A1258150222USEHt

1
Analysis of Hot Spring Microbial Mat Community
by DGGE
Wed 7/8 1-130pm Pour gradient gels Read
through protocols (Students will be divided
into 2 groups) 130-230pm Lecture on
DGGE 230-3pm 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 gt 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 (GC content) and Melting Characteristi
cs
Single strands
Electrophoresis
Or with GC-clamp
Double strand
8
PCR Amplification
Mixed Population of DNA
GC-Tailed Product
PCR Primers
Separate on Denaturing Gradient Gel

GC-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
65C
60C
68C
55C
50C
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
Mushroom Spring
S1
S2
S4
S5
S3
Mel