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
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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
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 domains1824C>T is aa conservative (G608G) but - in 300 con.1824C>T creates a cryptic donor site at 1819, -50 aa del
Best guess
Most diseases are probably interactions between polygenic heritable events, and environmental pressures leading to somatic epigenetic changes.
Translation: diseases are complicated.
Gene by Environment Interaction
DNA
Predisposition Event Disease
FAPMSHBRCALDLr
hydrocarbonsradiationestrogenslow fiber
colon CAcolon CAbreast CAatherosclerosis
Microarrays-the big net.Ideal disease-hunter: genomic scale protein quantitation andsequencing.
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 genesare then followed by proteomics and SNPs.
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)
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)
Base-pairing (the stuff of life)
T
CA
G
TC
A
G
Lewin. Genes VII page 8.
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 TmBase 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.
Melting Curves-Tm measured
TmTm
PCR Primer design
www.oligo.net
Array Choice Factors
Expression profiling:
Sequence known? Not known?
Oligo arrays cDNA arrays
High confidence Clone drift/cross hyb
Immediate ID sequence clones
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.
Laser Capture Microdissection
The importance of purity
Human colon cancer
Blue are normal cells
Red are tumor cells
Assessing sample qualityAmount > 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 nmthus, increasing impurity reduces ratio
Better: agarose gel electrophoresis, EtBR stainedif total RNA, 28s = 2 x 18s ribosomal (Lab-on-
chip)or
Q-PCR of a low and high gene, against standard
Best: test chip
GeneChip® Probe Arrays
Image of Hybridized Probe ArrayImage of Hybridized Probe Array
1.28cm1.28cm
GeneChipGeneChip Probe ArrayProbe Array
Millions of copies of a specificMillions of copies of a specificoligonucleotide probeoligonucleotide probe
Single stranded, Single stranded, labeled RNA targetlabeled RNA target
Oligonucleotide probeOligonucleotide probe
**
**
*Hybridized Probe CellHybridized Probe Cell
11 µm
>1 million probes
George Washington
Genomics Core Facility
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
Light(deprotection)
T T O O O
C A T A TA G C T GT T C C G
MaskMask
SubstrateSubstrate
MaskMask
SubstrateSubstrate
T –T –
C –C –REPEATREPEAT
GeneChip® Expression Array Design
GeneGeneSequenceSequence
Probes designed to be Probes designed to be Perfect MatchPerfect Match
Probes designed to be Probes designed to be MismatchMismatch
Egr-1 mRNA and protein in lesions vs normal cells.
Egr
-1 m
RN
A
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
GeneChip® Array Advantages – Specificity
Gene “on”Gene “on”
Gene “off”Gene “off”
Oligo arraysOligo arrays cDNA arrayscDNA arrays
Detection PatternDetection Pattern Single SpotSingle Spot
24 µm24 µm
~ 150 µm~ 150 µm
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-translationallyglycosylation, phosphorylation, etc.