DNA probe method for the detection of specific microorganisms in the soil bacterial community. [Bradyrhizobium japonicum]

Journal Article · Tue Mar 01 04:00:00 EST 1988 · Appl. Environ. Microbiol.; (United States)

OSTI ID:6946302

Holben, W E; Jansson, J K; Chelm, B K; Tiedje, J M

We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridization. Using single-stranded, /sup 32/P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10/sup 4/ cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1..mu..g DNA sample.

• Research Organization: Michigan State Univ., East Lansing (USA)
• OSTI ID: 6946302
• Journal Information: Appl. Environ. Microbiol.; (United States), Journal Name: Appl. Environ. Microbiol.; (United States) Vol. 54:3; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

510101* -- Environment
Terrestrial-- Basic Studies-- Radiometric Techniques-- (-1989)
54 ENVIRONMENTAL SCIENCES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
DNA
DNA SEQUENCING
HYBRIDIZATION
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
POPULATION DENSITY
RADIOASSAY
RADIOISOTOPES
RHIZOBIUM
SOILS
STRUCTURAL CHEMICAL ANALYSIS