I. Overview

Isolation of DNA from mycorrhiza poses a formidable task. Protocols must be designed to accommodate small amounts of starting material, since the dry weight of an individual mycorrhiza may be only a few mg. Pooling samples from several mycorrhiza may suffice to achieve sufficient dry weight, but these samples may contain DNA from more than one mycobiont. We present procedures for isolating DNA from one mycorrhizal root tip. However, to extract enough DNA to produce an RFLP (see Section IV.D), one needs 10-15 mg fresh weight (2-3 mycorrhizal root tips). Protocols must permit removal of DNA from the complex chemicals associated with the host and soil (e.g. polysaccharides and tannins). Plant contaminants can impede extraction of high molecular weight DNA (Murray and Thompson, 1980) and interfere with restriction nucleases. Polysaccharide-like contaminants can lead to incomplete digestion of DNA by restriction endonucleases and can cause anomalous reassociation kinetics (Merlo and Kemp, 1976).

2. Methods

The CTAB method of Rogers et al. (1989) (Section II.A.2.) was designed to extract DNA from field samples of mycorrhiza. We modified the CTAB procedure of Armstrong et al. (1989) (Section II.A.2.) to extract DNA from mycorrhiza receiving minimal treatment to remove adhering soil. This protocol is a modification of the method developed by Wagner et al. (1987) and Saghai-Maroof et al. (1984), who also based their methods on Murray and Thompson (1980).

(a) Modified CTAB procedure for mycorrhiza:

1. Harvest mycorrhiza from soil. Gently shake mycorrhiza to remove adhering soil, or wash gently with sterile distilled water if soil is fine. Store frozen in capped, glass vials.

2. While keeping sample frozen, grind 15-200 mg fresh weight in the glass vial with a plastic pestle (Pellet Pestle™, Kontes, Vineland, NJ, USA). Prevent thawing of tissue by periodically immersing vial in liquid nitrogen during grinding.

3. Immediately add powdered sample to 1 ml ice-cold extraction buffer [50 mM Tris-HCl (pH 8.0), 50 nw EDTA, 0.5% 2-mercaptoethanol (v/v), 0.1% bovine serum albumin (BSA) 0.35 m sorbitol, 10% polyethylene glycol 4000, 0.1% spermine tetrachloride, 0.1% spermidine trihydrochloride, 0.1 m diethyldi-thiocarbamic acid (DEDTC), 0.1 m PVP; buffer must be refrigerated and used within one week of preparation]. Homogenize for 1 min with a mechanical tissue homogenizer, especially if the mycorrhiza are thick or woody. If minimum capacity of available homogenizer is more than 1 ml, scale up the volume and quantity of tissue.

4. Filter through one layer of fine-mesh nylon cloth (e.g. Mira-Cloth™, Calbiochem Corp., La Jolla, CA, USA) and collect in a plastic or siliconized glass tube (Sambrook et al., 1989). Rinse cloth with 500 /A of extraction buffer.

6. Discard supernatant. Resuspend pellet in 500 fA ice-cold wash buffer [50 mM Tris-HCl (pH 8.0), 50 mM EDTA, 0.5% 2-mercaptoethanol, 0.1m DEDTC, 0.1m PVP, 0.35 m sorbitol; buffer must be used within one week of preparation, refrigerate]. Transfer to a microfuge tube. Keep on ice.

7. Add 100 ¡A 5% n-lauroyl sarcosine and shake vigorously. If doing multiple samples, keep them on ice until n-lauroyl sarcosine is added to all samples.

8. Incubate at room temperature for 10 min.

10. Add 60 /¿I CTAB solution [8.6% CTAB, 0.7 m NaCl], and shake vigorously.

12. Add 1.5 volume of chloroform/octanol (24:1), and shake vigorously.

13. Centrifuge (10 min, 25 ± 2 °C, maximum speed in a microfuge).

14. Transfer upper phase to a new microfuge tube.

15. Concentrate by alcohol precipitation (Section III. D).

16. Centrifuge (5 min, 25 ± 2 °C, 11000 g; or for 5 min at maximum speed in a microfuge).

17. Dry nucleic acid pellet (under vacuum or with lyophilizer) and resuspend in TE. Purify further according to procedures described in Section III.

Porteous and Armstrong (1991) developed a bulk DNA extraction method which yields up to 200 /xg DNA per gram of soil. The procedure has not been used with mycorrhiza. It offers promise as a means to reduce chemical constituents associated with field samples, but must be tested to determine its suitability as an additional procedure for mycorrhiza.

(b) Procedure of Porteous and Armstrong (1991):

1. Add 6 ml mixing buffer per gram of sieved soil [mixing buffer: 0.5 m sorbitol, 15% polyethylene glycol 4000, 2% DEDTC, 100 mm EDTA, 50 dim Tris-HCl (pH 8.0); prepare just prior to use]. Mix with a tube mixer for 1 min at room temperature.

2 Add 500 mg polyvinylpolypyrrolidone (PVPP) and mix with a tube mixer.

3. Add 100 fi\ lysozyme solution [50mgml-1; prepared just prior to use] and 120/¿I Novozym 234 (Novo Biolabs, Bagsvaerd, Denmark) solution [50mgml_1; prepared just prior to use]. Mix with a tube mixer for 15 s at room temperature, and incubate on ice for 1-2 h.

4. Add 3.8 ml lysis buffer [4% SDS, 100 mM EDTA, 500 /¿g proteinase Kml-1, 50 mM Tris-HCl (pH 8.0); prepared just prior to use], mix by slowly inverting and return to ice for 16 h.

