PNNL-13501 - Pacific Northwest National Laboratory

Automated DNA Fingerprinting Microarrays for Environmental Epidemiology of
Pathogenic Microorganisms
Study Control Number: PN00011/1418
Darrell P. Chandler, Douglas R. Call, Cynthia J. Bruckner-Lea
If we are to successfully develop and apply diagnostic instruments in environmental sciences, we will need to more
capably discriminate between closely related microorganisms. Such instruments must be rapid, easy-to-use, and
conducive to automation; capable of producing statistically rigorous data with relative ease; and highly reproducible. The
objective of this work is to develop new instruments for discriminating between microorganisms based on their DNA
fingerprints.
Project Description
The objective of this project is to develop an integrated
DNA fingerprinting microarray system for the identification,
characterization, location, and tracking of pathogenic
microorganisms in environmental systems and nonhuman
vectors. Nonamer- and decamer-oligonucleotide
arrays were constructed and used to interrogate repetitive-
DNA polymerase chain reaction (PCR) products from
closely related E. coli and Xanthomonas isolates. Seven
unique microarray fingerprints were obtained with a
simple five-probe nonamer array, where gel
electrophoresis of polymerase chain reaction products
showed no genetic discrimination. A 50-probe nonamer
array was subsequently constructed and is being used to
investigate signal variation due to polymerase chain
reaction, hybridization, and signal generation. The
50-probe array is now being used to fingerprint 50 to 100
different strains of E. coli and Xanthamonas, respectively.
Introduction
Current epidemiological investigations of pathogenic
microorganisms use fairly standard techniques for DNA
“fingerprinting,” or discriminating between closely
related isolates. These include pulsed field gel electrophoresis,
variations on southern hybridization or
polymerase chain reaction-based techniques such as
randomly amplified polymorphic DNA typing, restriction
fragment length polymorphism, single-stranded
conformation polymorphism, denaturing gradient gel
electrophoresis, or combinations thereof. In all cases,
these typing methods access a limited complement of
genetic information, the analytical method is technically
challenging and time-consuming, and parallel processing
with many independent restriction enzymes or probes is
required to achieve statistical rigor and confidence in the
resulting pattern of DNA fragments. Still, standard
fingerprinting techniques fail to answer fundamental
epidemiological questions. For example, Hancock et al.
(1998) identified multiple sources of E. coli O157:H7 in
feedlots and dairy farms, but were unable to discriminate
between isolates. In the absence of adequate resolving
power, it is not possible to determine how pathogens
disseminate in the environment, enter into and distribute
between environmental reservoirs, or impact human
health. Similar limitations persist for most other
epidemiological studies of pathogenic (and nonpathogenic)
microorganisms.
The objective of this research is to develop and demonstrate
DNA fingerprinting microarrays for epidemiological
identification and discrimination of closely related
microorganisms, focusing on the occurrence, distribution,
and transmission of E. coli O157:H7 found in cattle, dairy
farms, and feedlots. The scope of the work involves the
development of novel hybridization conditions and
procedures for decamer probes (DNA or polynuclear
aromatic hydrocarbons) attached to a two-dimensional
support and genetic discrimination between clonal isolates
of E. coli O157:H7 and/or Xanthomonas. The expected
results of this research include immediate application of
the fingerprint array to better understand the dissemination
of E. coli O157:H7 (and other microorganisms)
within and between environmental reservoirs.
Results and Accomplishments
Initial experiments focused on reproducible printing and
hybridization of decamer probes to polymerase chain
reaction-amplified DNA targets. Biotinylated repetitive
DNA primers (REP, ERIC) were used to prime a
Biosciences and Biotechnology 35