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Rapid DNA extraction protocol from soil for

polymerase chain reaction-mediated
amplification. J Appl Bacteriol 74: 78-85


Impact Factor: 2.48 DOI: 10.1111/j.1365-2672.1993.tb02999.x


221 694


Kornelia Smalla Neil Cresswell

Julius Khn-Institut Manchester Metropolitan University


Leda C Mendona-Hagler Jan Dirk van Elsas

Federal University of Rio de Janeiro University of Groningen


Available from: Leda C Mendona-Hagler

Retrieved on: 12 October 2015
Journal of Applied Bacteriology 1993, 74, 70-05

Rapid DNA extraction protocol from soil for polymerase chain

reaction-mediated amplification
K. Smalla, N. Cresswell', L.C. Mendonca-Hagler', A. Wolters3 and J.D. van EIsas3
Institute for Biochemistry and Plant Virology, Biologische Bundesanstalt, Braunschweig, Germany, Department '
of Biological Sciences, University of Warwick, Coventry, UK, Institute of Microbiology, Federal University of Rio
de Janeiro, llha do Fundao, Rio de Janeiro, Brazil and Institute for Soil Fertility Research, Wageningen Branch,
The Netherlands
4197/04/92: accepted 24 July 1992

K . S M A L L A , N . C R E S S W E L L , L . C . M E N D O N C A - H A G L E R . A. W O L T E R S A N D J . D . V A N E L S A S . 1993. A
simple and rapid method of DNA extraction from soil was developed and DNA was made
suitable for subsequent efficient amplification by the polymerase chain reaction (PCR). Key
features of the extraction and purification were cold lysozyme- and SDS-assisted lysis with
either freezing-thawing or bead beating, cold phenol extraction of the resulting soil
suspension, CsCl and KAc precipitation and, finally, spermine-HC1 or glass milk purification
of DNA. Crude DNA preparations contained 4-20 p g DNA per g of soil extracted, and at
least 50% of this was recovered in the final purified DNA preparations. T h e resulting DNA
was pure enough to be restricted by various enzymes, and was amplifiable at concentrations
of up to 20 ng of soil-derived DNA per 50 p1 reaction mix.
Amplification of a 683 bp target sequence, put, was performed with different Taq DNA
polymerases. Application of the protocol enabled us to detect target DNA derived from
roughly lo3 introduced Pseudomonaspuorescens (RP4 : : put) cfu per g of soil. The fate of an
introduced population in the soil could be followed to this limit with PCR-assisted detection
of target DNA. In addition, target DNA was detected in soil 5 months after release, when the
introduced organism was no longer detectable on selective agar plates.
The extraction and purification protocol applied to various different soil types resulted in
DNA of sufficient purity to permit amplification by PCR.

INTRODUCTION hybridization-based detection techniques, which use either

There is a need to develop sensitive methods for the detec- previous culturing of cells (e.g. colony hybridization) or
tion of micro-organisms in the environment, e.g. soil, given direct extraction of DNA (Ogram et al. 1987; Holben et a/.
the putative biohazards of released genetically modified 1988), offer obvious advantages, such as the possibility of
micro-organisms (GMOs). Microbial populations and their specific detection of micro-organisms, e.g. of living, non-
dynamics have traditionally been described with either culturable, ultramicro- (dwarf) or dead cells, and of genes,
cultivation-based techniques, e.g. by selective plate counts, by selecting an adequate probe.
or specific direct counts, e.g. vra immunofluorescence. Both The polymerase chain reaction (PCR), developed by
methods suffer from drawbacks. Plate counts monitor only Saiki et al. (1988) for the detection of target sequences in
culturable organisms, whereas immunofluorescence is often clinical settings, should lower the limit of detection of genes
plagued by lack of specificity and possibly also enumerates in the environment by enhancing the target copy number
dead cells. prior to analysis.
The application in microbial ecology of detection tech- T h e application of molecular techniques directly to soil,
niques based on DNA-DNA hybridization has increased however, has been hampered by problems caused by the
recently (Sayler et al. 1985; Ford and Olson 1988; Holben presence of impurities in soil-derived DNA samples, even
el a/. 1988; Trevors and van Elsas 1989). Such though successes in detection have been reported (Steffan
Correspondence to K Smalla, Institute for Biochemistry and Plant and Atlas 1988; Cresswell et al. 1991; Pillai et al. 1991;
Virology, Biologisrhe Bundesanstalt, Messeweg 11/12, Braunsrhweig, Selenska and Klingmuller 1991a, b ; van Elsas ef ul. 199la;
Germany Tsai and Olson 1992).

