Analysis of Hot Spring Microbial Mat Community by DGGE

Wed 7/8

1-1:30pm Pour gradient gelsRead 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 community

DNA extraction and PCR amplification

Mixed 16S rRNA gene copies

Separate by cloning in E. coli or DGGE

Sequence Phylogeneticidentification

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 MicroorganismsFigure 07-04

Brock Biology of MicroorganismsFigure 07-03

GC pairs > AT pairsStronger!

Denaturation of DNA= Melting

+TemperatureDenaturant

Brock Biology of MicroorganismsFigure 07-09

ds DNA ss DNA

Heat/Denaturant

Melting temperatureFunction of the GC content of the DNA

Separation Based on

Differences in Nucleotide Sequence

(G+C content) and Melting

Characteristics

Denaturant(Formamide/Urea)0% 100%

Electro

ph

oresis

Double strand

Partially melted Single strands

Orwith GC-clamp

Mixed Populationof DNA

+

16S rRNA Gene G+C-Rich“Clamp”

PCR Primers G+C-TailedProduct

Separate onDenaturing

Gradient Gel

PCR Amplification

Denaturing Gradient Gel Electrophoresis

Increasing Den

aturant

B CA D

Time Travel DGGE

Samples are loaded at regularintervals to determine optimum running time.

Hours1.5 3 4.5 6 7.5 9

Incr

easi

ng D

enat

uran

t

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

68°C 65°C 60°C 55°C 50°C

A’A B

C

S1 S2 S3 S4 S5

Mel

1 2 3 4 5 6 7 8 9 10 68 65 60 55

Mushroom SpringS1 S2 S3 S4 S5

DGGE Gel 1: Loaded by students

B

CA

A’