Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

ELSEVIER FEMS Microbiology Ecology 20 (1996) 25 I-260

MICROBIOLOGY ECOLOGY

Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA ( RAPD) technique

C. Vettori a, D. Paffetti a, G. Pietramellara b, G. Stotzky ‘, E. Gallori a,*

a Department of Animal Biology and Genetics, University of Florence. c;ia Romana 17, 50125 Florence, Italy b Department of Soil Science, Unic~ersir?; of Florence, Florence, Italy

’ Laborato? of Microbial Eco1og.v. Department of Biology New York lJnir~ersi@. New York, USA

Received 20 November 1995; revised 12 April 1996; accepted 18 April 1996

Abstract

Chromosomal DNA from 3ucilfus S&~&S, bound on the clay minerals, montmorillonite (Wyoming (W) and Apache County (Ap)) and kaolinite (RI, was subjected to the random amplified polymorphic DNA (RAPD) technique. DNA bound on the clays was not amplified with 0.625, 1.875, 6.25, and 12.5 U of Tuq DNA polymerase, but amplification occurred

when the clay-DNA complexes were diluted IO- and 20-fold or when 21 U of Taq DNA polymerase was added. DNA desorbed from the Ap-DNA and K-DNA equilibrium complexes was amplified with 0.625 U of Taq DNA polymerase, whereas amplification of DNA desorbed from the W-DNA complex occurred only after a IO-fold dilution or when 1.875 U of Tuq DNA polymerase was used. These observations indicate that clay minerals differentially affect the amplification process, probably by inhibiting the activity of Taq DNA polymerase.

Kqvwords: RAPD; Environmental DNA; Clay-DNA complex

1. Introduction DNA can persist for long periods, apparently as the

Extracellular DNA, mostly of microbial origin, has been detected in natural habitats that have been examined [1,2]. Lysis of dead bacterial cells, e.g., following phage infection [3,4], and the spontaneous release of both chromosomal and plasmid DNA during different phases of bacterial growth [5-71 have been suggested to be among the sources of such environmental DNA. Despite the abundance in natural habitats of DNases and microorganisms that can utilize DNA as a nutritional source, extracellular

* Corresponding author. Tel: +39 (55) 220498; Fax: + 39 (55)

222565; E-mail: [email protected]

result of the interaction of DNA with various compo- nents of the environment [ 1,8,9]. The adsorption and binding of DNA on particles, especially on clay minerals, have been shown to result in the protection of DNA against degradation by nucleases, such as DNases and restriction endonucleases, without inhibiting the ability of DNA to transform recipient cells [lo- 191.

Many factors [1,8,9] influence the adsorption and binding of DNA on clay minerals: e.g., the type of clay [ 14,201, the type of charge-compensating cations on the clay [15,20,21], the pH [ 14,211, the tertiary structure and size of the DNA [19,22]. The mecha- nisms involved in adsorption and binding are not

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252 C. Vettori et al. / FEMS Microhioloy~ Ecology 20 (19961251-260

known, although hydrogen bonding may be impor- tant [14], as it appears to be involved in the binding of proteins on clay minerals 1231. Nevertheless, extracellular DNA in soil and sediment appears to be associated with clay minerals [l&9,24], and clay- DNA complexes are stable for long periods, as demonstrated by the difficulty to desorb DNA from clay with solutions of different ionic strength, chelat- ing agents, and detergents [ 1,141 and by the resistance of bound DNA to degradation [ 14-16.191.

New methods for the identification of environmental DNA [2,25,26] have been developed in recent years, partially as a result of the concern about the release of genetically modified organisms (GMOS) to the environment [8,27]. Among these techniques, amplification of DNA by the polymerase chain reaction (PCR) has emerged as one of the most useful for the investigation of nucleic acids in environmental samples, because of the sensitivity and specificity of the PCR [28,29].

The PCR and its variant, the random amplified polymorphic DNA (RAPD) technique, which uses a single short oligonucleotide of random sequence for the amplification process [30,3 11, has been applied in natural habitats, e.g., the identification of microorganisms and the determination of target nucleotide sequences, including paleontological DNA sequences [32-361. However, no data are available on the amplification of DNA bound on clay minerals.

We report here the results of studies on the application of the RAPD technique to the direct amplification and detection of DNA from the soil bac- terium, Bacillus subtilis, bound on the clay minerals, montmorillonite and kaolinite.

