PNNL-13501 - Pacific Northwest National Laboratory

polymerase chain reaction from genomic DNA from eight
strains of E. coli. Polymerase chain reaction products
were then hybridized to a low-density microarray
consisting of five random decamer probes. The ERIC
polymerase chain reaction primer generated distinguishable
agarose gel fingerprints for the eight isolates;
hybridization of ERIC-polymerase chain reaction
fragments to a random five-probe decamer array
generated reproducible and distinct DNA fingerprints for
three of the eight isolates. More exciting results are
shown in Figure 1, where the REP polymerase chain
reaction primers showed no variability in agarose gel
fingerprint profiles, but the five-probe decamer array was
able to identify at least three unique fingerprints among
the isolates. We have improved hybridization buffers and
procedures and investigated the printing, binding, and
hybridization properties of nine-mer probes in this
context. Overcoming probe cross-contamination during
printing was a significant technical obstacle that is now
resolved. We verified that nonamer probes can be
successfully printed and hybridized to REP/ERIC
polymerase chain reaction products, which provides
approximately five times greater sampling of the E. coli
genome and polymerase chain reaction products than does
an array of 10-mer probes. Using nine-mer capture
probes, the REP fingerprinting array generated seven
unique fingerprint profiles from the eight strains of
E. coli.
Figure 1. REP polymerase chain reaction DNA fingerprinting
by agarose gel electrophoresis (top) and random
DNA fingerprinting array (bottom). This microarray/
polymerase chain reaction primer combination generated
three unique profiles where gel electrophoresis was
ineffective in genetic discrimination. Switching to a random
nonamer array resulted in seven unique array profiles.
36 FY 2000 Laboratory Directed Research and Development Annual Report
Based on these results, we constructed a 50-probe
nonamer fingerprinting array. Initial hybridization results
with E. coli REP polymerase chain reaction products
illustrate the need for improved image analysis and
statistical capabilities for routine implementation of
microarrays in epidemiological investigations. Even with
four replicate arrays in two independent hybridization
chambers, there was sufficient signal variation at certain
probe spots (Figure 2, squares) to question their assignment
as “positive” or “negative” signals. Phoretix image
analysis software allowed us to assign an arbitrary
threshold for positive signal and scoring purposes. This
procedure was used to fingerprint a small set of
Xanthomonas isolates (Figure 3) and demonstrated that
unambiguous identification of closely related isolates can
be obtained with a DNA fingerprinting microarray.
Current research is focused on a more comprehensive
fingerprinting analysis of Xanthomonas isolates.
5
11
Print Pattern
47
B 6 12 42
Summary and Conclusions
We have demonstrated that random DNA fingerprinting
arrays can be successfully used to identify closely related
microorganisms. Improved statistical techniques will be
required to assess variability in signal intensity at
individual spots and compare profiles between organisms.
Reference
Hancock DD, TE Besser, DH Rice, ED Ebel, DE Herriott,
and LV Carpenter. 1998. “Multiple sources of
Escherichia coli 0157 in feedlots and dairy farms in the
northwestern USA.” Prev. Vet. Med. 35:11-19.
Array 1
Array 2
Well 1 Well 2
Figure 2. 50-probe random nonamer array hybridized to
E. coli REP polymerase chain reaction products. Four
independent arrays were printed in two hybridization wells.
Circles indicate hybridization signals easily visible to the
unaided eye. Rectangles highlight regions of signal
variability at identical probe locations.
Biotin
Biotin