Tracking the fate of introduced GMOs and their genetic basic protocol used 5 g soil samples. Samples were sus-
material in soil has been the interest of the different labor- pended in 5 ml of 0.12 mol I - ' phosphate buffer (pH 8.0)
atories involved in the development of the simple and rapid supplemented with freshly added lysozyme at a final con-
DNA extraction/PCR protocol described here (Cresswell et centration of 5 mg ml-'. T h e resulting suspensions were
al. 1991 ; Smalla et al. 1991; van Elsas et at. 1991a, b; shaken for 5 rnin at room temperature and incubated at 4C
Waalwijk et al. 1991). Pseudomonas fluorescens R2f, carrying (incidentally 37C) for 30 min. After incubation 600 p1 of
the RP4 : : pat element, was used as a model as PCR and 20% sodium dodecyl sulphate (SDS) was added and sus-
tracking systems were available (Smit et al. 1991; van Elsas pensions were kept on ice. T o complete cell lysis, 2.5 g of
et al. 1991a). acid-washed glass beads ( 0 . 1 7 4 1 8 mm diameter) were
added and the suspensions were shaken in a Braun's
homogenizer three times for 90 s at the highest speed. The
MATERIALS AND METHODS resulting suspensions were mixed well with equal volumes
of cold phenol equilibrated with 0.5 mol I - ' Tris (final pH
Organisms and plasmid 7.8; Sambrook et al. 1989), after which they were centri-
PseudomonasPuorescens R2f, a grassland isolate (van Elsas et fuged for 15 min at 12000 g at room temperature. After
al. 1988) containing plasmid RP4 : : pat, was used. Plasmid transferring the aqueous phases to new tubes the pellets
RP4 : : pat was constructed by inserting the 683-bp potato were treated with 5 ml of 0.12 mol 1-' phosphate buffer
'patatin' cDNA sequence, pat, from pPATB2, into the (pH 8.0) and re-extracted with Tris-equilibrated phenol.
unique Hind111 site of RP4 (van Elsas and Trevors 1990; The resulting aqueous phases were combined with the first
van Elsas et al. 1991a). extracts, and these mixtures were extracted twice with
Pseudomonas Juorescens R2f (RP4 : : p a t ) was cultured in equal volumes of chloroform-isoamylalcohol (24 : 1, v/v).
LB broth supplemented with 50 p g ml-' tetracycline at The aqueous phases recovered from these extractions were
28"C, overnight, in a gyratory shaker. For introduction into ethanol-precipitated and the DNA pellets were washed
soil cells were washed twice in sterile demineralized water. with 70% ethanol. Dried pellets were resuspended in Tris-
EDTA ( T E ; Sambrook et al. 1989), giving a final volume
of 1000 p l .
Soil and soil inoculation
Ede loamy sand (van Elsas et al. 1986; Heijnen et al. 1992),
Purification steps
obtained from a field microplot at Ital, Wageningen, was
inoculated with Ps. jluorescens R2f (RP4 : : pat) in accord- The crude DNA preparations were subjected to several
ance with van Elsas et al. (1991a). Population densities sequential purification steps. Caesium chloride (CsCI) and
applied in the different experiments varied from 10 to lo7 potassium acetate (KAc) precipitation steps were first used
colony-forming units (cfu) per g of dry soil. to selectively remove proteins, RNA and humic substances.
Flevo silt loam (described by van Elsas et al. 1986) and Before and after each purification step, DNA samples were
different soil samples of undefined texture obtained from used in PCR and restriction assays.
Schonebeck, Germany, were also used in some DNA In the first purification step, CsCl (1 g) was added to
extraction and PCR experiments. 1000 pl of crude DNA extract. T h e resulting suspensions
were mixed well, kept for 3 h at room temperature, and
centrifuged for 20 rnin at 14000 g at room temperature.
Enumeration of inoculant cells from soil
After recovering the supernatant fluids (about 1 ml), 4 ml
Pseudomonaspuorescens R2f (RP4 : : pat) cfu numbers were of demineralized water and 3 ml of isopropanol were added.
determined by selective plating as described by van Elsas et The mixtures were kept at room temperature for 15 min
al. (1991a). and then centrifuged for 15 min at 14000 g. The resulting
T o obtain a total cell count of Ps. Juorescens R2f pellets were dissolved in 1000 p1 of T E buffer.
(RP4 : . pat), immunofluorescence of soil samples was In the next step, the CsC1-treated DNA preparations
applied ; specific FITC-labelled anti-R2f antibodies, as (1000 p l ) were mixed with 200 pl of 8 mol I - ' KAc solu-
described by Trevors et al. (1990), were used. tion and kept at room temperature for 15 min. Suspensions
were centrifuged for 15 rnin at 14 000 g and the supernatant
fluids recovered were treated with 600 pl of isopropanol.
Extraction of DNA from soil
After mixing well and keeping at room temperature for 5
In analogy to Ogram et al. (1987) we developed a direct min, the suspensions were centrifuged (14000 g, 15 min)
DNA extraction protocol, given its high efficiency in recov- and the resulting DNA pellets were washed extensively
ering bacterial DNA from soil (Steffan et al. 1988). T h e with 70% ethanol and dissolved in 1000 pl of T E buffer.
80 K. S M A L L A E T A L .