2. Materials and methods

2.1. Preparation of bacterial DNA

Badlus subtilis strain BD170 (thr-5 trpC2) [ 14,191 was the source of chromosomal DNA, which was prepared as described by Khanna and Stotzky

[141.

2.2. Preparation of homoionic clay

The < 2-pm fraction of montmorillonite, a 2:l (Si:Al) swelling clay, from two sources, Wyoming

Fig. 1. Procedure for the amplification of samples by the RAPD

technique.

(W) (Crook County, WY) and Apache County (Ap) (Arizona), and of kaolinite (K), a 1:l non-swelling clay (Zettliz, Germany), were made homoionic to calcium (Cal, as described by Fusi et al. [37]. The three clays differ in their cation-exchange capacity (W = 76.4 cmol kg-‘, Ap = 120 cmol kg- ‘, K = 5 to 10 cmol kg- ’ ) and specific surface area (W = 800 to 850 m’ gg’, Ap=700to750m’g-‘,K=30to 50 m2 gg I).

2.3. Preparation of clay-DNA complexes ,for amplification

Clay-DNA complexes, supematants after equilibrium adsorption, and each wash of the clay-DNA complexes after equilibrium adsorption were subjected to amplification by the RAPD technique ac- cording to the protocol in Fig. 1.

DNA (30 pg> was mixed with 100 pl of a suspension of the different clays (22 mg ml-’ of deionized distilled water (ddH20)) in a total volume of 1 ml of ddH,O. After 120 min of shaking (40 rpm), maximum adsorption at equilibrium was obtained [ 14,191, and the mixtures were centrifuged at 85 000 X g for 20 min at 20°C. The DNA concentration in the supematant was determined by measuring the absorbance at AzhO. Samples (1 ~1) of both the

C. Vettori et al. / FEMS Microbiology Ecology 20 f 19961 251-260 253

undiluted equilibrium supematant (Fig. 1, a) and the equilibrium supematant diluted IO-fold (Fig. 1, b) were subjected to the RAPD technique. The pellets of clay-DNA complexes formed after equilibrium adsorption were resuspended in 1 ml of ddH,O and centrifuged at 85 000 X g for 20 min at 20°C. This procedure was repeated four times (no free DNA was detected after the second or third wash [14,19]). Samples (1 ~1) of the first and second washes (Fig. 1. c> were subjected to the RAPD technique. The clay-DNA pellets containing DNA that was tightly bound were resuspended in 1 ml of ddH,O, and 1 ~1 of suspensions of the clay-DNA complexes undiluted (Fig. 1, d) or diluted lo- and 20-fold (Fig. 1, e) were subjected to the RAPD technique.

The clay minerals were subjected to the same centrifugation and washing procedures and used as controls in the experiments. All amplification experiments were performed in duplicate, and each experiment was repeated four times.

2.3. Preparation of clay-Taq DNA polvmerase complexes

Clay-Taq DNA polymerase complexes were prepared similar to the clay-DNA complexes, except that 200 pg of AmpliTaq DNA polymerase (Perkin Elmer, USA) was added to 100 ~1 of a suspension of the different clays (22 mg ml-’ ) in a total volume of 1 ml of ddH,O. The mixtures were centrifuged at 4”C, and the concentration of Taq DNA polymerase present in the supematants was determined by the micro assay procedure of the Bradford method (Pierce Inc., USA) [38] and by absorption at 280 nm (A,,,) using bovine serum albumin as the standard. The lower limit of detection of proteins by these methods was 1 kg/pi. However, amounts of polymerase less than 3 pg (0.625 U) did not amplify free DNA.

The clay-Taq DNA polymerase complexes after equilibrium adsorption were resuspended in 1 ml of ddH,O and centrifuged at 85 000 X g for 20 min at 4°C. This procedure was repeated until no more Taq DNA polymerase was detected in the supematants (after two washes), and the complexes were then washed twice more. The amounts of Taq DNA polymerase adsorbed at equilibrium and bound on the different clays were calculated as described by Stotzky [23].

The bound clay-Taq DNA polymerase complexes were used in the RAPD experiments. Either free DNA (10 ng) or clay-DNA complexes were added to an amount of clay-Taq DNA polymerase

complex containing about 0.625 U of tightly bound polymerase just before the addition of the amplification mixture, which did not contain Tag DNA polymerase. In some studies, free Taq DNA polymerase (0.625 to 21 U) was also added. All amplification experiments were performed in duplicate, and each experiment was repeated two times.

