Analysis of Hot Spring Microbial Mat Community by DGGE Wed 7/8 1-1:30pmPour gradient gels Read...
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Transcript of Analysis of Hot Spring Microbial Mat Community by DGGE Wed 7/8 1-1:30pmPour gradient gels Read...
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
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