Table of Contents

Microarrays: a brief history ....................................................................................... 2

DNA microarrays and microbes ................................................................................ 3

Phylogenetic oligonucleotide arrays .......................................................................... 4

Functional gene arrays ............................................................................................... 5

Data analysis .............................................................................................................. 6

Potential users ............................................................................................................ 6

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Microarrays: a brief history

Nucleic acid microarrays are based on the unique inherent specificity that is embedded in the
structure of the DNA duplex molecule since complimentary single strands can recognize each
other and hybridize to form a very stable duplex. It was Ed Southern in 1975 who first realized
that this specificity could be used to detect specific sequences in a complex mixture by labeling a
known DNA fragment (the probe) and using this to detect similar sequences in genomic DNA.
The Southern blot technique was soon being adjusted so that specific RNA molecules could be
similarly detected using Northern blots and consequently the routine analysis of mRNA
transcripts was established. At that point the concept of using a labeled probe fragment to identify
complimentary sequences was adapted for parallel processing of DNA clones. These methods and
their subsequent developments provided the foundation for virtually all aspects of current
molecular genetics and above all the basis for DNA microarray technology.

Microarray technology derives from two complimentary approaches developed in the 1990s. The
first cDNA microarrays were produced in Patrick Brown’s laboratory in Stanford, using robots to
“print” DNA from purified cDNA clones on glass microscope slides. The slides were hybridized
with fluorescently labeled RNA samples and the specific hybridization between a cDNA clone on
the slide and the labeled RNA in the sample used to infer the expression level of the gene
corresponding to each cDNA clone. In parallel work at Affymetrix, in situ synthesis of defined
oligonucleotides probes at very high density on glass substrates was shown to provide a reliable
route for measuring gene expression. The scene for the development of the current generation of
ultra-high density microarrays now employed for gene expression, genome tiling and genotyping
was set.

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2
DNA microarrays and microbes

Microorganisms play important and distinctive roles in ecosystem functioning including

biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus, and metals, as well as
degradation of contaminated environments. Microbes can also impact innumerable and valuable
agricultural crops, causing hundreds of millions of dollars in damage every year. For humans and
animals they can be the origin of devastating diseases changing the route of history. In short they
are the foundations of the Earth’s biosphere. Microorganisms are the most diverse groups of
organisms known both in terms of phylogeny and in functional abilities, and can be found in even
the most inhospitable environments. For these reasons an important role in microbial ecology is to
understand how microorganisms impact their environment, and how the microbial community
structure, function, interactions, and populations change temporally and spatially.

The study of microbial ecology faces several obstacles such as: (a) the huge diversity, for example
it has been estimated that one gram of soil contains 2,000 – 50,000 microbial species, and (b) the
vast majority of microorganisms (99%) have not been cultured yet. While studies focused on
populations that can be cultured or on isolates are still important, they provide an extremely
limited view of the microbial community diversity and function. In the culture-independent
studies, approaches like the 16S rRNA libraries, denaturating gradient gel electrophoresis
(DGGE), terminal-restriction fragment length polymorphism (T-RFLP), quantitative PCR, and in
situ hybridization can be utilized. The resolution power and coverage of these methods are limited.
For example, 16S rRNA libraries may underestimate the true diversity of microbial communities
by at least a factor of >10.

Microarrays, which can be used to examine thousands of genes at one time, can overcome many
of these obstacles. Because of their design, microarrays can provide information on a microbial
community in a simple, rapid, high-throughput and parallel manner. They can provide specific
and sensitive detection at a high resolution for a broad range of target microorganisms. Because
arrays have a defined set of genes or microorganisms that all samples are treated against, they are
ideal for comparing environmental samples from:

 different sites
 conditions
 times

These features make microarrays excellent tools for assessing microbial community structure,
functions, activities and dynamics in natural settings.

3
Phylogenetic oligonucleotide arrays

Phylogenetic oligonucleotide arrays (POA) are designed to determine community composition or

phylogenetic relatedness using 16S rRNA, or other conserved genes. The most comprehensive
POA is the PhyloChip, which uses the Affymetrix format. The PhyloChip contains 297,851
perfect-match (PM) and mismatch (MM) 16S rRNA gene probes for the detection of 842
subfamilies or 8741 taxa, covering 121 bacterial and archaeal orders, while 109,093 probes are
control probes. The PhyloChip has been shown to provide identification resolution at the family to
subfamily levels and it has been used in many microbial community studies because it provides a
quick and high-throughput analysis of the community composition.

The PhyloChip has been successfully used for the study of:

 the effect of abiotic factors on microbial communities

 time on microbial communities
 detection of pathogens
 microbial communities in extreme environments
4

4
While the 16S rRNA gene is the most commonly used phylogenetic marker, POAs have also been
designed using other gene markers. For example, the 23S rRNA gene was used to detect
pathogens in municipal wastewater, because this gene provides greater sequence variation than
the 16S rRNA. When unamplified DNA was used the detection limit was approximately 1 μg of
DNA, much too high for pathogen detection in natural environments. Amplification of the 23S
rRNA gene prior to hybridization increased the detection limit to 100 fg, and several pathogenic
microorganisms like Klebsiella pneumoniae, Pseudomonas aeruginosa and Clostridium
perfringens were detected in municipal wastewater using this method.

Functional gene arrays

Phylogenetic markers, such as 16S rRNA genes and DNA gyrase (gyrB) genes are commonly
used to examine microbial community structure. While these genes provide phylogenetic
information on the structure and diversity of a microbial community, they provide minimal
information on the community functional ability and activity. Functional genes can be used to
determine phylogenetic or functional relatedness. The most comprehensive functional gene array
(FGA) is the GeoChip with 24,243 50-mer oligonucleotide probes; targeting ~10,000 functional
genes from 150 gene families involved in the geochemical cycling of C, N, and P cycling, sulfate
reduction and resistance, and organic contaminant degradation.

5
Data analysis

Data analysis is the most challenging aspect of

POA and FGA because of the large amounts of
data generated. Several methods have been
frequently used in PhyloChip and GeoChip studies
(Figure 1). These include various diversity indices
(e.g. richness, evenness, diversity). For statistical
analysis several methods are commonly used.
These include ordination techniques like principal
component analysis (PCA) and cluster analysis. If
environmental data is available, several statistical
method are available to correlate environmental variables with the functional community structure.
These include canonical correspondence analysis (CCA), variance partitioning analysis (VPA) or
other correlation analyses.

Potential users

Great advances in microarray development, technology, applications, and analysis have been
made in the decade since microarrays were first developed. This exciting field has revolutionized
the study of molecular biology and microbial ecology. With the development of the DNA
microarrays a rapid, comprehensive and accurate identification of microbes within any living
organism or environmental sample without the need for culturing can be completed. The capacity
of the PhyloChip to monitor public health and environmental cleanup initiatives is unprecedented.

Target groups that could be interested in the unique properties of the microbial DNA microarrays
are:

 Academia

 Small and Medium Enterprises (SMEs) that are active in bioremediation, composting,
agriculture, quality control

 Governments for implementing National and European policies on public health

 Farmers for identifying pathogens and for quality control

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Figure 1. Microarray data analysis methods