Analysis of Hot Spring Microbial Mat Community by DGGE

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Analysis of Hot Spring Microbial Mat Community by DGGE. Wed 7/8 1-1:30pm Pour gradient gels Read through protocols (Students will be divided into 2 groups) 1:30-2:30pm Lecture on DGGE 2:30-3pm Pour staking gels Prepare samples for loading 3-4pm Load samples!.
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Analysis of Hot Spring Microbial Mat Community by DGGEWed 7/81-1:30pm Pour gradient gels Read through protocols (Students will be divided into 2 groups)1:30-2:30pm Lecture on DGGE2:30-3pm Pour staking gels Prepare samples for loading3-4pm Load samples!Different microbial populationsin a communityDNA extraction and PCR amplificationMixed 16S rRNA gene copiesSeparate by cloning in E. coli or DGGEPhylogeneticidentificationSequenceDenaturing Gradient Gel Electrophoresis (DGGE)
  • Separate DNA fragments of the same length but with different sequences
  • Separation is based on the melting behavior of double-stranded DNA
  • Melting behavior depends on base-pair composition of the DNA
  • DNA StructureBrock Biology of MicroorganismsFigure 07-04Stronger! GC pairs > AT pairsBrock Biology of MicroorganismsFigure 07-03Brock Biology of MicroorganismsFigure 07-09Denaturation of DNA = Meltingss DNA ds DNA Heat/Denaturant Melting temperatureFunction of the GC content of the DNA+TemperatureDenaturantDenaturant(Formamide/Urea)100%0%Partially meltedSeparation Based onDifferences in Nucleotide Sequence (G+C content) and MeltingCharacteristicsSingle strandsElectrophoresis Orwith GC-clampDouble strandPCR AmplificationMixed Populationof DNAG+C-TailedProductPCR PrimersSeparate onDenaturingGradient Gel+G+C-Rich“Clamp”16S rRNA GeneDenaturing Gradient Gel Electrophoresis BA C DIncreasing DenaturantHours1.5 3 4.5 6 7.5 9Time Travel DGGESamples are loaded at regularintervals to determine optimum running time.Increasing DenaturantWhat can you know from DGGE?
  • Community complexity
  • Identify community members by sequencing
  • Distribution of microbial populations inhabiting different environments (e.g. temperatures)
  • Monitor community changes
  • Example: Mushroom Spring Mat communityA’BAC65°C60°C68°C55°C50°CDGGE Gel 1: Loaded by studentsA’BAC1 2 3 4 5 6 7 8 9 10 68 65 60 55 S1S2S3S4S5S1S2S4S5Mushroom SpringS3Mel
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