Plant Molecular Biology Reporter 17: 1–8, 1999.

© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

Publish by Abstract

An Improved and Rapid Protocol for the Isolation of

Polysaccharide- and Polyphenol-Free Sugarcane DNA

S. M. ALJANABI∗, L. FORGET and A. DOOKUN

Biotechnology Department, Mauritius Sugar Industry Research Institute, Reduit, Mauritius

Abstract. We have optimized a simple and rapid method for isolating milligram quantities
of high quality DNA from polysaccharide- and polyphenolic- rich tissue such as sugarcane,
lettuce and strawberry. The protocol utilizes fresh tissue without making use of liquid ni-
trogen or freeze-drying for initial grinding of the tissue and it significantly minimizes the
use of lab materials. At least one hundred samples can be processed daily by one person.
The isolated DNA is essentially free of polysaccharides, polyphenols, RNA and other major
contaminants, as judged by: its clear color, its viscosity, A260/A280 ratio, digestibility with
restriction enzymes, and suitability for Restriction Fragment Length Polymorphism (RFLP)-
and Polymerase Chain Reaction (PCR)- based techniques.

Key words: DNA extraction, polyphenols, polysaccharide, RAPD, Saccharum, sugarcane

Introduction

Sugarcane is one of the leading crops in the world, producing 70% of the
world’s sugar, which amounted to 126 M tons in 1997–1998. This production
level closely matches world sugar consumption for the same period. Any
step that can add to the understanding of sugarcane genome organization and
function will assist in increasing sugar production worldwide.
Like many other plant species, sugarcane tissues contain high levels of
polysaccharides and polyphenolic compounds, which present a major con-
tamination problem in the purification of plant DNA. When cells are dis-
rupted, these cytoplasmic compounds can come into contact with nuclei and
other organelles (Loomis, 1974). In their oxidized forms, polyphenols co-
valently bind to DNA giving it a brown color and making it useless for
most research applications (Katterman and Shattuck, 1983; Guillemaut and

∗ Author for correspondence. e-mail: biotechn@msiri.intnet.mu; fax: 230-454-1971;

ph: 230-454-1061.
2 S. M. Aljanabi, L. Forget and A. Dookun

Maréchal-Drouard, 1992; our observations). One method commonly used to

avoid problems with polyphenols involves freezing the tissue during or prior
to homogenization (Katterman and Shattuck, 1983; Leutwiler et al., 1984).
The presence of these compounds renders studies difficult due to long and
tedious extraction procedures and often does not result in good standards in
terms of yield and quality.
The need for a rapid and efficient procedure for plants having high polysac-
charides and polyphenols is necessary when hundreds of samples need to be
analyzed rapidly, such as in genome mapping and marker assisted selection
(MAS) programs. High purity DNA is required for PCR and restriction-based
techniques such as random amplified polymorphic DNA (RAPD), for mi-
crosatellite, RFLP and amplified fragment length polymorphism (AFLP), for
genome mapping and DNA fingerprinting.
Several protocols have been described for plants containing high amounts
of polyphenols and polysaccharides including extraction of DNA from iso-
lated nuclei (Hamilton et al., 1972) and purification by cesium chloride fol-
lowing the classic plant DNA protocol (Murray and Thompson, 1980) using
liquid nitrogen. Although these methods yield DNA of high purity, they are
tedious and require expensive reagents and equipment such as high-speed or
ultracentrifuges. Also, liquid nitrogen may be difficult to obtain in certain
regions of the world where most species of Saccharum are found. A rapid
DNA extraction method for sugarcane and its relatives (Honeycutt et al.,
1992) and recently, a modification of a CTAB DNA extraction protocol for
plants containing high polysaccharide and/or polyphenol components (Poreb-
ski et al., 1997; Peterson et al., 1997), have been published. These methods
give a low yield of DNA and the procedures are relatively long, thereby
limiting the number of samples that can be processed per day. In addition,
expensive reagents and tedious processes were used in these protocols, such
as RNase, spermidine, proteinase K and the filtration of the ground tissue
through cheesecloth and/or mira-cloth.
In our research, we need large quantities of highly pure sugarcane genomic
DNA for genome mapping and marker-assisted selection (MAS). After trying
published protocols and failing to obtain DNA that was not contaminated
with polysaccharides and polyphenolic compounds, we have optimized an
alternative rapid method that yields polysaccharide and polyphenol-free high
quality genomic DNA from the meristem cylinder of sugarcane. It also pro-
duced high quality DNA from mature fresh leaves of strawberry and lettuce.
These tissues contain high levels of polysaccharides and polyphenols and
were included for comparison.
Sugarcane DNA extraction 3