2.5. RAPD-PCR condihons

Amplifications were performed in a 25 ~1 volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4.5 mM MgCl?, 0.001% (w/v) gelatin, 200 PM of each dNTP, 6.4 PM of primer, 1 ~1 of different amounts of template DNA (either free or bound on the clays), and 0.625 to 21 U of AmpliTaq DNA polymerase (either free or bound on the clays). The oligonucleotide used as primer was a IO-mer random primer (1253: 5’-GTT TCC GCC C-3’) with a G + C content of 70% and a T,,, of 34°C [39]. After incuba- tion at 90°C for 90 s and at 95°C for 90 s, the reaction mixtures were cycled 45 times through the following temperature profile: 95°C for 30 s (de- naturation), 36°C for 1 min (annealing), and 75°C for 2 min (extension), followed by one extension step at 72°C for 10 min. A Perkin Elmer 9600 thermocycler was used.

Amplification products (10 /Al per lane) were analyzed by gel electrophoresis on 2% (w/v) agarose gels (Boehringer Mannheim, Germany) at 10 V cm- ’ for 2 h in Tris-acetate-EDTA buffer containing 0.5 pg ml-’ (w/v) of ethidium bromide [40]. The gels were photographed with an UVP gel scanner (GDS2000; Ultra Violet Product Ltd., Cambridge, UK).

2.6. Release of DNA bound on clay during ampl$ca- tiort by PCR

Two sets of samples containing IO ~1 of a suspension of bound clay-DNA complexes were subjected to the PCR, as above, in the absence of Taq DNA polymerase. The following mixtures were used: (1) MgC12 and amplification buffer; (2) MgCl? , amplification buffer, and primer; and (3) MgCl?, amplification buffer, primer, and the mixture of

254 C. Vettori et al. / FEMS Microbiolog?; Ecology 20 (1996) 251-260

Table 1

Amplification of DNA present in clay-DNA complexes

Experiment Conditions Tuy DNA

polymerase (U) a

Clay

w AP K

A: Clay-DNA complexes b

B: Clay alone d

Undiluted

Undiluted + free DNA ’

Diluted (lo-fold)

Diluted (20-fold)

Undiluted

Undiluted + free DNA ‘

0.625 _ _ -

1.875 _ _ _

6.250 - _ _

12.500 _ _ _

21.000 + + +

0.625 - _ _

0.625 _ + +

0.625 + + +

0.625 _ _ _

0.625 _ _ -

Symbols: - = no amplification, + = amplification.

Amount of free Taq DNA polymera& in the amplification mixture.

Amount of DNA bound on clay (in 1 ~1): 5.9 ng pgg’ of W; 7 ng wg-’ of Ap; 5 ng pg-’ of K.

10 ng of DNA added before addition of the amplification mixture. Clays were washed four times with ddH?O.

dNTP. After 45 cycles of amplification, one set of samples was electrophoresed as above, except that 25 ~1 was used per lane. The other set was centrifuged at 17 000 X g for 10 min at room temperature (which completely sedimented the complexes), 1 ~1 of the undiluted supernatants was subjected to 45 cycles of amplification in the presence of 0.625 U of Tuq DNA polymerase, and 10 ~1 of the products were electrophoresed.

2.7. X-ray diffrraction (X-RD) analysis of W and the supernatants of W and W-DNA complexes after equilibrium adsorption

Suspensions (1 ml) of W alone (2.2 mg ml-’ ) and of the supematants from W and W-DNA complexes were spread on glass slides and air-dried at room temperature [21]. Analysis at intervals of 2 0 from 3” to 16” was done at room temperature using

Table 2

Amplification of DNA, either free or bound on clay minerals, in the presence of Taq DNA polymerase bound on clay minerals

Experiment Taq DNA Clay

polymerase (U) a W AP K

A: Clay-polymerase

complexes h + free DNA ’

0.000 _ _ _

0.625 _ _ _

1.875 - _ _

21.000 + + +

B: Clay-polymerase complexes b +

clay-DNA

complexes d

0.000 _ - _

0.625 _ _ _

1.875 _ _ _

21.000 + + +

Symbols: - = no amplification, + = amplification.

a Amount of free Taq DNA polymerase (U) added to the amplification mixture. ’ Amount of polymerase bound mgg ’ of clay: W = 61 pg; Ap = 14 pg; K = 4 pg.

