GI tract composition analysis
13 important questions on GI tract composition analysis
What is the difference between a population and community of micro-organisms?
- Microorganisms function as populations assemblages of similar microbes
e.g. Lactobacillus population - Microorganisms function as communities mixtures of different microbial populations.
e.g. GI tract microbial community
How is ribosomal RNA used as an marker?
- One ore more rRNA gene copies present in all living systems.
- Protein synthesizing is an essential function --> results in a slow genetic evolution --> rRNA sequences are highly conserved.
- Mutation rate of rRNA corresponds to evolutionary divergence of micro-organisms.
- Low lateral gene transfer and sequence heterogeneity
- Multicopy expression (rRNA) reflects cellular activity
- Clasification: 16S rRNA sequence > 97% similarity likely to be the same species.
How does Random cloning ans sequencing work?
- DNA isolated directly from feces
- PCR with universal bacterial primers
- Clone and sequence
- Comparative analysis to 16S rRNA databases.
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How does the DNA isolation form feces go?
This is done multiple times so you get millions of copies.
How does cloning and sequencing go?
What kind of bacteria do you have in the culture?
A: A new bacteria with a summarily to Faecalibacterium prausnitzii.
What is high trueput sampeling?
- Streptavidin-coupled Dynabead can bind to the primer that will bind to the DNA.
- Whent it is denatured it will stick to the bead.
- The primer has a reconisable sequence that is specific.
- So when you are analysing that data you can see that this DNA has this primer and therefore is from which sample your product came.
- So barcode A is from sample 1.
- You can analyse all your data at the same time.
How does PCR-DGGE work?
They are separated by denaturing gradient gel electroscopes.
- Because the DNA has different sequences they will denature at a different point.
- When they start denaturing they will stop running tough the gel and this is how you can separate them.
- A GC clamp (part of dna that is always the same) is added. This has the same sequence and will not denture. It prevents your DNA form being complacently denatured and running trough the gel.
What is Real-time PCR?
It's a fluorescence based technique: emission of fluorescence is directly correlated to the amount of amplified DNA. `(this is not realy the case in real life but close enough)
What is the difference between standard PCR and real time PCR?
With real time you look at the times you have to multiply the DNA to get the the treshold level. This can be a reliable indicator of the initial copy number.
How does a DNA Array work?
What are the outcome of the experiments and what are the main limitations?
What is the information you can get from fingerprinting?
You can compere samples
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