Breeding Science 52 : 151-155 (2002)

Note

Rapid and Efficient DNA Extraction Method from Various Plant Species Using

Diatomaceous Earth and a Spin Filter

Junichi Tanaka*1) and Shigeru Ikeda2)

1) Breeding and Genetic Resources Team, Department of Tea Science, National Institute of Vegetable and Tea Science, 15451 Beppu,

Makurazaki, Kagoshima 898-0032, Japan2) Gene Research Center, Kagawa University, 2393 Ikenobe, Miki, Kagawa 761-0795, Japan

Key Words: DNA extraction, diatomaceous earth, phe-

nol extraction, chloroform extraction,

polysaccharide, polyphenol, DNA marker.

High quality DNA is necessary for genomic library

construction, Southern analysis, long-PCR and so on. The

CTAB method (Murray and Thompson 1980) is the most

popular technique for DNA extraction from plants, but it is

time-consuming and is not appropriate for DNA extraction

from polysaccharide- or polyphenol-rich plant species. Al-

though a modified method (Wagner et al. 1987) can be used

for DNA extraction from such species, it involves numerous

additional steps.

In addition, in marker-assisted breeding, an easy, rapid

and safe DNA extraction method is indispensable. Although

many DNA markers associated with agriculturally important

traits have been identified, to date there are few cases where

DNA marker-assisted breeding has been put into practice.

This is because screening based on phenotype is easier, more

familiar, less costly and safer than DNA marker detection,

which requires DNA extraction. Recently, some simplified

protocols have been published (Liu et al. 1995, Ikeda et al.

2000). These methods, however, cannot be used for DNA

extraction from plant species that have polysaccharide- or

polyphenol-rich tissues and hard organs, and in many cases

the extracted DNA is not of high quality and is unstable dur-

ing long-term storage.

Here, a method for rapid extraction of high-quality

DNA from plants is described. This method was designed

based on a plasmid preparation protocol from E. coli that

uses diatomaceous earth and a spin filter (Hansen et al.

1995, Kim and Pallaghy 1996) and a method for DNA ex-

traction from plants that does not use phenol or chloroform

(Marechal-Drouard and Guillemaut 1995). This method is as

follows. The first step requires grinding of the plant tissue.

The second step involves extraction in a buffer containing

sodium dodecyl sulfate (SDS) and digestion of RNA by

RNase. The third is deproteinization by using potassium ac-

etate. In the fourth step, the DNA is bound to diatomaceous

earth in a chaotropic buffer in a spin filter. In the fifth step,

the bound DNA is washed with a buffer containing ethanol.

Finally, the DNA is eluted from the diatomaceous earth.

This method has the following four advantages. (1) The

quality of the extracted DNA is high enough for PCR, re-

striction enzyme digestion, ligation reaction and long-term

preservation. (2) The procedure is simple and rapid, because

it does not require any centrifugation steps that pellet the

DNA. (3) The cost is as low as that of conventional plasmid

extraction kits. (4) No dangerous organic solvents such as

phenol or chloroform are used. In summary, this method will

not only provide a convenient, simple, rapid, low-cost and

safe DNA extraction method for molecular biological exper-

iments, but also make possible DNA marker-assisted breed-

ing with rapid and accurate DNA marker detection even in

some recalcitrant plant species.

Illustrative Protocol

Materials and Solutions

1) DNA extraction buffer

DNA extraction buffer contains 500 mM Tris-HCl

pH 8.0, 50 mM EDTA pH 8.0, 300 mM NaCl, 8 % (v/v)

SDS, 1 µg/ml of RNaseA and 0.2 % β-mercaptoethanol (op-

tional). The recipe for 100 ml of DNA extraction buffer is as

follows. Prepare sterile stock solutions of 1 M Tris-HCl

pH 8.0, 0.5 M EDTA and 5 M NaCl. These can be stored at

room temperature after autoclaving. Mix 50 ml of 1 M Tris-

HCl pH 8.0 with 10 ml of 0.5 M EDTA, and add 8 g of SDS.

After the SDS is dissolved, add 6 ml of 5 M NaCl and mix

completely. Bring the total volume to 100 ml with sterilized

water, and add 10 µl of 10 mg/ml solution of RNaseA. In or-

der to prevent discoloration caused by oxidation, β-mercapto-

ethanol can be added just before use.

