Transcript Application of Molecular Biology Techniques in Astrobiology

APPLICATION OF
MOLECULAR BIOLOGY TECHNIQUES
IN ASTROBIOLOGY
RICCARDO S. GATTA
Ph.D. Molecular Genetics, I.C.G.E.B. New Delhi, Reg’d Pharmacist
JULIAN CHELA-FLORES
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The Abdus Salam International Centre for Theoretical Physics
Strada Costiera 11; 34136 Trieste, Italy and
Instituto de Estudios Avanzados,
Caracas 1015A, Venezuela
INTRODUCTION
 CLASSIC MOLECULAR BIOLOGY
APPROACH TO
RECOGNISE MOLECULAR BIOSIGNATURES,
&
 APPLY THIS IN AN ISOLATED ENVIRONMENT.
 SELECT TARGET,
 SELECT TECHNIQUE,
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 ADAPT...
SEARCH FOR LIFE IN THE UNIVERSE
OBSERVATIONS
-
EVOLUTION
?
The Origin
MODERN
DARWIN
ANAXIMANDER
(VI BCE)
MACROSCOPIC
DEMOCRITUS
(460-370
BCE)
(1809-1882)
D
C
IVERGENT
ONVERGENT
PARALLEL
HAEKEL
MICROSCOPIC
(1834-1919)

WOESE
(1980’s)
ANCESTOR
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?

MOLECULAR
MODELS OF
EVOLUTION
LAST UNIVERSAL
COMMON
ANCESTOR
EVOLUTION & NATURAL SELECTION
UNIVERSAL CONCEPTS?
IF
Yes
No
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continue…
(…and skip next slide)
LIFE…?
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LIFE
& AS
NATURAL
A TARGET
SELECTION
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IDENTIFY & QUANTIFY
UNIVERSAL CONCEPTS?
Structure
IF
Complexity
continue…
Transduction
Yes
No
Replication
(…and skip next slide)
Energy
LIFE…?
Complex Structures
Specific Interactions
More Complexity
Metabolism
Chemistry
Emergent
Chirality
Driving
Forces
More
Disequilibrium
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Waste
to Polymers Molecular / Cellular
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CENTRAL DOGMA OF MOLECULAR BIOLOGY
Crick, 1958
DNA
RNA
PROTEINS
DO NOT
REPLICATION:
TRANSCRIPTION:
TRANSLATION:
code for :
DNA DNA mRNA
↕
↓
↓
“… to apply only to
present-day
andProtein
DNAorganisms,
mRNA
not to events in the remote past, such as the origin of
life or the origin of the code.” Crick, 1970
Figure from Labfrontier Corporation
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Voltage-gated channels
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EVOLUTIONARY CRITERIA
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Kv
TIME /
K ir
Ca v Na v
Modern Proteins
EVOLUTION
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Na
PHANEROZOIC
~800
million
570 Myo
- present
Example of divergence
years ago
PROTEROZOIC
BUT
Where can we seek homology?Ca
from LUCA at
2.5 Gyo – 570 Myo
Homology with
Check-Point
known molecules ARCHEAN
Intervals
K
4.6 – 2.5 Gyo
For:
Homology
Ancient
Proteins, with
membrane
knownchannels
molecules
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Early K channels to
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Moczydlowski, 1998
2: BUILD UP (CONSTRUCTION)
Homology in:
1: INDIVIDUAL ELEMENTS
1:
2:
2: BUILD UP (CONSTRUCTION)
COMPOSITION
Vassily
Kandinsky.
BLUE 2, 8,
Joan
Miro’.
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2:
3: THE
BUILD
PARADIGM:
UP (CONSTRUCTION)
FROM: NASA, JPL, UA, UC.
DECONSTRUCT
EUROPA
IDENTIFY BASIC ELEMENTS
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SAMPLE ANALYSIS
PRELIMINARY ANALYSIS
ENVIRONMENTAL SAMPLE ANALYSIS
 ASSESS ENVIRONMENTAL CONDITIONS,
 LEGITIMISE FURTHER ASSAYS,
 DIRECT CHOICE OF PLAUSIBLE ASSAYS,
 INDICATE OPTIMAL CONDITIONS FOR ASSAYS.
