1. Hutchinson-Guilford Progeria
premature aging
lifespan = 13.4 years
retarded growth
midface hypoplasia
micrognathia
alopecia
low adiposity
osteodysplasia
premature, severe
atherosclerosis
-death due to MI
De Sandre-Ciovannoli, Science express, 17 April 2003

2. Lamin A mutations in HGS
Exons 11 and 12 code the Lamin A tail (not lamin c)
Red is coiled-coil and blue is globular domains
1824C>T is aa conservative (G608G) but - in 300 con.
1824C>T creates a cryptic donor site at 1819, -50 aa del

3. Best guess
Most diseases are probably interactions between polygenic heritable events, and environmental pressures leading to somatic epigenetic changes.
Translation: diseases are complicated.

4. Gene by Environment Interaction
Predisposition
Event
Disease
DNA
FAP
MSH
BRCA
LDLr
hydrocarbons
radiation
estrogens
low fiber
colon CA
breast CA
atherosclerosis

5. Microarrays-the big net.
Ideal disease-hunter: genomic scale protein quantitation and
sequencing.
Imperfect solution A: genomic scale detection of mRNA level.
Problem: little information on protein level
Imperfect solution B: genome-wide SNP/haplotype.
Problem: statistical limits on patient populations
Common compromise: microarray profiling mRNA transcripts
(transcript profiling) to identity target areas. Target genes
are then followed by proteomics and SNPs.

6. Array flavors DNA detection (SNP, genotyping, etc.)
• short oligonucleotides to detect mismatches
RNA detection (transcript profiling)
• Plasmid
• Inserts
• Long oligonucleotides (60 mers)
• Short oligonucleotides (20 mers)

7. Hybridization-basic elements
Hybridization = Annealing - Melting
CRUCIAL: non-covalent, hydrogen bonds
-->equilibrium rules, binding is statistical
Best hybridization occurs with:
long sequences (no hyb when nt<4)
high salt concentration (hybrids melt in water)
low temperatures (hybrids melt with heat)
G and C (3 H) bind better than A and T (2 H)
self-complementarity is low (high GC is bad)

8. Base-pairing (the stuff of life)C T A C G
Lewin. Genes VII page 8.

9. Tm-a good thing.
Tm is a measure of the stability of DS-DNA under a given set of conditions. Stability, and therefore Tm, is affected by:
Strand length - the longer the strand, the higher the Tm
Base Composition - higher the GC content, the higher the Tm.
Ionic Strength - as the ionic strength increases, so does Tm. Double helical DNA is stabilised by cations.
Divalent cations (eg Mg2+) are more effective than
monovalent cations (+ or K+).
Organic Solvents - formamide for instance lowers the Tm by weakening the hydrophobic interactions.

10. Melting Curves-Tm measured

11. PCR Primer design

12. Array Choice Factors Expression profiling: Sequence known? Not known?
Oligo arrays cDNA arrays
High confidence Clone drift/cross hyb
Immediate ID sequence clones

13. Sample selection
isolate the purest phenotypic examples of test and control
laser capture microdissection (LCM)
always control for treatment and manipulation
people are the most meaningful, but least controllable
animals are highly controllable, but less meaningful
cell systems (in vitro) are controlled, but meaningful?
small amounts of RNA can be amplified
while purifying cells is good, the processing is bad.
The quality of the results are directly proportional to the samples that are chosen.

14. Laser Capture Microdissection

15. The importance of purity
Human colon cancer
Blue are normal cells
Red are tumor cells

16. Assessing sample quality
Amount > 5 ug total RNA or 500 ng of poly A+
Basic: O.D. 260/280 ratio >2.1,
nucleic acids absorb at 260, protein at 280 nm
thus, increasing impurity reduces ratio
Better: agarose gel electrophoresis, EtBR stained
if total RNA, 28s = 2 x 18s ribosomal (Lab-on-chip) or
Q-PCR of a low and high gene, against standard
Best: test chip

17. GeneChip® Probe Arrays
Millions of copies of a specific
oligonucleotide probe
Single stranded,
labeled RNA target
Oligonucleotide probe *
Hybridized Probe Cell
GeneChip Probe Array
11 µm
1.28cm
GENECHIP PROBE ARRAYS
The core of the platform is our unique arrays
Oligonucleotides synthesized de novo (photolithography & combinatorial chemistry)
Currently 65,000 (50 micron features) to 250,000 (24 micron) different oligos on commercially available products
Each oligo represented in 107 to 108 full-length copies
>1 million probes
Image of Hybridized Probe Array

