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
Sequence
Phylogenetic
identification

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
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

5. 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

6. 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

7. 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

8. 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

9. Time Travel DGGE Hours 1.5 3 4.5 6 7.5 9 Samples are loaded at regular
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 communityB A C
68°C
65°C
60°C
55°C
50°C

12. DGGE Gel 1: Loaded by studentsC S1 S2 S3 S4 S5 S1 S2
Mel S3 S4 S5
Mushroom Spring