Transcript Genomics

MCB 317
Genetics and Genomics
Topic 11
Genomics
Readings
Genomics: Hartwell Chapter 10 of full
textbook; chapter 6 of the abbreviated
textbook
Concept
“Genomics” and Genomic techniques” are
Often “High-throughput” versions of
Standard Techniques in Genetics,
Molecular Biology, Biochemistry or
Cell Biology
Single
gene/protein
Most/all
genes/proteins
in an Organism
Genomics
Biochemistry
Subunits of Protein
Complex
Genomics:
High-throughput
genetics
Protein
D
Orthologs and
Paralogs
E
H
Gene
Ab
B, G
A
F
Txn
Profile
C
Mutant Gene
B, G
Protein
Profile/
Localization
Mutant Organism
Genetics
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Northern Blots
Isolate RNA (mRNA) from 2 tissues e.g. liver and muscle
Probe = DNA from one gene
Lane 1 = liver mRNA
Lane 2 = muscle mRNA
1
2
Qualitative Change in Transcription
Analysis of Tissue Specific Transcription
Northern Blots
Same Approach: this time mix two probes
(two genes); look at relative change
Probe = DNA from two genes
A and B
Lane 1 = liver mRNA
Lane 2 = muscle mRNA
A
B
1
2
-> Quantitative Change in Transcription
DNA Microarrays
1
5
2
6
3
7
4
8
1=DNA from gene 1, 2 = DNA from Gene 2, etc…
Where get DNA??? PCR!
DNA Microarray Outline
1. Isolate mRNA from two samples (two tissues, or two
conditions- e.g. +/- hormone, glucose vs. galactose,
mutant vs. wild-type organism)
2. Label one mRNA population RED
Label the other mRNA population GREEN
(or convert to labeled DNA)
3. Mix both sets of labeled mRNA (or DNA) and
hybridize both to the DNA Microarray
Lodish 9-36
DNA Microarrays
Liver mRNA = RED
Muscle mRNA = GREEN
1
2
3
4
1.
2.
3.
4.
On in Liver, Off in Muscle = RED
On in Muscle off in Liver = GREEN
On in both = YELLOW (RED + GREEN)
Off in both = BLACK (no flourescence)
Intensity of color is a quantitative
measure of the amount of mRNA
present [extent of txn]
DNA on the array is in excess,
signal is proportional to the
amount of RNA produced in the
cell.
Hartl Fig 13.30
DNA Micro-arrays and Expression
Profiling
Array DNA from ORFs
“Read” and quantitated by fluorescence scanner
Examples of Microarray Color Schemes
Another way to view the data: computer conversion to fold effect
Red = condition 1, Green = condition 2
Fold change from condition 1 to
condition 2
+2
+1.5
+2
0
0
-1.5
-2
-4
0
+3
-3
+1.2
0
+3
+4
-2
Another way to view the data: computer conversion to fold effect
Red = condition 1, Green = condition 2
Fold change from condition 1 to
condition 2
+2
+1.5
+2
0
0
-1.5
-2
-4
0
+3
-3
+1.2
0
+3
+4
-2
> -4 fold change
-2 to -4 fold change
+2 to -2 fold change
+2 to +4 fold change
> +4 fold change
Another way to view the
data:
Important Note: Color
scheme = fold change in
condition 2 relative to
condition 1
0 change = white -> both
yellow and black in
previous color scheme =
white here
Four Yeast Experiments
A. Wild-type vs. hypomorphic allele of an RNAPII subunit
B. Wild-type vs. nonessential subunit of mediator
C. Wild-type vs. gene X
D. Wild-type vs snf2
Color scheme = fold
change in mutant relative
to wild-type
Coupling
Microarrays and Yeast
Genetics:
Mutant v. Wild-type
Cell type 1 = WT
Cell type 2 = Mutant
Gene Discovery via Expression Profiling
1. Micro-array
2. Rearrange data from array into a list so that genes with
with similar expression patters are adjacent to each
other in the list.
3. This arrangement = cluster analysis
4. Genes that display similar patterns of expression (txn)
often code for proteins that are functionally related
(that are involved in the same biological process)
Series of Experiments
Yeast cells can be “synchronized” so that they are
all in the same stage of the cell cycle
1.
2.
3.
4.
5.
Asynchronous vs. early M-phase
Asynchronous vs. mid M-phase
Asynchronous vs. late M-phase
Asynchronous vs. early G1
Asynchronous vs. mid G1
etc… throughout all stages of the cell cycle
Cluster Analysis
Yeast cell cycle
clusters
Yeast cell cycle
clusters part 2
A=
DNA
Replication
cluster
Expression
Profile
Identifies
Genes that
may play a
role in DNA
replication
in this
example
Candidate gene discovery by
expression pattern
DNA Arrays and Cancer
• Diagnostics
• Gene discovery and mechanism
• Many types of cancer
• Many subtypes of cancer
• 3-7 genes mutated depending on type of cancer
Cancer
Diagnostics and
Gene Discovery
Cancer
• 3-7 genes mutated
• Histology parallels genetic progression
Primary
Tumor
Metastasized
Tumor
Candidate Genes
for Involvement
in Metastasis
Concept
“Genomics” and Genomic techniques” are
Often “High-throughput” versions of
Standard Techniques in Genetics,
Molecular Biology, Biochemistry or
Cell Biology
Single
Gene/Protein
Most/All
Genes/Proteins
in an Organism
Genomics Summary
A.
B.
C.
D.
E.
F.
G.
H.
Microarrays: expression profiling and other uses
Global Gene Knockouts
Global protein localization in yeast
Global complex identification in yeast
Global two-hybrid analysis in yeast and other organisms
RNAi
Transgenics, gene “knock-outs” (genetics not genomics)
Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
YFG encodes a DNA binding protein
ChIP against epitope tagged YFG
Label ChIP’d DNA Red
Label total genomic DNA green
Hybridize both sets of DNA to
microarray that has intergenic
regions and ORFs
Scan array and analyze data
ChIP on a chip
ChIP Seq
Rap1 binding sites in the yeast genome
Other Uses of DNA Micro-arrays
1. SNP genotyping
2. Recombination
3. Replication timing
4. Other…..