’ 10 ng of DNA added before addition of the amplification mixture.

d See Table 1.

C. Vettori et al. / FEMS Microbiology Ecology 20 f 1996) 251-260 255

iron-filtered Co K, radiation (PW 14 1 O/20 Philips

X-ray diffractometer).

3. Results and discussion

The results of RAPD amplification of DNA (1) bound on the clay minerals (W, Ap, and K) (Fig. I, d and e), (2) present in the respective supematants after equilibrium adsorption (Fig. 1, a and b), and

(3) present in the washes of the clay-DNA complexes after equilibrium adsorption (Fig. 1, c> are shown in Tables 1 and 3 and Fig. 2. The repro- ducibility of the RAPD profiles was confirmed in four independent experiments, each performed in duplicate.

DNA bound on the three clays was not amplified with 0.625, 1.875, 6.250, and 12.500 U of Tuq DNA polymerase when the clay-DNA complexes were used directly (Table 1, Experiments A and B). There

Fig. 2. RAPD amplification patterns of different samples obtained with primer 1253 (see Materials and methods). Lanes 1 and 30, molecular

mass marker: 123 bp DNA ladder (Sigma. USA): lane 2. W alone; lane 3, Ap alone; lane 4. K alone: lane 5. undiluted bound W-DNA

complex: lane 6, undiluted bound Ap-DNA complex; lane 7. undiluted bound K-DNA complex; lane 8, diluted bound W-DNA complex

(20.fold); lane 9, diluted bound Ap-DNA complex (10.fold); lane 10, diluted bound K-DNA complex (10.fold); lane 11 and 29. DNA

alone: lane 12. undiluted supematant from W-DNA complex after equilibrium adsorption: lane 13. filtered undiluted supernatant from

W-DNA complex after equilibrium adsorption; lane 14, diluted (lo-fold) supematant from W-DNA complex after equilibrium adsorption:

lane 15, undiluted supernatant from Ap-DNA complex after equilibrium adsorption: lane 16. undiluted supematant from K-DNA complex

after equilibrium adsorption; lane 17, undiluted supernatant from W-DNA complex after equilibrium adsorption + free DNA; lane 18.

undiluted supematant from Ap-DNA complex after equilibrium adsorption + free DNA: lane 19, undiluted supematant from K-DNA

complex after equilibrium adsorption + free DNA; lane 20, undiluted supematant from W alone; lane 21, undiluted supematant from Ap

alone; lane 22. undiluted supernatant from K alone; lane 23. undiluted supematant from W-DNA complex after equilibrium adsorption

mixed (ratio ]:I) with filtered undiluted supernatant from W-DNA complex after equilibrium adsorption; lane 24, undiluted supematant

from W-DNA complex after equilibrium adsorption mixed (ratio 1:lO) with filtered undiluted supematant from W-DNA complex after

equilibrium adsorption: lane 25. undiluted supernatant from W-DNA complex after equilibrium adsorption mixed (ratio I:]) with filtered undiluted supematant from Ap-DNA complex after equilibrium adsorption; lane 26, undiluted supematant from W-DNA complex after equilibrium adsorption mixed (ratio 1:lO) with faltered undiluted supematant from Ap-DNA complex after equilibrium adsorption: lane 27.

undiluted supematant from W-DNA complex after equilibrium adsorption mixed (ratio 1: 1) with filtered undiluted supematant from

K-DNA complex after equilibrium adsorption: lane 28, undiluted supematant from W-DNA complex after equilibrium adsorption mixed (ratio 1:lO) with filtered undiluted supematant from K-DNA complex after equilibrium adsorption.