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MOLECULAR TARGETS
RNA
DNA
Protein
Other:
STRUCTURAL
HIGHLY
CONSERVED
SCAFFOLDS
MOTIFS
 TATAAA,
conserved Shine-Dalgarno,
charged residues,
 essential
rRNA sub-types,
enzymes,
 functionally
shifts in pyrimidine/purine
divergent… ratios…
 superfamilies traceable to LUCA…
TRANSLATIONAL - ENZYMATIC
lipids,
peptidolipids,
 messenger
RNA, lipopolysaccharides,
special
 inhibitory
fatty acids
RNA…
or sugar residues…
chirality…
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MOLECULAR RECOGNITION TECHNIQUES
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
Taqman Applied Biosystems
PROBE
PULSE FIELD GEL ELECTROPHORESIS
FLUORESCENCE IN SITU HYBRIDISATION
FRAGMENT
PCR
SINGLE NUCLEOTIDE POLYMORPHISMS (SNP)
RESTRICTION
FLUORESCENCE
R
EXPRESSED
FRAGMENT
IN
I SEQUENCE
HYBRIDISATION
L
LENGTH
TAGS (EST)
PPOLYMORPHISM
PULSE
F
P
F
FIELD GEL
GF
ELECTROPHORESIS
ESSITU H
WHOLE GENOME
PHYLOGENETIC
STUDIES, DIAGNOSTICS…
VERSATILE
TECHNIQUE
RIBOTYPING
SMALLER FRAGMENTS
of rDNA (5S,(10
16S,
– 1000
23S etc.
kb)rRNA genes)
LABEL
WITH FLUORESCENT
RELATIONSHIP
- BACTERIAL
STRAINSDYE
RIBOSOMAL
GENE
SEQUENCES
LARGE
RESOLUTION
FRAGMENTS
IN 2D (1,000 – 20,000 kb)
POLYMERASE
CHAIN REACTION
GENOME SIZE – PHYLOGENETIC
STUDIES…
DENATURE
DNA
R
&
A
B
↓
HYBRIDISE
2D gelRFLPs
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From: Pillai SD
National Human Genome Research Institute (NHGRI) Darryl Leja
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From: Hernandez-Rivas and Scherf, 1997
MOLECULAR RECOGNITION TECHNIQUES
DNA
RNA
ONCE THE TARGET SEQUENCE IS IDENTIFIED
SEQUENCING
RT-PCR
BLOTTING
REAL TIME REVERSE
TRANSCRIPTION
NORTHERN
BLOT PCR
↓
↓
↓
SINGLE
CELLBLOT
mRNA RESOLUTION
WESTERN
SOUTHERN
BLOT
DIFFERENTIAL DISPLAY
REVERSE
TRANSCRIPTION PCR
proteins
RNA EXPRESSION PROFILES
Protein
PROBES: fluorescent technology
 green fluorescent protein…
ANTIBODIES, mono-poly…
 labeling environment…
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 limited number of conformations…
From: Alberts, Bray, Johnson, Lewis, Raff, Roberts, Walterc 1999
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ANALYTICAL DEVICES
to
FLUORESCENCE
TECHNIQUES
TECHNIQUES
– IMAGE ENHANCING
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BASIC TECHNIQUE,
MINIMAL SAMPLE PREPARATION,
APPLICATIONS MAY BE LIMITLESS...
Olympus Inc.
MetaMorph® Universal Imaging Corp.
Gatta,Interference
ICGEB
New Delhi
Phase
Differential
Contrast,
Contrast
Remove
some
‘out-of-focus’
effects:(DIC),
Nikon Eclipse TE2000
(optic
ganglion tissue
from rat
retina)
(3T3 fibroblast
monolayer,
NIH)
Digital
deconvolution.
MOL-BIO
LAB
USE:
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Fluorescence illumination:
-4',6-diamidino-2-phenylindole
salt fluorochrome (fast
(DAPI),
blue).
AS A -diazonium
LIGHT
MICROSCOPE:
- phase rings on fluorite
forobjective.
cell counting, etc.
