1. 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.

2. 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

3. MOLECULAR BIOLOGY TECHNIQUES III. analysis of gene expression
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY TECHNIQUES III.
analysis of gene expression

4. MOLECULAR BIOLOGY – Gene expression analysis
HYBRIDIZATION
RNA detection Y
ANTIBODY
protein detection

5. 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

6. Green Fluorescent Protein
MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
DNA
Gen X
Green Fluorescent Protein
Other reporter genes e.g. LacZ
Drosophila – GFP and embryonic development:

7. MOLECULAR BIOLOGY – Gene expression analysis
Mouse embryo histone H2B-GFP reporter

8. Green Fluorescent Protein
MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
DNA
Gen X
Green Fluorescent Protein
Other reporter genes e.g. LacZ
How?
Enhancer Trap, Knock-in ….
Drosophila – GFP and embryonic development:

9. 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
Viral intergration elemets
Transposon elements
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
Enhancer-, gene- and promoter-trap vectors, which all contain a lacZ reporter gene and a NEOMYCIN RESISTANCE GENE (neo) that is driven by an autonomous promoter, are shown trapping an endogenous gene 'X'. Successful integration of the trap vectors into the embryonic stem (ES)-cell genome will confer neomycin resistance permitting selection whether the insertion has occurred intergenically or intragenically. a | The p3LSN enhancer-trap vector contains a truncated heat-shock inducible minimum (hsp68) promoter immediately upstream of lacZ. Insertion of the enhancer-trap vector close to the enhancer of gene X will lead to the transcription and translation of the lacZ reporter when the enhancer of gene X is activated. This vector usually generates hypomorphic rather than null mutations.
a) Enhancer trap vectors

10. 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 | The pGT4.5 gene-trap vector contains a splice acceptor (SA) site immediately upstream of a promoterless lacZ gene. Its integration in an intron leads to a fusion transcript being generated from the upstream exon of gene X and lacZ upon transcriptional activation of gene X.
b) Gene trap vectors

11. 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 | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated.
( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.)
c) Promoter trap vectors

12. Targeting specific genes - BACTERIAL RECOMBINEERING
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter in mammalian cells
Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted
STOP
3’UTR
PCR generated
TARGETING construct
GFP
hygr
neor
E-coli prom
Mammalian prom
50bp of homlogous sequence flanking STOP codon
+ plasmids encoding RECOMBINASE enzymes from bacteriophage 
TRANSFORM the targeting construct into the genomic library bacterial clone
STOP
GFP
hygr
neor
E-coli prom
Mammalian prom
RECOMBINASE enzymes recognise homologous sequences and induce DNA double strand breaks and strand CROSSOVER
3’UTR
c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated.
( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.)

13. Targeting specific genes - BACTERIAL RECOMBINEERING
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter
Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted
STOP
3’UTR
PCR generated
TARGETING construct
GFP
hygr
neor
E-coli prom
Mammalian prom
50bp of homlogous sequence flanking STOP codon
+ plasmids encoding RECOMBINASE enzymes from bacteriophage 
TRANSFORM the targeting construct into the genomic library bacterial clone
c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated.
( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.)
Recombinase mediates the successful INSERTION/ INTEGRATION of the targeting construct into the genomic DNA library DNA creating the GFP FUSION REPORTER GENE
STOP
GFP
hygr
neor
E-coli prom
Mammalian prom
3’UTR
Successfully recombined bacterial clones can be selected for with antibiotics

14. MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
GFP FUSION REPORTER GENE DNA construct isolated and purified from bacterial DNA
GFP
hygr
neor
E-coli prom
Mammalian prom
3’UTR
TRANSFORM (TRANSFECT) GFP fusion construct into mammalian cells e.g. ES cells
GFP
hygr
neor
E-coli prom
Mammalian prom
3’UTR
STOP
Low frequency mammalian ENDOGENOUS RECOMBINATION enzymes induce strand cross over
Successfully recombination can be selected for with second antibiotic resistance gene
c | The p -gal promoter-trap vector needs to be inserted into the coding sequence of gene X to activate transcription of lacZ. On activation of gene X, a fusion transcript and protein between the upstream gene X sequence and lacZ will be generated.
( -gal, -galactosidase; -geo, -galactosidase–NeoR fusion; HSV-tk, herpes simplex virus thymidine kinase; h -actin, human -actin; pA, polyadenylation; PGK, phosphoglycerate kinase 1.)
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

