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!

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

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

DNA Structure Brock Biology of Microorganisms Figure 07-04 Caption: DNA structure. Complementary and antiparallel nature of DNA. Note that one chain ends in a 59-phosphate group, whereas the other ends in a 39-hydroxyl. The red bases represent the pyrimidines cytosine (C) and thymine (T), and the yellow bases represent the purines adenine (A) and guanine (G). Brock Biology of Microorganisms Figure 07-04

Stronger! GC pairs > AT pairs Brock Biology of Microorganisms Figure: 07-03 Caption: Specific pairing between adenine (A) and thymine (T) and between guanine (G) and cytosine (C) via hydrogen bonds. These two base pairs are the base pairs typically found in double-stranded DNA. Atoms that are found in the major groove of the double helix and that interact with proteins are highlighted in red. The deoxyribose phosphate backbones of the two strands of DNA are also indicated. Brock Biology of Microorganisms Figure 07-03

Function of the GC content of the DNA 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 Figure: 07-09 Caption: Thermal denaturation of DNA. The DNA absorbs more ultraviolet light as the double helix is denatured. The transition is quite abrupt, and the temperature of the midpoint, Tm, is directly related to the GC content of the DNA. Although the denatured DNA can be renatured by a slow cooling, the process does not follow a similar curve. Renaturation becomes progressively more complete at temperatures well below the Tm, and then only after a considerable incubation time. Temperature + Denaturant

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

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

Time Travel DGGE Hours 1.5 3 4.5 6 7.5 9 Samples are loaded at regular 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

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

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

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