Transcript IntroductoryDay1LectureMarch17

Large Scale Gene Expression with
DNA Microarrays
Vermont Genetics Network
Microarray Outreach
Program
Vermont Genetics Network (VGN)
Founded at the University of Vermont in 2001
through an NIH BRIN grant.
Purpose:
• Encourage biomedical research in Vermont
• Create a “network” of researchers and students
• Give outreach lectures to 4-year institutions
• Provide research grants to faculty and students
• Mentoring for students interested in research
VGN Microarray Outreach Program
• Introduce microarray technology to VT colleges.
• Develop microarray outreach module.
• Team of scientists to serve as instructors
Ahmad Chaudhry, Rebecca Guy, Tim Hunter,
Brian McElhinney, Pat Reed
Kathy Seiler, Scott Tighe
Microarray Experiment
• The effect of a chemical dimethyl sulfoxide (DMSO)
on gene expression in yeast
• DMSO is an environmental contaminant from the
paper industry and from pesticides
• Grow the yeast and treat one group with plain water
(control group) and the other group with 10% DMSO
• Isolate RNA from the yeast grown in two different
conditions, prepare target from it and use it on
microarrays to see changes in gene expression
Expressed Genes = mRNA
DNA (genes)
messenger RNA
Protein (effector molecules)
What are Microarrays?
• Microarrays are simply small glass or silicon
slides upon the surface of which are arrayed
thousands of genes (usually between 500-20,000)
• Via a conventional DNA hybridization process,
the level of expression/activity of genes is
measured
• Data are read using laser-activated fluorescence
readers
• The process is “ultra-high throughput”
Why use Microarrays?
• What genes are Present/Absent in a cell?
• What genes are Present/Absent in the experiment vs.
control?
• Which genes have increased/decreased expression in
experiment vs. control?
• Which genes have biological significance?
Why analyze so many genes?
• Just because we sequenced a genome doesn’t
mean we know anything about the genes.
Thousands of genes remain without an
assigned function.
• Patterns/clusters of expression are more
predictive than looking at one or two
prognostic markers – can figure out new
pathways
Creating Targets
mRNA
Reverse Transcriptase
cDNA
PCR amplification of DNA
More
cDNA
in vitro transcription
cRNA
RNA-DNA Hybridization
Targets
(RNA)
probe sets on chip
(DNA)
(25 base oligonucleotides of known sequence)
Non-Hybridized Targets are Washed Away
Targets
(fluorescently tagged)
“probe sets” (oligos)
Non-bound ones are washed away
The 6 steps of a DNA microarray
experiment (1-3)
1. Manufacturing of the microarray
2. Experimental design and choice of
reference: what to compare to what?
3. Target preparation (labeling) and
hybridization
The 6 steps of a microarray experiment (4-6)
4. Image acquisition (scanning) and
quantification (signal intensity to
numbers)
5. Database building, filtering and
normalization
6. Statistical analysis and data mining
Experimental Design
• Choice of reference: Common (nonbiologically relevant) reference, or paired
samples?
• Number of replicates: How many are
needed? (How many are affordable?).
Are the replicate results going to be
averaged or treated independently?
Is this a “fishing expedition” or a hypothesisbased experiment?
Why Use Yeast ??
• easily manipulated in the laboratory
• simple eukaryote, unicellular
• rapid growth (doubling 1.5 - 2.5
hours)
• non-pathogenic
• stable haploid and diploid states
• complete genome sequenced
E. coli
Yeast
Human
~ 1 x 3 m
~ 5 m dia.
~ 1.7 m
1 chromosome
16 chromosomes
23 chromosomes
4 x 10 6 bp
12 x 10 6 bp
3.3 x 10 9 bp
~ 4,377 genes
~ 5,726 genes
~ 30, 000 genes
Yeast Life cycle
S. Cerevisiae Genome
• DNA ~ 60% A + T
• 16 chromosomes (haploid)
• chromosomes have centromeres and
telomeres
• also have mitochondrial genome
• plasmid: 6 kb, 60-100 copies/cell
• entire genome has been sequenced
Yeast Genetic Nomenclature
• genes names are 3-letters + a number
• genes names are derived from
phenotype of the mutant, i.e. ste 3
mutants have sterile phenotype
• gene names are written in italics or
underlined
• wild type alleles: Uppercase STE 3
• mutant alleles: lowercase ste 3
Growth and Metabolism
+ O2 / no O2
CARBON + NITROGEN + BIOTIN + MINERALS
Prototroph: requires no additional nutrients (can grow in minimal
media).
Auxotroph: requires additional nutrients from the environment or
media.
Our strain, NRRL Y-12632 (or ATCC 18824) is a wild type,
prototrophic, Matα strain isolated from brewing yeast.
Yeast Microarray Experiment
untreated
Changes in gene expression?
Which genes are up regulated?
versus
Which genes are down
regulated?
What do the results say about
yeast biology?
DMSO treated
What parallels (if any) can we
make to human biology?