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Plant Molecular Biology Reporter 22: 71–81, March 2004

© 2004 International Society for Plant Molecular Biology. Printed in Canada.

Protocols

A Simple Extraction Method Suitable for PCR-

Based Analysis of Plant, Fungal, and Bacterial
DNA

GEORGE S. MAHUKU*
Centro Internacional de Agricultura Tropical (CIAT), Apartado Aereo 6713, Cali,
Colombia, South America

Abstract. A simple and easy protocol for extracting high-quality DNA from microorgan-
isms and plants is presented. The method involves inactivating proteins by using
SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further
purification is based on differential solubility of DNA and high-molecular-weight polysac-
charides in aqueous media. The procedure does not use the toxic and potentially hazardous
phenol and chloroform, and as many as 100 samples can be processed per day. Absor-
bency ratios (A260/A280) of 1.6-2.0 indicated a minimal presence of contaminating metab-
olites. The DNA was completely digested with 5 restriction enzymes: EcoR I, Rsa I, Taq I,
EcoR V, and Hind III. PCR analysis using enterobacterial repetitive intergenic consensus
(ERIC) sequence, sequence-characterized amplified region (SCAR), and random amplified
microsatellite (RAMS) primers showed the DNA’s compatibility with downstream applica-
tions. This procedure is applicable to a range of pathogens and plants and thus may find
wide application in quarantine services and marker-assisted selection (MAS) breeding.

Key words: DNA extraction, DNA purification, marker-assisted selection, RAMS, restric-
tion enzyme digestion

Abbreviations: ERIC, enterobacterial repetitive intergenic consensus; MAS, marker-

assisted selection; RAMS, random amplified microsatellite; SCAR, sequence-
characterized amplified region.

Introduction A simple DNA extraction method Mahuku

Molecular markers have become the method of choice for plant pathologists who
are characterising pathogens to understand or elucidate the principles or factors
underlying molecular evolution, population genetics, plant-fungus interactions, or
pathogen evolution at the molecular level (Leung et al., 1993; Milgroom and Fry,
1997). Because molecular markers are selectively neutral, relatively easy to assay,
and sample the genome randomly, the genetic differences that are detected reflect
the variability existing in the whole genome (Leung et al., 1993; Mitchell et al.,
1995). Where PCR-based techniques such as random amplification of polymor-
phic DNA (RAPD) (Grajal-Martin et al., 1993), random amplified microsatellite

*
Author for correspondence. e-mail: g.mahuku@cgiar.org; fax: 57-2 445 0073;
ph: 57-2 445 0000.

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72 Mahuku

(RAMS) (Hantula et al., 1996), or amplification of defined genomic regions

(Hsaing and Mahuku, 1999) are used, a prerequisite is the rapid isolation of rela-
tively pure genomic DNA of high molecular weight from all compartments of the
cell by using strategies that minimise interruptions in the genes or sequences of
interest.
A major limitation of applying molecular techniques in developing coun-
tries, particularly those in Africa, is the lack of well-equipped laboratories for nu-
cleic acid extraction and analysis. Heavy reliance for this type of analysis must
therefore be placed on laboratories in developed countries. Compounding this lim-
itation are quarantine restrictions on carrying living organisms across borders.
However, to the best of our knowledge, no such restrictions exist for the transna-
tional movement of DNA samples of pathogens. This means that pathogen isola-
tion and purification can be done in developing countries, and the DNA can be
sent to well-equipped laboratories in other countries for analysis. Such an alterna-
tive is desirable because it avoids the bio-risks associated with moving living
organisms across borders while providing the much-needed information on patho-
gen genetic variation.
The lack of an easy DNA extraction method suited to conditions commonly
encountered in many laboratories in developing countries has limited the imple-
mentation of DNA analytical services for developing countries. Alternative meth-
ods must be found because most laboratories are not equipped to handle toxic
organic substances such as phenol and chloroform, which are commonly used in
DNA extractions (Graham et al., 1994; Zhang et al., 1996). These methods must
avoid using these chemicals but still yield the high-molecular-weight DNA
needed for most DNA analyses. Most methods begin with freezing the tissue sam-
ple with liquid nitrogen and then pulverizing it with a mortar and pestle. Extrac-
tions with phenol-chloroform or chloroform-isoamylalcohol follow, and, finally,
precipitation occurs using a hexadecyltrimethylammonium bromide (CTAB) solu-
tion that either is low in salt or contains sodium dodecyl sulphate (SDS) and high
salt to remove contaminating proteins, hydrates, polysaccharides, and other debris
(Garber and Yoder, 1983; Kim et al., 1990, Möller et al., 1992). These methods
can be used by well-equipped and developed laboratories that have the facilities
for handling the toxic and hazardous phenol and chloroform compounds (Möller
et al., 1992; Riana and Chandlee, 1996) but are not suitable for most laboratories
found in National Agricultural Research Stations (NARS) in developing countries.
The method that we developed for extracting DNA does not use toxic and po-
tentially hazardous reagents, such as phenol and chloroform, but yields DNA of
high quality and purity that is suitable for restriction digestion and PCR-based anal-
ysis. This method was developed by combining and modifying the protocols re-
ported by Cenis (1992), De Boer et al. (1995), and Möller et al. (1992). The
procedure involves inactivating proteins by SDS/proteinase K and precipitating
polysaccharides in the presence of high salt (Kim et al., 1990). The removal of
polysaccharides and other contaminating hydrates is based on the differential solu-
bility of DNA versus the high-molecular-weight polysaccharides in aqueous media
(Rozman and Komel, 1994). This method was targeted to provide our partners in
developing countries with a means to address their needs for molecular character-
ization of pathogen populations and extraction of plant DNA for MAS breeding.

