A SIMPLE AND RAPID TECHNIQUE FOR THE ISOLATION OF DNA FROM MICROALGAE 1

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A SIMPLE AND RAPID TECHNIQUE FOR THE ISOLATION OF DNA
FROM MICROALGAE
1
Marvin W. Fawley
2
and Karen P. Fawley
Department of Biological Sciences, North Dakota State University, Fargo, North Dakota 58105, USA
A simple method for the purification of PCR-
quality DNA from microalgae is presented. This
method uses the detergent dodecyltrimethylammo-
nium bromide coupled with cell breakage by
agitation in the presence of glass beads and chloro-
form. A final purification step involves a commer-
cial cartridge system. The procedure requires only
about 1–2 mL of algal culture and can be completed
in about 20 min. DNA suitable for PCR has been
obtained from several algal lineages using this
method, including numerous green algae and
stramenopiles.
Key index words: DNA isolation; microalgae
Abbreviations: CTAB, hexadecyltrimethylammo-
nium bromide; DTAB, dodecyltrimethylammonium
bromide
Diversity studies of microalgae require the purifica-
tion of PCR-ready DNA from a large number of
isolates. In our ongoing investigation of the diversity of
chlorophyte microalgae, we are purifying DNA from
over 1000 isolates. With such a large number of
isolates, it is essential that the method used for DNA
purification is rapid, inexpensive, requires little cellular
material, results in DNA that can be used directly for
PCR, and is effective across a broad range of algal
lineages. Moreover, the use of highly toxic material
such as phenol should be avoided, and the procedure
should be simple enough for undergraduate students
to perform.
DNA purification techniques that are commonly
used for microalgae are mostly based on the hexade-
cyltrimethylammonium bromide (CTAB) method of
Doyle and Doyle (1990), which requires grinding a
fairly large quantity of cells in liquid nitrogen. Several
modifications of the basic CTAB method have been
published, but they are generally fairly time consum-
ing. At the other extreme, some studies have foregone
the isolation step altogether and instead have used
whole cells in the PCR (Hoham et al. 2002), a process
that can only work for some organisms.
Some coccoid microalgae, such as Chlorella vulgaris,
can present special problems for DNA isolation,
primarily because of the mechanical strength of their
cell walls coupled with their very small size. A technique
useful for some coccoid algae was developed by Friedl
(1995). We previously modified this technique to make
it somewhat simpler (Fawley et al. 1999, Phillips and
Fawley 2000). This technique used an initial extraction
with the detergent dodecyltrimethylammonium bro-
mide (DTAB) with cell disruption using a MiniBead-
Beater (Biospec Products, Bartlesville, OK, USA), a
chloroform extraction, followed by precipitation using
CTAB in low salt solution, and a final ethanol pre-
cipitation. Very little starting material was required for
this procedure, and several samples could be processed
simultaneously. However, several steps were used and
required about an hour to complete. We also found
that results with this technique were not consistent for
many algal isolates (unpublished observations), al-
though we were able to purify PCR-quality DNA from
isolates from the Chlorophyceae, Trebouxiophyceae,
and Prasinophyceae using this technique (Fawley et al.
1999, 2000, Phillips and Fawley 2000). This technique
has also been successfully used for at least some strepto-
phytes (Turmel et al. 2002).
Our new technique takes some of the procedures
from Friedl’s (1995) method and our (Fawley et al.
1999, Phillips and Fawley 2000) modification and
replaces the precipitation steps with a single step using
a standard DNA purification spin cartridge. The result
is a DNA purification procedure that typically requires
only 1.5–2mL of algal culture, works with a wide range
of algal lineages, and takes about 20 min to complete.
Multiple samples can be processed simultaneously, and
the process is simple enough that students learn it in
only one session. The resulting DNA is suitable for PCR
with a variety of primers and provides enough DNA for
at least 30 25-mL reactions.
DNA purification procedure. One to 2 mL of algal
culture was centrifuged in a conical-bottom 2-mL
screw-top microcentrifuge tube at approximately
16,000 gfor 1 min. The supernatant was discarded
and 200 mL extraction buffer (1 M NaCl, 70 mM Tris,
30 mM Na
2
EDTA, pH 8.6) added and vortexed
briefly. This suspension was then centrifuged at
16,000 gfor 1 min, the supernatant discarded, and
200 mL fresh extraction buffer added. A quantity of
glass beads (G-8772, Sigma Chemical Co., St. Louis,
1
Received 30 April 2003. Accepted 24 October 2003.
2
Author for correspondence: e-mail marvin.fawley@ndsu.nodak.edu.
223
J. Phycol. 40, 223–225 (2004)
r2004 Phycological Society of America
DOI: 10.1046/j.1529-8817.2004.03081.x

