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MOLECULAR BIOLOGY – Basic Genetics
EXAMS – SAVE THE DATE!
KMB 758 MCQ KMB 758 MCQ
The exam format will be a multiple choice question (MCQ) test with 40 questions. There will be two possible exam dates. Please mark them in your diaries. Should you fail or are not satisfied with your grade you can have an aural retake exam with me.
Potential revision Class next time!!
Alexander Bruce
Ph.D.
Aleksandar Mihajlovic
M.Sc.
Vasanth Thamodaran
M.Sc.
USUAL TIME & PLACE 10 am in lecture theatre
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY TECHNIQUES III.
analysis of gene expression
MOLECULAR BIOLOGY – Gene expression analysis
HYBRIDIZATION
RNA detection
ANTIBODY
protein detection
MOLECULAR BIOLOGY – Gene expression analysis
Where in organism is the gene expressed?
Detection of RNA by
in situ hybridization
:
Stage 5 Stage 10 Stage 11 Stage 13
DNA MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
Gen X Green Fluorescent Protein Other reporter genes e.g.
LacZ
Drosophila – GFP and embryonic development:
http://www.youtube.com/watch?v=MMLVqGEOD58
MOLECULAR BIOLOGY – Gene expression analysis Mouse embryo histone H2B-GFP reporter
DNA MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
Gen X Green Fluorescent Protein Other reporter genes e.g.
LacZ
How?
Enhancer Trap, Knock in ….
Drosophila – GFP and embryonic development:
http://www.youtube.com/watch?v=MMLVqGEOD58
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for
random
integration into cell genomes (e.g. ES cells) leading to production of transgenic animals
Reporter has minimal promoter that can be activated by a functioning proximal enhancer
Transposon elements Viral intergration elemets random integration
Antibiotic resistence allows selection of successfully integrated transgenes
Reporter activity if integration site is proximal to an active gene enhnacer i.e. cell marked/ highlighted
a) Enhancer trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for
random
integration into cell genomes (e.g. ES cells) leading to production of transgenic animals
Splice acceptor site/ sequence (no promoter)
random (intronic) integration
Gene fusion ... Simultaneously reports expression and usually inactivates function (potential cytolocalization analysis in marked cells) - hypomorphic
b) Gene trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for
random
integration into cell genomes (e.g. ES cells) leading to production of transgenic animals
No splice acceptor site or promoter
random (exon) integration
Active promoter also leads to gene fusion thus reports and usually inactivates gene function (also potential cytolocalization analysis in marked cells)
c) Promoter trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter in mammalian cells
GFP E-coli prom hyg r Mammalian prom neo r
PCR generated
TARGETING
construct Bacterial
GENOMIC LIBRARY CLONE
containing the gene to be targeted
TRANSFORM
the targeting construct into the genomic library bacterial clone
STOP
50bp of homlogous sequence flanking STOP codon
3
’
UTR
+ plasmids encoding
RECOMBINASE
enzymes from bacteriophage
GFP E-coli prom hyg r Mammalian prom neo r RECOMBINASE
enzymes recognise homologous sequences and induce DNA double strand breaks and strand
CROSSOVER STOP 3
’
UTR
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter
GFP E-coli prom hyg r Mammalian prom neo r
PCR generated
TARGETING
construct Bacterial
GENOMIC LIBRARY CLONE
containing the gene to be targeted
TRANSFORM
the targeting construct into the genomic library bacterial clone
STOP
50bp of homlogous sequence flanking STOP codon
3
’
UTR
+ plasmids encoding
RECOMBINASE
enzymes from bacteriophage Recombinase mediates the successful
INSERTION/ INTEGRATION
of the targeting construct into the genomic DNA library DNA creating the
GFP FUSION REPORTER GENE GFP E-coli prom hyg r Mammalian prom neo r
Successfully recombined bacterial clones can be selected for with antibiotics
STOP 3
’
UTR
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes BACTERIAL RECOMBINEERING GFP FUSION REPORTER GENE
DNA construct isolated and purified from bacterial DNA
GFP E-coli prom hyg r Mammalian prom neo r 3
’
UTR TRANSFORM (TRANSFECT)
GFP fusion construct into mammalian cells
e.g.
