Chapter 13 - Nucleic Acid–Based Methods of Analysis

Abstract

This chapter discusses the theories and applications of various nucleic acid-based approaches such as gene probes, microarrays, conventional and real-time polymerase chain reaction (PCR), whole genome amplification, sequencing, comparative genomics, and metagenomics. Gene probes are single-stranded DNA, and can be used to identify a particular nucleic acid sequence within an environmental sample through colony hybridization, Southern and Northern hybridization (used for DNA and RNA respectively), Fluorescent in situ hybridization (FISH), microarrays, and phyloarrays. PCR is extremely sensitive and is able to detect a single target molecule in some cases. The use of PCR based techniques—such as reverse transcriptase–PCR (RT–PCR), introduced integrated cell culture-PCR (ICC-PCR), arbitrarily primed-PCR (AP-PCR), and Real-Time PCR has enabled the researchers to detect specific nucleic acid sequences in isolates as well in environmental samples. Recombinant DNA technology or DNA cloning has been widely used to examine the genetics of bacterial populations. Metagenomics is another nucleic acid-based approach that allows analysis of multiple genes and screening of large environmental fragments for functional activities. Several computer software sequence analysis programs and tools—such as Basic Local Alignment Sequence Tool (BLAST) or Clustal W—are available for sequences searches, and comparative genomics in such cases allows the comparison of subsets of genes or whole genomes of interest. Restriction fragment length polymorphism (RFLP) and its modifications such as amplified fragment length polymorphisms (AFLP) and terminal-restriction fragment length polymorphism (T-RFLP) can characterize or differentiate the whole genomes or specific PCR products from bacterial isolates. Denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE) has been employed to assess the microbial diversity. Plasmid analysis has been used to detect specific bacteria or genes such as in Bacillus anthracis .