Download DNA probe technology - Woods Hole Oceanographic Institution

Detection of Protistan
Genomes in the Environment
Rebecca J. Gast
Woods Hole Oceanographic Institution
Identification….
Detection …..
Enumeration…….
………Of marine protists
Identification of cells
• Morphology
- Most useful for larger protists
- Motility of cells also diagnostic
• Sequences
- Small subunit ribosomal RNA gene
- Phylogenetically/taxonomically informative
Detection of organisms
• Indirect - detection without visual
confirmation of cell
- Sequences
- Proteins
- Lipids
• Direct
- Microscopy
- In situ probe hybridization
Oligonucleotide probes
• Different levels of specificity - Extensive
sequence databases are an important resource for designing
probes.
- Kingdom (archea, bacteria, eukaryote)
- Group (methanotroph, prymnesiophyte)
- Individual organism (E. coli,
Paraphysomonas)
• Hybridization
- Southern blots, dot blots
- Arrays
- Whole cell - In situ (whole cell) hybridization is
often considered the “gold standard” because it
provides visual confirmation of a cell.
Reverse Dot Blot Hybridization (aka arrays)
Detection and identification of acanthamoeba
ribotypes in environmental samples
–Ribotypes recovered by enrichment culture are potentially
not representative of the natural sample
–New ribotypes can be detected with this method
Fluorescence in situ hybridization
Litaker et al., 2002. Journal of Phycology, 38 (3):442-463.
Still difficult to address
• Abundance
- Quantitative analyses require the ability to retain
and recover cells.
• Monitoring over scales relevant to the microbial
system
- Spatial - there may be significant changes over very
small distances
- Temporal - community changes may occur rapidly
in response to physical or chemical perturbation.
New Technology
• Automated water samplers
• New probe types
- Peptide nucleic acid (PNA)
• New instrumentation
- ESP
- In situ flow cytometers
What is a PNA?
Behave like nucleic acid, but lack the negatively charged backbone.
PNA probes are desirable for in situ hybridization
shorter probes can be used (increased % mismatch)
lower salt concentration (increased specificity)
better cell penetration
Flow cytometric detection of hybridized
cells
In situ
hybridization
chamber
Increase spatial sampling
capability
Couple to flow cytometer
Quantitative
Collaborators and support
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Robert Olson
Heidi Sosik
Mark Dennett
Alexi Shalapyonok
Ken Doherty
WHOI Green Technology Award
NSF Biocomplexity IDEA