2) 4 M potassium acetate pH 4.8

Dissolve 23.55 g of potassium acetate in sterilized wa-

ter to a volume of 66 ml. Add 28.5 ml of glacial acetic acid,

mix and titrate with about 1.5 ml of concentrated HCl to a

pH of 4.8. Bring the final volume to 100 ml with sterilized

water. If necessary, the solution can be sterilized by auto-

claving or filter sterilization. Store at room temperature.

Communicated by K. Harada

Received August 27, 2001. Accepted January 7, 2002.

*Corresponding author (e-mail: tanajun@affrc.go.jp)

Tanaka and Ikeda152

3) 8 M guanidine hydrochloride solution

Dissolve 76.4 g of guanidine hydrochloride in distilled

water and bring the final volume to 100 ml. 8 M guanidine

hydrochloride solution (Wako 071-02891, Osaka) is a con-

venient commercial preparation. Guanidine hydrochloride is

poisonous, and should be handled with caution. It must not

be autoclaved. Store at 4°C.

4) Spin filter

Quantum Prep Mini Spin Filters (BioRad 732-6027,

Hercules CA) can be used. Store at room temperature.

5) Diatomaceous earth emulsion

Quantum Prep Matrix (BioRad 732-6110, Hercules

CA) is a useful form of diatomaceous earth. An emulsion of

diatomaceous earth can be prepared according to the proto-

col of Kim and Pallaghy (1996). Store at room temperature.

Resuspend completely just prior to use.

6) Washing buffer

Washing buffer contains 50 % ethanol, 20 mM Tris-

HCl, 0.4 M NaCl. Quantum Prep Wash Buffer (BioRad 732-

6024, Hercules CA) mixed with ethanol can be used. Store at

room temperature.

7) TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA)

Quantum Prep Miniprep Kit (BioRad 732-6100, Her-

cules CA) includes 4) Quantum Prep Mini Spin Filters, 5)

Quantum Prep Matrix and 6) Quantum Prep Wash Buffer.

Procedure

1) Grind 50-200 mg of plant tissue into a fine powder us-

ing a mortar and a pestle under liquid nitrogen. If the

quality of extracted DNA is not critical, grinding under

liquid nitrogen is not necessary. Grind samples at room

temperature with the extraction buffer.

2) Add the powdered plant tissue to 500 µl of extraction

buffer at 65°C in a microcentrifuge tube.

3) Incubate for 15 minutes at 65°C. After incubation, the

sample can be preserved for at least one year at −20°C.

4) Centrifuge at 12,000 × g for 10 minutes at room tem-

perature.

5) Transfer the upper clear layer (about 350 µl) to a clean

microcentrifuge tube. Recover the upper layer without

disturbing the lower layer. If the volume of the upper

layer is very small, additional buffer should be used to

re-extract.

6) Add 0.45 volumes of 4 M potassium acetate pH 4.8,

and mix gently.

7) Incubate on ice for 30 minutes.

8) Centrifuge at 12,000 × g for 20 minutes at 4°C.

9) Transfer the upper clear layer (about 400 µl) to a spin

filter set in a 2 ml microcentrifuge tube. Recover the

upper layer without disturbing the lower layer. If the

volume of the upper layer is very small, additional buff-

er should be used to re-extract.

10) Add 0.6 volumes of 8 M guanidine hydrochloride solu-

tion and mix.

11) Add 0.4 volumes of diatomaceous earth emulsion and

mix.

12) Centrifuge the 2 ml microcentrifuge tube at 12,000 × g

for 30 seconds at room temperature to remove the liq-

uid.

13) Remove the spin filter from the 2 ml microcentrifuge

tube and discard the waste fluid. After that, set the spin

filter back into the same 2 ml-tube, add 500 µl of wash-

ing buffer onto the filter, and centrifuge at 12,000 × g

for 2 minutes at room temperature. After a few minutes

of centrifugation, if some solution remains on the spin

filter, remove it with a pipet. Then, centrifuge again for

another 5 minutes, and proceed to the next step.

14) Repeat step 13), but increase the centrifugation period

to 2 minutes. Repeat until the diatomaceous earth be-

comes colorless.

