mcb 317 genetics and genomics topic 11 genomics. readings genomics: hartwell chapter 10 of full...

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…..