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0099-2240/91/041070-05$02.00/0
Copyright 1991, American Society for Microbiology
A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous
microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate
was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency
(>90%), the yields of DNA were high (38 and 12 ,ug/g [wet weight] from sediments and soil, respectively). This
method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an
Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required,
which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h)
period.
Isolation of bacterial DNA from natural environments has inated with polynuclear aromatic hydrocarbons (6.5 lug/g
become a useful tool with which to study the ecological [wet weight]) and mercury (0.07 ,ug/g), and the ORT sedi-
functions of certain characterized genes that encode impor- ments were contaminated with mercury (up to 80 ,ug/g) and
tant metabolic pathways (25), allow tracking of genetically other heavy metals (Global Geochemistry Corp., Canoga
engineered organisms (8, 10), and reveal bacterial DNA Park, Calif.; Oak Ridge National Laboratory, Oak Ridge,
diversity (21, 22) in microbial ecosystems. The application of Tenn.). The heterotrophic bacterial population in both sam-
DNA extraction methods to environmental samples can ples was enumerated on plate count agar (Difco, Detroit,
obviate the need for cell cultivation, since cell cultivation Mich.). The indigenous mercury-resistant strains were re-
has the disadvantage of obtaining only a very small propor- covered on plate count agar amended with 25 ,ug of Hg2+ as
tion of the total microbial community. DNA extraction HgCI2 per ml. Naphthalene degraders were selectively cul-
methods can also help researchers to understand the occur- tivated on a minimal medium saturated with naphthalene
rence of particular bacterial genes in situ through applica- vapor (18). Sterile soil and sediment samples were produced
tions of nucleic acid technology. by autoclaving at 121C for 30 min. All soil and sediment
Several techniques have been described for direct detec- quantities are expressed as wet weight unless otherwise
tion and extraction of DNA from aquatic environments (3, 6, indicated. The percentage of water was determined to be
13-15, 19), but there are only a few reports related to 51% in the ORT sediments and 10% in the SC soil (2).
extraction of DNA from soil and sediments (8, 12, 23). Two Organisms. Two strains (V55 and VNM43) isolated from
current methods used to obtain microbial DNA from soils or SC soil were used as seed organisms. They were identified as
sediments are (i) bacterial cell extraction followed by cell Pseudomonas luteola and Pseudomonas putida, respec-
lysis and DNA recovery (8, 23) and (ii) direct extraction by tively, by the API Rapid NFT? kits (Analytab Products,
alkaline lysis (12). The direct extraction method produces a Plainview, N.Y.). In the remainder of this paper these
better DNA yield than the cell extraction method (20). strains are referred to as P. luteola V55 and P. putida
However, both approaches share three disadvantages, i.e.,
they (i) are lengthy and laborious, (ii) require a relatively VNM43. Both strains have the ability to degrade naphtha-
large sample size (50 to 100 g), and (iii) produce low yields of lene and are resistant to mercuric ions. They were grown to
DNA. late exponential phase in 50% plate count broth (Difco) with
In this study we have developed a rapid method for direct 25 pLg of Hg2+ per ml and were washed once with 10 mM
extraction of DNA from soil and sediments by applying a phosphate buffer (pH 7.0) and resuspended in phosphate
freeze-thaw approach. This method overcomes the draw- buffer before inoculation into the soils or sediments. The
backs of the previous techniques. The high yield from a inoculated soils and sediments were shaken at 100 rpm
small sample size (1 g [wet weight]) means that the method (Orbit Shaker; Lab-Line Instrument, Inc., Melrose Park,
described in this report can be used efficiently to study in situ Ill.) at 23C for 30 min prior to DNA extraction.
