High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction.
Soil is a complex environment considered as one of the main reservoirs of microbial diversity. However, the inability to cultivate most soil bacteria hampered fundamental attempts to determine the diversity of the prokaryotic world and limited its industrial exploitation. In the last 20 years, new methods have been developed to overcome these limitations based on the direct extraction of DNA from bacteria in their natural environment. In addition to fundamental research, the cloning of the extracted DNA for the development of metagenomic DNA clone libraries offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria in surrogate bacterial hosts. However, such objectives require adapting DNA extraction methods and cloning strategies in order that entire gene clusters encoding biosynthetic pathway for secondary metabolites can be cloned. In this paper, we report that the size of DNA fragments extracted from soil varied in a range between less than 100 kb and more than 400 kb depending on the soil. The relatively limited size of DNA fragments extracted from some soil was not only due to mechanical, chemical or enzymatic shearing of the DNA during the extraction process but partly to the microbial growth status. Stimulating bacteria in situ by providing nutrients to the soil improved the size of extracted DNA, but it modified the bacterial community structure.
Bertrand
Hélène
H
Ecologie Microbienne, UMR CNRS 5557, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, France. h.bertrand@libragen.com
Poly
Franck
F
Van
Van Tran
VT
Lombard
Nathalie
N
Nalin
Renaud
R
Vogel
Timothy M
TM
Simonet
Pascal
P
D001419:Bacteria / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field; D016680:Genome, Bacterial; D015698:Genomic Library; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D025341:Principal Component Analysis; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012988:Soil Microbiology
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S