Transcript Application of Molecular Biotechnologies to Remediation

Application of Molecular
Biotechnologies to Remediation
Shu-Chi Chang, Ph.D., P.E., P.A.
Assistant Professor1 and Division Chief2
1Department of Environmental Engineering
2Division of Occupational Safety and Health,
Center for Environmental Protection and Occupational Safety and Health
National Chung Hsing University
Wednesday, June 13, 2007
Categories
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Molecular biological methods
Biochemical methods
Microbiological methods
Molecular biological methods
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PCR based
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A PCR animation from “Molecular Biology of
the Cell”
Probe hybridization
PCR based
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ARDRA (amplified ribosomal DNA restriction analysis): Separates amplified 16S
molecules by restriction patterns
DGGE (denaturing gradient gel electrophoresis): Separates amplified 16S molecules by
%G-C content
TGGE (temperature gradient gel electrophoresis): Separates amplified 16S molecules
by %G-C content;
T-RFLP (terminal-restriction fragment length polymorphism): Separates amplified 16S
molecules by restriction patterns
LH-PCR (length heterogeneity polymerase chain reaction): Separates amplified 16S
molecules by length
RISA (ribosomal intergenic spacer analysis): Separates amplified 16S-23S intergenic
region by length
SSCP (single-strand conformation polymorphism): Separates amplified 16S ssDNA by
sequence-dependent higher order structure
RAPD (randomly amplified polymorphic DNA): Sequence-independent profiling based
on random PCR priming,
Sequencing of cultured isolates: Sequencing of PCR amplicons derived from cultured
isolates
Functional PCR: Several PCR-based analyses using amplified catabolic genes; indirect
functional assay
Direct cloning and sequencing: Direct sequencing of isolated and cloned fragments
ARDRA
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Amplify community rDNA
Add combinations of restriction enzymes
Assumption: if right enzymes were used, each
species will have a unique pattern (fingerprint).
However, it is hard to differentiate from each other.
Usually only one fingerprint for one community
BY incorporating probe hybridization, more detail
information can be obtained
Disadvantage: need optimized combination of
restriction enzymes.
Advantage: fast and cost-effective
DGGE
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Different G-C contents render different mobility in
DNA-denaturing gel which is prepared to have a
concentration gradient of denaturant.
Probably most widely applied method for community
characterization.
Limitation
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Need to optimize the gradient and electrophoresis duration
DNA fragment < 500bp
Need large quantity of DNA
Statistical method may help to resolve some
problems associated with DGGE.
T-RFLP
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Modified form of ARDRA using
fluorescent PCR primers
Limitation of database (only prokaryotic)
Can only observe 50 or so populations
Sensitivity ~0.5%
Potential bias from PCR
Probably more quantitative than other
methods
RISA
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Ribosomal intergenic region
Utilizing natual variability of rrl operon
in rRNA
Can be used to distinguish different
strains and closely related species
Rapid and simple but biases from PCR
and secondary structure.
RAPD
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Is able to generate a unique set of
amplicons for each species.
random short PCR primer
Usually 5~15 sets per species
Cannot be complemented by other
method
Comparison of methods
Probe hybridization
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General probe hybridization: Identifies
presence of desired sequences using
labeled probes
DNA microarrays: Extremely highthroughput multiple probe hybridization
Probe hybridization
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Purposes
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Presence of various taxanomic groups
Measure relative abundance
Determine their spatial distribution
Type
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FISH
CISH
CARD-FISH
MAR-FISH
Probe hybridization
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Advantages
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Great flexibility
Rapid and low cost
Good specificity, usually
Can aim at multiple targets
Disadvantages
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Probe design ->mismatch
Sensitivity
DNA microarray
Microarray data analysis
Microarray
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Related areas
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Bioinformatics : Online Services : Gene Expression and
Regulation at the Open Directory Project
Gene Expression : Databases at the Open Directory Project
Gene Expression : Software at the Open Directory Project
Data Mining : Tool Vendors at the Open Directory Project
Notable companies
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Affymetrix
Agilent Technologies
CombiMatrix
Eppendorf
Nanogen
Biochemical methods
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DNA composition and kinetics assays
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Metabolic assays
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DNA reassociation kinetics: Estimates sample diversity based on
rate of reassociation of denatured DNA
Bisbenzimidazole-CsCl-gradient fractionation: DNA fractionnation
based on %GC content
Community DNA hybridization: Estimates relative similarity of two
communities by cross hybridization kinetics
Metabolomics: Emerging technique to profile total metabolites
produced by a community
Lipid analyses
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Quinone profiling: Culture-independent community profile based on
distribution of quinones
PLFA (phospholipids fatty acids) + FAME (fatty acid methyl esters):
Culture-independent community profile based on distribution of
various membrane lipids
Metabolomics
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Systematic study of the unique chemical
fingerprints that specific cellular processes
leave behind
mRNA gene expression data and proteomic
analyses do not tell the whole story of what
might be happening in a cell, metabolic
profiling can give an instantaneous snapshot
of the physiology of that cell.
PLFA
PLFA
PLFA
Microbiological methods
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Metabolic assay
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CLPP (community-level physiological profiling): Creates a
profile of substrates metabolized by the microbial community
Cell counting techniques
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Direct cell counting: Microscopic counting of stained cells
Indirect cell counting: Counting of a culturable subset of the
microbial community
Morphological counting: Microscopic identification and
enumeration of the morphotypes in an environmental
sample
Flow cytometry and cell sorting: Physically separates
microbial assemblages on the basis of measurable properties,
CLPP
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BiologTM
Basics of flow cytometry
Side scatter light
Side Scatter : Blue
Fluorescence 1: Green
Fluorescence 2: Yellow
Fluorescence 3: Red
Fluorescence 4: Dark red
Light
source
Forward scatter light
Forward scatter : Blue
Basics of flow cytometry
Fluorescence 1
Side Scatter
530/30
Fluorescence 2
488/10
Fluorescence 4
585/42
661/16
Fluorescence 3
488nm
Blue Laser 635nm
Red Laser
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670LP
FSC
488/10
No.
FSC
SSC
FL1
FL2
FL3
FL4
1
0.50
1.20
2.23
0.31
0.54
0.33
2
3.11
1.22
0.45
0.39
0.51
3.33
3
0.27
3.20
0.38
1.24
3.61
3.44
4
0.06
0.01
1.14
0.71
1.67
0.69
5
1.27
1.92
0.06
2.30
3.07
2.74
6
3.14
3.33
1.18
0.16
2.74
3.44
7
3.13
3.28
0.55
0.21
2.55
0.27
8
3.88
0.84
3.37
2.94
0.52
0.55
9
0.88
0.43
1.51
1.85
3.88
2.86
10
2.07
1.64
0.92
1.12
1.92
1.83
Three major modules: Optics, Electronics, and Microfluidics.
Side scatter light
FL1: Green Fluorescence
High
Low
Flow cytometry output
Light
source
R2
R1
Transparent
Side scatter light
Opaque
SSC
FL1
FL2
FL4
FL3
Forward
scatter
light
FSC