From Event to Action: Accelerate Your Decision Making with Real-Time Automation
How To Analyze A New Gene
1. How to analyze a new gene
A novel gene
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Gene expression Gene Functions
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Linkage analysis
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Protein Location and complex
Biochemical and biological activity
Domain analysis and structure determination
2. Protein Expression is regulated at multiple levels
Gene copy number
DNA Chromosome structure
Methylation and acetylation
Transcription Promoter activity
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Inducible transcription factor
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Alternative splicing
RNA
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mRNA transport and stability
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Ribosome binding site
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Translation Codon usage
Termination
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Protein
Protein stability
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Phosphorylation
Post-translational Glycosylation
modification Ubiquitination
Sumonylation
Palmolylation …
Biological
Cellular process
Activity
3. Methods for Analysis of Gene Expression
Analyzing promoter:
Analyzing transcription:
--- Nuclear Run-on assay
--- Northern blots
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--- DNA Foot-printing assay
--- RNase protection assay
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--- EMSA
--- Reverse transcription PCR
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--- Reporter gene assay
--- Real Time PCR
--- DNA truncation and
--- In situ hybridization
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site-directed mutagenesis
--- Primer extention assay
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--- in vitro reconstitution assay
--- CHIP assay
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Comparing transcriptomes: Translational analysis:
--- Differential screening --- Western Blots
--- Subtractive hybridization --- Immunocytochemistry
--- Differential display Immunohistochemistry
--- Array-based methods --- Protein modification
--- Proteomics
4. Topics covered in this lecture
Promoter analysis
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Western Blot
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RT-PCR
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Real Time PCR
RNA interference (RNAi)
5. Promoter analysis
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Promoters: DNA element(s) or sequence which determines
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the site of transcription initiation of RNA polymerase and
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may also affect the efficiency of initiation
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Promoters are and should be defined functionally as
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sequences that direct transcription initiation in vivo or in vitro
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using a functional assay (cause transcription of an RNA).
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6. Components of a typical gene involved in gene regulation
Matrix Attachment region
Matrix Attachment region
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Boundary element
Boundary element
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Gene
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Regulatory
promoter
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Enhancer
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DNA
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Core promoter
7. Sequence elements in a typical core promoter
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Downstream core
Promoter element
TATA motif Initiator
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Py Py AN T/A Py Py R G A/T C G T G
TATAAA
G/C G/C G/C CGCC
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TF II D
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TF II B
TF II A
Polymerase II
8. A simplified model of enhanceosome formation
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9. Strategy for promoter analysis
Isolate putative promoter from genomic clone
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Sequence putative clone
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Determine transcription start site
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Establish functional assays
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Identify cis-acting DNA elements and trans-acting factors
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Determine relevance of these factors
Establish regulatory model of the promoter in vivo and in vitro
10. Nuclear Run-on Transcription Assay
---An indirect measure of the in vivo rate of transcription initiation from a given gene
---The amount of specific radiolabeled RNA observed is approximately proportional to the number
of polymerase molecules associated with the gene when the cells were lysed and chilled, which in
turn is proportional to the rate of transcription initiation from the gene.
