Fluorescence in situ hybridisation (FISH) Flashcards
understanding microbial communities (40 cards)
why was fluorescence in situ hybridisation developed?
limited morphological diversity
because existing stains didnt give enough information
we needed a way to separate out the bacteria at different levels
what ways are microorganisms classically characterised?
looked for physiological and biochemical characteristics
– Enrichment
– Pure culture
– Physiological characterisation
why was ribosomal RNA chose as a marker?
- its present in every living organism
- its present in high copy number
- same function in every organism (protein biosynthesis)
- evolutionary conserved molecule
- some regions highly variable
what makes up small subunits of prokaryotic ribosomes?
30S = 16S rRNA
what makes are large subunits of prokaryotic ribosomes?
50S = 5S and 23S rRNA
describe the full cycle rRNA approach to FISH
- DNA is extracted
- amplified via PCR
- cloned
- sequenced
- comparistive sequence analysis and phylogenetic affiliation
- probe design from sequence database
- probe testing
- in situ hybridisation
- detection
what is the only tool in microbiology to determine true cell numbers?
FISH
what are some features and pros to FISH?
- Target naturally amplified molecule rRNA within the cell (100 to 1000 copies per cell).
- Variable as well as conserved regions – Broad/narrow specificity.
- Fluorescently labelled oligonucleotide s (probes) – Detection of “stained” whole single cells in natural context (“in situ”).
breifly describe the process of FISH using monolabelled oligonucleotide probes
fix cells for permeabilisation, then hybridise with fluorescently labelled oligonucleotide. wash away excess, then quantify via epifluorescence or confocal microscopy, or flow cytometry
the prerequisite for probe design is a comprehensive database
what should it comprise?
- should comprise all three domains
- should comprise all types of RNA
give two examples of rRNA databases
- RDP
- SILVA rRNA database project
what are the limitations of rRNA databases?
- No comprehensive archaeal sequence database.
- No comprehensive 18S rRNA database.
- No 23S rRNA database.
- No 5S rRNA database.
- Tedious manual sequence retrieval from NCBI/EBI by blast/acc number.
- ARB can only handle ~150,000 sequences (for PT server calculation).
what are the key characteristics of probe design?
- probe length (15-25 nucleotides)
- sequences
- dissociation temperature (GC content, * Td = 4N(G+C) + 2N(A+T))
- number of diagnostics mismatches
- quality of mismatches
- position of mismatches
- neighbourhood bases (stacking, GS-stretches)
- intra-molecular base pairing (hairpins)
- secondary structure of target
- method of hybridisation (temp, ion strength, denaturants)
how is the position of mismatches significant in probe design
- In some cases, a single mismatch can still mean a probe will work and allow binding if central.
If central then either end of the probe still binds and you still get a signal
If the mismatch is at either end then there is more chance of the probe pulling apart and there is no signal
new probes need at least a single base mismatch to all non target organisms
why do you need to consider the quality of mismatches in probe design?
some are more destabilising than others
* Destabilising: A-A, A-C, T-T, T-C, C-C
* Slightly destabilizing: G-T, G-A, G-G
what things can effect probe efficiency?
- GC stretches
- Formation of hairpin structures.
what significance does temperature and concentration of components have in FISH optimisation
normally FISH probes are DNA, which is pretty suseptible to temperature
temperature is important for the binding (dissociation temperature)
concetration of components is important (conc of formamide can make the difference between good discrimination)
describe the competitor concept that was attempted to overcome non specific binding
a second probe is used to bind to the non-target species
its not really used because it makes things messy
what are the limitations of FISH?
- Permeabilisation of cell membrane.
- Low rRNA content affecting sensitivity. (slow growing species have lower content)
- Problems accessing target site.
- Optimisation of hybridization/washing conditions.
- Quantification.
- Species differentiation.
- Physiological information missing.
- Signal can be reduced by nucleotides acting as quenchers.
- Unspecific binding of probes producing background noise
what are helper probes?
- Are unlabeled oligonucleotides that bind adjacent or near the target region. – by binding they open up the inaccessible unit by altering he structure
what do helper porbes do?
- Can increase accessibility.
- Can allow inaccessible target regions based on secondary structures to become accessible.
why do helper probes need to be a few nucleotides longer?
to ensure tight binding beyond the melting point of the probe