5. Centrifuge (5 min, 4 ± 2 °C, 5000 g) to separate nucleic acids (aqueous phase) from PVPP, humic compounds, soil particles and other debris. Store aqueous phase in sterile tube on ice.

6. Add 3 ml wash buffer [50 mM Tris-HCl (pH 8.0), 100 mM EDTA] to pellet, invert, mix with a tube mixer for 2 s, then centrifuge and pool aqueous phase with aqueous phase from Step 5.

7. Repeat Step 6.

8. Centrifuge (5 min, 4 ± 2 °C, 15 000 g) and transfer supernatant to sterile tube and hold at room temperature.

9. Add 5 m potassium acetate to a final concentration of 0.5 m, incubate on ice for 1-2 h.

11. Add 2 volumes 95% ethanol (room temperature), mix and centrifuge (10 min, 10-15 °C, 15000 g).

12. Dry nucleic acid pellet (under vacuum or with lyophilizer) and resuspend in TE. Purify further according to procedures described in Section III.

The lysis procedures we describe for mycorrhizal fungi and mycorrhiza do not depend on cell wall-degrading enzymes (e.g. chitinase, cellulase, hemicellulase; Peberdy, 1989). Researchers may want to add these enzymes to improve cell lysis. However, the available enzyme preparations are typically contaminated with nucleases which degrade DNA after its release from cells. The nuclease activity can be inhibited with an increased EDTA concentration (e.g. 100 mM EDTA) in the extraction buffer, as was done by Porteous and Armstrong. These investigators (Porteous and Armstrong, 1990) also used pulverized glass (Gene-clean®; Section III.F) to separate soil chemicals from the partially purified DNA at Step 10.

C. Comments

Depending on the type of sample and use of the DNA, additional procedures may be required to increase DNA purity. Some uses generally may not require highly purified DNA (e.g. amplification by the polymerase chain reaction; Innis et al., 1990), so the DNA may not need extensive purification. However, endonuclease activity increases as DNA purity increases. The high concentration of phenolics in some species of fungi (e.g. Pisolithus tinctorius and Paxillus involutus) may require purification using CsCl density gradient ultracentrifugation (Section III.E.) or another procedure (Section III.F.) before the DNA is usable. Katterman and Shattuck (1983) overcame the effects of secondary host and soil-borne chemicals while extracting DNA from nuclei of Gossypium samples from the field by including a high concentration of glucose in the maceration buffer (citrate buffer, Triton X-100, and 1 m glucose). The glucose was a reducing agent to suppress formation or activity of oxidized phenolic groups.

As cells lyse and DNA is released, an increase in solution viscosity is indicative of the presence of DNA. The optimal buffer-.tissue ratio permits the suspension to be mixed to homogeneity (visual check) by gentle shaking. If the suspension does not move freely in the tube during the shaking, add more buffer. Our CTAB procedure (Section II.B.2.) yields high amounts of DNA when the first CTAB extraction buffer:tissue ratio is 10-20 /nlmg-1 dry wt tissue. Some experimentation may be needed to find the optimum buffer:tissue ratio for various types of mycorrhiza. Generally, from 10 to more than 500 mg powder can be extracted as long as the buffer:tissue ratio is maintained. Depending on the use of the extracted DNA, it can be treated with RNase and proteinase K at various steps during the extraction procedure (see Sections III.B and III.C).

Many polysaccharides are insoluble at the salt concentrations used in the CTAB procedures to precipitate nucleic acids, possibly requiring additional effort to eliminate these contaminants if DNA is isolated from host and mycobiont. Differential precipitation with high acetate salts selectively removes many carbohydrates with a high pectin content. When isolating DNA from small amounts of tissue (i.e. expected DNA yield is approximately lOngml-1), the alcohol precipitation can be done at lower temperatures (~80°C) for longer times (overnight), or a carrier nucleic acid (e.g. 25 /tig tRNAmg-1 dry wt tissue) can be added during alcohol precipitation.

The requirement of sorbitol in the extraction and wash buffers of our CTAB procedure to isolate total DNA from mycorrhiza has not been investigated. The original Wagner et al. (1987) and Saghai-Maroof et al. (1984) protocols used sorbitol as an osmotic stabilizer to isolate chloro-plast DNA from needles of coniferous species.

Additional protocols utilizing CTAB for isolating DNA from filamentous fungi have been developed by Jahnke and Bahnweg (1986) and Manicom et al. (1987). Readers may consider the work of Verma (1988) who studied Neurospora crassa and used an alternative way to lyse cells with guanidine hydrochloride and guanidinium isothiocyanate.

III. Techniques to purify DNA

A. Overview

After cell lysis and deproteinization by extraction with phenol, pheno-chloroform, isoamyl alcohol or chloroform (e.g. Step 3 in Leach et al. procedure, Section II.A.3.), the DNA should be essentially protein-free. The absence of a denatured protein layer at the aqueous-organic interface is diagnostic of protein-free DNA. This can be achieved after repeated deproteinizations, followed by dialysis (Sam-brook et al., 1989) to remove the chemicals. Other options to purify DNA are CsCl density gradient ultracentrifugation (Section III.E), Geneclean® (Section III.F.) and chromatography (Malliaros, 1988; Mer-ion, 1988, 1989). Digestion with ribonuclease (RNase) is a convenient means to obtain RNA-free DNA.

During the cell lysis procedures, DNA is not degraded as the constituents of the lysis solutions inactivate nucleases. However, once isolated, DNA is susceptible to degradation by nucleases associated with the skin. In general, use of sterile glassware and solutions and aseptic handling of the DNA extract is recommended. Gloves should be worn whenever one expects to touch surfaces which will contact the extract (e.g. during preparation of dialysis tubing).

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