DNA isolation from soil samples for cloning in different hosts

• 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
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-003-1528-8

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.