Genomics Technology to Assessing Microbial Activity in the Environment

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Genomics Technology to Assessing Microbial
Activity in the Environment
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MICRO ARRAY TECHNOLOGY

• Array technology and how it works
• How it is likely to be applied and the
  implications
• What WILL be gained from its application
• Advantages and cost benefits

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Diverse Chemicals and Microorganisms

• 10 x 106 Chemicals
• 8 x 106 Xenobiotic
• 1 x 106 Recalcitrant
• 0.4 x 106 traded at over 50 tonnes per year
• Toxicological/ biodegradative data on only around
  5000-6000
• MICROORGANISMS IN COMPLEX COMMUNITIES IN THE
  ENVIRONMENT
• Difficult to assess their effectiveness
• Some cannot be cultured

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Some soil numbers
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The tree of life rRNA sequence-based phylogeneti
c tree
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Assessing microbial diversity

• These are all nucleic acid (principally rRNA or
  rDNA) based methods. More direct, analytical
  methods
• (ie. FAME analysis / MS fingerprinting) can
  also be used to estimate diversity.
• These address only diversity (Whos there?)
  and not metabolic potential or activity unless
• metabolic genes or their transcripts are
  targeted by gene probes etc.

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Overview of array technology for environmental
samples
Environmental sample Extract total DNA or RNA.
Use PCR to amplify gene copies (convert RNA to
DNA with reverse transcriptase) and
apply fluorescent dye label
Robotic arraymaker makes many multigene arrays.
The genes present hybridse to their counterparts
on the array and can be detected.
Use laser fluorescence reader to scan slide. Two
dyes can be used. One to target genes that
identify the bacteria and the other to detect
active biodegradation genes.
Hybridise to complimentary sequence in array
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Preparation of a DNA Microarray
Robotic arm compresses micro samples onto slide
Silylated Slides have reactive aldehyde groups
and covalently bind tissue, cells or single or
double-stranded DNA directly to the glass surface
of a high quality microscope slide via the Schiff
base aldehyde-amine chemistry (lysine residues of
proteins, primary amines of DNA bases, or via
synthetic DNA bases bearing amino-modifications).
DNA samples may be 1. Oligonucleotides 2.
Clones 3. Total DNA 4. PCR products
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Sample Preparation and hybridisation
PCR to amplify target gene copies
Hybridise to complimentary DNA on the array and
wash off excess
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Reading a DNA Microarray after hybridisation
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INVOLVES APPLICATION OF ADVANCED DNA ARRAY
TECHNOLOGY
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• A DNA microarray
• 20,000 features, printed at 170µm pitch. Spot
  diameter approx 145µm.
• A different DNA probe can be printed on each
  spot

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Primary structure (sequence) and secondary
structure (folding, loops etc) dictate tertiary
(3D) structure of ribosome
ribosome
Bacterial SSU rRNA variability map red highly
conserved
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16S rDNA gene

• 1500bp gene - encodes 16S (small subunit) rRNA
  molecule
• Parts of the sequence are conserved among all
  living things
• Central metabolism inherited from the last
  common ancestor
• 40000 sequences in the Ribosomal Database
  Project DB
• Conserved blocks can be exploited to amplify
  the gene in vitro
• Variable regions are used for taxonomy
  phylogenetics

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How it is likely to be applied and the
implications.

• Large amount of gene sequence data
• Estimated 10 years worth now added in 10 weeks!
• Gene expression studies
• e.g. New drug targets sought
• Rapid detection of pathogenic bacteria and
  viruses from complex samples
• Organisms detected AND their active genes
• Rapid detection of biodegrading bacteria in the
  environment
• NO SINGLE AREA OF BIOSCIENCES WITH REMAIN
  UNAFFECTED BY THIS TECHNOLOGY (Recent US
  government report)

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What will be gained from its application?

• For the first time it will be possible to analyse
  a complex microbial population and its relative
  performance in a SINGLE step.
• The implications are enormous!
• This will become the standard diagnostic tool
• Legislation will follow on from this development
• THE DESIGN AND SUITABILITY OF THE ARRAYS WILL BE
  THE MAIN LIMITING STEP IN APPLICATIONS

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Variability in dioxygenase genes in the
Rhodococci (unpublished data)
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Advantages and cost benefits

• Revolutionises environmental analysis
• Analysis of MANY organisms / genes handled in
  single step.
• DNA/RNA extracted on site can remain stable
• High throughput back at the laboratory within one
  day
• Fluorescence methodology is sensitive and
  quantitative
• We can take advantage of our existing databases
  and expertise