0123456789
relative frequency
% tot biomass
Mycorrhizal Diversity in an Abies concolor Forest
Fungal species in ranked order
Work by Antonio IzzoBased on 36 soil cores from a total of 9 plotscontained within a 2.5 hectare region
Assumptions of clone and sequence approaches
• No extraction bias
• No amplification bias
• No cloning biases
• Sequences retrieved were from living organisms
• Cloning and PCR artifacts are unimportant
• Phylogenetic placement is predictive of functional attributes
Chimera formation via partial extensions
partial extension
denature
Heterologous partial fragments anneal
Extension creates chimara
Further rounds of amplificationcreate many copies
Assumptions of clone and sequence approaches
• No extraction bias
• No amplification bias
• No cloning biases
• Sequences retrieved were from living organisms
• Cloning and PCR artifacts are unimportant
• Phylogenetic placement is predictive of functional attributes
What can we say about unique sequences?
Giovannoni et al. 1996
Achenbach and Coates 2000
photosynthetic
Fe reducing, obligate anarobe
Non-Fe reducing, facultative anarobe
From Achenbach and Coates 2000
*
*
Advantages and Disadvantages of Sequence approach to community analysis
• Still is one of the best ways to identify a pool of total unknowns
• Produces an imperfect quantitative picture
• Doesn’t tell you much about function
• Cost and effort limit the number of replicate samples
O’Brien et al. 2005
O’Brien et al. 2005
O’Brien et al. 2005
• Amplify portion of rDNA gene using a primer with a 5’ GC clamp
• Load pool of amplicons onto denaturing gradient gel
• Slightly different products are separated by sequence differences that cause different levels of partial denaturation.
DGGE - Denaturing gradient gel electrophoresis& TGGE - temperature gradient gel electrophoresis
From Ward et al 1998. Mol. Biol. Rev
DGGE gel From Ward et al 1998. Mol. Biol. Rev
denature
extension
annealing of primers
extension
denature
Reannealing of strands
heteroduplex
homoduplex
Heteroduplex formation: a feature of all PCR reaction with complex mixtures of similar products
Amplify pool of sequences with one of the primers labeled
Digest with a restriction enzyme
A B C
B
A
C
Each ampliconproduces a singledetected fragment
T-RFLP (terminal restriction fragment length polymorphism)
An example of t-RFLP data from a very simple community
T-RFLP analysis & gel
Moeseneder et al 1999
How can t-RFLP analysis separate as many 16S sequences as DGGE?
Because many of the differences are based on indels rather than base substitutions in restriction sites
What can’t you do with t-RFLP that you could do with DGGE or TGGE?
Retrieve the entire sequence by cutting the fragment out of the gel and sequencing it
SSCP - Single stranded conformational polymorphisms
• Amplify target (100-600 bp), with one of the primers phosphorylated
• Digest products with Lambda exonuclease (only phosphorylated strand is digested)
• Separate remaining single-stranded products on non-denaturing gel
• Migration of fragments due to conformation rather than size
SSCP gel from soil microbial communitySchmalenberger & Tebbe Mol. Ecol. 2003 12:251-262
Excised bands were cloned and sequenced and found to be complex pools of sequences
Comparison of finger printing methodsMethod Unique advantages Unique Disadvantages
DGGE, TGGE Can excise, clone and sequence bands
Specialized gel or equipment needed, heteroduplex bands
Results difficult to reproduce between labs
T-RFLP Can be run on an automated sequencer
Highly reproducible size estimates
Can not excise and sequence bands easily, may not be very useful for protein coding sequences
SSCP Can excise, clone and sequence bands
Single fragments may have multiple conformers
Results difficult to reproduce between labs
For higher resolution the same methods canbe applied to the spacer region, but no database exists!
• Size typically 540-950 bp• Specific primers allow fungal sequences to
be easily amplified from complex environments
• Usually highly variable between species groups
• Variation is often rich in IDELs
The ITS (internal transcribed spacer) region in fungi
Top Related