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