Genomics Technology to Genomics Technology to Assessing Microbial Activity in Assessing Microbial Activity in
the Environmentthe Environment
MICRO ARRAY TECHNOLOGYMICRO 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
• 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
Diverse Chemicals and Microorganisms
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
• 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
Some soil numbers…
Mean particle Per gram
Soil type Diameter (mm) Volume (mm3) Number of Particles
Surface area (cm2)
Very coarse sand
2.00-1.00 4.18 x 101 9.0 x 101 1.1 x 101
Coarse sand 1.00-0.50 5.24 x 10-1 7.20 x 102 2.3 x 101
Medium sand 0.50-0.25 6.55 x 10-2 5.70 x 103 4.5 x 101
Fine sand 0.25-0.10 8.18 x 10-3 4.60 x 104 9.1 x 101
Very fine sand 0.10-0.05 5.24 x 10-4 7.22 x 105 2.27 x 102
Silt 0.05-0.002 6.50 x 10-5 5.78 x 106 4.54 x 102
Clay <0.002 4.20 x 10-9 9.03 x 1010 8.0 x 106
The ‘tree of life’rRNA sequence-basedphylogenetic tree
You are here…
Most of the biomass,and most of the diversity is here – there are many missing branches…
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 (“Who’s there?”) and not metabolic potential or activity – unless metabolic genes or their transcripts are targeted by gene probes etc.
Pure cultures
Amplified DNA
Nucleicacids
WholeCells
FISH /microarray
Molecularfingerprinting
Gene probe
DNAsequencing
Sequencedatabase
‘Evenness’(relative abundance
of each taxon)
‘Richness’(number of unique
taxa)
Microbial community
Clonelibrary
Overview of array technologyfor environmental samples
Overview of array technologyfor environmental samples
Slide or membrane base
Robotic arraymaker makesmany multigene arrays.
Environmentalsample: Extract totalDNA or RNA.
Use PCR to amplifygene copies (convert RNA to DNA with reversetranscriptase) and applyfluorescent dye label
Hybridise tocomplimentarysequence in array
Use laser fluorescencereader to scan slide.Two dyes can be used. One to target genes that identify the bacteria andthe other to detect activebiodegradation genes.
The genes present hybridse to their counterparts on the array and can be detected.
Preparation of a DNA MicroarrayPreparation of a DNA Microarray
DNA samples may be1. Oligonucleotides2. Clones3. Total DNA4. PCR products
Robotic armcompresses micro samplesonto slide
Silylated Slides have reactive aldehyde groups and covalently bind tissue, cells or single or double-strandedDNA 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).
Sample Preparation and hybridisation Sample Preparation and hybridisation
Complex community of microorganisms
Extract total DNAOR RNA
Reverse Transcriptaseto give DNA
PCR to amplifytarget gene copies
Hybridise to complimentary DNA on the array and wash offexcess
Reading a DNA Microarray after hybridisation
Reading a DNA Microarray after hybridisation
Dual labelled fluorescencethat can be quantified
OR other chromogeniclabelling method
SLIDE PLACED INAUTOMATED LASERREADER
DATA LOGGED ON COMPUTER
INVOLVES APPLICATION OF ADVANCED DNA ARRAY TECHNOLOGY
• A ‘DNA microarray’
• 20,000 features, printed at 170µm pitch. Spot diameter approx 145µm.
• A different DNA ‘probe’ can be printed on each spot
ribosome
Bacterial SSU rRNA variability map; red = highly conserved
Primary structure(sequence) and secondary structure (folding, loops etc) dictate tertiary (3D) structure of ribosome
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’
27f primer
519r primer
Highly conserved sequence
Variable sequence
V1 V2 V3 V5V4 V7V6
How it is likely to be applied and the implications.
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)
• 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)
What will be gained from its application?
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
• 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
Variability in dioxygenase genes in the Rhodococci (unpublished data)
STRAIN edoA edoB edoC edoD bphC-RHA1
Rhodococcus sp. Acr33 ++ (95%) ++ (99%) - ++ (98%) + (97%)
Rhodococcus sp. A1 + (90%) ++ (97%) - - + (96%)
Rhodococcus sp. B1 - ++ (92%) - - -
Rhodococcus sp. C1 - ++ (92%) - - + (97%)
Rhodococcus sp. E0 + (100%) ++ (98%) ++ (100%) + (97%)
Rhodococcus sp. E3 - ++ (98%) ++ (92%) + (97%)
Rhodococcus sp. I1 + (95%) ++ (100%) ++ (100%) ++ (100%) + (97%)
Rhodococcus sp. P5 - + (89%) - - -
Advantages and cost benefitsAdvantages 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
• 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