ES cells
GFP E-coli prom hyg r Mammalian prom neo r
Low frequency mammalian
ENDOGENOUS RECOMBINATION
enzymes induce strand cross over Successfully recombination can be selected for with second antibiotic resistance gene
STOP 3
’
UTR 3
’
UTR
The
GFP FUSION REPORTER GENE
for a specific gene has now replaced the endogenous copies of the original gene in the mammalian cell genome. These cells can then be used to create
TRANSGENEIC
animals The same approaches can be used to disrupt genes by insertion or deletion of DNA sequence to generate
GENETIC KNOCKOUTS
to asses specific gene function
MOLECULAR BIOLOGY – Gene expression analysis
To what extent is the gene expressed?
RNA DNA ... Southern blot
DNA
PROMOTER exon 1 ATG TRANSCRIPTION
Pre-mRNA
AUG intron RNA SPLICING
mRNA
AUG REVERSE TRANSCRIPTION exon 2
cDNA MOLECULAR BIOLOGY – Gene expression analysis
exon 3 TAA AATAAA TAC intron UAA AAUAAA UAA AAUAAA ATT TTATTT HIV
MOLECULAR BIOLOGY – Gene expression analysis cDNA synthesis Some reverse transcriptases initiate 2nd stand synthesis via addition of hairpins Ordinary 1st strand synthesis
GENERATION OF A cDNA THAT CAN BE AMPLIFIED USING SPECIFIC PRIMERS BY PCR
RP49 control (house-keeping) MOLECULAR BIOLOGY – Gene expression analysis Quantitative
RT-PCR
(reverse transcription)
light-cycler SYBR- green dsDNA intercalation and flourescence MOLECULAR BIOLOGY – Gene expression analysis
Real-Time PCR
MOLECULAR BIOLOGY – Gene expression analysis Molecular Beacon Probes Potential for multiplex
i.e.
more than one probe per sample
MOLECULAR BIOLOGY – Gene expression analysis
Norhern blot, RT-PCR to study one (couple) gene(s) at once ...
... in genomic era?
MOLECULAR BIOLOGY – Gene expression analysis Genomic expression analysis using
MICROARRAY
(CHIP)
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
Chromatin remodeling
methylase (CG) + histone deacetylases (HDACs) histone acetyltransferases (HATs)
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip
Ch
romatin
i
mmuno
p
recipitation combined with DNA microarrays (
chip
) or
ChIP-seq
Ch
romatin
i
mmuno
p
recipitation combined with DNA
seq
uencing
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip
e.g.
of transcription factor binding site discovery
REST
RE1 NEW TARGET GENES COMBINE WITH HISTONE ChIP on Chip for extra structure function data
MOLECULAR BIOLOGY – Gene expression analysis
cDNA
more RNA in original sample ...
... more times the particular fragment is sequenced
quantifiable without knowledge of sequence
(microarray needs to know the sequences)
MOLECULAR BIOLOGY – Gene expression analysis
ChIP-seq
Ch
romatin
i
mmuno
p
recipitation combined with DNA
seq
uencing
MOLECULAR BIOLOGY – Gene expression analysis
... at the protein level
immunolocalization
ANTIBODY
protein detection
ANTIBODY
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
MONOCLONAL ANTIBODY
polyclonal serum
MOLECULAR BIOLOGY – Gene expression analysis
POLYACRYLAMIDE-GEL ELECTROPHORESIS
(PAGE)
Figure 8-18a
Molecular Biology of the Cell
(© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
RNA
PROTEINS
WESTERN BLOTTING DNA
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
1
st
dimension: Isoelectric focusing
Figure 8-22
Molecular Biology of the Cell
(© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
Two-dimensional electrophoresis
MOLECULAR BIOLOGY – Gene expression analysis Figure 8-23
Molecular Biology of the Cell
(© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
PROTEOMICS
What is the difference on B?
identification by mass spectroscopy
MOLECULAR BIOLOGY – Gene expression analysis Figure 8-21
Molecular Biology of the Cell
(© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
GENOME TRANSCRIPTOME PROTEOME
MOLECULAR BIOLOGY – Gene expression analysis
Good Luck
with molecular biology!
RNA
PROTEINS
RT-PCR Real-Time PCR Inverse PCR Nested PCR DNA
Potential revision Class next time!!
Alexander Bruce
Ph.D.
Aleksandar Mihajlovic
M.Sc.
Vasanth Thamodaran
M.Sc.
USUAL TIME & PLACE 10 am in lecture theatre