Extraction lysozyme treatment of 5 g soil Fig. 1 Outline of extraction and

L purification protocol. SDS, Sodium
addition of SDS (1.25%) and dodecyl sulphate; PCR, polymerase chain
bead-beating reaction
phenol extraction ( 2 x )
chloroform extraction (2 x )
ethanol precipitation
resolved DNA pellet -crude extract
Purification of the DNA crude extract
CsCl precipitation (1 g mi-') +centrifugation -+ isopropanol precipitation
(0.6~ 0 1 %o)f DNA in the supernatant fluid
KAc precipitation (1.3 mol I-') +centrifugation + isopropanol precipitation
(0.6 vol'X) of DNA in the supernatant fluid
PCR glass milk spermine

When necessary, final purification was performed by two Slot blot hybridization
alternative methods. The first was based on the capacity of
DNA preparations (100-200 pl) were applied, both directly
spermine-HC1 to precipitate selectively DNA in a low-salt
and in serial twofold dilution, to a nylon membrane
environment (Hopwood et al. 1985); 25 pl of spermine-HC1
(Hybond, Amersham RPN 303N, or Gene-Screen plus)
(pH 84L8.5) at 100 mmol 1-' was added to 500 pI KAc-
treated DNA preparations. The mixtures were kept at room
temperature for 5 min, after which the DNA was precipi-
tated by centrifugation (3 min, room temperature, 14000 1 2 3 4 5 6 7 8

g). The resulting pellets were washed twice with 1 ml of

ice-cold 70Y0 ethanol supplemented with 100 p1 of 3 mol
1 sodium acetate (NaAc) and 10 pl of 1 mol 1- MgCl, ,
~ '
followed by dissolution in 500 p1 of T E buffer. As an alter-
native method we used DNA purification with glass milk
(Geneclean 11; Bio 101 Inc). The procedure was performed
according to the manufacturer's instructions. For additional
purity, the spermine- or glass milk-purified DNA was again
treated with spermine or glass milk. An outline of the pro-
tocol is shown in Fig. 1.

Determination of DNA recovery rate

Initially, DNA recovery was estimated from the intensity of
ethidium bromide-stained DNA bands in agarose gels and
comparison with controls of known concentration. In later
work Polaroid pictures of DNA bands in ethidium
Fig. 2 DNA (10 pl sample per well) obtained from two unseeded
bromide-stained agarose gels were scanned with a Bio-
soils using the modified procedure. Lanes: 1-3, DNA extracted
Image scanner (Millipore). For quantification it was neces- from Flevo silt loam ; 4-6, DNA extracted from Ede loamy sand ;
sary to have an internal standard of known DNA content 1 and 4, crude preparations; 2 and 5, after KAc precipitation; 3
on the gel. For calculation, the integrated optical densities and 6, after glass milk purification; 7, pUC19 D N A ; 8, I-kb
were used. The Gibco-BRL 1-kb ladder (No. 52M615SA; ladder (see Materials and Methods, arrow indicates I-kb hand).
molecular size range 0.3-12.2 kb) was used to determine Note the 'cloud' caused by humic substances in the crude DNA
sizes of fragments on agarose gels (Figs 2 and 3). from the Ede loamy sand soil

I 2 3 4 5 6 7 8 9 10 II 12 13 Restriction analysis was performed on soil DNA extracts

