DNA Isolation Protocol for Seaweeds

YOLAINE JOUBERT and JOEL FLEURENCE*

Laboratoire Ecophysiologie Marine Intégrée, Faculté des Sciences et Techniques, 2 rue de la

Houssinière, 44072 Nantes cedex 03, France

Abstract. We report a DNA isolation protocol for red seaweeds. Recovering DNA of high quality
and quantity is a prerequisite for ensuring suitable applications, such as polymerase chain reaction
(PCR), restriction fragment length polymorphism (RFLP) analysis, and sequencing. Isolation of DNA
from seaweeds has proven difficult because of coprecipitation of polysaccharides. Our process
minimizes this contamination, which is mostly due to the highly hydrocolloidal content of algal
cell walls. This protocol, using 2 steps, is based on a preliminary enzymatic digestion of cell wall
with specific enzymes (Novozymes) followed by centrifugation, allowing isolation of DNA on the
pellet. This provides a higher yield of DNA, in the range of 40 µg (Palmaria palmata) and 18 µg
(Gracilaria verrucosa) from 50 mg of fresh frozen pellet.

Full text†: This article, in detail, is available only in the electronic version of the Plant Molecular
Biology Reporter.

Key words: DNA extraction, enzymatic digestion, Gracilaria verrucosa, ITS rDNA gene, Palmaria
palmata, RFLP Abbreviations: CTAB, cetyltrimethylammonium bromide; DEAE, diethylaminoethyl;
ITS, internal transcribed spacer; RFLP, restriction fragment length polymorphism; SDS, sodium
dodecyl sulfate.

Introduction

DNA Isolation Protocol for Seaweeds Joubert and Fleurence

Isolation of DNA from land plants and seaweeds is difficult because of their cellulosic walls and
abundant polysaccharide content, which differs among species. The liberation of such compounds
during cell lysis leads to highly viscous supernatants, the main source of DNA contamination.
Inhibition of Taq DNA polymerase activity by such extracts from different species was reported
(Fang et al., 1992; Jin et al., 1997). Several protocols have been used: lysis by Sarkosyl and sodium
dodecyl sulfate (SDS) (Sambrook et al., 1989), cetyltrimethylammonium bromide (CTAB) extraction
(Doyle and Doyle, 1987), lithium chloride (Hong et al., 1997), CsCl gradient ultracentrifugation
(Goff and Coleman, 1988), hydroxyapatite column purification (Dutcher et al., 1990),
diethylaminoethyl (DEAE) purification (Sharma and Anjaiah, 2000), cellulose agarose gel
electrophoresis purification (Saunders, 1993), and pectinase (Rogstad et al., 2001; Rether et al.,
1993). We report a genomic DNA isolation protocol registered as Fleurence and Joubert (2004). To
minimize polysaccharide contamination, we used enzymatic digestion, which modifies the
substances so that they are removed more easily after cell lysis. The solution was centrifuged,
allowing a clean pellet without polysaccharide and cellulose contamination to be obtained. DNA
extraction was achieved from 50 mg of frozen pellet. This protocol was applied with success to
red seaweeds Palmaria palmata and Gracilaria verrucosa and to diatoms. No signs of degradation
were observed after agarose gel electrophoresis, and PCR amplification and restriction fragment
length polymorphism (RFLP) analysis were successful. In addition, this protocol enhances the DNA
yield.
Materials and Methods

P. palmata specimens were collected from the french Brittany coast at Belle-Ile island; G. verrucosa
specimens were collected from the Tunisian lagoon at Tunis. Epiphytes and sand were removed by
successive washings with seawater and distilled water. The algae were then frozen and kept at –
20° C.

