Genomics and transcription 1

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Genomics and transcription 1 2
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Overview

• mRNA measurement techniques
• Genomics
• DNA microarrays

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Northern blot
Advantages it works separation on size too
Disadvantages requires relatively large amounts
RNA (ie sensitivity) time-consuming one gene at a
time not easily quantifiable not easily
comparable across many different exps (requires
work with radioactive probes)
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Quantitative real time PCR

1. Isolate total RNA
2. cDNA synthesis (reverse transcription)
3. Real time PCR

Advantages Semi-quantitative Better comparable
across experiments Higher throughput of samples
possible More sensitive than Northern (No
radioactivity)
Disadvantages Semi-quantitative Only few genes
at a time Always coupled to use of a control mRNA
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Genomics
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Sequencing and the birth of genomics

• 1953 structure of the DNA double helix (Watson,
  Crick Franklin)
• 1975 dideoxy sequencing method (Sanger)
• 1980s 1990s automated sequencing
• 1990s genome centers
• 1997 1st eukaryotic genome sequence
• S. cerevisiae, 12x106 base-pairs (Mbp), 6200
  genes
• 2003 human genome completed (3000 Mbp, 25 000
  genes?)

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The importance of (the human) genome sequence

• Several new beginnings
• insight into all our genetic material
• insight into the importance of even more
  efficient sequencing methods
• whole genome sequences have made other new
  technologies possible
• DNA microarrays for expression-profiling
• DNA microarrays for ChIP on chip
• (but also pivotal for protein mass spectrometry
  approaches)

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What is a DNA microarray?

• An array of spots that contain DNA, allowing
  measurement of
• 10 000 s of different mRNA levels through
  hybridization

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DNA microarrays
Each spot contains 100 000s of DNA molecules
attatched to the surface The DNA molecules are
identical within a spot, but differ between
spots, allowing each spot to measure the
expression level of a different gene
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Microarrays exploit DNA hybridisation
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Microarrays exploit DNA hybridisation

• Two complementary single-strand DNA molecules
  bind to each other through hydrogen bonds between
  the complementary base pairs

G C
A T
T A
C G
C G
T A
G C
A T
G C
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Microarrays exploit DNA hybridisation
Micorarray
DNA or RNA from cells
G
A
T
C
C
T
G
A
G
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Microarrays exploit DNA hybridisation
G
A
T
C
C
T
G
A
G
microarray
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How does a DNA microarray work?
fluorecently labeled cDNA reaction
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
probes gene B (100 000s)
probes gene A (100 000s)
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How does a DNA microarray work?
labeled cDNA is hybridized on the microarray
cDNA gene B
cDNA gene A
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
A T C A T G G T T G C A C G T G C
probes gene B
probes gene A
After hybridization, fluorescence of spot gene A
measure of amount of cDNA gene A measure of
amount of gene expression (mRNA) gene A
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cDNA is amplified if there are few cells

• Information
• DNA mRNA protein

cell
cDNA
cRNA
microarray
experiment
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5
3
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
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annealing of double anchored primer
70C
5
3
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
NVTTTTTTTTTTTTT
3
T7
5
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first strand synthesis
48C
5
3
SS II
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
NVTTTTTTTTTTTTT
3
T7
5
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48C
5
3
SS II
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
NVTTTTTTTTTTTTT
T7
5
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5
3
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
NVTTTTTTTTTTTTT
3
T7
5
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ds cDNA synthesis nicking of RNA
16C
5
3
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
RNase H
RNase H
RNase H
NVTTTTTTTTTTTTT
3
T7
5
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ds cDNA synthesis nicking of RNA
16C
5
3
AAAAAAAAA AAAAAAAAAAAAAAAAAAAAA

NVTTTTTTTTTTTTT
3
T7
5
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ds cDNA synthesis second strand synthesis
16C
5
3
DNA pol I
DNA pol I
AAAAAAAAA AAAAAAAAAAAAAAAAAAAAA
NVTTTTTTTTTTTTT
3
T7
5
25
16C
AAAAAA AAAAAA
5
T7
NVTTTTTTTTTTTTT
3
T7
3
5
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ds cDNA synthesis repair of nicks
16C
AAAAAA AAAAAA
5
DNA ligase
DNA ligase
T7
NVTTTTTTTTTTTTT
3
T7
3
5
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AAAAAAAAAAAAA
T7
3
5
TTTTTTTTTTTTT
T7
5
3
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in vitro transcription linear amplification incor
poration of amino-allyl UTP
37C
T7 RNA polymerase
AAAAAAAAAAAAA
T7
3
5
TTTTTTTTTTTTT
T7
5
3
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37C
AAAAAAAAAAAAA
T7
3
5
3
TTTTTTTTTTTTT
T7
5
3
UUUUUUUUUUUUU
5
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3
5
UUUUUUUUUUUUU
37C
3
5
UUUUUUUUUUUUU
AAAAAAAAAAAAA
T7
3
5
3
TTTTTTTTTTTTT
T7
5
3
UUUUUUUUUUUUU
5
3
5
UUUUUUUUUUUUU
3
5
UUUUUUUUUUUUU
3
5
UUUUUUUUUUUUU
To avoid bias in vitro transcription 4 hours,
not longer (our experience and in lit.)
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Are we ready to hybridize?
Need to label cRNA Direct incorporation
Cy5-dUTP during cRNA synthesis Indirect
labeling amino-allyl dUTP during cRNA synthesis
followed by covalent coupling of Cy5 later
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The devil is in the detail
From cells to array image 160 steps (3-4) days
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Different sorts of microarrays
short oligo arrays (eg Affymetrix) probe 25
bases (each gene represented by multiple
probes) long oligo arrays (eg Agilent or
home-made) probe 60 bases cDNA arrays
(home-made) probe PCR product of the
transcript also single channel
(Affymetrix/Ilumina) vs two channel (Agilent/HM)!
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Distinguish between what is fixed (covalently) on
array beforehand during manufacture (probe) and
what is hybridized to array (target) For
example both can be cDNA!
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How do whole genome sequences come into this?
Selection of probe sequence
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How powerful are microarrays?