of different degrees of purification, with HindIII
(Boehringer 656321), EcoRl (Boehringer 703737), BamHl
(Boehringer 567604), PvuII (Boehringer 642690) and Bgfl
(Boehringer 404101). The reaction mixtures (20 p l ) con-
tained the recommended buffer, 5 p1 of soil-derived DNA
and 5-10 units of the enzyme, and were incubated at 37C
for at least 3 h. The same mixtures were also incubated
with an additional 200 ng of the cloning vector pUC19 to
follow more readily the inhibition of different restriction
enzymes by the soil DNA extract. After incubation the
digests were precipitated with ethanol. T h e washed pellets
were resuspended in 10 p1 of T E and analysed after agarose
gel electrophoresis and ethidium bromide staining.
The pat probe was prepared according to van Elsas et al.
(1991a). Nick translation was used to label probes for initial
Fig. 3 Restriction by HindIII of soil DNA with and without
hybridizations, following the protocol of Sambrook et al.
added pUC19 DNA at different purification steps. Lanes: 1 and
2, crude; 3 and 4, after KAc precipitation; 5 and 6, after glass (1989). As a non-radioactive alternative for nick translation,
milk purification; 7 and 8, after spermine-HCI purification; 9 and labelling was performed with the digoxigenin (Dig) label-
10, after spermine purification followed by glass milk; 11, pUC19 ling kit (Boehringer 1175033). Prehybridization and hybrid-
digestion product (2.7 kb); 12, pUC19 undigested; 13, I-kb ization were done at 68C according to the Boehringer
ladder (see Materials and Methods, arrow indicates 1-kb band). protocol. After washing at high stringency (68"C,
Uneven numbers, no pUC19 added; even numbers, pUC19 added 0.1 x SSC), chemiluminescent detection was performed
with the Dig luminescent detection kit (Boehringer 136514)
as described by the manufacturer.
held in a Minifold-1 apparatus (Schleicher and Schuell,
Dassel). The hybridization procedure was according to
standard protocols (Sambrook et al. 1989), with the 683-bp RESULTS AND D I S C U S S I O N
pat fragment as a probe.
Development of rapid DNA extraction-PCR protocol
Amplification of the pat sequence in soil DNA extracts
In a previous paper (van Elsas et al. 1991a), the pat frag-
Polymerase chain reaction was used to amplify the pat
ment was amplified from Ps.Jluorescens (RP4 : : pat)-inocu-
sequence in different soil extracts. For each enzyme used,
lated Ede loamy sand after purification of crude DNA
i.e. AmpliTaq, the Stoffel fragment (both from Perkin-
extracts from soil via agarose gel electrophoresis and
Elmer/Cetus) and SuperTaq (Sphaero-Q), the reaction
electro-elution of fragments of desired size. In addition,
mixes (50-100 p1) were prepared according to the manufac-
background pat or other DNA amplifiable with pat-specific
turer's instructions. Primers used were as described by van
primers, were shown to be absent from this soil. A dis-
Elsas et al. (1991a). One pl of each DNA preparation was
advantage of this method might be the inherent limits to
added to the reaction mixture following denaturation. The
DNA recovery in gel electrophoresis and the bias in
hot start, as suggested by d'Aquila et al. (1991), was used in
electro-elution towards smaller DNA fragments. We there-
the final protocol. Routinely, 35 cycles (94"C, 1 min; 45 or
fore decided to base a modified protocol on more quantitat-
50C, 1 min; 72"C, 1-2 min; final extension at 72C for 9
ive extraction and purification, omitting gel electrophoresis
min) were run. Polymerase chain reaction products,
as well as time-consuming CsCl gradients and polyvinyl
expected to be about 0.7 and, incidentally, 1.2 kb in size
poly-pyrrolidone adsorption of humics.
(van Elsas et al. 1991a), were analysed on gels after agarose
gel electrophoresis (0.8%) and ethidium bromide staining. Cell lysis and DNA extraction and recovery
In addition, detection was performed on Southern blots
(Sambrook et al. 1989) to nylon membranes (see above) The experiments performed in the development of the pro-
with the pat fragment as a probe. tocol used soil samples seeded with roughly lo6 or lo7 cfu
of Ps.Jluorescens per g, which contained up to 10-fold less
Molecular biology techniques applied to detection cfu at the time of sampling. Cell lysis was originally based
from soil on freezing/thawing of a soil suspension in the presence of
The manual of Sambrook et, al. (1989) was used for all lysozyme and SDS, followed by rapid extraction of the soil
molecular work. lysate with cold phenol. In later work lysis was improved
82 K . S M A L L A E T A L

Table 1 Effect of purification of

Digestion by Amplification by soil-derived DNA on restrictability and
Purification step Hind111 E/Bt Sau3A BamHl PvuII PCR polymerase chain reaction (PCR)
I . Crude - - +/- - - -
2. After caesium chloride precipitation - - +/- +/- - -
3. After potassium acetate precipitation +/- +/- +/- +/- - -
4. After spcrmine-HCI + +I- + +/- + +
5. After glass milk + + + ND + +
* Soil crude DNA extracts were derived from soil containing about lo6 cfu g- using the
modified protocol described with Braun's lysis. pUC19 was added to DNA preparations to
serve as a marker of the activity of restriction enzymes.
t E/R, EcoRI/Bgfl double digestion.
-, No digestion or amplification; / -, partial digestion; +,
complete digestion or posi-
tive amplification; ND, not determined.

