application of molecular biotechnologies to remediation
DESCRIPTION
Application of Molecular Biotechnologies to Remediation. Shu-Chi Chang, Ph.D., P.E., P.A. Assistant Professor 1 and Division Chief 2 1 Department of Environmental Engineering 2 Division of Occupational Safety and Health, Center for Environmental Protection and Occupational Safety and Health - PowerPoint PPT PresentationTRANSCRIPT
Application of Molecular Biotechnologies to Remediation
Shu-Chi Chang, Ph.D., P.E., P.A.Assistant Professor1 and Division Chief2
1Department of Environmental Engineering2Division of Occupational Safety and Health,
Center for Environmental Protection and Occupational Safety and HealthNational Chung Hsing University
Wednesday, June 13, 2007
Categories
Molecular biological methods Biochemical methods Microbiological methods
Molecular biological methods
PCR based A PCR animation from “Molecular
Biology of the Cell” Probe hybridization
PCR based ARDRA (amplified ribosomal DNA restriction analysis): Separates amplified 16S mol
ecules by restriction patterns DGGE (denaturing gradient gel electrophoresis): Separates amplified 16S molecule
s by %G-C content TGGE (temperature gradient gel electrophoresis): Separates amplified 16S molecul
es 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 16
S molecules by length RISA (ribosomal intergenic spacer analysis): Separates amplified 16S-23S intergeni
c 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 bas
ed on random PCR priming, Sequencing of cultured isolates: Sequencing of PCR amplicons derived from cultur
ed isolates Functional PCR: Several PCR-based analyses using amplified catabolic genes; indir
ect functional assay Direct cloning and sequencing: Direct sequencing of isolated and cloned fragments
ARDRA Amplify community rDNA Add combinations of restriction enzymes Assumption: if right enzymes were used, each spec
ies 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 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 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 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
Ribosomal intergenic region Utilizing natual variability of rrl opero
n in rRNA Can be used to distinguish different st
rains and closely related species Rapid and simple but biases from PCR
and secondary structure.
RAPD
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 m
ethod
Comparison of methods
Probe hybridization
General probe hybridization: Identifies presence of desired sequences using labeled probes
DNA microarrays: Extremely high-throughput multiple probe hybridization
Probe hybridization Purposes
Presence of various taxanomic groups Measure relative abundance Determine their spatial distribution
Type FISH CISH CARD-FISH MAR-FISH
Probe hybridization
Advantages Great flexibility Rapid and low cost Good specificity, usually Can aim at multiple targets
Disadvantages Probe design ->mismatch Sensitivity
DNA microarray
Microarray data analysis
Microarray Related areas
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 Affymetrix Agilent Technologies CombiMatrix Eppendorf Nanogen
Biochemical methods DNA composition and kinetics assays
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
Metabolic assays Metabolomics: Emerging technique to profile total metabolites produced by
a community Lipid analyses
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 Systematic study of the unique chemical fin
gerprints 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
Metabolic assay CLPP (community-level physiological profiling): Creates a
profile of substrates metabolized by the microbial community
Cell counting techniques Direct cell counting: Microscopic counting of stained cells Indirect cell counting: Counting of a culturable subset of t
he microbial community Morphological counting: Microscopic identification and e
numeration of the morphotypes in an environmental sample
Flow cytometry and cell sorting: Physically separates microbial assemblages on the basis of measurable properties,
CLPP
BiologTM
Basics of flow cytometry
Light source
Side scatter light
Forward scatter light
Forward scatter : Blue
Fluorescence 3: RedFluorescence 4: Dark
red
Fluorescence 2: Yellow
Side Scatter : BlueFluorescence 1:
Green
Basics of flow cytometry
Three major modules: Optics, Electronics, and Microfluidics.
488nmBlue Laser
FSC
488/10
Fluorescence 1
488/10
530/30
585/42
661/16
670LP
Side Scatter
Fluorescence 2
Fluorescence 4
Fluorescence 3
635nmRed Laser
No. FSC
SSC FL1 FL2 FL3 FL4
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
1 0.50
1.20
2.23
0.31 0.54 0.33
Side scatter light
FL
1: G
reen
Flu
ore
scen
ce
Flow cytometry outputLight
source
Side scatter light
Forward scatterlight
FSC
FL4FL3
FL2
SSCFL1
Lo
wH
igh
Transparent Opaque
R1
R2