PNNL-13501 - Pacific Northwest National Laboratory

esolved (Gabriel et al. 1988, 1989; O’Donnell and Gray
1995; O’Donnell et al. 2000; Vauterin et al. 1995).
More than 100,000 fungal species are known. Most are
saprophytic living off dead/decaying organic matter.
While approximately 50 fungal species can cause disease
in humans and animals, more than 10,000 fungal species
can cause disease in plants (Kingsley 2000). Therefore
problems of identification of specific organisms are often
large.
This work is an outgrowth of a prior project that isolated,
characterized, and cloned rhizosphere inducible bacterial
promoter elements for use in bioremediation
(phytoremediation) applications and biocontrol.
Approach
Our approach involved attaching a series of 47 9-mer
(9 bases long) oligonucleotides (probes) to a glass slide to
which biotinylated polymerase chain reaction products
(targets) are hybridized. Probes are spotted in a high pH
buffer to facilitate covalent attachment. Spotting is
accomplished with a Genetic Microsystems 417 arrayer,
and we used 12-well Teflon-masked slides (Eri Scientific
Co., Portsmouth, New Hampshire). This permitted
printing 12 full arrays on a single slide, useful for running
replicate reactions and multiple samples. The microbial
polymerase chain reaction products were labeled using
biotinylated primers (REP, ERIC, BOX bacterial repeat
primers).
Denatured, biotin-labeled polymerase chain reaction
products were applied to individual wells (40 µL
maximum volume), hybridized, and washed.
Streptavidin-alkaline phosphatase is then added
(AMDEX, Amersham Piscataway, New Jersey) to each
well and incubated, washed, and reacted with
fluorescence visualization reagents. The fluorescent
emissions were captured using a BioRad Fluor-S imager.
Fluorescent array images were quantified using Phoretix
software (Ver. 1.00, Phoretix International, Newcastle,
United Kingdom). The polymerase chain reaction
fingerprint products were also visualized by traditional
gel electrophoresis, and the gel image was captured and
analyzed using GelComparII (Version 2, Applied Maths,
Kortrijk, Belgium) to produce a cluster analysis of the
similarity of the samples. This approach provided a
means for comparing the resolving power of the array
versus the traditional gel-based approach.
92 FY 2000 Laboratory Directed Research and Development Annual Report
Results and Accomplishments
Polymerase Chain Reaction Amplifications, Labeling of
Microbial DNA, Analysis of REP-PCR Gels
Xanthomonas (bacteria, Figures 1 through 4) and
Fusarium (fungus, Figure 5) genomic DNA was labeled
with a biotin-tagged forward REP primer by standard
REP-PCR amplification. Prior to array analysis, and to
provide a reference between traditional gel-based analyses
of the reactions and the analysis performed on the 47target
9-mer array(s), all reactions were analyzed by gelelectrophoresis.
To visualize the reactions run in the gel,
the gels were stained with the fluorescent dye ethidium
bromide, illuminated with ultraviolet light, which
visualizes the DNA bands, and photographed. Figure 1
shows an example of 17 Xanthomonas REP-PCR DNA
MW 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Figure 1. Agarose gel electrophoresis analysis of 17
Xanthomonas isolates. The banding patterns were generated
using REP-PCR primers that recognize a conserved DNA
repeat sequence. The forward primer included a biotin tag
that labeled the products for DNA array analysis (see
Figure 3). Lanes 1-3 contain reactions from control strains
and lanes 4-17 from various X. axonopodis pv. citri isolates
from diverse geographical locations worldwide.
fingerprints analyzed by gel electrophoresis. The digital
image of the gel was loaded into the analysis program
(GelComparII) for homology analysis of the REP-PCR
DNA fingerprints. This analysis is based on the
similarity/differences of the banding patterns between and
among the various samples included in the analysis. The
results of the analysis can be displayed as a cluster
dendrogram that indicates the relatedness of the analyzed
patterns. The strains of Xanthomonas are highly related
and this is reflected in the level of clustering and the
linkage points (Figure 2).