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A simple DNA extraction method 73

Materials and Methods

Fungal and bacterial isolates
Microorganisms used in this study included 2 fungal plant pathogens, Colleto-
trichum lindemuthianum and Phaeoisariopsis griseola, and the bacterium, Xan-
thomonas campestris pv. phaseoli. The isolates were collected from naturally
infected bean tissues and purified according to the protocol of Pastor-Corrales et
al. (1998).

Production of fungal mycelium

Mycelium of P. griseola and C. lindemuthianum were produced in V8 juice me-
dium as outlined by Mahuku et al. (2002). Briefly, Erlenmeyer flasks (200 mL)
containing 60 mL of liquid V8 juice medium were inoculated with ten 1-cm discs
removed from actively growing monosporic cultures. The cultures were placed on
a rotary shaker (115 rpm) and incubated at room temperature (~24°C) for 7-10 d.
Mycelia were harvested by filtration through cheesecloth, blotted dry with sterile
paper towels, and used immediately for DNA extraction.

DNA extraction protocol

• Transfer fresh mycelium (150 mg) to a sterilized 1.5-mL Eppendorf (microcen-
trifuge) tube containing 300 µL of TES extraction buffer (0.2 M Tris-HCl
[pH 8], 10 mM EDTA [pH 8], 0.5 M NaCl, 1% SDS) and acid-washed, steril-
ized sea sand or 0.5-mm glass beads.
• Macerate mycelium for 2 min with a hand-held disposable homogenizer that
fits the 1.5-mL microcentrifuge tube.
• Vortex samples for 30 s and add an additional 200 µL of TES extraction buffer
containing proteinase K (final concentration of 50 µg/mL).
• Vortex to thoroughly mix and place tubes in a water bath at 65°C for 30 min.1
• Add one-half volume (250 µL) of 7.5 M ammonium acetate.
• Mix and incubate the samples on ice or at ~5ºC in the refrigerator for 10 min.
• Centrifuge for 15 min at 20,800g.
• Transfer the supernatant to a new tube and add an equal volume (500 µL) of
ice-cold isopropanol.
• Incubate tubes at –20ºC for 1-2 h.
• Centrifuge for 10 min at 20,800g to pellet the DNA.
• Decant the supernatant and wash DNA pellet with 800 µL of cold 70% etha-
nol.2
• Turn tubes upside-down on clean sterile paper towels for 10-15 min to air-dry
DNA.
• Elute DNA from the pellet with twice-repeated extractions with 250 µL of 1×
TE buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA), each time centrifuging to
avoid collecting pelleted polysaccharides.
• Transfer DNA solution to a 1.5-mL microcentrifuge tube, add 5 µL of RNase A
(20 mg/mL), and incubate at 37°C for 60 min.
• Recover DNA and air-dry as described above.

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74 Mahuku

• The dry DNA pellet can be shipped to other laboratories for analysis or dis-
solved in 100 µL of 1× TE buffer.
Notes
1
Mycelium can be lyophilised or frozen in liquid nitrogen before grinding with a
hand-held pestle that fits in a 1.5-mL microcentrifuge tube.
2
If the DNA pellet is loose, centrifuge the sample for 3 min at 3800g.

