Application of Molecular Biology Techniques in Astrobiology

1
PRELIMINARY ANALYSIS
ENVIRONMENTAL SAMPLE ANALYSIS

• ASSESS ENVIRONMENTAL CONDITIONS,

• LEGITIMISE FURTHER ASSAYS,

• DIRECT CHOICE OF PLAUSIBLE ASSAYS,

• INDICATE OPTIMAL CONDITIONS FOR ASSAYS.

2
MOLECULAR TARGETS
DNA
RNA
Protein
Other
HIGHLY CONSERVED MOTIFS
STRUCTURAL SCAFFOLDS

• TATAAA, Shine-Dalgarno,

• conserved charged residues,

• rRNA sub-types,

• essential enzymes,

• shifts in pyrimidine/purine ratios

• functionally divergent

• superfamilies traceable to LUCA

TRANSLATIONAL - ENZYMATIC
lipids, peptidolipids, lipopolysaccharides,

• messenger RNA,

• inhibitory RNA

special fatty acids or sugar residues
chirality
3
MOLECULAR RECOGNITION TECHNIQUES
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
DNA
PROBE
PROBE
TaqmanRApplied Biosystems
PULSE FIELD GEL ELECTROPHORESIS
FLUORESCENCE IN SITU HYBRIDISATION
PCR
FRAGMENT
FRAGMENT
SINGLE NUCLEOTIDE POLYMORPHISMS (SNP)
RESTRICTION FRAGMENT LENGTH POLYMORPHISM
R
F
L
P
FLUORESCENCE IN SITU HYBRIDISATION
S
H
I
EXPRESSED SEQUENCE TAGS (EST)
PULSE FIELD GEL ELECTROPHORESIS
P
F
G
E
F
WHOLE GENOME
PHYLOGENETIC STUDIES, DIAGNOSTICS
VERSATILE TECHNIQUE
RIBOTYPING of rDNA (5S, 16S, 23S etc. rRNA genes)
SMALLER FRAGMENTS (10 1000 kb)
RELATIONSHIP - BACTERIAL STRAINS
RIBOSOMAL GENE SEQUENCES
LABEL WITH FLUORESCENT DYE
LARGE FRAGMENTS (1,000 20,000 kb)
RESOLUTION IN 2D
POLYMERASE CHAIN REACTION
GENOME SIZE PHYLOGENETIC STUDIES
DENATURE
A
B
HYBRIDISE
?
2D gel
RFLPs
National Human Genome Research Institute
(NHGRI)   Darryl Leja  
From Hernandez-Rivas and Scherf, 1997
4
MOLECULAR RECOGNITION TECHNIQUES
DNA
RNA
TARGET SEQUENCE IS IDENTIFIED
SEQUENCING
RT-PCR
BLOTTING
REAL TIME REVERSE TRANSCRIPTION PCR
NORTHERN BLOT
?
?
?
SINGLE CELL mRNA RESOLUTION
WESTERN BLOT
?
SOUTHERN BLOT
DIFFERENTIAL DISPLAY REVERSE TRANSCRIPTION PCR
proteins
RNA EXPRESSION PROFILES
Protein
PROBES fluorescent technology

• green fluorescent protein

ANTIBODIES, mono-poly

• labeling environment

• limited number of conformations

From Alberts, Bray, Johnson, Lewis, Raff,
Roberts, Walterc 1999
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ANALYTICAL DEVICES
FLUORESCENCE TECHNIQUES IMAGE ENHANCING
FLUORESCENCE TECHNIQUES

• BASIC TECHNIQUE,

• MINIMAL SAMPLE PREPARATION,

• APPLICATIONS MAY BE LIMITLESS...

Olympus Inc.
MetaMorph Universal Imaging Corp.
Differential Interference Contrast (DIC),
Phase Contrast,
Remove some out-of-focus effects
Gatta, ICGEB New Delhi
(optic ganglion tissue from rat retina)
(3T3 fibroblast monolayer, NIH)
Digital deconvolution.
MOL-BIO LAB USE
Fluorescence illumination -diazonium salt
fluorochrome (fast blue).
Fluorescence illumination
-4',6-diamidino-2-phenylindole (DAPI),

• AS A LIGHT MICROSCOPE
• for cell counting, etc.

- phase rings on fluorite objective.
IN FLUORESCENCE MODE to highlight
transfection efficiency...
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