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Biofilms & Microbial communities > Fluorescence in situ hybridisation (FISH) > Flashcards

Fluorescence in situ hybridisation (FISH) Flashcards

understanding microbial communities

1
Q

why was fluorescence in situ hybridisation developed?

A

limited morphological diversity
because existing stains didnt give enough information
we needed a way to separate out the bacteria at different levels

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2
Q

what ways are microorganisms classically characterised?

A

looked for physiological and biochemical characteristics
– Enrichment
– Pure culture
– Physiological characterisation

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3
Q
A
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4
Q
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5
Q

why was ribosomal RNA chose as a marker?

A
  • 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
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6
Q

what makes up small subunits of prokaryotic ribosomes?

A

30S = 16S rRNA

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7
Q

what makes are large subunits of prokaryotic ribosomes?

A

50S = 5S and 23S rRNA

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8
Q

describe the full cycle rRNA approach to FISH

A
  1. DNA is extracted
  2. amplified via PCR
  3. cloned
  4. sequenced
  5. comparistive sequence analysis and phylogenetic affiliation
  6. probe design from sequence database
  7. probe testing
  8. in situ hybridisation
  9. detection
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9
Q

what is the only tool in microbiology to determine true cell numbers?

A

FISH

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10
Q

what are some features and pros to FISH?

A
  • 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”).
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11
Q

breifly describe the process of FISH using monolabelled oligonucleotide probes

A

fix cells for permeabilisation, then hybridise with fluorescently labelled oligonucleotide. wash away excess, then quantify via epifluorescence or confocal microscopy, or flow cytometry

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12
Q

the prerequisite for probe design is a comprehensive database
what should it comprise?

A
  • should comprise all three domains
  • should comprise all types of RNA
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13
Q

give two examples of rRNA databases

A
  • RDP
  • SILVA rRNA database project
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14
Q

what are the limitations of rRNA databases?

A
  • 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).
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15
Q

what are the key characteristics of probe design?

A
  • 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)
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16
Q

how is the position of mismatches significant in probe design

A
  • 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

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17
Q

why do you need to consider the quality of mismatches in probe design?

A

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

18
Q

what things can effect probe efficiency?

A
  • GC stretches
  • Formation of hairpin structures.
19
Q

what significance does temperature and concentration of components have in FISH optimisation

A

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)

20
Q

describe the competitor concept that was attempted to overcome non specific binding

A

a second probe is used to bind to the non-target species
its not really used because it makes things messy

21
Q

what are the limitations of FISH?

A
  • 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
22
Q

what are helper probes?

A
  • Are unlabeled oligonucleotides that bind adjacent or near the target region. – by binding they open up the inaccessible unit by altering he structure
23
Q

what do helper porbes do?

A
  • Can increase accessibility.
  • Can allow inaccessible target regions based on secondary structures to become accessible.
24
Q

why do helper probes need to be a few nucleotides longer?

A

to ensure tight binding beyond the melting point of the probe

25
Q

when is flow cytometry favoured for quantification following FISH?

A

For single cells in a suspension,

26
Q

when is microscopy favoured for quantification following FISH?

what else can it tell us?

A

for complex samples/ biofilms

tells us numbers, biovolume or surface coverage

27
Q

what is LNA-FISH?

A

LNA = locked nucleic acid
a synthetic probe is used to increase sensitivity and specificity

  • LNA nucleotides can be mixed with DNA or RNA residues.
  • Hybridise according to Watson-Crick base pairing rules.
28
Q

what is PNA-FISH?

A
  • PNA = peptide nucleic acid.
  • Synthetic DNA analogues,
    negatively charged sugar phosphate backbone is replaced with non-charged peptide backbone. (no charge effects)
    allows rapid and specific binding
29
Q

what are the advantages of PNA-FISH

A
  • Allows rapid and specific binding.
  • Shorter probe lengths, allows easier accessibility to target sequences.
  • Less need for stringent hybridisation conditions e.g. temp, ionic changes, enzymatic changes.
30
Q

compare DNA and PNA probes for FISH

A
  • PNA hydridisation affinity with DNA is at least 1 degree higher per base
  • PNA is 100-5000 times faster
  • independent of salt concentration for hybridisation (compared to DNA which is dependent)
  • Tm single mismatch lowering 15 (rather than 10degree)
  • PNA is chemically stable (whereas DNA is unstable in acid and base)
  • PNa is stable to nuclease and protease (DNA is degraded by nucleases)
  • PNA shorter probe length 13-18nt (DNA 20-30nt)
31
Q

how does the back bone of PNA and DNA differ

A

DNA/RNA have a negatively charged sugar-phosphate backbone
PNA has a non-charged polyamide or peptide backbone

32
Q

how does the DNA and PNA probe binding to RNA targets differ

A

DNA probes must overcome a destabilising electrostatic repulsion during hybridisation to complementary nucleic acid targets

The non-charged backbone of PNA probes allow for tighter and more specific hybridisation to nucleic acid targets

33
Q

what is CARD-FISH?

A

CARD = catalysed reporter deposition

horseradish-peroxidase (HRP-) labelled oligonucleotides used to boost the signal.

Bound peroxidase molecules catalyse deposition of fluorescently labelled tyramide within cells targeted by HRP-probe.

results in 20 times brighter signal

*Staining results from secondary incubation with fluorescently-labelled tyramide.

34
Q

what is the disadvantage to CARD-FISH

A

more stringent cell permeabilisation steps are needed to enable larger enzyme-labelled oligonucleotides to cross cell membrane – can result in cell lysis and alter community structure.

35
Q

what is CLASI-FISH?

A
  • CLASI = Combinational Labelling And Spectral Imaging.
  • Uses combinations of probes to label bacteria in a mixed population and spectral imaging to visualise.
  • Overcomes the problem of reduced number of fluorophores.
36
Q

what are the disadvantages of CLASI-FISH?

A

limitations due to multiplex hybridisation protocols, need for stringent conditions, increased permeabilisation.

37
Q

what FISH techniques were developed to provide more information about physiology and metabolic state?

A
  • MAR-FISH
  • ISRT-FISH
  • Raman-FISH
  • NanoSIMS-FISH
38
Q

what is MAR-FISH?

A
  • Uptake radioactively labelled substrates into cells.
  • Detected by MicroAutoRadiography (MAR) with simultaneous bacterial identification with FISH.
  • Decay from incorporated substrate can be visualized along with the cells metabolising it.
39
Q

what is ISRT-FISH?

A
  • ISRT - In Situ Reverse Transciption
  • Specific mRNA are amplified by qPCR to cDNA.
  • Targeted by fluorescent probes.
  • Used to study metabolically active groups and control of gene expression in populations.
40
Q

give some examples of FISH applications

A
  • food production facilities
  • rapid tests for diagnosis

Biofilms & Microbial communities (11 decks)

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