DNA isolation from soil samples for cloning in different hosts

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2004-06, Vol.64 (5), p.665-670
Main Authors: Kauffmann, I.M, Schmitt, J, Schmid, R.D
Format: Article
Language:English
Subjects:
Actinobacteria - genetics
Actinomycetes
Arthrobacter
Biological and medical sciences
Biotechnology
Cloning
Cloning, Molecular
Deoxyribonucleic acid
DNA
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
DNA, Bacterial - isolation & purification
extraction
Fundamental and applied biological sciences. Psychology
Gram-negative bacteria
Gram-positive bacteria
Methods
Microorganisms
Polymerase Chain Reaction
Pseudomonas
Rhodococcus
RNA, Ribosomal, 23S - chemistry
RNA, Ribosomal, 23S - genetics
soil bacteria
soil biology
Soil Microbiology
Specimen Handling - methods
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Summary:Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-003-1528-8