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Add Hybridize specific
Isolate
NTPs Incubate radiolabeled RNA
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Radio-
and at 37
Lyse cells on ice Pellet nuclei to unlabeled cDNA
labeled
Buffer for several immobilized on
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RNA
(One minutes filter
labeled)
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Little or no
transcription of gene
Uninduced cells
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(e.g plasmid backbone)
Induced cells
Negative control
gene of interest
Positive control
Active transcription
(e.g β-actin)
cDNA from
11. Primer Extension assay
5‘ 3‘ (20-25 bases)
Synthesize primer and label at
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5’ end with [γ-32p]ATP and T4
polynucleotide kinase
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Hybridize hot primer
to specific mRNA
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mRNA
50-150 bases
Extended primer
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5‘ 3‘
3‘ 5‘
Extend primer to 5’ end
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of mRNA using reverse
transcriptase G A T C
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mRNA
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cDNA
3‘ 5‘
Analyze radiolabeled
DNA on sequencing gel
Free
excess primer
12. RNase protection assay
+1 +1
Screen genomic DNA library to +59 +59
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find the DNA fragment spanning HindIII
Sub-clone Hind III
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Sp6 promoter Sp6 promoter
the region thought to contain the
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transcription start site for the
gene of interest
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en
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Sp6 RNA poltmerase
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3‘
NTPs (α-32P)UTP
ist
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G A T C mRNA
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5‘
3‘ 5‘
3‘ 5‘
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RNase T1 Hybridize
and/or
RNase A
13. 5‘ RACE Procedure
(Rapid Amplification of cDNA Ends)
3‘
mRNA
5‘
AAAAA
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3‘
mRNA Hybridize primer 100-200 bases
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AAAAA
from 5’ end of mRNA
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3‘
mRNA
5‘ Reverse Transcription
AAAAA
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3‘ 5‘
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Ligate oligo of known sequence
3‘
mRNA to 3’ end using RNA ligase or
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AAAAA
Extend cDNA using terminal
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5‘
3‘
transferase(TdT) and dGTP
PCR
5‘
3‘
Insert PCR produt into vector
Sequence individual clones
14. Establishing functional assays for promoter analysis
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Transient transfection assay
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Stable Transfection assay
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In vitro transcription assay
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Transgenic assay
Homologous recombination assay
15. Transient transfection assay
Constitutive promoter
(SV40, HSV-TK)
Promoter
of interest Reporter
Reporter Reporter gene
gene gene
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or
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Distant control
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region of interest
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Transfect effective and control cell
(Inducible genes or cell type specific genes)
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Stimulation Grow 24-72 hours
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Harvest cells.
Prepare protein extract or mRNA
Measure enzymatic activity Measure reporter
of reporter gene product mRNA levels
16. Stable Transfection assay
Measure enzymatic activity
Promoter
of reporter gene product
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of interest
Dominant selectable
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marker gene Reporter
gene
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Measure reporter
Constitutive promoter
mRNA levels
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(SV40, HSV-TK)
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Transfect cultured cells
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Drug selection for 1-4 weeks
Harvest cells.
Expand several cell clones or
Prepare protein extract or mRNA
pools of drug resistant cells
17. In vitro transcription assay
Promoter
A 360-bp sequence devoid of G
of interest
residues on the noncoding strand.
G-less cassette
The resulting message is G-less.
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Transcription should in principle
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terminate at the end of the G-less
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cassette.
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Incubate plasmid in nuclear extract
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with ATP, CTP, [32P]UTP
In crude extract, despite extensive dialysis,
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Including 3’-O-Methyl-GTP there are enough contaminating GTP to
allow nonspecific RNA synthesis.
Acting as chain terminator
Gel electrophoresis analysis
18. Choice of Reporter genes
CAT (chloramphenicol acetyltransferase)
Transfers radioactive 14C acetyl groups to chloramphenicol.
detection by thin layer chromatography and autoradiography
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GAL (β-galactosidase)
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Hydrolyzes colourless galactosides to yield coloured products. Assay
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change/production of colour
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SEAP (secreted human placental alkaline phosphatase)
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highly-sensitive bioluminescent alkaline phosphatase assay
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LUC (luciferase)
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Oxidizes a luciferin emitting photons. Count photons by luminometer or photon-
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counting camera. Different luciferases avaiable
GFP (green fluorescent protein)
Fluoresces on irradiation with UV – observed upon fluorescence microscopy or
measure fluorescence. Use destabilised forms
19. Common transfection methods
Calcium phosphate:
DNA is mixed with calcium chloride in a phosphate buffer, resulting in the formation of a
DNA-calcium phosphate precipitate, which is layered on cells and taken up by endocytosis.
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For both transient and stable transfection of adherent cells
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DEAE-dextran:
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The soluble complex between negatively charged DNA and cationic DEAE-dextran is
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taken up by endocytosis into cells.
For transient transfection of both adherent and non-adherent cells.
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Rarely useful for stable transfection because of toxicity.
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Electroporation:
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A high voltage electroshock induces or stabilizes pores in the plasma membrane through
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which the DNA enters the cell.
