PROTOCOLS

DNA Extraction From Different Plant Species 137

A Fast, Simple, and Reliable High-Yielding Method

for DNA Extraction From Different Plant Species
Raul Tapia-Tussell,1 Andres Quijano-Ramayo,1 Rafael Rojas-Herrera,2
Alfonso Larque-Saavedra,1 and Daisy Perez-Brito1,*

Abstract
Genetic studies and pathogen detection in plants using molecular methods require the isolation of DNA
from a large number of samples in a short time span. A rapid and versatile protocol for extracting high-
quality DNA from different plant species is described. This method yields from 1 to 2 mg of DNA per gram
of tissue. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. A minimal presence of con-
taminating metabolites (as polymerase chain reaction [PCR] inhibitors) in samples and a considerable sav-
ings in reagents are characteristics of this protocol, as well as the low cost of the analysis per sample. The
quality of the DNA was suitable for PCR amplification.
Index Entries: Agavaceae; Cariacaceae; Cucurbitaceae; DNA isolation; Graminaeae; Palmae; PCR;
Rutaceae; Solanaceae; SSR.

1. Introduction DNA purification. Hence, the objective of this re-

The isolation of genomic DNA from different search was to develop a fast, reliable, and low-cost
plant species, suitable for polymerase chain re- protocol for DNA extraction suitable for PCR am-
action (PCR)-based molecular analysis, is diffi- plification from different plant species.
cult because of the high content of interfering
2. Materials
compounds in plant extracts, such as polysaccha-
rides, polyphenols, tannins (1). Although some 2.1. Plant Material
protocols have been reported for genomic DNA Foliar tissue from Nicotiana spp, Citrus auran-
isolation in many crops, most of them use large tium, Cucumis sativus (var. Pikkle), Carica papaya
quantities of fresh or lyophilized tissue (2,3), or (var. Maradol), Lycopersicum esculentum (var.
they do not allow the processing of large amounts Union), Capsicum chinense Jacq, Zea mays (var.
of samples in a short time span (4–6), or they are Naltel), Cocus nucifera, and Agave fourcroydes were
species specific (7,8). Furthermore, the presence taken from the Regional Botanical Garden at the
of PCR inhibitors in samples has been reported Centro de Investigación Científica de Yucatan,
frequently (9,10). Mérida, Mexico, and tuber tissue from Solanum
In a molecular biology laboratory that intends tuberosum was obtained from potatoes purchased
to provide external services, sample output is cru- from a local market. All samples were stored at
cial and it depends, in part, on methods used for –80°C, lyofilized, and ground for analysis.

*Author to whom all correspondence and reprint requests should be addressed. 1Laboratorio GeMBio, Centro de Investigación Científica de
Yucatán, Calle 43, #130, Col. Chuburná de Hidalgo, Mérida 97200, Yucatán, México. E-mail: [email protected]. 2Unidad Sureste, Centro de
Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A. Normalistas 800 S.H. Colinas de la Normal 44270, Guadalajara,
Jalisco, México.

Molecular Biotechnology  2005 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2005/31:2/137–140/$30.00

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 138 Tapia-Tussell et al.

Table 1
Quantitative Data
DNA conc. Amplicon size
Lane Species Family (µg/µL) A260/280 (bp)
2 Nicotiana spp. Solanaceae 0.445 1.78 180
3 Citrus aurantium L. Rutaceae 0.220 1.93 196
4 Cucumis sativus L Cucurbitaceae 1.335 1.80 171
5 Carica papaya L var. Maradol Caricaceae 1.535 1.80 164
6 Lycopersicon esculentum var. Unión Solanaceae 0.685 2.00 187
7 Capsicum chinense Jacq Solanaceae 1.260 1.60 196
8 Zea mays var. Naltel Graminaeae 0.890 1.80 183
9 Cocus nucifera Palmae 1.210 1.70 200
10 Solanum tuberosum Solanaceae 1.170 1.75 181
11 Agave fourcroydes Agavaceae 0.765 1.80 183

