Molecular Microbial Ecology Manual-Section 1精品分析.doc

Molecular Microbial Ecology Manual Kluwer Academic Publishers 2004 Section 1 - Isolation of Nucleic Acids 1.01 Simplified protocols for the preparation of genomic DNA from bacterial cultures Edward Moore1, Angelika Arnscheidt1, Annette Krüger1, Carsten Str?mpl1 and Margit Mau1 (1) Division of Microbiology, GBF – German National Research Centre for Biotechnology, Mascheroder Weg 1, D38124, Braunschweig, Germany Introduction The development of methodologies for the analysis of microorganisms and microbial ecology, at the molecular level (i. e., nucleic acids, proteins, lipids, and their genes), has progressed phenomenally in recent years. Each methodology has specific advantages and disadvantages, or complications. However, the advances in PCR, cloning, gene probing, sequencing and fingerprinting have enabled techniques exploiting nucleic acids to be utilised extensively for the analysis of microorganisms. Often, such protocols require, firstly, that the nucleic acids are extracted in a form which can be employed for the analyses. This may, in some cases, be more difficult than anticipated initially, since many bacteria are extremely resistant to cell disruption. Typically, these are Gram-positive bacteria (e.g., Mycobacterium spp., Peptococcus spp., Rhodococcus spp., etc.), as well as some Archae (e.g., methanogens), with thick cell walls of polysaccharide or pseudopeptidoglycan, and many species of fungi and algae. General considerations Several protocols have been developed and described for the preparation of genomic DNA from bacteria, beginning with the prototypal method of Marrnur [HYPERLINK "/content/n140586252p88013/fulltext.html#CR16_1-4020-2177-1_1"16], which involved: a) cell disruption by an enzyme-detergent lysis; b) extractions with organic solvents; and c) recovery of the DNA by alcohol precipitation. Subsequent protocols have usually involved some modification of one or more of these general steps. Cell disruption The most difficult and