Materials and methods

Five hundred sugarcane hybrids, their parents, five strawberry and five lettuce
plants were used for DNA extraction. Plant samples were transferred from
the field to the laboratory in plastic bags without the need to place them on
ice and they were processed on the same day. A modification of the Doyle
and Doyle (1990) CTAB extraction procedure was adopted, which uses a
higher CTAB and NaCl concentration to remove polysaccharides (Lodhe,
et al., 1995), thereby preventing their interaction with DNA. PVP and sodium
sulfite were also added to prevent the oxidation of phenolic compounds that
results in brown colored DNA (Loomis, 1974). All the chemicals used in this
experiment were from Sigma Chemical Co., St. Louis MO, USA.

Solutions

Homogenization buffer: 200 mM Tris-HCl, 50 mM EDTA, 2.2 M NaCl, 2%

CTAB, 0.06% sodium sulfite1 , pH 8.0
phenol:chloroform:isoamyl alcohol (25:24:1)
6 M NaCl
10% polyvinylpyrrolidone (PVP)
5% N-lauroyl-sarcosine
20% CTAB

Protocol

• Slice about 10 cm (2–4 g) of sugarcane fresh meristem cylinder (after

removing outer leaf sheaths) and place in 50 mL Falcon tubes. Similarly,
slice fresh lettuce and strawberry leaves to about 10 mm2 and place in
Falcon tubes.
• Homogenize the fresh tissue with homogenization buffer (4 mL/g fresh
tissue) using an Ultra-Turax homogenizer with a large dispersing ele-
ment, for a few seconds2 .
• Add 2 mL of 5% N-lauroyl-sarcosine, 2 mL of 10% PVP3 and 2 mL of
20% CTAB. Mix well by inversion.
• Incubate for 30–60 min at 65 ◦ C in a water bath.
• Mix samples by inversion 3–4 times during incubation.
• Take samples from the water bath and cool down to room temperature.
• Add an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol,
mix by inversion, then centrifuge the tubes at 3000 g for 10 min at 4 ◦ C.
• Recover aqueous phase4 and transfer to a fresh tube.
• Add an equal volume of isopropanol followed by 2 mL of 6 M NaCl.
• Incubate at −20 ◦ C for at least 1 h.
4 S. M. Aljanabi, L. Forget and A. Dookun

Figure 1. Agarose gel electrophoresis of total uncut and Hind III digested genomic
DNA isolated as described in materials and methods. Five to ten µg of sugarcane hy-
brids (Saccharum ssp., lanes 2–5), strawberry (Fragaria sp., lanes 6–7) and lettuce (Lactuca
sativa. Lanes 8–9) were loaded in each lane. Lane 1, 100 bp molecular weight marker XIV
(Boehringer Mannheim).

• Fish out the DNA with a glass pipette and transfer to a fresh tube con-
taining 10 mL of 70% ethanol. Alternatively, spin down the DNA pellet
briefly, wash with 10 mL of 70% ethanol, air dry, then resuspend in 2–
3 mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Notes
1. Prepare just before use.
2. Longer homogenization time is not recommended as it can result in partial degraded
DNA.
3. PVP adsorbs polyphenols thereby preventing their interaction with DNA (Loomis, 1974);
sodium sulfite inhibits oxidation of polyphenols.
4. Avoid disturbing the interface while pipetting the aqueous phase.

