Plant Molecular Biology Reporter pages 143-148 14 (2) 1996

Protocol

A Simple Method of DNA Extraction from Soil for Detection of Composite Transgenic Plants by

PCR

Dieter Ernst, Evi Kiefer, Alain Drouet, and HeiHrich Sandermam~, Jr.

E-mail: ernst@gsf.de (DE, EK, HS) GSF-Institut fi_ir Biochemisclne Pflanzenpathologie, D-85758

Oberschleil?heim, Germany (AD) Universit6 d'Orl6ans, Laboratoire de Biologie des Ligneux Forestiers,

BP6749 45067, Orl6ans Cedex 2, France

Keywords: DNA extraction, PCR, soil, transgene

Abstract: This new and simple method of DNA extraction from composite soil allows the isolation of plant DNA with high efficiency, quality and reprod uctiviO,. The method is based on a simple CaCl,-precipitation step and requires no additional purification steps to eliminate humic acids. The extracted DNA was obtained in sufficient purity and quantity to allow direct detection of transgenes bv PCR. Furthermore, the simple procedure allows the assay of many samples at ttqe same time.

F or an adequate assessment of introducing transgenic plants into the environment, information is required on the persistence and transfer of the genetic material (Casper and Landsmann, 1992, and

articles cited therein). It has been shown that plasmid DNA persisted over several weeks in different soil samples (Romanowski et al., 1992). Polymerase chain reaction (PCR) technology has been shown to be a very sensitive method to detect traces of DNA. In soil samples, however, the coextraction of humic acids and metal ions frequently resulted in the inhibition of Taq polymerase, the key enzyme of PCR (Tsai and Olsen, 1992; Tebbe and Vahjen, 1993). Purification protocols are often time consuming and have been used with varying degrees of success (Ogram et al., 1987; Jacobsen and Rasmussen, 1992; Romanowski et al., 1992; Tsai

144 Ernst et al.

and Olsen, 1992b; Smalla et al., 1993; Tebbe and Vahjen, 1993; Young et al., 1993). The logical consequence was to develop a simple DNA extrac- tion procedure that also allows one to remove PCR-interfering sub- stances.

In the present article, we describe a simple direct DNA extraction procedure applicable to composite soil samples. A single CaC12 precipi- tation step efficiently removed humic acids, and a subsequent dilution step resulted in DNA that was suitable for PCR amplification.

Materials

Soil material Soil samples were taken randomly from a field in Olching, Bayern, where transgenic maize were growing. Phophinothricin-resistant maize plants, containing pat, the gene encoding phosphinothricin acetyl transferase, under the control of the 35S promoter (Wohlleben et al., 1988; Donn et al., 1992), were grown during sun~rner 1994. Composite soil samples were collected in autumn 1994 and spring 1995. Controls were taken from parcels where no transgenic maize was growing. Soils do not require pre- treatments, such as drying, grinding, sieving and lysis.

Extraction buffer: 120 mM Na2HPO 4, pH 6.8; 5% SDS (w/v); PVPP; 1-3% CaC12 (w/v)

DNA precipitation buffer: 5 M NaC1; 50% PEG 8000 lxTE buffer: 10 mM Tris-HC1, 1 mM EDTA, pH 8.0 tris-saturated phenol, pH 8.0 chloroform/isoamylalcohol 24:1 (v/v) ethanol, 70%(v/v) 5 M sodium acetate, pH 5.2

Procedures

D N A extraction �9 Mix I g of composite soil with 2 mL of 120 mM Na2HPO 4 (pH 6.8),

then add immediately 0.4 mL 5% SDS (w/v) and 0.04 g PVPP. �9 Precipitate humic acids by the addition of CaC12 to a final concen-

tration of 2 % (w/v). Incubate the mixture for I h at 65 ~ in a water bath with occasional stirring, and then centrifuged at 8,000 g for 20 min at 10 ~

�9 For DNA precipitation, add NaC1 to a final concentration of 500 mM and then add 0.5 volume of 50 % (w/v) PEG 8000. Incubate overnight at 4~

Detection of Transgenic DNA in Soil 145

�9 After centrifugation at 6,000 g for 30 min at 4 ~ dissolve the resulting pellet in 0.5 mL lxTE. Extract with 0.5 volume of Tris- saturated phenol and then with 1 volume of chloroform/isoamyl alcohol, and precipitate the DNA with 2 volumes of ethanol and 0.1 volume of 5 M sodium acetate.

�9 After centrifugation at 16,000 g for 30 min, wash the pellet with 70% ethanol, centrifuge again, and then dissolve the pellet in water (10 to 50 ~tL).

