Primer Selection. For any process, the primer selected for use in PCR to amplify some piece of DNA is the first point that determines what sort of information you will be working with Universal for ID, targeted for group selection, gene group targeting (NOT necessarily expressed though). Bias.
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Starting DNA sequence:
Liu et al. (1997) Appl Environ Microbiol 63:4516-4522
Run DGGE animation here – from http://www.charite.de/bioinf/tgge/
Very sensitive to variations in DNA sequence
Can excise and sequence DNA in bands
”One band-one species” isn’t always true
Cannot compare bands between gels
Only works well with short fragments (<500 bp), thus limiting phylogenetic characterization
Relatively easy to do
Results can be banked for future comparisons
Less sensitive phylogenetic resolution than sequencing
Each fragment length can potentially represent a diversity of microorganisms
Cannot directly sequence restriction fragments,making identification indirectRFLP vs. DGGE