Studies of the Intestinal Microflora by Traditional, Functional and Molecular Techniques
Elisabeth Norin, Cecilia Jernberg, Hans-Olof Nilsson and Lars Engstrand
from: Lactobacillus Molecular Biology: From Genomics to Probiotics (Edited by: Åsa Ljungh and Torkel Wadström). Caister Academic Press, U.K. (2009)
To resolve the complexity of a bacterial population sophisticated analytical methods and alternative genetic techniques are required. Traditional cultivation, microscopy and determination of fermentation/degradation products are still important. Future studies of the microbe-microbe and the microbe-host cross-talk will strengthen our knowledge about the composition and function of the microflora. We will discuss functional studies including the GAC-MAC concept, and the development of different molecular techniques, which provide microbiologists complementary methods to study complex microbial communities. The most commonly utilized targets are the genes coding for small subunit ribosomal (r) RNAs. The 16S and 23S rRNA genes contain conserved, variable and hypervariable sequences, making them suitable for studies of bacterial evolution and microbial community composition. Denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis are gel-based techniques that separate double-stranded DNA molecules of identical size that vary in sequence and composition. Terminal-Restriction Fragment Length Polymorphism also provides a reproducible fingerprint of complex microbial communities. The recent development of sequence based methods including 454-pyrosequencing has improved sequencing technology and enabled genomics to take a quantum leap in terms of high throughput DNA sequence analysis facilitating completely new types of investigations. Finally, metagenomics, i.e. analyses of microbial populations by the use of genetic/molecular techniques as well as functional analyses, will be discussed read more ...