by using a Braun's homogenizer. Crude DNA preparations between 1.2 and 1.4, indicating the presence of large
obtained with cold phenol were consistently less brown amounts of contaminating matter. T h e approximate DNA
than those obtained after a hot phenol extraction step amount was determined from agarose gels, because O.D.260
(60C, 30 min), as in the Ogram protocol, indicating that values as well as the values obtained by fluorimetric DNA
these contained less humic substances. However, crude measurements indicated that the latter two methods were
DNA prepared in this way and pure pUC19 DNA added to unsuitable.
the preparations were insensitive to restriction by HindIII, '
T h e DNA recovery values (4-20 pg g - of soil) obtained
EcoRl Bgll, BamHl and Pvu1; only Sau3A produced by us were comparable to those reported by Steffan et al.
partial cuts in the DNA (Table 1 and Fig. 3). In addition, (1988), Porteous and Armstrong (1991) and Selenska and
crude DNA was not amplifiable. This suggested the pres- Klingmiiller (1991b). Considering cell numbers (direct
ence of substances hindering both digestion by restriction counts) commonly found in soil (109-10'0 g - I ) , and
enzymes and amplification of the DNA. Figure 2 shows the assuming a cellular DNA content of 5-8 fg (McCoy and
appearance of DNA and humic substances following gel Olson 1985), theoretically between 5 and 80 pg of bacterial
electrophoresis and ethidium bromide staining. DNA per g of soil should be extractable from a 'common'
Several attempts to improve purity at this stage, i.e. the soil. Assuming the likely interference by some fungal DNA,
use of mercapto-ethanol/spermidine during lysis, the addi- and neglecting DNA from non-microbial sources, this still
tion to soil, prior to lysis, of salmon sperm DNA to suggests that our DNA yields, and those of the other
complex humic substances and the use of RNase and workers, represent sizeable parts of the bacterial popu-
pronase digestion following extraction, did not lead to lations in soil.
improved purity as evidenced via agarose gel electro-
phoresis and 0.D.260/0.D.280 ratios (1.2). Purification steps, restriction and amplification
The amount of DNA recovered in the crude prep- The objective of the purification procedure was to obtain
arations, estimated from agarose gels, was initially about DNA clean enough to be amplifiable, and at the same time
1-5 pg of DNA per g of dry soil, vs up to fourfold less for to attain quantitative recovery of DNA. As criteria for
parallel preparations obtained with the protocol of Ogram judging the effectiveness of each purification step, we used
et al. (1987). DNA obtained by freeze/thaw lysis appeared restrictability with various restriction enzymes (Steffan et
in a band of about 1&25 kb size, whereas DNA obtained al. 1988), and direct amplifiability in the PCR. Most of the
following lysis in a Braun's homogenizer was somewhat work was carried out with DNA samples derived from soil
more sheared (Fig. 2). However, the degree of shearing was microcosms containing about lo6 cfu of Ps.jluorescens R2f
less than that described by Ogram et al. (1987). Cell lysis (RP4 : : pat) per g at the time of sampling.
induced in the Braun's homogenizer resulted in a consider- Initial purification via CsCl precipitation of impurities
able improvement of DNA recovery, i.e. between 4 and 20 (protein, RNA, humic substances) resulted in a substantial
pg of DNA was routinely obtained in the crude prep- loss of brown colour and a drastically improved efficiency
arations. In one representative experiment, recovery, as of the following KAc precipitation step. Little to no loss of
determined by scanning, was 11 pg DNA per g of soil after DNA was recorded. However, DNA after CsCl clean-up
mechanical lysis and 7 pg DNA following freeze/thaw lysis. was not restrictable with most enzymes tested (except for
The 0.D.260/0.D.280 values of all crude preparations were partial digestion with Sau3A) and, in addition, was not