Extracting bacterial DNA

Bacterial DNA was extracted from the bean common blight pathogen, Xantho-
monas campestris pv. phaseoli. Bacterial cells were harvested from 24-hour-old
cultures grown on YDC broth and suspended in 1 M NaCl by vigorous vortexing
as described by Chan and Goodwin (1995). The tubes were centrifuged at 2700g
for 10 min, supernatant was discarded, and the process was repeated to reduce
and separate the cells from the polysaccharide xantham gum. The cells were fi-
nally washed twice in sterilized, distilled, and deionized water to reduce the salt
concentration, the pellet was suspended in 500 µL of TES extraction buffer con-
taining 50 mg/mL proteinase K, and the nucleic acids were extracted as described
under “DNA extraction protocol.”

Extracting plant DNA

Young trifoliate bean leaves were harvested from 2-week-old seedlings of segre-
gating F2 populations derived from crossing G 10474 (resistant to P. griseola) and
Sprite (susceptible to P. griseola) (Mahuku et al., 2003). The top of the tube was
used to sever 2 discs of leaf tissue directly into sterilized 1.5-mL Eppendorf
microcentrifuge tubes; the tubes were placed on ice and taken to the laboratory
for DNA extraction. TES extraction buffer and sterilized sea sand or glass beads
(0.5 mm) were added, and the tissue was macerated for 2 min with a hand-held
mortar that fits into the 1.5-mL Eppendorf tube. Plant DNA was then extracted as
described under “DNA extraction protocol.”

DNA analysis
The quality of the extracted DNA was obtained by means of electrophoresis in
0.7% agarose gels, followed by staining with ethidium bromide. The purity of the
DNA was estimated from the A260/A280 ratio, whereas the yield was obtained by
measuring absorbance at 260 nm with a spectrophotometer. DNA purity was fur-
ther confirmed by digestion with 5 restriction enzymes (EcoR I, Rsa I, Taq I,
EcoR V, Hind III) followed by gel electrophoresis.
To check the suitability of extracted DNA for downstream analysis, a vari-
ety of PCR-based assays were done. The ribosomal intergenic spacer region was
amplified as described by Mahuku et al. (2002), using the conserved primers CLN
12 and CNS1 (Hsaing and Mahuku, 1999), and analysed by means of digestion
with the restriction endonuclease Rsa I. Microsatellite sequences in P. griseola
and C. lindemuthianum were amplified by using the (ACA)n RAMS primer (Han-
tula et al., 1996) and conditions described by Mahuku et al. (2002). Bacterial
DNA was amplified by using the ERIC-PCR primers as described by Sulzinski et

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A simple DNA extraction method 75

Figure 1. Fungal, bacterial, and plant DNA extracted by using the new method reported here and the
phenol-chloroform method. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 are DNA samples
extracted with phenol-chloroform. Lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 are DNA
samples extracted with the new method, which does not use phenol or chloroform. Lanes 1-10
represent Phaeoisariopsis griseola; lanes 11-18, Colletotrichum lindemuthianum; lanes 19-22,
Xanthomonas campestris pv. Phaseoli; and lanes 23-26, Phaseolus vulgaris DNA. Lane M is DNA
molecular marker (λ DNA digested with Hind III). All DNA samples were treated with 20 mg/mL
RNAse to remove contaminating ribonucleic acids (RNA).

al. (1997), while plant DNA was amplified by the SCAR marker linked to the re-
sistant gene in the germplasm accession G 10474. The PCR reaction for the
SCAR was done in 12.5-µL reaction volumes with 1× PCR buffer (10 mM Tris-
HCl, 50 mM KCl, 0.1% triton X-100), 2 mM MgCl2, 0.1 µM of each dNTP,
0.5 µM of each SCAR primer (PF5-F; 5′-CTTGTTCTGAGTCATTTACCTTGC-
3′, PF5-R; 5′-GAATTCACAGTCCAAACTACTCTAATC-3′), 20 ng of DNA, and
1.25 U Taq DNA polymerase (Promega, Madison, WI, USA). Amplification was
performed in an MJ Research Thermocycler (Watertown, MS, USA) programmed
for 35 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, followed by a final
extension for 7 min at 72°C. The PCR products were run on 1.5% agarose gels.

Results
DNA yield and purity
Large quantities of high-molecular-weight DNA were extracted from 3 bean
pathogens and bean leaves by using the simple DNA extraction method described
here (Figure 1). DNA yields ranged from 20-50 µg per 250 mg of starting mate-
rial, and these yields were comparable with the phenol-chloroform method nor-
mally used in our laboratory. The ratios from absorbency at A260/A280 ranged from
1.63-1.99, showing that the DNA was of high purity (Table 1). DNA was com-
pletely digested with 5 different restriction enzymes (EcoR I, Rsa I, Taq I, EcoR
V, Hind III), further confirming the purity of the extracted DNA. Figure 2 is an
example of DNA digested with restriction endonucleases Rsa I and Taq I.

