a phylogenetic tree. Similarly to representative operational taxonomic units (OTU) sequences, all sequenced 16S fragments can be assigned to a species or taxon in a database. Community comparison by genetic fingerprinting techniques: This chapter discusses community fingerprinting analyses that can be used to compare multiple microbial communities. It focuses on denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and automated rRNA intergenic spacer analysis (ARISA) methods. DGGE discriminates DNA fragments having different sequences and different AT/GC content. Terminal restriction fragment length polymorphism (T-RFLP) involves the extraction of DNA from environmental samples. The ARISA technique uses the highly variable internal transcribed (ITS) regions of rDNA. All fingerprinting techniques have the advantage of being able to process many samples at the same time and to compare bacterial communities among different samples or in a single sample after certain treatments. DGGE, T-RFLP and ARISA share similar steps but require different materials and equipment. The three methods involve (i) sampling of the biofilms; (ii) DNA extraction and quantification; and (iii) PCR using specific primers. Metagenomics: This chapter focuses classical and next-generation metagenomics methods. These are limited to bacterial artificial chromosome (BAC) and Fosmid libraries and Sanger and next-generation 454 sequencing, as these methods are currently the most frequently used in research. The chapter discusses the special handling of deoxyribonucleic acid (DNA) needed to construct BAC and Fosmid libraries from marine water samples. It also briefly addresses the related topics of library archiving, databasing, and screening. The chapter provides a high-level overview of the special handling methods required to prepare DNA for BAC library construction. No special handling is needed for Fosmid library construction.
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