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IN FLUORESCENCE MODE:
to highlight transfection efficiency...to
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ION CHANNELS AS TARGETS
HOW CAN THEY BE RECOGNISED?
RATIONAL DESIGN
WHOLE / LYSED CELLS
ExtraCellular
IntraCellular
From: Miller, 2000.
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Aplysialividans
K+ channel
(Kv),Moczydlowski,
Miller, P
2000.
,californica
DIABODIES
, B(KcsA),
IOMIMETIC
ROBES
… to
Streptomyces
K+ channel
1998.
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ANALYTICAL DEVICES
Potential
for Size
Optimisation
Potential
for and
Size Power
and Power Optimisation
“Hyphenation” with other techniques:
Confocal Microscope,
 Scanning Near-Field
Optical Microscope,
 Atomic Force Microscope.
Micro-Tech Scientific, Inc.
…other devices:
Chromatography (CZE),
MS
Spectroscopy (MS, IMS).
Teepe et al., 2001 SNOM/ATM
Raman
IMS
From: MBARI.
CE
NMR
(Figure: Bruker BioSpin AVANCETMICE® )
UnityNOVA Varian Inc.
SNOM/ATM, Seiko Instruments, Inc
TM WITek
SPM200
(Figure:
Bruker
BioSpin Minispec® )
(Figure: Haskin
Research
Group)
From: JPL
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ANALYTICAL DEVICES
LABORATORY-ON-A-CHIP
GENERAL STRUCTURE:
CE
From:Wang J, NMSU
From: MBARI.
Gourley and Sasaki, 2001
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and Fiehn, 2003
From:WangGast
J, NMSU
ANALYTICAL DEVICES
Potential
FOR Afor
PRELIMINARY
Size and PowerANALYSIS
Optimisation
µ-Capillary Electrophoresis
“Hyphenation”
with other techniques:
Ion Mobility
Spectrometer
Confocal
Microscope,
 chip-based : ITP / CZE,
 Scanning
Near-Field
 combination with detection :
Optical Microscope,
- electrochemical
(Φ, Ω, MS etc.),
 Atomic
Force
Microscope.
- optical
(diode-LIF),
- for devices:
analysis of sample / environment.
…other
Applications:
Chromatography (CZE),
MS
 field investigation of VOCs,
Spectroscopy (MS, IMS).
CE
From: MBARI.
 multi-organic component analysis,
IMS separation.
 macromolecule
Teepe et al., 2001
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From: JPL
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ANALYTICAL DEVICES
ES
MICRO-ARRAYS / BIO-CHIPS
Bio-Chip “reading” techniques:
 automation and miniaturisation,
 choice of techniques and materials,
- antibodies / fluorescence…
- novel detection methods,
- cantilever biosensors,
- surface plasmon resonance,
- new materials…
Daniel T. Colbert
Multi-Protein
Specific Analysis:
Analysis :
NanoReader BioForce
NanoSciences
Courtesy
of Flavio
Robles / PDB
Figure:
Biacore
Corporation
Few
More
Observations
Variables ==
Silica from Tethya aurantia,
moreMore
hypotheses
DATA
Weaver
J. and Morse D
TM
®
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Biacore®
(Figure: ©1998(Figure:
by Alberts,
GeneChip
Bray, ®Johnson,
Affymetrix)
Lewis, Raff,
Roberts, Walter Published by Garland Publishing.)
SUMMARY
 REVIEWED AVAILABLE TECHNIQUES,
 APPLICATION TO ASTROBIOLOGY PARADIGM,
 RATIONALE OF TARGET SELECTION,
 SELECTION OF ANALYSIS TECHNIQUES.
FUTURE CHALLENGES
 MICROFLUIDIC ENVIRONMENT FOR ASSAYS,
 PERMIT MULTIPLE SIMULTANEOUS ASSAYS (tens of 1000’s),
 SEQUENTIAL NANO-MODELING OF PROBES,
 ... SECURE
RETURN MISSION.
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ACKNOWLEDGEMENTS
the
abdus salam
international centre for theoretical physics
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END
Participants of the Seventh Trieste Conference on
Chemical Evolution and the Origin of Life
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