PCR For PCR, two gene-specific primers (23- and 25- mer) were designed. PCR amplification of a 606-bp fragment, including the pat coding region was performed in total volume of 50 ~tL. Reaction solutions contained 2.5 U of Taq polymerase (Perkin Elmer) and lxPCR buffer (Perkin Elmer). Primer and dNTP-concentrations were 0.2 and 100 ~tM, respectively. The final MgC12 concentration was increased to 3.75 mM (Tebbe and Vahjen, 1993). The extracted soil DNA was diluted with water (1:50, v /v ) and 10 I-tL were used for PCR. DNA was denatured at 92 ~ for 2 rain, and primers were annealed at 56 ~ for 2 min. Extension was at 72 ~ for 2 min during 30 cycles, terminated at 72 ~ for 10 rain. PCR products were analyzed by horizontal gel electrophoresis in 1% agarose, followed by ethidium-bromide staining of the gels.

Resul t s and D i s c u s s i o n

Contamination of extracted soil DNA with humic acids is a serious problem for PCR amplification (Tsai and Olsen, 1992; Tebbe and Vahjen, 1993). Humic acids were precipitated by CaCI_~ at concentrations higher than 1% (w/v). However, CaCI~ concentration higher than 4 % resulted in DNA precipitation. Consequently, to eliminate humic acid contamina- tion of extracted soil DNA, a CaCL concentration within a range of 1 to 3 % (w/v) should be used. Under our experimental conditions 2 % CaCL turned out to be the best concentration, resulting in a clear supernatant. DNA preparations contained 2 to 20 I-tg DNA per g of extracted soil. Fig. 1 shows amplifications of pat after different dilutions steps of the extracted soil DNA. A single band at 606 bp clearly demonstrates the good quality of the extracted DNA used for PCR amplification. The transgenic DNA was detectable in composite samples randomly taken from the field. Even seven months after maize had been ploughed into the soil, pat could still be detected (Fig. 1). This is a longer period than the two months found for ploughed transgenic petunia plants (Becker et al., 1993).

146 Erns t et al.

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Fig.1. Agarose gel electrophoresis of a PCR-amplified pat sequence from total DNA extracted from soil. m~ DNA, reaction mixture without DNA; 1994, composite samples from autumn l~tq4 used at indicated dilutions; 1995 2.1r composite sample from sprin~ 1~)c)5 at; 2-10-'dilution; co,tirol DNA, soil sample from a control field without transgenic maize at 2. l t) 2 dilution; 4.5 H,e, pat, reaction mixture containing 4.5 ng of the Fal plasmid. PCR products were visualized by staining with ethidium bromide. Arrows mark the position of the expected PCiR product of 000 bp, as estimated with DNA molecular weight marker 111 from Boehringer.

The removal of launlic acids b \ a simple CaCl~-precipitation step early ill tile DNA extraction p rocedure obviates the requi rement in other procedures for further purification, which are oftent imes consuming and expensive: ion-exchange or gel permeat ion ch roma tog raphy (Tsai and Olson, 1992; Picard et al., 1992;Tebbe and Vahjen, 1993; Leung et aI. 1995), dialysis (Romanowski et al., 1902), glass milk (Smalla et al., 1993; Leung et al., 1995), PVP-agarose gel electrophoresis (Young et al., 1993), or CsCI gradient centr ifugat ion (Ogram et al., 1987; Jacobsen et al., 1992) fol- lowed by hydroxyapa t i te ch roma tog raphy (Ogram et al., 1987). Addi- tion of PVPP to the extraction buffer resulted in the elimination of interfering phenolic compounds (Picard et al., 1992; Romanowski et al., 1992; Young et al., 1993).

Detection o fTransgen ic D N A in Soil 147

N o PCR p r o d u c t s cou ld be obse rved w h e n u n d i l u t e d D N A extracts were used (Fig. 1). This m i g h t be due to the pers i s tance of subs t ances that interfere wi th the Taq p o l y m e r a s e (Tsai a nd Olsen, 1992) and pe r tu rb PCR. To r educe the concen t r a t i on of these subs tances , the extracts were rou t ine ly d i lu ted p r io r to PCR at a ratio of of l:50, wh ich tu rned ou t to be best (Fig. 1). MgC12, h o w e v e r , had to be ad jus ted to 3.75 m M to obta in op t ima l a m o u n t s of ampl i f ied p r o d u c t s (Tebbe and Vahjen, 1993).

The D N A extrac t ion p r o c e d u r e desc r ibed here s h o u l d a l low the de tec t ion of o ther genet ica l ly eng inee red c o m p o s i t e p lan t D N A s f rom soil samples . The in te r fe r ing effect of h u m i c acids in PCR-ampl i f i ed D N A detec t ion was efficiently e l imina ted by one CaCI : p rec ip i ta t ion step. This p r o c e d u r e is s imple and fast and can be rou t ine ly app l ied for D N A extrac t ion f rom mul t ip le soil s amples at the same time.

Acknowledgments : We thank W. Heller for stimulating discussions during the preparation of the manuscript, and H. Eckev-Kaltenbach and D. G~irtner for critically reading the manuscript. This work lnas been supported by the Bayerische Forschungsstiftung.

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