1 2 3 4 5 6 7 8 9 Further development of PCR applled to soil samples

The PCR was optimized with pure pPATB2 DNA (van

Elsas and Trevors 1990) as a target. Variations in the PCR
cycle, the number of cycles, the enzyme, the buffer used
and the primers were tested using different concentrations
of target DNA. T h e protocol described (Materials and
Methods) summarizes the results of these efforts. With this
optimized protocol and either one of the three enzymes
mentioned, it was possible to obtain amplification of the
highest dilution used, i.e. 0.01 fg of target DNA, corre-
sponding to 16 copies of the target.
AmpliTaq was used in initial amplifications from soil,
but was easily inhibited by soil compounds. SuperTaq was
shown to be relatively insensitive to soil compounds, per-
Fig. 4 Southern blot of polymerase chain reaction (PCR) mitting the amplification of up to 2 pl of soil DNA in 50 jd
products amplified with the Stoffel fragment using DNA from reaction mix ; PCR products were often smeared, however,
Ede loamy sand. DNA preparations were purified twice with glass making interpretation difficult. T h e cause of this smearing
milk. Lanes contain PCR product from soil containing per g: 1, is unknown, and PCR applied to a different target sequence
about lo6 cfu of Pseudomonasjuorescens R2f (RP4 : : p a t ) ; 2, as in produced distinct bands of defined size (not shown). T h e
1, plus 20 ng of free RP4 : : put DNA; 3, as in 1, plus 0.2 ng of
use of the Stoffel fragment avoided this problem with the
free RP4 : : put DNA; 4, 3 x lo3 cfu of Ps.j?uorescens R2f
pat amplification system, and also allowed for efficient
(RP4 : : p ut ); 5 , as in 4, plus 20 ng of free RP4 : : p a t ; 6 ; 20 ng of
free RP4 : : pat DNA ; 7 ; 0.2 ng of free RP4 : : par DNA ; 8,
amplification, but a second round of glass milk or spermine
positive control (Ps.juorescens R2f (RP4 : : put) added); 9, was sometimes necessary to obtain consistent amplification.
negative control. Amplification product (indicated by arrow) was T o determine the limit of detection of the PCR the two
0.7 kb as expected (van Elsas el al. 1991a) purification protocols were applied to day-1 soil samples
inoculated with 0, 3 x lo2, 3 x lo3, 3 x lo4, 3 x lo,
3 x lo6 and 3 x lo7 cfu of Ps. fltlorescens R2f (RP4 : : pat)
per g of soil. Colony-forming unit counts at sampling were,
amplifiable (Table 1). Potassium acetate precipitation of respectively, 0, 1.3 x lo, 3.0 x lo2, 5.4 x lo3, 4.4 x lo4,
contaminating matter was then applied to CsC1-treated 4.5 x lo5, and 1.0 x lo7 per g dry soil. T h e application of
DNA preparations. This, again, removed humic substances the PCR resulted in the appearance of 683-bp amplification
and other impurities a t negligible loss of DNA. DNA prep- products only in the soil DNA preparations with counts of
arations were only partially restrictable with Sau3A and 3 x 10 cfu (inoculum level 3 x lo3) per g of dry soil or
BamH 1, and only incidentally amplifiable after dilution to higher. Theoretically, at a level of lo3 inoculant cfu per g
0.5-1 ng per PCR reaction mixture. As final purification of soil, and three copies of RP4 : : pat per cell, 1.5 x lo4
steps, two strategies were followed. The first was based on copies of pat would be present in the final volume (1000
DNA precipitation by spermine-HCI, and the second used pl). U p to 1 p1 of this final volume, corresponding to 20 ng
precipitation of DNA on glass milk. Both methods resulted total soil DNA, could serve as a template for amplification.
in virtually complete removal of the brown colour from the Therefore, assuming 100% recovery of DNA, and the
DNA preparations at recovery values of 50-100% for necessity of 10-20 copies of the target for consistent posi-
spermine-HC1 and 75-100% for glass milk. In addition, the tive amplification, about lo3 inoculant cfu per g of soil
DNA was now restrictable with HindIII, EcoRI/Bgll, would still be detectable, which is about the limit of detec-
PvuII, Sau3A and BamHl (partially for spermine-HC1; tion found. Tsai and Olson (1992), targeting a 7-copy 16s
Table 1). Application of the PCR to spermine- and glass ribosomal DNA sequence by PCR, reported a limit of
milk-cleaned preparations routinely produced the expected detection of 5 x 10 Esrherichia coli cells per g of soil, or
683-bp amplification product, as evidenced by gel electro- the equivalent of about three cells (21 copies of the target)
phoresis and Southern blot analysis (Fig. 4). Both DNA in the PCR reaction mixture. Pillai et at. (1991) reported
from soil containing about lo6 and 3 x lo3 cfu g-, and PCR-assisted detection of 1-10 transposon Tn5-labelled
target DNA freely added to soil (0.2 and 20 ng g - of soil), Rhizobium leguminosarum cells per g of soil with PCR on
and mixes of both, were detected via PCR amplification pellets obtained from soil-derived suspensions. However,
and Southern hybridization (Fig. 4); unseeded soil did not two rounds of PCR were needed which enhances the risks
show an amplification product. of non-specific amplification. Moreover, signals of low
84 K. S M A L L A ET A L .