PCR analysis
Successful and reproducible amplifications of simple sequence repeat regions
were obtained by using the BDB (ACA)5 RAMS primer (Figure 3), demonstrating

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76 Mahuku

Table 1. Spectrophotometric measurements of concentration (conc.) and purity of

DNA* isolated from different bean pathogens.

Fungal
Species Isolate Number A260 A280 A260/A280 DNA Conc., ng

PG PG 16 MWI 0.990 0.496 1.99 4950

PG 1 MWI 0.851 0.434 1.96 4255
PG 2-5 KEN 0.821 0.418 1.96 4105
PG 2-8 UGD 0.890 0.449 1.98 4450
XCP XCP 180 COL 0.239 0.125 1.91 1195
XCP 362 CR 0.171 0.090 1.89 855
XCP 44 GTM 0.236 0.122 1.93 1180
XCP 148 BRA 0.220 0.118 1.87 530
CL CL 10 ECU 0.033 0.021 1.63 165
CL 75 ARG 0.044 0.026 1.69 220
CL 32 GTM 0.035 0.019 1.84 175
CL 143 CR 0.099 0.054 1.85 495

µ
*In all cases, DNA was dissolved in 100 L of 1× TE buffer. Phaeoisariopsis griseola (PG) DNA

was extracted from 300 mg of mycelium, Colletotrichum lindemuthianum (CL) DNA was ex-
tracted from 100 mg of mycelium, and Xanthomonas campestris pv. phaseoli (XCP) was extracted
from a pellet obtained from 5 mL of a 1010 cells/mL solution.

Figure 2. Fungal and bacterial DNA digested with 2 restriction endonucleases, Rsa I (lanes 1-8) and
Taq I (lanes 9-16), according to the manufacturer’s instructions. Phaeoisariopsis griseola isolates are
in lanes 1 and 9 (PG 16 MWI), 2 and 10 (PG 1MWI), 3 and 11 (PG 2-5 KEN), and 4 and 12 (2-
8 UGD). Isolates of Xanthomonas campestris pv. phaseoli are in lanes 5 and 13 (XCP 180 COL), 6
and 14 (XCP 362 CR), 7 and 15 (XCP 44 GTM), and 8 and 16 (XCP 148 BRA). Lane M is the λ
DNA digested with Hind III molecular marker. The DNA was extracted by using the simplified
method described in this article.

that DNA extracted with this procedure is suitable for PCR-based assays. No dif-
ferences in amplification patterns were obtained for the same isolates when DNA
was extracted with the phenol-chloroform method or the procedure reported here
or column purification of the DNA extracted using this procedure (Figure 3);

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A simple DNA extraction method 77

Figure 3. Reproducibility of PCR amplifications of DNA extracted from Phaeoisariopsis griseola by

using the microsatellite primer BDB(ACA)5. DNA was extracted at 3 different times, and
amplifications were performed simultaneously. The DNA of each isolate was extracted according to
the following methods: Lane 1, the phenol-chloroform method; lane 2, the protocol described in this
article; and lane 3, the protocol described here and an additional column purification. Lane M is the
100-bp molecular weight marker, and lane C is the negative control.

Figure 4. Bacterial DNA extracted by using the new protocol and amplified with the enterobacterial
repetitive intergenic consensus (ERIC) primers. Lanes 1-10 are Xanthomonas campestris pv. phaseoli
isolates, lane 11 is the negative control (no bacterial DNA added), and lane 12 is the 100-bp DNA
stepladder.

therefore, further purification of extracted DNA is not necessary for PCR-based

assays. Bacterial DNA was successfully amplified by using ERIC primers (Fig-
ure 4), and amplifications were reproducible for different DNA extraction times.
Successful amplifications of plant DNA were obtained by using a SCAR marker
linked to the angular leaf spot resistance gene in the germplasm accession G
10474 (Figure 5).

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78 Mahuku

Figure 5. Marker-assisted selection (MAS) based on sequence-characterized amplified region

(SCAR)–PCR with plant DNA. PCR products from F2 population showing homozygous resistant
plants (301 bp), homozygous susceptible plants (280 bp), and heterozygous plants with both
fragments. Lane 19 is negative control (no plant DNA) while 20 is the 100-bp molecular size marker.