15. MOLECULAR BIOLOGY – Gene expression analysis
To what extent is the gene expressed?
NORTHERN BLOT
RNA
DNA ... Southern blot

16. MOLECULAR BIOLOGY – Gene expression analysis
PROMOTER
exon 1
exon 2
exon 3
DNA
ATG
TAA
AATAAA
TRANSCRIPTION
intron
intron
Pre-mRNA
AAUAAA
AUG
UAA
RNA SPLICING
mRNA
AUG
UAA
AAUAAA
REVERSE TRANSCRIPTION
TTATTT
TAC
ATT
cDNA
HIV

17. 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

18. MOLECULAR BIOLOGY – Gene expression analysis
Quantitative RT-PCR (reverse transcription)
RP49 control
(house-keeping)

19. Real-Time PCR light-cycler
MOLECULAR BIOLOGY – Gene expression analysis
Real-Time PCR
light-cycler
SYBR- green dsDNA intercalation and flourescence

20. MOLECULAR BIOLOGY – Gene expression analysis
Molecular Beacon Probes
Potential for multiplex i.e. more than one probe per sample
Diagram of molecular beacon. This beacon is 33 nucleotides long with a reporter dye attached to the 5' end and a quencher attached to the 3' end. The nine 5' bases are able to form base pairs with the nine 3' bases which brings the reporter and quencher in very close proximity. Therefore, when the reporter is excited by the appropriate light, its emission is absorbed by the quencher and no fluorescence is detected. The pink lines represent nucleotides that can form base pairs with the PCR product under investigation.
As the PCR continues, the newly synthesized PCR products are denatured by high temperatures. As each strand of the product are separated, the molecular beacon also is denatured so the hairpin structure is disrupted. As the temperatures cool for the next round of primer annealing, the molecular beacon is capable of forming base pairs with the appropriate strand of the PCR product (Figure 3). Any molecular beacons that do not bind to PCR product reform the hairpin structures and thus are unable to fluoresce. However, molecular beacons that bind to PCR product remove the ability for the quencher to block fluorescence from the reporter dye. Therefore, as PCR product accumulates, there is a linear increase in fluorescence.

21. MOLECULAR BIOLOGY – Gene expression analysis
Norhern blot, RT-PCR
to study one (couple) gene(s) at once ...
... in genomic era?

22. MOLECULAR BIOLOGY – Gene expression analysis
Genomic expression analysis using MICROARRAY (CHIP)

23. MOLECULAR BIOLOGY – Gene expression analysis

24. Chromatin remodeling methylase (CG) +
MOLECULAR BIOLOGY – Gene expression analysis
Chromatin remodeling
methylase (CG) +
histone deacetylases (HDACs)
histone acetyltransferases (HATs)
Nucleosomes consist of DNA (black line) wrapped around histone octomers (purple). Post-translational modification of histone tails by methylation (Me), phosphorylation (P) or acetylation (Ac) can alter the higher-order nucleosome structure. Nucleosome structure can be regulated by ATP-dependent chromatin remodellers (yellow cylinders), and the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Methyl-binding proteins, such as the methyl-CpG-binding protein (MECP2), target methylated DNA (yellow) and recruit HDACs. a | DNA methylation and histone deacetylation induce a closed-chromatin configuration and transcriptional repression. b | Histone acetylation and demethylation of DNA relaxes chromatin, and allows transcriptional activation.

