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Analysis of Hot Spring Microbial Mat Community by DGGE

Delve into the diversity of microbial populations using Denaturing Gradient Gel Electrophoresis (DGGE). This workshop includes gel pouring, DNA extraction, amplification, and phylogenetic identification. Learn to separate DNA fragments based on melting behavior and sequence differences in this interactive session. Understand the importance of GC content and melting temperature in DNA structure while exploring the complexity and dynamics of microbial communities. Participants will load samples on the DGGE gel to study community changes and environmental adaptations. Join us to unravel the mysteries of microbial diversity in hot springs.

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Analysis of Hot Spring Microbial Mat Community by DGGE

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  1. 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!

  2. Different microbial populations in a community DNA extraction and PCR amplification Mixed 16S rRNA gene copies Separate by cloning in E. coli or DGGE Phylogenetic identification Sequence

  3. Denaturing 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

  4. DNA Structure Brock Biology of Microorganisms Figure 07-04

  5. Stronger! GC pairs > AT pairs Brock Biology of Microorganisms Figure 07-03

  6. Brock Biology of Microorganisms Figure 07-09 Denaturation of DNA = Melting ss DNA ds DNA Heat/Denaturant Melting temperature Function of the GC content of the DNA + Temperature Denaturant

  7. Denaturant (Formamide/Urea) 100% 0% Partially melted Separation Based on Differences in Nucleotide Sequence (G+C content) and Melting Characteristics Single strands Electrophoresis Or with GC-clamp Double strand

  8. PCR Amplification Mixed Population of DNA G+C-Tailed Product PCR Primers Separate on Denaturing Gradient Gel + G+C-Rich “Clamp” 16S rRNA Gene Denaturing Gradient Gel Electrophoresis B A C D Increasing Denaturant

  9. Hours 1.5 3 4.5 6 7.5 9 Time Travel DGGE Samples are loaded at regular intervals to determine optimum running time. Increasing Denaturant

  10. What 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

  11. Example: Mushroom Spring Mat community A’ B A C 65°C 60°C 68°C 55°C 50°C

  12. DGGE Gel 1: Loaded by students A’ B A C 1 2 3 4 5 6 7 8 9 10 68 65 60 55 S1 S2 S3 S4 S5 S1 S2 S4 S5 Mushroom Spring S3 Mel

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