inoculum densities were only positive in dot-blots of PCR sample. T h e possibility of detecting such cells or the target
reaction mixes. represents the major advantage of the use of PCR detection
A more precise method for quantification of PCR seems for monitoring of G M O s following release into soil.
to be possible using temperature gradient gel electro-
phoresis (TGGE), as developed by DIAGEN. We are cur-
Concluding remarks
rently looking for possibilities to apply this methodology to
soil DNA extracts. D N A extraction and purification methodology from soil
was developed to obtain, via a simple, inexpensive and
rapid protocol, D N A preparations suitable for PCR
Application of PCR to the description of the fate of
analysis. T h e protocol described here represents an
introduced Pseudomonas fluorescens (RP4 : :pat)
improvement over a formerly applied method (van Elsas rt
As shown in Table 2, introduced Ps. Puorescens R2f al. 1991a), as the gel electrophoresis and electro-elution
(RP4 : : pat) showed a progressive decline in cfu numbers steps could be replaced by three simple, rapid and quanti-
in the Ede loamy sand soil. T h e corresponding imrnuno- tative precipitation steps. Furthermore, hydroxyapatitc
fluorescence specific cell counts also showed a progressive purification, which often resulted in poor (2.5'XI) D N A
decline, albeit at a significantly lower rate. This suggested recovery, and tirne-consuming CsCl gradients could be
the (temporary) occurrence of non-culturable cells in the omitted. Our scheme for extraction and purification
system, which might be either viable, non-viable or even permits amplification and restriction enzyme analysis of
dead. D N A derived from different soils. For instance, Flevo silt
Attempts to track the introduced cells by specific detec- loam as well as different Schonebeck soil samples extracted
tion of put via slot-blot hybridization of crude extracts by the protocol presented also showed amplification by
showed that only at cfu and cell counts around or above PCR (not shown); the number of purification steps needed
lo6 per g of dry soil, a (weak) hybridization signal was after KAc precipitation was dependent on soil type. Therc-
detected with the pat probe. In some cases, dilution of the fore, the developed protocol is flexible in that it allows for
DNA preparations was needed in order to obtain a positive sufficient purity of D N A for PCR and restriction analysis
signal. As shown in Table 2, PCR accurately detected the after varying final purification steps, depending on the soil
presence of the pat sequence throughout the course of the type (Fig. 1).
microcosm experiment, accompanying and complementing Purification of soil D N A oia different steps was devel-
the cfu and total specific cell counts. oped for routine testing in GMO field releases as well as for
We also applied detection via PCR to a study over a control investigations in areas surrounding contained use of
longer period in the same soil, with a starting inoculum of GMOs.
about lo5 cfu of Ps. jluore~censR2f (RP4 : : pat) per g of A major factor limiting the usefulness of PCR for
soil. Total DNA was extracted from the soil 5 d and 5 environmental studies is that it is still qualitative or, at best,
months after inoculation. Both D N A preparations purified semi-quantitative. T h e possibility of using end-point
with glass milk could serve as a template for PCR. Whereas dilution/most-probable-number quantification or of ampli-
Ps.Juorescens (RP4 : : pat) cfu were still detectable after fication in the presence of internal standards, e.g. cia
5 d, no cfu were obtained after incubation for 5 months. T G G E , still has to be explored for soil-derived DNA. A
These observations suggested the presence of non- second factor is the interpretation of the appearance of
culturable cells or liberated target D N A in the latter PCR products, i.e. whether these indicate the presence of
whole living or possibly dead bacterial cells or even free
Table 2 Survival of PseudomonasJluorescens R2f (RP4 : : pat) in DNA. However, the analysis presented here suggests the
soil, using selective plating, immunofluorescenceand usefulness of PCR for the detection of G M O s (and/or
molecular-based techniques sequences) of interest in a non-culturable state or at
reduced levels in soil.
Log cfu or cells (IF) per g dry soil on (day):
tested 0 1 3 7 14 28 42 49 ACKNOWLEDGEMENTS
Cfu 6.62 6.64 6.71 6.08 5.83 4.48 4.08 3.93 We thank A. Hagler, G. Berben, L. van Overbeek and C.
Cells (IF) 6.70 ND ND 6.62 6.08 ND 5.30 <4.2 Clegg for contributing to some of the experiments. This
Slot-blot + + + + ND - - -
work was supported by an E C grant awarded to J.D.v.E.
PCR + N D + + N D N D + + and by O E C D short-term fellowship awards to K. Smalla
IF, I mmunofluorescence; ND, not determined ; PCR, polymerase and N. Cresswell. L.C. Mendonca-Hagler was supported
chain reaction. by an EC: fellowship.