Discussion

The purpose of this study was to improve and simplify the currently available
DNA extraction methods (Chow and Käfer, 1993; De Boer et al., 1995; Möller et
al., 1992; Rozman and Komel, 1994) for use by researchers in developing coun-
tries. In these countries, suitable facilities for handling toxic and hazardous mate-
rials commonly used in most DNA extractions do not exist or are insufficient,
thus precluding studies such as pathogen detection and characterization and germ-
plasm analysis at the molecular level, where DNA extraction is a prerequisite.
This method is expected to enhance germplasm and pathogen characterization by
facilitating the movement of DNA to laboratories where equipment for DNA anal-
ysis exists while circumventing the bio-risks associated with moving living organ-
isms across borders.
The DNA extraction protocol described here is rapid and technically easy
for preparing nucleic acid that is useful as an amplification target. Sufficient
quantities of pure and high-molecular-weight DNA were extracted from fungal,
bacterial, and plant tissues, and this DNA was suitable for both PCR-based assays
and digestion with restriction enzymes. Successful amplifications were obtained
by using RAMS, SCARs, RAPD primers, and conserved primers targeting ribo-
somal intergenic regions. This method provides another effective nucleic acid ex-
traction procedure to complement several existing methods (Baldrian et al., 1999;
Borgia, et al., 1994; Graham et al., 1994). Moreover, this method does not require
the toxic and potentially hazardous organic solvents (e.g., phenol, chloroform)
that are normally used in other methods, making it suitable for use in areas where
facilities for handling such chemicals do not exist.
To demonstrate the usefulness of this simple DNA extraction method for
bean researchers in developing countries, courses to introduce this method were
held at Kawanda Agricultural Research Organization in Uganda for bean re-
searchers in Africa and at CIAT headquarters for bean researchers in Colombia,

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A simple DNA extraction method 79

Bolivia, Peru, and Ecuador. DNA was successfully extracted from 3 bean patho-
gens: P. griseola, C. lindemuthianum, and X. campestris pv. phaseoli. P. griseola
DNA extracted during the course in Africa was shipped to CIAT headquarters as
dried pellets and successfully analysed with RAMS and RAPD primers. This
demonstrated that DNA extractions could be performed in one country, and the
DNA could subsequently be analysed in laboratories of other countries, thus of-
fering an alternative for successful pathogen population studies for countries lack-
ing this capacity.
Because our method can be applied to a wide variety of organisms, includ-
ing fungi, bacteria, and plants, it can find wide application, not only in bean-
pathogen interactions, but in other host-pathogen interactions and MAS breeding
(Miklas et al., 1996). In addition, this DNA extraction method can be scaled up to
handle large samples, making it useful for MAS, where large-scale screening of
field samples is required.
Other important features of this protocol are that (1) small amounts of tis-
sues (mycelium, plant leaves, bacterial cells) is sufficient for successful extrac-
tion; (2) large numbers of samples can be processed in parallel; (3) the procedure
works well with mycelia or fruiting bodies (spores), bacterial cells, and plant
leaves; and (4) the method is simple and not labour intensive. Because the extrac-
tions can be carried out in Eppendorf tubes, the chances of contamination and loss
of DNA are minimized (Cenis, 1992; Stewart and Via, 1993). In addition, this
method omits the need for many surface areas such as mortars, pestles, spatulas,
and other equipment that cells and their DNA contact in other methods (Graham
et al., 1994; Michaels et al., 1994). Therefore, the likelihood that contaminating
DNA will enter the process is significantly reduced.
The development of a rapid DNA extraction method that does not use toxic
and potentially hazardous substances such as phenol and chloroform should now
make practical the large-scale characterization of plant pathogenic fungi and bac-
teria from developing countries that lack the capacity for PCR-based analysis.
This method could find wide application in quarantine purposes and in MAS
when genes for several traits need to be pyramided into the same background. A
single person can process up to 100 samples per day. Because only DNA is
moved between countries, this procedure has the advantage of offering a way to
eliminate moving organisms across borders, thus safeguarding quarantine regula-
tions, while facilitating pathogen characterisation and diversity studies. In addi-
tion, the quick DNA extraction protocol is useful for laboratories that offer DNA
analysis services and have depended on the transfer of frozen or lyophilised sam-
ples.

Acknowledgments

The technical support of Maria del Carmen Hernadez is greatly appreciated. The
bean networks ECABREN (Africa) and PROFRIZA (South America) provided fi-
nancial support for workshops to introduce this DNA extraction method to bean
researchers. The support of Drs R. Buruchara and O. Voysest in organising and
running the workshops to test this DNA extraction is greatly appreciated.

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80 Mahuku

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