Introducing students to DNA: Identifying nutritional plants in a simple tried and tested laboratory experiment

Authors


Abstract

In this paper we report on a practical experiment, containing isolation, purification, and characterization of plant DNA, which is designed to give students unexperienced in DNA handling and molecular biology a glimpse of the field. Based on well established procedures, genomic DNA of a nutritional crop is isolated, and a non-coding region of chloroplast DNA is amplified by use of PCR with universal primers. The origin of the sample is revealed by the size of the amplified fragment and in case of indistinguishable fragment sizes of wheat and barley by selective digestion with a suitable restriction endonuclease.

It is an ongoing challenge for those involved in teaching laboratory courses to design experiments that awake students interest and at the same time allow thorough evaluation of the experimental results.

The experiment described here was set up for the biochemistry course of fourth year chemistry students well trained in synthetic chemistry, both organic and inorganic, and physical chemistry but without any previous experience in handling of the small quantities, in terms of both molarity and weight, usually under investigation in biochemistry or molecular biology labs. There are several points we especially had to focus on. 1) No previous experience should be required. 2) It should be possible to successfully complete the experiments within one full or two half-days. 3) Costs should be kept at a minimum; no special equipment beyond standard biochemistry labs should be required. 4) The experiment should be robust, regarding both the procedures themselves and their reproducibility. 5) The use of kits should be avoided as they are, although an indispensable tool in research labs, of very limited didactic value. 6) To attract maximum attention, the origin of the sample investigated should be unknown to the students, thus at the same time giving them a problem to solve and making copying of results impossible.

EXPERIMENTAL PROCEDURES

Instructor Preparations—

Barley (Hordeum vulgare), wheat (Triticum aestivum), maize (Zea mays), and oats (Avena sativa) are grown from commercial seeds to an age of about 4 weeks. Leaves are harvested, cut into small pieces of about 1 cm2 to prevent visual identification, and portions of ∼1 g are immediately frozen and kept at −80 °C.

Buffer solutions were prepared according to Dellaporta [1] and Sambrook etal. [2] as follows: 1 × EB buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8, 50 mM (sterile) NaCl, 10 mM dithiothreitol), 5 × TE buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 7.5), 1 × TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), 1 × TBE buffer (10 mM Tris borate, pH 7.5, 1 mM EDTA) (for agarose gel), 4 × DNA gel loading buffer (50% glycerol, 0.5 mM TBE buffer, 0.2% SDS, 0.1 M EDTA, 0.2% bromphenol blue).

DNA Isolation—

DNA isolation is carried out according to the procedure of Dellaporta [1] as follows: 1 g of frozen leaves is powdered with the aid of liquid N2, mortar, and pestle. 7.5 ml of EB-buffer is added, and the mixture is allowed to thaw. 1 ml of SDS solution (10%) is added, and the mixture is incubated for 15 min at 65 °C. After adding 2.5 ml of KOAc solution (5 M) and mixing, the preparation is kept in an ice bath for 30 min. The suspension is centrifuged (8500 × g, 20 min) and filtered through cheesecloth into a 15-ml tube already containing 5 ml of cold 2-propanol. Filtrate and 2-propanol are mixed, and the solution is centrifuged (5000 × g, 30 min, 4 °C). The supernatant is decanted carefully, and the grayish-white pellet formed is allowed to dry in air for about 30 min. (Fig. 1).

RNA Digestion—

Contaminating RNA is digested according to standard protocols [2]. The pellet is dissolved in 900 μl of 5 × TE buffer, 8 μl of RNase-solution (10 mg/ml) are added, and the mixture is incubated for 90 min at 37 °C. The solution is divided into two 1.5-ml Eppendorf vials, and 450 μl of extraction solution (phenol:chloroform:amyl alcohol = 25:24:1) are added in the hood. After mixing the solution by gently inverting the vials several times, it is centrifuged for 1 min in a standard table top centrifuge. The upper (aqueous) layer is transferred to another vial with the aid of a Pasteur pipette, and the extraction step is repeated twice, the last time with pure chloroform. Sodium acetate solution (3 M, 1/10th of the estimated volume of the aqueous layer) and ethanol (twice the estimated volume of the aqueous layer) are added, and the DNA is allowed to precipitate for at least 2 h at −20 °C. The vials are centrifuged at 12,000 × g for 10 min and decanted carefully, and the DNA pellet is allowed to dry in air for 15 min. Purified DNA is finally dissolved in buffer or double distilled water, and the DNA concentration is determined photometrically.