15) Transfer the spin filter to a clean 1.5 ml microcentri-

fuge tube.

16) Add 100 µl of TE or sterilized water at 65°C, and mix

gently to re-suspend diatomaceous earth completely.

17) Centrifuge the 1.5 ml microcentrifuge tube containing

the spin filter at 12,000 × g for 1 minute at room tem-

perature, and recover the eluted DNA solution.

Depending on the samples, steps 4) to 6) can be substi-

tuted with one step as follows; add 0.45 volumes of 4 M po-

tassium acetate pH 4.8 and mix completely. This shortcut

has been found to be efficient in tea, yellow camellia, camel-

lia, rice and sorghum. However, this shortcut was not suc-

cessful with leaves of soybean and clover.

Quality and Amount of Extracted DNA

DNA samples were extracted from the leaves and roots

of tea (Camellia sinensis) and rice (Oryza sativa) of OD260/

OD280 ratios between 1.70 to 1.82, indicating that small

amounts of contaminants were present. The yield of extract-

ed DNA from 200 mg of tea leaves (flesh weight) typically

ranged between 7 and 30 µg. The extracted DNA could be

used for long-PCR, restriction digestion, ligation reaction af-

ter restriction digestion and PCR-based DNA marker detec-

tion (Fig. 1a, b, c). Moreover, even after incubation for one

month at room temperature, the extracted tea DNA could be

used as a template for Random Amplified Polymorphic

DNA (RAPD) analysis (Williams et al. 1990) (Fig. 1d).

DNA extraction trials with leaves from 48 plant species

including many that are recalcitrant were performed. The

DNA samples could be extracted from 44 species and used

as templates for RAPD PCR. The four exceptions were

strawberry (Fragaria × ananassa), feijoa (Feijoa sellowiana),

Japanese yew (Podocarpus macrophyllus) and kiwi fruit

(Actinidia deliciosa) (Table 1). Figure 2 shows the results

of the electrophoresis of extracted DNA and products of

RAPD analysis. The Breeding and Genetic Resources Team,

Department of Tea Science, National Institute of Vegetable

and Tea Science, has used a modified CTAB method for

tea (Tanaka et al. 2001). The DNA of yellow camellia

(Camellia crysantha) or camellia (C. japonica) could not

DNA extraction method from plants using diatomaceous earth and spin filter 153

be extracted by a modified CTAB method for tea or by

Marechal-Drouard and Guillemaut’s method without phenol

and chloroform for plants (data not shown), but the method

presented here resulted in good DNA preparations.

The Breeding and Genetic Resources Team, Depart-

ment of Tea Science, National Institute of Vegetable and

Tea Science, in a DNA marker-assisted breeding of tea

study, was able to extract nearly one thousand DNA samples

using this method, almost all of which could be used for

PCR-based DNA marker detection.

Application to Breeding

In general, it is required 2-3 days to extract high-quality

DNA from tea leaves using a modified CTAB method. In

particular, dissolving pellets after each centrifugation step

required several hours. In this regard, the DNA extraction

method described here does not include any centrifugation

or DNA re-dissolving steps. With this method, DNA marker

information can be obtained on the same day as DNA extrac-

tion even in recalcitrant plant species like tea.

This DNA extraction method will enable to conduct

molecular biological experiments, as well as rapid, high-

Fig. 1. Quality of extracted DNA samples using diatomaceous earth and a spin filter from tea

leaves.

M: DNA size marker (Mixture of l/HindIII digest and fX174/HincII digest)

a: Results of electrophoresis of long-PCR products using the extracted DNA as a template.

1: amplified fragments (5.6 and 5.0 kb) of dihydroflavonol 4-reductase gene including the

introns, 2: amplified fragment (2.7 kb) of sequence-tagged site.

b: Results of electrophoresis of the extracted DNA digested with restriction enzymes. 1:

HincII, 2: Sau3AI.

c: HincII or Sau3AI digest of the extracted DNA was ligated with HincII or Sau3AI adapt-

er, respectively. Results of electrophoresis of PCR products using the ligation products of

HincII (lane 1) or Sau3AI (lane 2) adapter as a template.

d: Effect of incubation on the quality of the extracted DNAs. Results of electrophoresis of

products of RAPD analysis of 8 different individuals (1-8) in a segregating population be-

fore or after incubation of the extracted DNAs for one month at room temperature.