Equipment

• Water bath with shaking

• 15-mL tubes, 20-mL Nalgene tubes, 2- and 1.5-mL Eppendorf tubes

• High-speed refrigerated centrifuge

• Spectrophotometer

Chemicals, solutions, and buffer

• Enzymatic digestion buffer: 50 mM sodium acetate (pH 5) stocked at 4° C

• Enzymes from Novozymes: Ultraflow L (β-glucuronidase), Finizym 250L (β-glucanase), Shearzyme

500L (xylanase), Celluclast 1.5L (cellulase); from Sigma: agarase from Pseudomonas atlantica

• Stock DNA extraction buffer (pH 8): 100 mM Tris HCl, 100 mM NaCl, 10 mM EDTA; autoclave
and store at 4°C

• SDS solution, 20% w/v in water; store at room temperature

• Sarcosyl, 10% w/v in water; store at room temperature

• Proteinase K, 20 mg/mL of deionized water; store at –20° C

• Phenol-chloroform-isoamylalcohol (pH 7.7)

• Isopropanol

• 70% ethanol

• H2O, molecular grade

• Agarose

• Tris-acetate-EDTA 0.5× buffer

• Ethidium bromide

• Long-wave UV light

Protocol

• Defrost and remove samples from ice water.

• Cut 2 g of seaweed plant material.

• Add 12.5 mL of enzymatic digestion buffer.

• Add enzymes at various concentrations.

• Incubate for 6 h in a water bath at 40° C with shaking.

• Centrifuge at 28,600g for 30 min at 4° C.

• Transfer supernatant to a new tube.

• Frost the pellet at –20° C.

• Transfer 50 mg of pellet in a 2-mL Eppendorf tube.

• Add 2 mL of stock DNA extraction buffer.

• Incubate for 2 h in a water bath with shaking.

• Separate into 2-mL Eppendorf tube.

• Add 1 mL of phenol-chloroform-isoamylalcohol.

• Mix by inversion to form an emulsion.

• Centrifuge at 20,800g for 15 min.

• Carefully pipet the upper aqueous phase (700 µL) to a new Eppendorf tube.

• Add 700 µL of isopropanol at room temperature and mix by inversion for 3 min.

• Spin at 20,800g for 15 min.

• Discard isopropanol.

• Wash pellet twice with cold 70% ethanol.

• Spin at 13,000 rpm at 4° C.

• Dry pellet at 37° C in a thermobloc for 10 min.

• Redissolve the DNA in distilled water molecular grade.

• Allow enough time for complete resuspension.

• Calculate the DNA concentration by measuring absorbance at 260 nm and purity by measuring
absorbance at 280 nm.

Notes

1. Small pieces were obtained by cutting samples with a blade.

2. The nature of enzymes varied in regard to their cellular walls: For P. palmata, we use a mixture
of Shearzyme (24 U/g) and Celluclast (24 U/g); for G. verrucosa, a mixture of Finizym (30 U/g),
Celluclast (30 U/g), and agarose (50 U/g).

3. Supernatant is usually viscous and must be strictly removed from the pellet.

4. Vigorously invert the tubes for 3 min.

5. A large amount of protein is precipitated as salt complexes. Avoid taking this phase.

6. Resuspension of genomic DNA is difficult. To enhance this procedure, water should be warmed
to 50°C before being added to DNA. Avoid vortexing genomic DNA because it could be broken.
The solution is to place in a water bath at the same temperature for 30 min.

Amplification of internal transcribed spacer (ITS1 and ITS2) sequences

The 5.8s rDNA and flanking ITS1 and ITS2 sequences were amplified from total DNA. For both
seaweeds, oligonucleotides were complementary to the 3′ end of the small subunit 18s rDNA (ITS
forward) and to the 5′ end of the large subunit 28s rDNA (ITS reverse). For P. palmata, the
oligonucleotides used were as follows: ITS forward, 5′-GGGATCCGTTTCCGTAGGTGAACCTGC-3′; ITS
reverse, 5′ GGGATCCATATGCTTAAGTTCAGCGGGT-3′. For G. verrucosa the oligonucleotides were as
follows: ITS forward, 5′-GTGAACCTGCGGAAGGATC-3′; ITS reverse, 5′-
GGGATCCATATGCTTAAGTTCAGCGGGT-3′. PCR reactions were done in a PTC100 thermocycler (MJ
Research) with Qiagen PCR buffer and Taq DNA polymerase in the presence of Q solution. Q
solution is recommended for amplification of templates that are GC-rich or that have extensive
secondary structure. A DNA band was evident under long-wave UV light and was cut from the
gel. Agarose was digested and DNA purified according to the instructions from Qiagen (QIAquick
Gel Extraction Kit).