For cells resistant to transfection
Lipofection:
Positively charged cationic lipid compounds
Exceptionally high efficiency for some cell line
Polycations:
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Warming up the Basic PCR procedure
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21. Dissection of promoter cis-elements
(DNA motif mapping)
Identification of cis-elements using in vivo or in vitro
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protein-DNA interaction methods
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--- EMSA
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--- DNase Footprinting
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Identification of cis-elements by database analysis
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--- http://transfac.gbf.de/index.html
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Identification of cis-elements by comprehensive mutant
analysis
--- Deletion analysis
--- Generation of internal deletion mutants
--- Generation of substitution mutants (Linker scanning)
--- Site-directed mutagenesis
22. Deletion analysis
Long Promoter
Reporter
of interest
gene
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PCR or Restriction Enzyme
blunt ligation
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Reporter gene activity
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23. Generation of internal deletion mutants by PCR
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5‘ 3‘
3‘ 5‘
PCR
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5‘ 3‘
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3‘ 5‘
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3‘ 5‘
Denature and anneal
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3‘
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3‘ 5‘
Fill in with Taq and dNTPs
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3‘
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PCR +1
5‘ 3‘
3‘ 5‘
Clone into reporter plasmids and carry out reporter assay
24. Generation of substitution mutants by PCR
(Linker scanning)
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5‘ 3‘
6-10 base pairs
3‘ 5‘
Same length
Different sequence
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PCR
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Denature and anneal
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3‘
5‘
3‘ 5‘
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3‘
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3‘ 5‘
PCR +1
5‘ 3‘
3‘ 5‘
Clone into reporter plasmids and carry out reporter assay
25. Site-directed mutagenesis
Pfu turbo DNA polymerase
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Methylated nucleotide
Digest the methylated
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Transform the circular,
nicked dsRNA into
E.coli.. The bacteria
will repair the nicks in
the mutated DNA
26. Filter Blotting
1979: Towbin et.al Dot blot or Slot blot
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ECL detection of
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human serum transferrin
--- Providing quantitative information about protein expression levels
--- Useful for on multiple samples performed in parallel
--- Lacking information on protein molecular weight, its isoforms or
post-translational modification
27. Western Blotting
Protein mixture
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SDS-PAGE
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Blot
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ECL detection of
human serum transferrin
Detection
31. PVDF membranes offer better protein retention,
physical strength and chemical compatibility
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32. Pretreatment of PVDF membrane
PVDF is an inherently hydrophobic polymer and will not wet-out in
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aqueous solutions. In order for a PVDF membrane to be compatible
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with aqueous systems, it must first be wet in a 50% (v/v) or
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greater concentration of alcohol,methanol,or isopropanol. Complete
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wetting is evident by a change in the membrane’s appearance from
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opaque to semi-transparent.
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The alcohol is then removed from the membrane by extensive rinsing
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in water, and the membrane is equilibrated in the appropriate
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buffer.
Once the membrane is wet, protein binding can be achieved by
simply bringing the protein into contact with the membrane.
34. Tank Transfer System
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Tris/glycine transfer buffer: The methanol added to transfer buffers has two major functions:
25 mM Tris base --- Stabilizes the dimensions of the gel
192 mM glycine, --- Strips complexed SDS from the protein molecules
10% (v/v) methanol
pH 8.3
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Semi-dry Transfer System
36. Electrotransfer Timing:
the longer is not always the better
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12 hours 1.5 hours 12 hours 1.5 hours
Coomassie Blue Staining
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Long transfer times are best suited for tank systems,
which normally require cooling of the unit and
internal recirculation of the transfer buffer. In
semi-dry transfer, however, prolonged blotting may
result in buffer depletion, overheating and gel drying.
42. Chromogenic Detection:
Coupled enzyme (Alkaline phosphatase, AP) catalyzes a reaction
resulting in the deposit of an insoluble colored precipitate.
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(BCIP: 5-bromo-4-chloro-3-indolylphosphate
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NBT: nitroblue tetrazolium salt)
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Isolating RNA for RT-PCR
48. Purification of RNA from Cells and Tissues by Acid
Phenol-Guanidinium Thiocyanate-chloroformExtraction
cells are homogenized in guanidnium thiocyanate and the RNA is purified from the lysate
by extraction with phenol:chloroform at reduced pH. Many samples can be processed
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simultaneously and speedily. The yield of total RNA depends on the tissue or cell resource
and is generally in the range of 4-7 µg/ml starting tissue or 5-10 µg/106 cells.
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Prepare all reagents with DEPC-treated H2O.
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Guanidnium thiocyanate is a strong denaturing agent, preventing RNA degradation
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Proteinase K degrades protein contaimination
RNAase-free DNAase degrades DNA.