Results and Discussion

The protocol we used is a modification of Doyle and Doyle (1987) where

fresh leaf tissue is homogenized in an extraction buffer without making use
of liquid nitrogen or filtration of the homogenate through cheese cloth or
mira-cloth. In addition no RNase treatment is required, as RNA seems to be
degraded during extraction. This significantly reduces sample handling time
and wasting of laboratory materials, especially when hundreds of samples
need to be processed. Two 50 mL Falcon tubes per sample are needed to
finish the extraction of the DNA.
Sugarcane DNA extraction 5

To check the DNA quality, 5–10 µg of uncut and Hind III digested ge-
nomic DNA from each sample were run on a 1% agarose gel as shown in
Figure 1. There was neither RNA contamination nor any sign of degraded
DNA in all samples. There was no RNase treatment of any DNA sample
when the first batch of samples showed only traces of RNA or none at all
on the agarose gel. It seems that RNA is degraded during the process of
the extraction. Compounds that adsorb polyphenols or prevent oxidation re-
actions inhibit the interaction of genomic DNA with oxidized polyphenols,
which affects DNA quality. The purity of the DNA was determined from
the A260/A280 ratio, which averaged 1.76–1.96 in all samples. The yield of
DNA ranged from 0.5 to 0.8 mg/g of fresh weight. This amount of DNA is
sufficient to perform hundreds of RFLP experiments, which is still the method
of choice in many laboratories, and thousands of PCR-based techniques such
as RAPD, AFLPTM and simple sequence repeats (SSR) analyses. Figure 1
also shows that the DNA was completely digested with Hind III. Equivalent
results were obtained with the 3 other enzymes and all other sugarcane DNA
samples (data not shown).
The digested DNA was subjected to Southern blot analyses using the
non-radioactive Dig-labeling system (Boehringer Mannheim, Germany). The
quality of the Southern blot analysis using the sugarcane DNA extracted by
the above method is evident in Figure 2.
RAPD analysis of genomic DNA was performed, according to Williams
et al. (1990), using 10–30 ng genomic DNA, 200 µM each dNTP, 2 units of
Stoffel fragment Taq polymerase (Perkin-Elmer USA), 1× Taq polymerase
reaction buffer (supplied by the manufacturer) and 0.22 µM 10-mer random
primer (Operon Technologies, USA). Two hundred and forty primers were
screened against the parent’s DNA and the ones that detected polymorphisms
were selected for screening of the segregating population. Figure 3 shows an
example of RAPD analyses of sugarcane DNA, which indicates that the DNA
extracted by our method is suitable for PCR-based techniques.
Unlike other protocols mentioned above, our protocol does not make use
of expensive chemicals or equipment. Homogenization of fresh tissue was
carried out inside a 50 mL Falcon tube while other procedures used a mortar
and pestle to grind fresh tissues in the presence of liquid nitrogen. A sterile
mortar needs to be used for each sample to prevent cross contamination. This
becomes impractical when hundreds of samples need to be processed.
Unlike other DNA extraction protocols, our method is consistent and pro-
duces large amounts of high quality DNA necessary for studies requiring
relatively large quantities of DNA, such as RFLP. The extracted DNA was
stable and could be amplified by PCR both before and after 12 months of
storage at 4 ◦ C.
6 S. M. Aljanabi, L. Forget and A. Dookun

Figure 2. Southern blot analysis of sugarcane genomic DNA using the non-radioactive
Dig-labeling system. Ten µg of Hind III digested genomic DNA of the parents (R570 and
M555/60) and their progeny (M24-1 to M24-17) were loaded on an 0.8% agarose gel then
transferred to a nylon membrane and hybridized with the probe CDSR29.

Acknowledgements

The authors would like to thank Dr L. J. C. Autrey and Dr S. Saumtally for

critical review of the manuscript. Part of this work was funded by the Tertiary
Education commission.
Sugarcane DNA extraction 7

Figure 3. RAPD fingerprints of genomic DNAs from 48 sugarcane hybrids using primers
OPB-18 (50 -CCA CAG CAG T-30 ) and 2 units of Stoffel fragment Taq DNA polymerase.
The amplification program included an initial denaturation step of 94 ◦ C for 3 min followed
by 39 cycles of 94 ◦ C, 1 min; 35 ◦ C, 1 min and 72 ◦ C, 2 min. The last cycle was followed by
5 min incubation at 72 ◦ C and a hold at 15 ◦ C. The amplification products were resolved on
a 2% agarose gel dissolved in 0.5× TBE, stained with ethidium bromide and visualized under
UV light. M, molecular weight marker X (Boehringer Mannheim).

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