256 C. Vettori et al. / FEMS Microbio1og.v Ecology 20 (19961 X-260

Fig. 3. Gel electrophoresis of bound clay-DNA complexes sub-

jected to 45 thermal cycles in the absence of Taq DNA poly-

merase (mixture 3. see Materials and methods) (lanes 2 to 4), and

of supernatants of the amplification products of bound clay-DNA

complexes (lanes 5 to 7) and of DNA alone (lane 8) amplified in

presence of 0.625 U of Tuq DNA polymerase. The same results

were obtained with all three mixtures not containing DNA poly-

merase. Lanes 1 and 9, molecular mass markers: 123 bp DNA

ladder (Sigma. USA): lane 2, suspension of bound W-DNA

complex (5.9 ng of DNA pg-’ of W ~1~ ’ ): lane 3. suspension

of bound Ap-DNA complex (7 ng of DNA fig-’ of Ap PI-‘):

lane 4. suspension of bound K-DNA complex (5 ng of DNA

CLg-’ of K ~1~ ‘); lane 5. supernatant of the amplification

product of bound W-DNA complex: lane 6, supernatant of the

amplification product of bound Ap-DNA complex; lane 7, super-

natant of the amplification product of bound K-DNA complex;

lane 8, DNA alone.

was also no amplification when 10 ng of free DNA was added to the clay-DNA complexes and to the clays alone. Clay-bound DNA was amplified only after dilution of the clay-DNA complexes: a greater dilution (20-fold) was required to obtain amplification of W-DNA complexes than of complexes with Ap and K (IO-fold). Similar results were obtained

with W made homoionic to magnesium (Mg’+ ), indicating that the lack of amplification was not the result of a decrease in the concentration of Mg’+ in the amplification mixture, which could have occurred as a consequence of cation exchange between Ca’+ on the clay and Mg’+ in the mixture.

To investigate the activity of the DNA polymerase in the presence of the clay minerals, the equilibrium adsorption and binding of Taq DNA polymerase on W, Ap, and K was determined. The polymerase showed different percentage of binding on the clay minerals: 75% of that added on W: 30% on Ap; and 23% on K. These results suggested that the lack of amplification of the undiluted clay-DNA complexes was the result of the adsorption/binding of the Tuq DNA polymerase on the clay particles. This was supported by the fact that amplification occurred when the amount of Taq DNA polymerase in the amplification mixture was increased to 21 U (Table 1, Experiment A).

To verify that the adsorption/binding of Tug

DNA polymerase on the clays resulted in the inhibition of enzyme activity, bound clay-polymerase complexes, rather than free polymerase, were used to amplify DNA, both free and bound on the clay minerals. The clay-bound polymerase did not amplify DNA (Table 2). Amplification was again observed only when 21 U of free enzyme was added to

(a) (1 51 nm)

(b) (7.52 nm)

2.k

Fig. 4. X-ray diffractograms of: (a) W alone; (b) supematant from

W-DNA complex after equilibrium adsorption; and (c) water and supernatant from W alone.

C. Vettori et al./ FEMS Microbiology Ecology 20 (19961251-260 251

the samples. These findings were in agreement with other observations on the reduction in the activity of some enzymes when adsorbed or bound on clay minerals (e.g., [41]). This lack or reduction of enzymatic activity could have resulted from interference by the clay particles with the catalytic site of Tuq DNA polymerase or from a change in the conforma- tion of the enzyme when adsorbed or bound on the clays [23,41]. When the concentration of the clay minerals was reduced by dilution of the clay-DNA complexes, amplification of DNA was observed (Ta- ble 1, Experiment A).

were then subjected to amplification in the presence of 0.625 U of Tuq DNA polymerase, and again no bands were detected on the electrophoresed gels (Fig. 3, lanes 5 to 7). These data confirmed that the DNA remained tightly bound on the clays during the amplification process and that bound, rather than released. DNA was amplified.

To determine whether DNA bound on the clays was released during amplification and, therefore, the presumed amplification of clay-bound DNA was ac- tually of free DNA, the clay-DNA complexes were first subjected to 45 thermal cycles in the absence of Taq DNA polymerase, and the products were analyzed on electrophoresed gels. No DNA bands were detected on the gels (Fig. 3. lanes 2 to 4). The supernatants of the cycled clay-DNA complexes

The ability to amplify DNA present in the undiluted supernatant from the equilibrium adsorption of DNA on the clays varied with the type of clay. With 0.625 U of Tuq DNA polymerase, DNA in the equilibrium supematant from complexes with Ap and K was amplified, whereas there was no amplification of DNA present in the supematant from complexes with W; there was also no amplification when 10 ng of free DNA was added to the equilibrium supematant from W-DNA complexes (Table 3, Ex- periment A). Amplification of the equilibrium supernatant from W-DNA complexes occurred when 1.875 U of DNA polymerase was added or after a IO-fold dilution (Table 3, Experiment A). The lack of am-