PCR Analysis—

Amplification by PCR of the desired non-coding region of chloroplast DNA is carried out according to standard protocols, employing a pair of universal primers [2, 3]. The PCR mixture is as follows: 5 μl of DNA preparation (10–100 ng), 5 μl of sense primer (100 pmol), 5 μl of antisense primer (100 pmol), 16 μl of dNTP mixture (1.25 mM each dNTP), 10 μl of PCR buffer (10 × ), 56.5 μl of double distilled water, 2.5 μl of Pwo polymerase (2.5 units). The thermocycler program is as follows: 92 °C, 3 min (92 °C, 1 min; 47 °C, 1 min, 72 °C, 1 min); 25 cycles; 72 °C, 10 min.

Agarose Minigel—

15 μl of the PCR preparation are analyzed in a 2% agarose minigel [2] after staining with ethidium bromide. Results for barley, oats, maize, and wheat are depicted in Fig. 1.

Restriction Digest—

In case both barley and wheat are to be included in the experiment, the amplified fragments cannot be differentiated on a 2% minigel and have therefore to be identified by an additional restriction digest experiment. Upon treatment with DdeI, the fragment originating from barley is cut whereas the one from wheat is not thus enabling a clear separation on a 2% minigel (Fig. 2). The digestion mixture is as follows: 15 μl of the respective PCR mixtures are mixed with 0.5 μl (5 units) of DdeI, 2 μl of 10 × buffer NE4, and 2.5 μl of water and incubated for 2 h at 37 °C. The products are analyzed on a 2% agarose minigel.

MATERIALS AND METHODS

Chemicals—

Chemicals used are as follows: ethanol, chloroform, isopropanol (high pressure liquid chromatography grade), and agarose for DNA electrophoresis were from Sigma-Aldrich; liquid nitrogen; primers according to literature [3] were from MWG-Biotech AG, Ebersberg, Germany; dNTP solution, DNA size markers were from PEQLAB Biotechnologie GmbH, Erlangen, Germany.

Enzymes—

Enzyme buffers were used as supplied by the manufacturer. RNase, Pwo DNA polymerase, and DdeI restriction endonuclease were from Roche Molecular Biochemicals.

Hardware—

Water bath, tabletop centrifuge, thermocycler, DNA-calculator, refrigerable centrifuge, balance, agarose-electrophoresis set (minigel), microwave oven, UV-lamp (306 nm), Gel documentation system (optional), UV-protection, cheesecloth was from Merck-Eurolab GmbH, 64293 Darmstadt, Germany.

Feedback, Typical Problems, and Study Questions—

The experiment described, which has been carried out as a part of the 4th year biochemistry course for three years in a row, is generally welcomed by our chemistry students as instructive and interesting. Typical errors were accidental loss of DNA pellet (which can be replaced with an isolate previously made by the supervisor) and smeared bands in the gel (usually still good enough for identification when a reference mixture is given). On the average, about four of five teams (groups of two) have so far been able to fully and successfully complete the experiment. Regarding study questions it has been beneficial, apart from the concept of the polymerase chain reaction, to work out the chemistry and molecular structures behind DNA synthesis. The main purpose is to make it clear that nothing but synthetic organic chemistry is taking place in the test tube, albeit with enzymes, nature's catalysts, which are highly efficient both in molecular recognition and reaction turnover.

Figure FIGURE 1..

Products of PCR amplification of non-coding regions of chloroplast DNA of wheat, maize, oats, and barley by use of universal primers according to Taberlet et al. [3]. Lane 1, all four products; lane 2, wheat; lane 3, maize; lane 4, oats; lane 5, barley; lane 6, 0.1–10-kb molecular weight markers.

Figure FIGURE 2..

Restriction digest of PCR products of wheat and barley with Dde I.Lane 1, wheat + DdeI; lane 2, wheat − DdeI; lane 3, barley + DdeI; lane 4, barley − DdeI; lane 5, 0.1–10-kb molecular weight markers.

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