REFERENCES Selenska, S. and Klingmuller, W. (1991b) DNA recovery and

direct detection of Tn5 sequences from soil. Letters in Applied
Cresswell, N., Saunders, V.A. and Wellington, E.M.H. (1991)
Microbiology 13, 21-24.
Detection and quantification of Streptomyces violaceolatus
Smalla, K., Isemann, M., John, G., Weege, K.-H., Backhaus, H.
plasmid DNA in soil. Letters in Applied Microbiology 13, 193-
and Wendt, K. (1991) A risk assessment of industrial pro-
duction of alpha-amylase with an rDNA production strain. In
DAquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczca,
Biological Monitoring of Genetically Engineered Plants and
P. and Kaplan, J.C. (1991) Maximizing sensitivity and speci-
Microbes ed. MacKenzie, D.R. and Henry, S.C. pp. 205-220.
ficity of PCR by preamplification heating. Nucleic Acids
Bethesda, Maryland : Agricultural Research Institute.
Research 19, 3749.
Smit, E., van Elsas, J.D., van Veen, J.A. and de Vos, W. (1991)
Ford, S. and Olson, B.H. (1988) Methods for detecting genetically
Detection of plasmid transfer from Pseudomonas Jluorescens to
engineered microorganisms in the environment. Advances in
indigenous bacteria in soil by using phage @R2f for donor
Microbial Ecology 10, 45-73.
counterselection. Applied and Environmental Microbiology 57,
Heijnen, C.E., Hok-A-Hin, C.H. and Van Veen, J.A. (1992)
Improvement to the use of bentonite clay as a protective agent,
Steffan, R.J. and Atlas, R.M. (1988) DNA amplification to
increasing survival levels of bacteria introduced into soil. Soil
enhance detection of genetically engineered bacteria in environ-
Biology and Biochemistry (in press).
mental samples. Applied and Environmental Microbiology 54,
Holben, W.E., Jansson, J.K., Chelm, B.K. and Tiedje, J.M.
2 185-2 I9 1.
(1988) DNA probe method for the detection of specific micro-
Steffan, R.J., Goksoyr, J., Bej, A.K. and Atlas, R.M. (1988)
organisms in the soil bacterial community. Applied and Environ-
Recovery of DNA from soils and sediments. Applied and
mental Microbiology 54, 703-71 1.
Environmental Microbiology 54, 2908-291 5.
Hopwood, D.A., Bibb, M.J., Chater, K.F., Kieser, T., Bruton,
Trevors, J.T. and van Elsas, J.D. (1989) A review of selected
C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P., Ward, J.M. and
methods in environmental microbial genetics. Canadian Journal
Schrempf, H. (1985) Genetic Manipulation of Streptomyces: A
of Microbiology 35, 895-902.
Laboratory Manual. Norwich, U K : T h e John Innes Founda-
Trevors, J.T., van Elsas, J.D., van Overbeek, L.S. and Starodub,
M.E. (1990) Transport of a genetically engineered Pseudomonas
McCoy, W.F. and Olson, B.H. (1985) Fluorimetric determination
Jluorescens strain through a soil microcosm. Applied and
of the DNA concentration in municipal drinking water. Applied
Environmental Microbiology 56, 401408.
and Environmental Microbiology 49, 81 1-817.
Tsai, Y.-L. and Olson, B.H. (1992) Detection of low numbers of
Ogram, A,, Sayler, G.S. and Barkay, T.J. (1987) DNA extraction
bacterial cells in soils and sediments by polymerase chain reac-
and purification from sediments. Journal of Microbiological
tion. Applied and Environmental Microbiology 58, 754-757.
Methods 7, 57-66.
van Elsas, J.D. and Trevors, J.T. (1990) Plasmid transfer to
Pillai, S.D., Josephson, K.L., Bailey, R.L., Gerba, C.P. and
indigenous bacteria in soil and rhizosphere : problems and per-
Pepper, I.L. (1991) Rapid method for processing soil samples
spectives. I n Bacterial Genetics in Natural Environments ed. Fry,
for polymerase chain reaction amplification of specific gene
J. and Day, M.J. pp. 188-199. London: Chapman & Hall.
sequences. Applied and Environmental Microbiology 57, 2283-
van Elsas, J.D., Govaert, J.M., Dijkstra, A.F. and van Veen, J.A.
(1986) Survival of Pseudomonas Jluorescens and Bacillus subtilis
Porteous, L A . and Armstrong, J.L. (1991) Recovery of bulk
introduced into two soils of different texture in field microplots.
DNA from soil by a rapid, small-scale extraction method.
F E M S Microbiology Ecology 38, 151-160.
Current Microbiology 22, 345-348.
van Elsas, J.D., Trevors, J.T. and Starodub, M.E. (1988) Bac-
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R.,
terial conjugation between pseudomonads in the rhizosphere of
Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-
wheat. F E M S Microbiology Ecology 53, 299-306.
directed enzymatic amplification of DNA with a thermostable
van Elsas, J.D., van Overbeek, L.S. and Fouchier, R. (1991a) A
DNA polymerase. Science 239, 487494.
specific marker, pat , for studying the fate of introduced bacteria
Sambrook, J., Fritsch, E.F. and Maniatis, T . (1989) Molecular
and their DNA in soil using a combination of detection tech-
Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor,
niques. Plant and Soil 138,49-60.
New York.
van Elsas, J.D., van Overbeek, L.S., Feldmann, A.M., Dull-
Sayler, G.S., Shields, M.S., Tedford, E.T., Breen, A., Hooper, emans, A.M. and de Leeuw, 0. (1991 b) Survival of genetically
S.W., Sirotkin, K.M. and Davis, J.W. (1985) Application of
engineered Pseudomonas Juorescens in soil in competition with
DNA-DNA colony hybridization to the detection of catabolic
the parent strain. F E M S Microbiology Ecologv 85, 53-64.
genotypes in environmental samples. Applied and Environmental
Waalwijk, C., Dullemans, A.and Maat, C. (1991) Construction of
Microbiology 49, 1295-1303.
a bioinsecticidal rhizosphere isolate of Pseudomonas Jluorescens.
Selenska, S. and Klingmuller, W. (l991a) Direct detection of n f -
F E M S Microbiology Letters 77, 257-264.
gene sequences of Enterobacter agglomerans in soil. F E M S
Microbiology Letters 80, 243-246.