• mRNA measurement without microarrays
• eg Northern blot (or RT-PCR)

0 15 30 45 60 minutes
information on one gene several days work
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How powerful are microarrays?
mRNA expression measurement with DNA
microarrays yeast culture from lag- to
stationary phase
6200 genes S. cerevisiae
Information about ALL genes slightly more work
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Microarrays and studying gene expression
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Microarrays and studying gene expression
(there are many more applications of DNA
microarrays) describing the process finding
genes regulated under particular
conditions discovering regulatory motifs
responsible finding targets of regulatory
proteins determining processes controlled by
regulators using expression-profile of mutated
regulator as a phenotype to determine functional
relationships with other regulators
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Transcription factor targets
environment
signal transduction
transcription machinery
X
1000s of genes
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Transcription factor target genes
Transcription factor knock-out (y-axis) vs wild-ty
pe (x-axis)
wild-type (y-axis) vs wild-type (x-axis)
mRNA expression knock-out
mRNA expression wt 2
mRNA expression wt 1
mRNA expression wt 1
Expression of 150 genes is higher in TF KO gt TF
is a negative regulator Gene list can also give
insight into cellular process and regulatory
elements
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In terms of understanding mechanism, what is the
major drawback of this approach?
direct vs (different types of) indirect effects?
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Besides being useful for studying gene expression
mechanisms, microarrays are also useful for
studying and treating disease directly
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Types of (DNA) microarrays
DNA microarray DNA chip Expression profiling
measuring expression of all genes
cDNA Long oligo Short oligo
Expression-profiling Single Nucleotide
Polymorphisms (SNP) Comparative Genome
Hybridisation Chromosomal localisation
analysis etc.
DNA Protein Cell cultures Tissue
Arrays
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In terms of determining direct targets of TFs,
what is the major drawback of DNA microarray
expression profiling?
direct vs (different types of) indirect effects
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Chromosomal location analysis with microarrays
ChIP on chip
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Principles of ChIP
Grow cells
Crosslink proteins and DNA
Extract chromatin fraction
Sonication
Input fraction
Immunoprecipitation
Reverse crosslink
Hybridization on array with full genome coverage
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An example gene-specific TF
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Pol II binding upon exit from stationary phase
Whole-genome view
t 0
t 1
t 2
t 5
t 10
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Examples
HSP26
PAB1
TEF2
PHO89
t 0
t 1
t 2
t 5
t 10
SP-specific
de novo
Constitutively
5' ORF binding
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Chromosomal location analysis
In principle useful for delineating direct from
indirect effects Also extremely powerful in its
own right location on genome of TFs finding
regulatory motifs location of chromatin
modifications location of nucleosomes determining
dynamics of TFs on DNA
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Location analysis is not just useful for studying
transcription
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Location analysis is not just useful for studying
transcription
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If two TFs ChIP to same position ....
ChIP enrichment vs input
TF1
TF1
TF2
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If two TFs ChIP to same position ....
ChIP enrichment vs input
TF1
TF1
TF2
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If two TFs ChIP to same position ....
ChIP enrichment vs input
TF1
TF1
TF2
... may want to consider reChIP/sequential ChIP
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Chromosomal location analysis with microarrays
ChIP on chip
....is being replaced by ChIPseq ie HT/next
generation sequencing of ChIP material
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The importance of whole genome approaches

• Less biased research
• Discovering general principles
• Ability to discriminate between general rules and
  exceptions
• Start can be made to investigating the entire
  system of transcription regulation

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Measurement technologies

• Northern
• Q RT-PCR
• DNA microarray for expression-profiling

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Measurement technologies

• Northern
• Q RT-PCR
• DNA microarray for expression-profiling
• RNA seq

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How should you test any/new technology?
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Measurement characteristics
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Measurement characteristics
Precision (reproducibility) Accuracy
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Measurement characteristics
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Measurement characteristics
Precision (reproducibility) Accuracy Sensitivity
(precision and accuracy over the range
required)
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Technology optimisation precision and accuracy

• calibration standards to determine accuracy

sample 2x calibration controls
sample 1x calibration controls