2.2. Reagents and Solutions with 500 µL of 70% ethanol. Let it dry at RT.
1. Extraction buffer: 100 mM Tris-HCI (pH 7.5), Resuspended the pellet in 50 µL of TE buffer.
700 mM NaCl, 50 mM EDTA (pH 8.0), 1% Quantity and quality of DNA prepared must be
hexadecyltrimethylammonium bromide (CTAB) determined according to protocols previously
(w/v), 140 mM β-mercaptoethanol. Added 16 µL described (11).
of RNase A, 10 mg/mL (SIGMA) per milliliter For PCR amplification, 20 ng of DNA was
of buffer just before use. used. Amplification primers and reaction condi-
2. TE buffer: 10 mM Tris-HCI (pH 8.0), 1mM tion was according to a previous report (12), but
EDTA (pH 8.0), 10 mM NaCl. using 0.25 µM of primers as a final concentration.
3. 10X TBE gel buffer: 0.9 M Tris-borate, 20 mM
The isolated DNAs were quantified spectropho-
EDTA.
tometrically and yields ranged from 1 to 2 mg/g of
4. Chloroform.
lyophilized tissue, whereas the A260/A280 ratio
5. Isopropanol (chilled).
6. Ethanol 70%. ranged from 1.6 to 2.0 (Table 1) indicating low
amounts of contaminating proteins. The addition
3. Methods of RNase A in extraction buffer eliminates pipetting
steps and thus reducing the time for analysis. This
1. Weigh 30–40 mg of ground and lyophilized tis-
purification method also yields high molecular
sue into a 2-mL microcentrifuge tube, added
weight DNA, as can be seen in Fig. 1.
1 mL of warm (65°C) extraction buffer and
To verify the suitability of DNA for PCR ampli-
mix thoroughly in a vortex.
2. Incubated at 65°C for 15 min, with occasional fication, two conserved primers, designed for
inversion. Cooled the mixture to room tem- simple sequence repeat (SSR) analysis in chloro-
perature (RT). Added 600 µL of chloroform plast, were used. DNAs were consistently amplified
and invert gently for 10 min. and amplification patterns were highly reproduc-
3. Centrifuged for 10 min at 15,682g at RT. ible. Furthermore, amplicon sizes matched those
Transferred top aqueous layer to a new 2-mL previously reported (12).
microcentrifuge tube and added 700 µL of In summary, the protocol described is a fast,
chilled isopropanol. Mixed by very gentle inver- simple, and reliable high-yielding method for
sion and then incubated for 10 min at –80°C (see DNA extraction from various plant species belong-
Note 1). ing to different families and can be used in labora-
4. Centrifuged for 5 min at 15,682g at RT. Dis- tories where large amounts of samples require
carded the supernatant and washed the pellet processing.

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 DNA Extraction From Different Plant Species 139

Fig. 1. DNA of the different plant species on 1% agarosa gel (upper panel) and SSR patterns obtained by using
primer ccmp 2 (lower panel). Lane 1: Molecular markers; lane 2: Nicotiana spp.; lane 3: C. aurantium; lane 4: C.
sativus; lane 5: C. papaya; lane 6: L. esculentum; lane 7: C. chinense; lane 8: Z. mays; lane 9: C. nucifera; lane
10: S. tuberosum; and lane 11: A. fourcroydes.

4. Notes 5. Saghai-Maroof, M. A., Soliman, K., Jorgensen, R. A.,

and Allard, R. W. (1984) Ribosomal DNA spacer-
1. As a result of the low amounts of DNA needed length polymorphisms in barley: Mendelian inherit-
for PCR amplification, the incubation at –80°C ance, chromosomal location, and population dynamics.
after isopropanol precipitation may be negli- PNAS 81, 8014–8018.
gible and can be avoided. 6. De la Cruz, M., Whitkus, R., and Mota-Bravo, L.
(1995) Tropical tree DNA isolation and amplifica-
Acknowledgments tion. Mol. Ecol. 4, 787–789.
7. Li, H., Luo, J., Hemphill, J. K., Wang, J., and Gould,
The authors are grateful to Alberto Cortez and J. H. (2001) A rapid and high yielding DNA miniprep
Angel Nexticapan from Centro de Investigación for cotton (Gossypium spp). Plant Mol. Biol. Rep. 19,
Científica de Yucatán A.C. for providing some of 183a–183e.
the samples used in this study. 8. Keb-Llanes, M., Gonz·lez, G., and Chi-Manzanero,
B. (2002) A rapid and simple method for small-scale
DNA extraction in Agavaceae and other tropical
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