18. Synthesis of Ordered Oligonucleotide Arrays
O O O O O
Light
(deprotection)
HO HO O O O
T T O O O
T T C C O
C A T A T
A G C T G
T T C C G
Mask
Substrate
T –
C –
REPEAT
Light removes protecting groups at defined positions. Single nucleotide washed over the chip, binds where the protecting group removed. Through successive steps, any sequence can be built up in any position on the chip. The number of steps corresponds with length of oligo, so can increase # of genes without # of steps

19. GeneChip® Expression Array Design
Multiple oligo probes 5´ 3´
Gene
Sequence
Probes designed to be Perfect Match
Probes designed to be Mismatch

20. Procedures for Target Preparation
Cells
AAAA
Labeled transcript
IVT
(Biotin-UTP
Biotin-CTP)
Poly (A)+
RNA L L L L
cDNA
Fragment
(heat, Mg2+) L
Wash & Stain L
Hybridize
(16 hours) L
Target Preparation
RNA isolation is the first step. 1-2 hours
The messenger RNA is then reverse transcribed into cDNA (we then go on to make the second strand of cDNA).
4 hours
An in vitro transcription reaction using biotinylated nucleotides is then done to both amplify and label the transcripts hours
These are then fragmented in order to get a more efficient hybridization ( bases pairs is the goal).
1.0 hours
The fragmented target is then hybridized overnight to a GeneChip expression array. 16 hours
When washing and staining of the array is complete it can then be scanned hours L
Scan
Labeled fragments
Streptavidin-Phycoerythrin (SAPE)
Fluorescent stain-laser stimulated

21. Analysis of expression level from probe sets
A single, contiguous gene set for the rat B-actin gene.
Each pixel is quantitated and integrated for each oligo feature (range 0-25,000)
Perfect Match (PM)
Mis Match (MM) Control
PM - MM = difference score
All significant difference scores are averaged to create “average difference” = expression level of the gene.

22. Affymetrix® Instrument System
Platform for GeneChip® Probe Arrays
Integrated
Exportable
Easy to use
Versatile

23. GeneChip analysis of human atherosclerosis
Dissect normal media from atherosclerotic lesion
Prepare highly purified RNA
O.D. 260/280 = 2.0
Reverse transcribe w/poly dT + T7 = cDNA
Transcribe with T7 + biotin dUTP = cRNA
Purify probe/hybridize to chip
Wash and detect with avidin/PE + ab amplification
Read fluorescent label
And deconvolve genes

24. Basic Bioinformatics-Scatterplot

25. Transcript profiling of aged rat aorta.
Affymetrix GeneChip analysis of mo. vs. 3 mo.

26. FAQs: How many replicates?

27. Simple fold changes Crude, insensitive--but effective Criteria:
Present
1.5-fold up/down

28. Hierachical clustering

29. Statistical testing and ontology

30. Pathways of genetic information

31. Expression of Egr-1 mRNA in human lesions.

32. Egr-1 mRNA and protein in lesions vs normal cells.B)
Western blot
Media
E197
E221
E240
E243 20
Lesion M L M L M L M L 15
Egr-1
Egr-1 mRNA x x 10 5
Actin
E197
E196

33. Expression screening by GeneChip
• each oligo sequence (20 mer) is synthesized
as a 11 µ square (feature)
• each feature contains > 1 million copies of the oligo
• scanner resolution is about 2 µ (pixel)
• each gene is quantitated by 11 oligos and
compared to equal # of mismatched controls
• 44,000 genes are evaluated with 11 matching oligos
and 11 mismatched oligos = 4 x 106 features/chip
• features are photolithographically synthesized
onto a 2 x 2 cm glass substrate

34. GeneChip® Array Advantages – Specificity
Oligo arrays
cDNA arrays
Gene “on”
~ 150 µm
24 µm
Gene “off”
Detection Pattern
Single Spot

35. Limitations to all microarrays.
dynamic range of gene expression:
very difficult to simultaneously detect low and high
abundance genes accurately
- each gene has multiple splice variants
2 splice variants may have opposite effects (i.e. trk)
arrays can be designed for splicing, but complexity ^ 5X
- translational efficiency is a regulated process:
mRNA level does not correlate with protein level
- proteins are modified post-translationally
glycosylation, phosphorylation, etc.
- pathogens might have little ‘genomic’ effect

36. CardioChip in silico workup Lipoprotein genes/variants
Heart failure predictors
Atherosclerosis markers
Restenosis markers
Coagulation factors
Stress markers
Infectious agents
Inflammatory markers