MO, USA) sufficient to fill the conical portion of the
centrifuge tube was then added, followed by 25 mL
10% DTAB (Sigma Chemical Co.) and 200 mL chloro-
form. A MiniBeadBeater was then used to disrupt the
cells, with agitation for 20 s at top speed. The mixture
was then centrifuged at 2000 gfor 2 min to separate
the phases. If the cells were not well broken, as
evidenced by a cell layer at the interface of the
aqueous and organic phases and a lack of green
pigment in the organic phase, the MiniBeadBeater
step and centrifugation were repeated. The Mini-
BeadBeater step was never repeated more than once.
One hundred microliters of the aqueous phase was
removed to a 1.5-mL microcentrifuge tube, 500 mL
GeneClean salt (QBiogene, Carlsbad, CA, USA) was
added and mixed, and the resulting solution was
applied to a GeneClean Turbo (QBiogene) cartridge.
The cartridge was then used to purify the DNA
according the manufacturer’s instructions.
Test organisms. We used this DNA purification
technique with a wide range of organisms, including
green algae from the Chlorophyceae, Trebouxiophy-
ceae, Pedinophyceae, and Prasinophyceae; xantho-
phytes; eustigmatophytes; a red alga; a brown alga;
and diatoms. Most of these organisms are our own
isolates from our diversity studies, but we also tested
several organisms from culture collections. Table 1
presents a partial list of organisms that have been
examined. These organisms were grown using
standard methods for our laboratory (Fawley et al.
1990, Fawley 1992).
PCR. Universal primers specific for 18S rDNA
were used to evaluate the DNA purification proce-
dure. Primers and conditions were essentially those of
Phillips and Fawley (2000) or Fawley et al. (1999,
2000), except that the Qiagen Master Mix PCR Kit
was used (Qiagen, Valencia, CA, USA).
The 18S rDNA was successfully amplified using
template DNA isolated from all the test organisms by
the new procedure. Figure 1 shows an agarose gel of
the amplification products from a sampling of these
organisms. We also used this technique successfully
with over 300 additional isolates from our present
diversity studies. Additional loci, including rbcL and the
ribosomal internal transcribed spacer regions, were
also PCR amplified from many isolates using template
DNA purified with this procedure (data not shown).
The procedure also works with some organisms (Porph-
yridium and Ectocarpus) that possess polysaccharides
that can inhibit PCR amplification (Kitade et al. 1996).
Although Porphyridium DNA can be isolated using a
simple procedure that includes a standard kit (Yoon
et al. 2002), existing protocols for DNA isolation from
the Ectocarpales are quite involved (Siemer et al.
1998). The largest PCR product we obtained using
template DNA isolated with this procedure was about
4.3 kb, the 18S rDNA from a Choricystis sp. that
possessed multiple putative group I introns. However,
amplification of larger fragments may also be possible.
FIG. 1. Agarose gel electrophoresis of 18S rDNA PCR
products from select algal isolates (see Table 1). Sizes (in base
pairs) of pertinent markers from the 1-kb DNA standard (Life
Technologies, Rockville, MD, USA) indicated to the left.
TABLE 1. Algal isolates used to evaluate the new DNA
purification system.
Taxon Isolate
Eustigmatophyceae
Nannochloropsis limnetica Krienitz KR1998/3
Bacillariophyceae
Nitzschia palea (Ku
¨tz.) W. Smith FDCC L996
Nitzschia sigma (Ku
¨tz.) W. Smith FDCC L1546
Phaeophyceae
Ectocarpus siliculosus (Dillw.) Lyng. UTEX LB2008
Xanthophyceae
Gloeobotrys sp. Tow 6/3 P-16w
Chlorophyceae
Chlamydomonas reinhardtii Dang. CGC1691
Pseudodictyosphaerium sp. CCMP2217
Scenedesmus sp.
Monoraphidium sp. Itas 9/21 14-7w
Trebouxiophyceae Tow 8/18 P-2w
Chlorella vulgaris Beij. UTEX 30
Choricystis sp. CCMP2207
Nannochoris sp. CCMP2225
Oocystis sp. CCMP2249
Pedinophyceae
Pedinomonas minor Korsh. UTEX 1350
Prasinophyceae
Nephroselmis pyriformis (Carter) Ettl. CCMP717
Prasinococcus capsulatus Miyashita
et Chihara
CCMP1407
Pyramimonas parkeae Norris et Pearson CCMP725
Unidentified coccoid CCMP1413
Rhodophyta
Porphyridium aerugineum Geitler UTEX 755
Scenedesmus sp., Monoraphidium sp., and Gloeobotrys sp. are
from our collection. CCMP, Provasoli-Guillard National Center
for Culture of Marine Phytoplankton (Andersen et al. 1997);
CGC, Chlamydomonas Genetics Center (Harris 1984); FDCC,
Freshwater Diatom Culture Collection (Czarnecki 1994);
UTEX, Culture Collection of Algae at the University of Texas
at Austin (Starr and Zeikus 1993).
MARVIN W. FAWLEY AND KAREN P. FAWLEY224

With the new procedure, we typically purify DNA
from four to eight algal isolates at the same time, with
approximately 30 min required for purification. If
desired, the purification procedure could be scaled
up to process over 100 isolates per day. The main
advantages to this method over previous methods are
as follows:
1. Ease and simplicity. This DNA purification
procedure requires far fewer steps than most other
procedures. The process is easily mastered by anyone
able to use a micropipetter and microcentrifuge.
2. General applicability. Although we have not
attempted this procedure with some types of algae, it
has been applied successfully to different lineages of
green algae and stramenopiles and one rhodophyte
and is likely to work with many other kinds of algae.
3. Low exposure to hazardous materials. Only a
single chloroform extraction is used in this procedure.
4. Low cost. The only specialized equipment re-
quired is a low cost MiniBeadBeater, and the materials
used are inexpensive compared with commercial DNA
isolation kits.
This DNA purification procedure should enable
phycologists to examine many algal isolates by mole-
cular techniques much more rapidly than using
existing protocols. It should have wide applicability
for any studies involving PCR-based techniques, such
as PCR-RFLP, rapid analysis of polymorphic DNA, and
sequencing.
This material is based on work supported by the National
Science Foundation under grant nos. DBI-0070387 and MCB-
0084188. We thank Lothar Krienitz for providing the culture
of Nannochloropsis limnetica.
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