gene occurrence and to detect the distribution of genetically Direct extraction of DNA. SC soil or ORT sediment sam-
engineered microorganisms in the environment. ples (1 g) were mixed with 2 ml of 120 mM sodium phosphate
buffer (pH 8.0) by shaking at 150 rpm for 15 min. The slurry
was pelleted by centrifugation at 6,000 x g for 10 min. The
MATERIALS AND METHODS
pellet was washed again with phosphate buffer, resuspended
Soil characterization. Subsurface soil samples from a man- in 2 ml of lysis solution (0.15 M NaCl, 0.1 M Na2EDTA [pH
ufactured gas site in Southern California (SC) and sediment 8.0]) containing 15 mg of lysozyme/ml, and incubated in a
samples from a settling pond in Oak Ridge, Tenn. (ORT), 37C water bath for 2 h with agitation at 20- to 30-min
were used for DNA extraction. The SC soils were contam- intervals, and then 2 ml of 0.1 M NaCl-0.5 M Tris-HCl (pH
8.0)-10% sodium dodecyl sulfate was added. Three cycles of
freezing in a -70C dry ice-ethanol bath and thawing in a
*
Corresponding author. 65C water bath were conducted to release DNA from the
1070
VOL. 57, 1991 DIRECT DNA EXTRACTION FROM SOIL AND SEDIMENTS 1071
FIG. 5. Restriction enzyme digestion patterns of DNA extracted from ORT sediments. (A) Ethidium bromide-stained agarose gel. (B)
Autoradiogram of merA hybridization signals from Southern blot of gel in panel A. Lanes 2 to 4 and 5 to 7 each contained 1/30 of DNA extracts
from unseeded and seeded samples, respectively. Lanes 8 to 10 and 11 to 13 contained 1/20 of Elutip-d column-purified DNA from unseeded
and seeded samples, respectively. Lanes: 1, HindIll-cut lambda phage molecular weight marker with ladder of 23.1, 9.4, 6.5, 4.4, 2.3, and
2.0 kb; 2 and 8, unseeded and uncut; 3 and 9, unseeded and EcoRI cut; 4 and 10, unseeded and BamHI cut; 5 and 11, seeded and uncut; 6
and 12, seeded and EcoRI cut; 7 and 13, seeded and BamHI cut.
SC soil (Fig. 2B, lane 4), but the direct application of DNA versity of California, Riverside, Department of Soil and
to membranes by the slot blot hybridization technique Environmental Sciences; Oak Ridge National Laboratory).
showed that crude DNA extracted from the same soil did Figure 3A shows the merA hybridization signals from the
contain merA genes (Fig. 3A, slot 1). The discrepancy crude DNA extracted from sterile SC soil seeded with
between these two observations was due to incomplete different quantities of P. putida VNM43. Signals were de-
transfer of DNA from the gel to the filters during Southern tected in a 50% aliquot of total DNA extract when as few as
blotting. Therefore, the existence of mercury-resistant bac- 1.1 x 104 cells per g (wet weight) were inoculated into soil,
terial strains such as P. putida VNM43 and P. luteola V55 in indicating that the sensitivity was 5.5 x 103 cells per g. A
the polynuclear aromatic hydrocarbon-contaminated SC comparative sensitivity of 4.3 x 104 cells per g (dry weight)
soils was further supported by the gene probe data. The was reported for the cell extraction method (8). Figure 3B
DNA extracts from seeded sterile (Fig. 2B, lane 3) and illustrates hybridization signals of putative merA genes ex-
nonsterile (lane 5) soils revealed smaller DNA fragments tracted from nonsterile unseeded and seeded ORT sediments
(between 1 and 20 kb) containing homologous merA se- and nonsterile seeded SC soils. As the extracted DNA was
quences. This shearing is probably due to the grinding effect normalized at 1 ,ug, DNA from seeded samples (Fig. 3B,
of the sand particles in SC soil during the extraction proce- slots a6 and b2) demonstrated higher merA hybridization
dure because pure-culture controls (lane 6 and 7) did not signals (7,049 and 24,477 cpm) than that from unseeded
exhibit this effect. The two soils differ in cation exchange samples (800 cpm; slot a2). As would be expected, the signal
capacity and particle size distribution, with SC soils having for the SC soils inoculated with a higher cell density (slot b2)
low cation exchange capacity (9 mEq/100 g) and high sand had a higher-intensity signal of merA sequences than did
concentration (18.4%), and ORT sediments having high ORT sediments inoculated with a lower cell density (slot a6).
cation exchange capacity and low sand concentration (Uni- The pure-culture DNA control showed the highest signal
1074 TSAI AND OLSON APPL. ENVIRON. MICROBIOL.
(28,648 cpm) in 1 ,ug of DNA extract. The radioactivity orange epifluorescent technique for counting bacteria in natural
counts for hybridized DNA were lower from soil and sedi- waters. Trans. Am. Microsc. Soc. 92:416-421.
ment samples than from pure cultures, indicating that DNA 6. Fuhrman, J. A., D. E. Comeau, A. Hagstrom, and A. M. Chan.
had absorbed into the soil particles or that more nonhomol- 1988. Extraction from natural planktonic microorganisms of
ogous merA sequences were present in the former samples. DNA suitable for molecular biological studies. Appl. Environ.
The detection limit of homologous merA DNA was 10 ng of Microbiol. 54:1426-1429.
7. Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. Use of
total extracted DNA from both P. putida VNM43 pure Nuclepore filters for counting bacteria by fluorescence micros-
culture and P. putida VNM43-seeded SC soils (Fig. 3B). The copy microscopy. Appl. Environ. Microbiol. 33:1225-1228.
merA hybridization signals were found in 1 ,ug of total DNA 8. Holben, W. E., J. K. Jansson, B. K. Chelm, and J. M. Tiedje.
extracted from nonsterile unseeded ORT sediments. In 1988. DNA probe method for the detection of specific microor-
comparison, Ogram et al. (12) required 1.2 ,ug of total DNA ganisms in the soil bacterial community. Appl. Environ. Micro-
to detect nif nitrogen fixation genes from sediments other biol. 54:703-711.
than those used in the present study. In the present study 9. Ingraham, J. L., 0. Maaloe, and F. C. Neidhardt. 1983. Growth
there is no indication that humic materials in the crude of the bacterial cell, p. 1-48. Sinauer Associates, Inc., Sunder-
extract affect DNA-DNA hybridization (Fig. 3). land, Mass.
Figure 4 shows the restriction patterns of DNA extracts 10. Jansson, J. K., W. E. Holben, and J. M. Tiedje. 1989. Detection
from a pure bacterial culture and from a seeded SC soil. The in soil of a deletion in an engineered DNA sequence by using
DNA probes. Appl. Environ. Microbiol. 55:3022-3025.
unpurified DNA extract was clearly digested by Hindlll but 11. Medveczky, P., C.-W. Chang, C. Oste, and C. Mulder. 1987.
only slowly digested by EcoRI. The application of restriction Rapid vacuum driven transfer of DNA and RNA from gels to
enzyme analyses to directly extracted DNA may allow more solid supports. BioTechniques 5:242-246.
precise studies of in situ gene rearrangements or amplifica- 12. Ogram, A., G. S. Sayler, and T. Barkay. 1987. The extraction
tion. The impurities in ORT sediments did affect the restric- and purification of microbial DNA from sediments. J. Microb.
tion enzyme digestion (Fig. 5). EcoRI and BamHI did not Methods 7:57-66.
digest DNA extracts from either unseeded or seeded sam- 13. Paul, J. H., and D. J. Carlson. 1984. Genetic material in the
ples within the 5-h incubation period. The DNA yield marine environment: implications for bacterial DNA. Limnol.
suffered a 40% loss during the Elutip-d column purification Oceanogr. 29:1091-1097.
step, and the purified DNA (A260/A280 = 1.8) was cut by 14. Paul, J. H., W. H. Jeffrey, and M. DeFlaun. 1985. Particulate
EcoRI but not by BamHI. Therefore, for studying in situ DNA in subtropical oceanic and estuarine planktonic environ-
ments. Mar. Biol. 90:95-101.
gene transfer the purification step might be needed depend- 15. Paul, J. H., and B. Myers. 1982. Fluorometric determination of
ing on the soil type, but it is not necessary for determination DNA in aquatic microorganisms by use of Hoechst 33258. Appl.
of gene occurrence. Environ. Microbiol. 43:1393-1399.
In conclusion, this rapid method for the direct extraction 16. Roszak, D. B., and R. R. Colwell. 1987. Survival strategies of
of DNA needs only 1 g of soil or sediments and can detect bacteria in the natural environment. Microbiol. Rev. 51:365-
the presence of a target gene, such as merA, from a minimum 379.
of 5,000 bacterial cells. It is simple and can be completed in 17. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
7 h. In addition, because of the small sample size (1 g), a cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
large number of soil or sediment samples can be analyzed at Laboratory, Cold Spring Harbor, N.Y.
one time. Finally, the high yield in combination with the high 18. Sayler, G. S., M. S. Shields, E. T. Tedford, A. Breen, S. W.
quality of DNA will enable microbial ecologists to study Hooper, K. M. Sirotkin, and J. W. Davis. 1985. Application of
DNA-DNA colony hybridization to the detection of catabolic
DNA from environmental samples in a more detailed fashion genotypes in environmental samples. Appl. Environ. Microbiol.
by molecular biology techniques. 49:1295-1303.
19. Somerville, C. C., I. T. Knight, W. L. Straube, and R. R.
ACKNOWLEDGMENTS Colwell. 1989. Simple, rapid method for direct isolation of
nucleic acids from aquatic environments. Appl. Environ. Mi-
This study was funded by grant 8000-25 provided by the Electric crobiol. 55:548-554.
Power Research Institute. 20. Steffan, R. J., J. Goks0yr, A. K. Bej, and R. M. Atlas. 1988.
We are grateful to Marie Park for developing the autoradiograms. Recovery of DNA from soils and sediments. Appl. Environ.
We also thank 0. A. Ogunseitan, P. A. Rochelle, and C. C. Tebbe Microbiol. 54:2908-2915.
for valuable discussions and suggestions and R. Turner for provision 21. Torsvik, V., J. Goks0yr, and F. L. Daae. 1990. High diversity in
and analysis of ORT sediments. DNA of soil bacteria. Appl. Environ. Microbiol. 56:782-787.
22. Torsvik, V., K. Salte, R. S0rheim, and J. Goks0yr. 1990.
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