Tanaka and Ikeda154

throughput DNA marker detection experiments for practica-

ble DNA marker-assisted breeding even in recalcitrant plant

species.

Acknowledgments

The authors thank Mrs. M. Iwata, Assistant, Breeding

and Genetic Resources Team for her technical assistance,

Mr. S. Matsumoto, Department of Physiology and Quality

Science, National Institute of Vegetable and Tea Science,

Table 1. Performance of the DNA extraction method using diatomaceous earth and a spin filter

Scientific name Common name

Plant species to which this method could be successfully applied

Equisetum arvence field horsetail

Athyrium niponicum Japanese painted fern

Cycas revoluta Japanese sago palm

Ginkgo biloba ginkgo

Laurus nobilis laurel

Camellia sinensis tea

Camellia crysantha yellow camellia

Camellia japonica camellia

Brassica oleracea broccoli

Eruca sativa arugula

Lobularia maritima sweet alyssum

Luffa cylindrica sponge gourd

Hydrangea macrophylla hydrangea

Prunus persica peach

Prunus mume Japanese apricot

Trifolium repens clover

Glycine max soybean

Citrus natsudaidai natsumikan

Oxalis corniculata yellow oxalis

Sapium sebiferum Chinese tallow tree

Ficus benjamina benjamin fig

Portulaca oleracea summer purslane

Ipomoea batatas sweet potato

Pharbitis nil morning glory

Quamoclit pennata star-glory

Phlox subulata moss phlox

Lycopersicon esculentum tomato

Ocimum basilicum sweet basil

Perilla frutescens perilla

Coffea arabica coffee

Tagetes patula marigold

Artemisia princeps mugwort

Erigeron canadensis horseweed

Solidago altissima tall goldenrod

Commelina communis common spiderwort

Musa cross paradisiaca banana

Crocosmia crocosmiiflora montbretia

Chlorophytum comosum spider plant

Epipremunum aureum golden pothos

Cymbidium goeringii

Oryza sativa rice

Sorghum vulgare sorghum

Digitaria ciliaris

Miscanthus floridulus giant silver grass

Plant species to which this method could not be applied

Feijoa sellowiana feijoa

Podocarpus macrophyllus Japanese yew

Fragaria × ananassa strawberry

Actinidia deliciosa kiwi fruit

DNA extraction method from plants using diatomaceous earth and spin filter 155

for informing us of Marechal-Drouard and Guillemaut’s

method (which was useful for the development of our meth-

od) and Dr. Y. Takeda, head of the Breeding and Genetic

Resources Team for the critical review of the manuscript.

This work was supported by a grant from the Ministry of

Agriculture, Forestry and Fisheries of Japan (Rice Genome

Project DM-2107).

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Fig. 2. Results of electrophoresis of DNA extracted from plant species using diatomaceous

earth and a spin filter (upper row) and RAPD PCR products generated with the ex-

tracted DNA (bottom row). M: DNA size marker (upper row: l/HindIII digest, bot-

tom row: fX174/HincII digest), 1: field horsetail (Equisetum arvence), 2: Japanese

painted fern (Athyrium niponicum), 3: Japanese sago palm (Cycas revoluta), 4: laurel

(Laurus nobilis), 5: tea (Camellia sinensis), 6: yellow camellia (Camellia crysantha),

7: camellia (Camellia japonica), 8: broccoli (Brassica oleracea), 9: hydrangea

(Hydrangea macrophylla), 10: peach (Prunus persica), 11: clover (Trifolium repens),

12: soybean (Glycine max), 13: natsumikan (Citrus natsudaidai), 14: Chinese tallow

tree (Sapium sebiferum), 15: morning glory (Pharbitis nil), 16: star-glory (Quamoclit

pennata), 17: moss phlox (Phlox subulata), 18: tomato (Lycopersicon esculentum),

19: coffee (Coffea arabica), 20: marigold (Tagetes patula), 21: montbretia (Cro-

cosmia crocosmiiflora), 22: rice (Oryza sativa), 23: sorghum (Sorghum vulgare).