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Acidic pH
Neutral pH
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Aqueous layer
Aqueous layer
( RNA)
(DNA and RNA)
Protein precipitate
Protein precipitate
Phenol layer
Phenol layer
(DNA)
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Getting Rid of DNA
52. Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography
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Chromatography on oligo(dT) columns is the preferred method for large-
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scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian
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cells. A combination of temperature and ionic strength to maximize binding
and recovery of polyadenylated RNA. Typically, between 1% and 10% of
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the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA.
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53. Synthesis of First-strand cDNA Catalyzed
by Reverse Transcriptase
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58. The Evolution of PCR to Real-Time PCR
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PCR has completely revolutionized the detection of RNA and DNA. Traditional PCR
has advanced from detection at the end-point of the reaction to detection while the
reaction is occurring.
59. Limitations of Traditional PCR
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Poor Precision
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Low sensitivity
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Short dynamic range < 2 logs
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Low resolution
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Size-based discrimination only
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Results are not expressed as numbers
Ethidium bromide for staining is not
very quantitative
Post PCR processing
60. What is Real-Time PCR
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A PCR system in which we can monitor the amplification reaction
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as it is occuring. It incorporates the ability to directly measure and
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quantify the reaction while amplification is taking place.
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A basic PCR run can be broken up into three phases:
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Exponential: Exact doubling of product is accumulating at every cycle (assuming 100%
reaction efficiency). The reaction is very specific and precise.
Linear (High Variability): The reaction components arebeing consumed, the reaction is
slowing, and products are starting to degrade.
Plateau : The reaction has stopped, no more products are being made and if left long
enough, the PCR products will begin to degrade.
65. The Threshold line is the level of
threshold
detection or the point at which a reaction
reaches a fluorescent intensity
above background. The threshold line is
set in the exponential phase of
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the amplification for the most accurate
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reading. The cycle at which the
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sample reaches this level is called the
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Cycle Threshold, Ct.
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66. Common Standards for Real Time PCR
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--- Glyceraldehyde-3-phosphate dehydrogenase mRNA
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--- Beta-actin mRNA
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--- MHC I (major histocompatability complex I) mRNA
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--- Cyclophilin mRNA
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--- mRNAs for certain ribosomal proteins RPLP0
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28S or 18S rRNA
67. REAL TIME PCR
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• kinetic approach
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• early stages
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• while still linear
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www.biorad.com
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3.
5. ccd
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intensifier
detecto
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1. halogen
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tungsten lamp
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2b. emission 350,00
filters 0 pixels
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2a. excitation
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filters
4. sample
plate
www.biorad.com
69. The 5’ exo-nuclease activity of AmpliTaq® Polymerase and
FRET (Fluorescent Resonant Energy Transfer) makes it possible
to detect PCR amplification in Real-Time.
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The Scorpions uni-probe consists of a single-stranded bi-labeled fluorescent probe sequence held in a
hairpin-loop conformation (approx. 20 to 25 nt) by complementary stem sequences (approx. 4 to 6 nt)
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on both ends of the probe. The probe contains a 5’end reporter dye and an internal quencher dye
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directly linked to the 5’end of a PCR primer via a blocker. The blocker prevents Taq DNA
polymerase from extending the PCR primer. The close proximity of the reporter dye to the quencher
dye causes the quenching of the reporter's natural fluorescence. At the beginning of the real-time
quantitative PCR reaction, Taq DNA polymerase extends the PCR primer and synthesizes the
complementary strand of the specific target sequence. During the next cycle, the hairpin-loop unfolds
and the loop- region of the probe hybridizes intramolecularly to the newly-synthesized target
sequence. The reporter is excited by light from the real-time quantitative PCR instrument (hg ). Now
that the reporter dye is no longer in close proximity to the 1 quencher dye, fluorescence emission
may take place (hg ).
74. SYBR Green chemistry is an alternate
method used to perform real-time
PCR analysis. SYBR Green is a dye
that binds the Minor Groove of double
stranded DNA. When SYBR Green dye
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binds to double stranded DNA, the
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intensity of the fluorescent emissions
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increases. As moredouble stranded
amplicons are produced, SYBR Green
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dye signal will increase.
