Post on 13-Jan-2016
MCB 317Genetics and Genomics
Topic 11Genomics
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
Singlegene/protein
Most/all genes/proteinsin an Organism
Ab
Protein
TxnProfile
Gene
Orthologs and Paralogs
Mutant Gene
Biochemistry
Genetics
Mutant Organism
A
C
F
Subunits of Protein Complex
B, G
D E
ProteinProfile/Localization
Genomics:
High-throughput genetics
Genomics
B, G
H
Genomics Summary
A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. Human Genome Project, Next Generation Sequencing,
and Comparative Genomics
Northern Blots
Qualitative Change in TranscriptionAnalysis of Tissue Specific Transcription
Isolate RNA (mRNA) from 2 tissues e.g. liver and muscle
Probe = DNA from one geneLane 1 = liver mRNALane 2 = muscle mRNA
1 2
Northern Blots
-> Quantitative Change in Transcription
Same Approach: this time mix two probes(two genes); look at relative change
Probe = DNA from two genes A and BLane 1 = liver mRNALane 2 = muscle mRNA
1 2
A
B
DNA Microarrays
2
1
4
3
6
5
8
7
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 REDLabel 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
2
1
4
3
Liver mRNA = RED Muscle mRNA = GREEN
1. On in Liver, Off in Muscle = RED2. On in Muscle off in Liver = GREEN3. On in both = YELLOW (RED + GREEN)4. 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 effectRed = condition 1, Green = condition 2
Fold change from condition 1 to condition 2
+2
+3
+3
+4
+2
0
0 0
-4
-3
-2 -1.5
+1.5
+1.2
0
-2
Another way to view the data: computer conversion to fold effectRed = condition 1, Green = condition 2
Fold change from condition 1 to condition 2
+2
+3
+3
+4
+2
0
0 0
-4
-3
-2 -1.5
+1.5
+1.2
0
-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 subunitB. Wild-type vs. nonessential subunit of mediatorC. Wild-type vs. gene XD. Wild-type vs snf2
Color scheme = fold change in mutant relative to wild-type
CouplingMicroarrays and Yeast Genetics:Mutant v. Wild-type
Cell type 1 = WTCell type 2 = Mutant
Gene Discovery via Expression Profiling
1. Micro-array2. 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 analysis4. 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. Asynchronous vs. early M-phase2. Asynchronous vs. mid M-phase3. Asynchronous vs. late M-phase4. Asynchronous vs. early G15. 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
PrimaryTumor
MetastasizedTumor
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
SingleGene/Protein
Most/All Genes/Proteinsin an Organism
Genomics Summary
A. Microarrays: expression profiling and other usesB. Global Gene Knockouts C. Global protein localization in yeast D. Global complex identification in yeast E. Global two-hybrid analysis in yeast and other organismsF. RNAiG. Transgenics, gene “knock-outs” (genetics not genomics)H. 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 genotyping2. Recombination3. Replication timing4. Other…..