Table 3

Amplification of DNA present in supernatants of clay-DNA complexes after equilibrium adsorption and in washes of clay-DNA complexes

Experiment Conditions Taq DNA Clay

polymerase (U) a W AP K

A: Supematants of Undiluted 0.625 _ + +

clay-DNA 1.875 + + +

complexes h Undiluted + free DNA ’ 0.625 _ + +

Diluted (IO-fold) 0.625 + + +

Filtered undiluted d 0.625 + + +

B: First wash Undiluted 0.625 _ + +

of clay-DNA

complexes ’

B: Second wash

of clay-DNA

complexes r

Undiluted 0.625 + + +

C: Supernatant of

clay alone

Undiluted

Undiluted + free DNA ’

0.625 - - -

0.625 + + +

Symbols: - = no amplification, + = amplification. a Amount of free Taq DNA polymerase (U) in the amplification mixture. ’ Amount of DNA in I ~1 of supematants from: W = 14.4 ng: Ap = 12.7 ng; K = 16.9 ng.

’ 10 ng of DNA added before addition of the amplification mixture.

’ Supematant was filtered through a 0.2-Frn pore-size nitrocellulose membrane.

’ AmountofDNAinl ~l:W=1.7ng;Ap=l.3ng;K=l.6ng.

f Amount of DNA in 1 ~1: W = 0.9 ng; Ap = 0.6 ng; K = 0.5 ng.

258 C. Vettori rt al. / FEMS Microbiology Eco1og.v 20 (1996) 251-260

plification with 0.625 U of polymerase probably resulted from the presence of clay particles in the equilibrium supernatant from the W-DNA complexes, as amplification occurred after filtration of the supernatant (Table 3, Experiment A). The presence of W particles in the supematant was demonstrated by an X-ray diffraction peak in the same position as of W alone (Fig. 4). Moreover, after filtration, the A,,, of the equilibrium supematant of W-DNA complexes was reduced by about 50%, whereas the AzGO of the DNA solutions did not change after filtration.

The inhibition of amplification by clay particles present in the equilibrium supematant from W-DNA complexes was also observed when the supematant from W-DNA complexes was mixed (1: 1) with filtered equilibrium supematants from W-DNA, Ap- DNA, and K-DNA complexes (Fig. 2, lanes 23, 25, and 27). However, when the concentration of W particles was decreased IO-fold by mixing (1: 10) with filtered equilibrium supematants, from all complexes, amplification occurred (Fig. 2, lanes 24. 26. and 28).

When free DNA was added to the equilibrium supematant of the clays alone, amplification was observed (Table 3, Experiment C), indicating that sedimentation of W by centrifugation was more complete when W was not complexed with DNA. The absence of particles of W in the equilibrium supematant was confirmed by the lack of a peak in the X-ray diffractogram (Fig. 4). Amplification of DNA in the first two washes of the clay-DNA complexes after equilibrium adsorption (Fig. 1, c) was observed with all complexes, exception for the first wash of the W-DNA complex (Table 3. Experi- ment B).

The results of the present study confirmed that the RAPD technique can be used for the amplification of bacterial DNA, even when bound on clay minerals. However, its applicability was influenced by the presence of clay particles, as it has been reported for humic substances [42,43]. Chromosomal DNA bound tightly on three clay minerals (W, Ap. and K) was amplified when the concentration of clay particles present in the clay-DNA complexes was decreased or the amount of Tuq DNA polymerase in the amplification mixture was increased. The degree of inhibition of amplification differed with the type of

clay mineral. Inhibition by W was greatest, as W- DNA complexes required a greater dilution for amplification than complexes with Ap and K, and there was no amplification in the presence of the supernatant from the equilibrium adsorption of DNA on W and the first wash of the W-DNA complex. The lack of amplification appeared to have been caused by inactivation of the Tuq DNA polymerase. probably as a result of its adsorption/binding on the clay particles and/or interference of the particles with its catalytic site. The conditions for the direct amplification of DNA in natural habitats by the RAPD technique must apparently be optimized, case-by-case, on the basis of the clay content of the habitat under study, to take advantage of the usefulness of the technique.

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Amplification of bacterial DNA bound on clay minerals by the random amplified polymorphic DNA (RAPD) technique

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