25. ChIP on chip ChIP-seq Chromatin immunoprecipitation
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip
Chromatin immunoprecipitation
combined with DNA microarrays (chip) or
ChIP-seq
Chromatin immunoprecipitation
combined with DNA sequencing
The ChIP–chip method can be used to study many of the epigenomic phenomena discussed in this Review. The example presented here shows how ChIP–chip can be used to study histone modifications. Modified chromatin is first purified by immunoprecipitating crosslinked chromatin using an antibody that is specific to a particular histone modification (shown in green). DNA is then amplified to obtain sufficient DNA. The colour-labelled ChIP DNA, together with the control DNA prepared from input chromatin and labelled with a different colour, is hybridized to a DNA microarray. The microarray probes can then be mapped to the genome to yield genomic coordinates.

26. ChIP on chip e.g. of transcription factor binding site discovery
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip e.g. of transcription factor binding site discovery
REST
RE1
The ChIP–chip method can be used to study many of the epigenomic phenomena discussed in this Review. The example presented here shows how ChIP–chip can be used to study histone modifications. Modified chromatin is first purified by immunoprecipitating crosslinked chromatin using an antibody that is specific to a particular histone modification (shown in green). DNA is then amplified to obtain sufficient DNA. The colour-labelled ChIP DNA, together with the control DNA prepared from input chromatin and labelled with a different colour, is hybridized to a DNA microarray. The microarray probes can then be mapped to the genome to yield genomic coordinates.
NEW TARGET GENES
COMBINE WITH HISTONE ChIP on Chip for extra structure function data

27. 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)

28. ChIP-seq Chromatin immunoprecipitation combined with DNA sequencing
MOLECULAR BIOLOGY – Gene expression analysis
ChIP-seq
Chromatin immunoprecipitation
combined with DNA sequencing

29. MOLECULAR BIOLOGY – Gene expression analysis
... at the protein level
immunolocalization Y
ANTIBODY
protein detection

30. MOLECULAR BIOLOGY – Gene expression analysis
ANTIBODY

31. MOLECULAR BIOLOGY – Gene expression analysis
MONOCLONAL ANTIBODY
Monoclonal antibodies are produced by immunizing mice with antigen X, to stimulate the production of antibodies targeted against X.  The B-cells or antibody secreting cells are isolated from the mouse's spleen.  Antibody secreting B-cells that secrete antigenX-specific antibodies are then selected using a B-cell selection assay.  These B-cells are then fused with Tumour cells grown in culture, in the presence of PEG (poly-ethylene glycol). This results in a hybridoma. Hybridomas are then screened for antibody production against antigenX. Each hybridoma cell produces relatively large quantities of identical antibody molecules. Hybridomas are then allowed to multiply in culture,thus generating a large population of cells, each of which produces identical antibody molecules.
polyclonal serum

32. POLYACRYLAMIDE-GEL ELECTROPHORESIS
MOLECULAR BIOLOGY – Gene expression analysis
POLYACRYLAMIDE-GEL ELECTROPHORESIS
(PAGE)
Figure 8-18a Molecular Biology of the Cell (© Garland Science 2008)

33. MOLECULAR BIOLOGY – Gene expression analysis
RNA
PROTEINS
WESTERN BLOTTING
DNA

34. MOLECULAR BIOLOGY – Gene expression analysis

35. MOLECULAR BIOLOGY – Gene expression analysis
1st dimension: Isoelectric focusing
Figure Molecular Biology of the Cell (© Garland Science 2008)

36. MOLECULAR BIOLOGY – Gene expression analysis
Two-dimensional electrophoresis

37. MOLECULAR BIOLOGY – Gene expression analysis
Figure Molecular Biology of the Cell (© Garland Science 2008)

38. MOLECULAR BIOLOGY – Gene expression analysis
PROTEOMICS
What is the difference on B?
identification by
mass spectroscopy

39. MOLECULAR BIOLOGY – Gene expression analysis
Figure Molecular Biology of the Cell (© Garland Science 2008)

40. MOLECULAR BIOLOGY – Gene expression analysis
GENOME
TRANSCRIPTOME
PROTEOME

41. MOLECULAR BIOLOGY – Gene expression analysis
Good Luck with molecular biology!
DNA
PROTEINS
RNA
SAGE, CAGE, PAGE ...
GENOMICS
TRANSCRIPTOMICS
PROTEOMICS
RT-PCR
Real-Time PCR
Inverse PCR
Nested PCR
RFLP
AFLP

42. 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