Microbial Community Analysis I Flashcards
What are some examples of culture-independent sequence techniques?
Sequencing of 16S rDNA/RNA
DNA fingerprinting, still based on 16S rRNA gene (includes DGGE, ARISA, T-RFLP)
Sequencing of metagenome, entire genome, metatranscriptome, entire transcriptome
What is community fingerprinting used for?
To profile the diversity of a microbial community
What are the advantages and disadvantages of community fingerprinting?
Adv: doesn’t rely on cultivating microbes in the lab; usually quick and cheap on a number of samples simultaneously
Dis: no identification of microbes unless a further sequencing step is performed on bands of interest; not always reproducible, unable to capture rare taxa, some bands can be comprised of more than one sequence type
Draw out DGGE process
DO IT
What is the process of DGGE?
Denaturing Gradient Gel Electrophoresis
The PCR amplicons (16S rRNA sequences of the microbial community) are separated on a gel based on the properties of the sequence
These properties have mainly to do with nucleotide content, in particular, GC content which exerts a strong influence on the ability of the sequence to denature
A DGGE gel used a gradient denaturant - a mixture of urea and formamide
Some researches use a linear temperature gradient
When a sequence fragment reaches a position on the gel that equals to its melting point, it stops moving through the gel
This is due to the GC clamp
What is the GC clamp for?
Anchors DNA together once it’s been denatured
The GC-clamp portion of primers for DGGE amplicon preparation provides a key component in resolving fragments of similar size but different sequence
What are the advantages of DGGE?
[same as before]
What are the disadvantages of DGGE?
[same as before]
Some bands can comprise more than one sequence type
The GC clamp can be variable because different investigators use different one which leads to different DGGE profiles, therefore, DGGE patterns are not comparable between different studies
Draw out T-RFLP process
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What is the process of T-RFLP?
Terminal Restriction Fragment Length Polymorphism
The PCR amplicons (16S rRNA of microbial communities) are fluorescently labelled during PCR, so after each PCR, each amplified copy of DNA carries the fluorescent label
Restriction enzymes are then used to cut the amplified DNA at specific recognition sites
Different sequences (belonging to different microbes) will thus be cut at different locations
Each different restriction site is considered to represent an OTU
Unlabelled fragments are not recorded in the final analysis
The fragments are then separated by size on a gel
Lasers are then used to detect the size and fluorescent intensity of fragments
What are the advantages of T-RFLP?
[same as before]
What are the disadvantages of T-RFLP?
[same as before]
Some bands may be comprised of more than one sequence type because different types of microorganisms may share the same restriction site in the gene of interest being amplified, therefore, these organisms would be represented as one peak on the electropherogram
Draw out ARISA process
DO IT
What is the process of ARISA?
Automated Ribosomal Intergenic Spacer Analysis
ARISA takes advantage of the fact prokaryote DNA encodes for 2 highly conserved genes (16S rRNA and 23S rRNA - the small and large subunits of prokaryote ribosomal RNA)
In between these genes is an Internal Transcribed Spacer (ITS) region (a non-coding region), that is a highly variable nucleotide sequence and variable in terms of length
This ITS sequence of the microbial community is amplified by PCR either by
1) Using fluorescent-labelled PCR primers for ARISA
- the fluorescent PCR fragments are then run on a gel and translated into peaks based on length and intensity of the bands = abundance of respective organisms
- the banding pattern is translated into community diversity
2) Using non-labelled PCR primers for RISA
- the PCR fragments are then run on a gel and the banding patterns are translated into community diversity
What are the advantages of ARISA?
[same as before]
Can provide higher resolution in detecting microbial diversity compared to DGGE and T-RFLP due to hypervariable ITS region