RFLP analysis

Nucleotide sequencing of the amplified regions was done, and theoretical restriction maps were
obtained with Infobiogen software, permitting the choice of restriction enzymes that would
discriminate each species. PCR fragments (200 ng) were digested at 37° C for 2 h with 1 U of
restriction enzymes from Boehringer (AluI, 20 U/µL; EcoRI, 12 U/µL) in a final volume of 20 µL.
Products of the digestion were visualized on 1.5% agarose gels.

Results

About 800-900 µg of DNA is obtained from 1 g of pellet from P. palmata pellet and 300-400 µg
from G. verrucosa. The ratio of absorbance at 260 to that at 280 nm (A260/280) of the DNA
ranged from 1.8-1.9 for P. palmata and 1.7-1.8 for G. verrucosa. Figures 1 and 2 show results with
DNA extracted according to our protocol: PCR-amplified ITS rDNA and RFLP fragments from P.
palmata and G. verrucosa, respectively. The DNA was completely digestible with 0.5 U of AluI for P.
palmata and EcoRI for G. verrucosa. The DNA of both species wassequenced, with a mean length
of 910 bp for P. palmata (Figure 2) and 1104 bp for G. verrucosa (Figure 1).

Discussion

In an attempt to isolate pure nuclear DNA from red seaweeds, we tried several published DNA
isolation protocols. Most of them use a CTAB extraction technique, which derives from an earlier
publication from Doyle and Doyle (1987). In our case, such a protocol produces coprecipitation of
polysaccharides with the DNA; this DNA is not of suitable quality for PCR amplification. We
describe an efficient protocol for DNA extraction from 2 species of red seaweeds, P. palmata and
G. verrucosa. The enzymatic digestion of cell wall was useful for depolymerizing polysaccharide
contaminants into smaller molecules that are more easily released from the pellet following
centrifugation. An enzymatic process was reported by Rogstad et al. (2001); however, this process
differs from our protocol. Firstly, by using the typical CTAB extraction protocol, Rogstad and
colleagues obtained a contaminated viscous DNA solution. Secondly, they incubate this DNA
solution with a pectinase solution overnight or longer, until the DNA is watery (nonviscous). This
protocol requires more steps and is more timeconsuming than our protocol. Routine yields on a
per-gram basis (wet weight of cells) from P. palmata (800 µg) and G. verrucosa (300 µg) were
compared with those obtained from red algae. Our yields appear higher than those reported by
Wattier et al. (2000) and Antoine and Fleurence (2003) for the dry mass of these seaweeds.
Therefore, expression of the DNA quantity in regard to dry weight could be multiplied by a factor
of 5. Purity of DNA was monitored by the A260/280 ratio. For P. palmata, our value (1.8-1.9) is
higher than that reported by Antoine and Fleurence (2003). For G. verrucosa, the value is in the
same range. For both species, the DNA was of suitable quality for PCR amplification. Sequencing
of ITS1, 5.8 rRNA gene, and ITS2 fragments was successful and was submitted to NCBI BLAST
software (GenBank). The sequences obtained for P. palmata were aligned with the sequence
Y11506 and for G. verrucosa with the sequence GVU21342. No differences have been noted
between theoretical restriction map (lengths of restriction fragments were 122, 602, and 282 for G.
verrucosa and 138, 603, and 281 for P. palmata) and experimental electrophoresis of RFLP
fragments.