MicrobeGR Leaflet DNA Micro Arrays

8/6/2019 MicrobeGR Leaflet DNA Micro Arrays

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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 Browns 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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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 Earths 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.


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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

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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 g of

DNA, much too high for pathogen detection in natural environments. Amplification of the 23SrRNA 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.


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Data analysis

Data analysis is the most challenging aspect of

POA and FGA because of the large amounts ofdata 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

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MicrobeGR Leaflet DNA Micro Arrays