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78. RNA interference (RNAi)
post-transcriptional gene silencing (PTGS)
Antisense-mediated silencing: large amounts of a nucleic acid whose sequence is
complementary to the target messenger RNA are delivered into the cytoplasm of a
cell. Base pairing between the 'sense' mRNA sequence and the complementary
'antisense' interfering nucleic acid is thought to passively block the processing or
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translation of mRNA, or result in the recruitment of nucleases that promote mRNA
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destruction.
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Unexpected results:
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---Both the antisense and the control sense RNA preparations induced silencing of C.
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elegans par-1 mRNA to block par-1 expression.
---Silencing effect could be transmitted in the germ line.
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---The silencing effect could also spread from tissue to tissue within the injected
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animal.
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Hypothesis:
---The properties of RNAi demands the existence of cellular mechanisms that initiate
and amplify the silencing signal, and suggest that the RNAi mechanism represents an
active organismal response to foreign RNA.
---dsRNA, often encountered by cells during viral infection, might itself be the initial
trigger. dsRNA proved to be an extremely potent activator of RNAi — at least 10-
fold and perhaps 100-fold more effective than purified preparations of single-stranded
RNA.
79. Natural mechanism of RNA interference
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Dicer: RNase III enzyme
RISC: RNA-induced silencing complex
80. Advantages and Disadvantages of Different
Gene Suppression Strategies
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Advantage Disadvantage
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Transfection-dependent
RNAi Potent, specific “Knockdown” not
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“knockout”
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Antisense DNA Variable efficacy and specificity
Simple, inexpensive
oligonucleotides Transfection-dependent
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dominant negative Transfection-dependent
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discrete regions of a protein
Unanticipated side effects
mutant gene
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Stable suppression possible
Small molecule Often nonspecific
Ease of administration May not be available
inhibitors
Laborious to produce
Expensive
“Knockout mouse” 100% gene silencing
Lethal mutants may prevent
embryonic development
81. siRNA design
There are expanding libraries of validated, commercially
available siRNAs directed toward some commonly
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targeted genes. These may be of use, if available. However,
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if the gene of interest has not been targeted using siRNA
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before, a novel siRNA must be developed.
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Selection of the targeted region is currently a trial-and-
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error process, but with the likelihood of 80–90% success ,
given a large enough random selection of target genes
82. Methods to generate short RNAs
that silence gene expression
Silencing by RNAs that
are generated in vitro
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Silencing by RNAs that
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are generated in vivo
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(H1 and U6)
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83. Advantages and Disadvantages of
Different siRNA Delivery Strategies
Advantage Disadvantage
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Rapid synthesis
Transient RNAi
Chemical and in vitro
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High purity using chemical
enzymatic synthesis expensive for multiple siRNA
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synthesis
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More economical for
More labor intensive to
multiple sequences
DNA plasmid vector or
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cassette Stable RNAi achievable using
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Transfection-dependent
selection marker
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May be effective in cells
resistant to transfection with
More labor intensive to
dsRNA and plasmids
generate
Virus-mediated Integration produces stable
Potential biohazard
RNAi even in the
absence of a selection
pressure
84. Selection of siRNA duplexes
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[1]Select the target region from the open reading frame of a given cDNA sequence preferably
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50 to 100 nt downstream of the start codon. Avoid 5′ or 3′ untranslated regions (UTRs) or
regions close to the start codon as these may be richer in regulatory protein binding sites.
[2]Search for sequences 5′-AA(N19)UU . Choose those with approximately 50% G/C content
( from 32 to 79% G/C content ). If not, follow (B).
[3] Blast-search the selected siRNA sequences against EST libraries or mRNA sequences of
the respective organism to ensure that only a single gene is targeted.
[4] It may be advisable to synthesize several siRNA duplexes to check the silencing effectivity.
A nonspecific siRNA duplex is needed as control.
85. Preparation of siRNA duplexes by Chemical Synthesis
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Proligo (Hamburg, Germany, www.proligo.com)
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Dharmacon Research (Lafayette, CO, www.dharmacon.com)
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Pierce Chemical (www.perbio.com)
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Glen Research (Sterling, VA, www.glenres.com)
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ChemGenes (Ashland, MA, www.chemgenes.com)
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Cruachem (Glasgow, UK, www.cruachem.com).
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Analysis of siRNA duplex formation
86. Schematic for a typical RNAi experiment
using chemically synthesized siRNA
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87. Silencing of a green fluorescent protein
(GFP) reporter in C. elegans
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RNAi Control
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19 bases
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Vector Based RNAi
