Download No Slide Title

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Copy-number variation wikipedia , lookup

Genome evolution wikipedia , lookup

Epigenetics of human development wikipedia , lookup

Metagenomics wikipedia , lookup

Messenger RNA wikipedia , lookup

Primary transcript wikipedia , lookup

Saethre–Chotzen syndrome wikipedia , lookup

Genetic engineering wikipedia , lookup

Gene expression programming wikipedia , lookup

Genomics wikipedia , lookup

Protein moonlighting wikipedia , lookup

Gene desert wikipedia , lookup

History of genetic engineering wikipedia , lookup

Gene wikipedia , lookup

Epitranscriptome wikipedia , lookup

Gene therapy wikipedia , lookup

Neuronal ceroid lipofuscinosis wikipedia , lookup

Nutriepigenomics wikipedia , lookup

Point mutation wikipedia , lookup

Epigenetics of diabetes Type 2 wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Gene therapy of the human retina wikipedia , lookup

Genome (book) wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Microevolution wikipedia , lookup

Helitron (biology) wikipedia , lookup

Epigenetics of neurodegenerative diseases wikipedia , lookup

Gene expression profiling wikipedia , lookup

RNA-Seq wikipedia , lookup

Therapeutic gene modulation wikipedia , lookup

Gene nomenclature wikipedia , lookup

Designer baby wikipedia , lookup

NEDD9 wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

Transcript
DNA Microarrays for Biomedical Research:
Wonders and Cautions
Daniel R. Salomon, M.D.
Department of Molecular and Experimental Medicine
The Scripps Research Institute
If you don’t understand anything
today - it is my fault.
This is just technology.
What is a gene?
30-35,000
Open Reading Frames
(ORFs)
Genome
Transcriptosome
Proteosome
300-500,000
Proteins
Environment: ECM, Ischemia, Stress
Growth Factors
Cytokines
Development/Differentiation Programs
Baseline/Constitutive
Activated
What happens immediately, what happens next?
Tissues are complex mixtures
of different cell types.
Infiltrating
Inflammatory
Cells
Endothelium
Epithelium
Mesenchymal Elements
How are signal pathways
regulated by transcription?
Gene Discovery vs. Custom
Arrays
• “Gene Discovery Arrays”
Affymetrix GeneChips
Incyte
Rosetta
Filter arrays
• “Custom DNA Microarrays”
Low-cost
Replicate data points
Focused sets of genes
Affymetrix Chip Technology
Affymetrix: Photolithography-based Gene Chip Printing
Affymetrix Chip System
Chip
Hybe/Wash
Station
Array
Scanner
Informatics
Work Station
Processing Microarray Image Data - Step 1
Scanning Image
Red - Cy5
Green - Cy3
Yellow - Both
Processing Microarray Image Data - Step 2
Creating an image mask
Processing Microarray Image Data - Step 3
Analyzing probe intensity at a single spot
Processing Microarray Image Data - Step 4
Comparing multiple time points or conditions
Image Analysis
• ImaGene (Biodiscovery)
• QuantArray (GSI Lumonics)
• Affymetrix GeneChip software
Data Analysis
•
•
•
•
•
Excel
GeneSpring (Silicon Genetics)
Affymetrix GeneChip software
Cluster/Treeview (Stanford)
NFUEGO, Promoter Cruncher,
Chip Annotation databases
Image files
(from Affymetrix or
Custom slide arrays)
To
Web
Server
Microarray specs
Patient/Clinical data
Database
Annotation
Database
Promoter
Cruncher
User Workstations
ImaGene, GeneSpring, Cluster/Treeview
GeneChip, NFUEGO
A
B
C
Affymetrix Chip HG U95A
X60957
Human mRNA for putative receptor tyrosine kinase
X65962
H.sapiens mRNA for cytochrome P-450
X68149
Homo sapiens BLR1 gene for Burkitt''s lymphoma receptor 1
X68149
Homo sapiens BLR1 gene for Burkitt''s lymphoma receptor 1
X68277
H.sapiens CL 100 mRNA for protein tyrosine phosphatase
X07820
Human mRNA for metalloproteinase stromelysin-2
U48705
Human receptor tyrosine kinase DDR gene, complete cds
U50648
Human interferon-inducible RNA-dependent protein kinase (Pkr) gene
U51004
Homo sapiens protein kinase C inhibitor (PKCI-1) mRNA, complete cds
Y09305
H.sapiens mRNA for protein kinase, Dyrk4, partial
U53442
Human p38Beta MAP kinase mRNA, complete cds
U70981
Human interleukin-13 receptor mRNA, complete cds
U85611
Human DNA-PK interaction protein (KIP) mRNA, complete cds
J00219
Human immune interferon (IFN-gamma) gene, complete cds
Incyte Genomics: Human UniGene I Array
(8393 unique genes or ESTs)
Clone ID
Accession #
Gene Name
1362728
1363074
1363684
1363832
1364004
1364225
1365434
1365507
1365962
1365975
1366043
1366085
1366602
1366614
1366817
1366978
1367201
1367516
1367527
1367862
1368173
1368319
1368493
1368653
AF068236
BF035921
NM_014823
NM_006405
AA778107
AW136140
AI627624
BE045743
U75898
AA595575
BE043061
NM_001129
AI416967
N68666
R58925
AF007170
AI754198
AA813998
AF072164
H39214
Y00698
AA348317
AK000005
X57548
nitric oxide synthase 2A (inducible, hepatocytes)
lymphocyte cytosolic protein 1 (L-plastin)
KIAA0344 gene product
transmembrane 9 superfamily member 1
Homo sapiens mRNA; cDNA DKFZp586O2124
Homo sapiens cDNA: FLJ23053 fis, clone LNG02858
zinc finger protein 195
LBP protein 32
heat shock 27kD protein 2
Homo sapiens cDNA: FLJ23516 fis, clone LNG04848
Homo sapiens cDNA FLJ12366 fis, clone MAMMA1002411
AE-binding protein 1
MUM2 protein
f-box and leucine-rich repeat protein 7
EST
DEME-6 protein
KIAA0076 gene product
ESTs
HsHomo sapiens HSFE-1 mRNA, partial cds
ESTs
phosphofructokinase, muscle
ESTs
FLJ00005 protein
cadherin 2, type 1, N-cadherin (neuronal)
http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/
Human Gene Nomenclature Database: Genew3 Search
Search of Approved Symbols AND Literature Aliases from this page [help]
This public copy of the database was last updated on Wed Apr 11, 2001
Now containing 12892 active gene symbols
and 8700 literature aliases
and 2634 withdrawn symbols
Quick search by first letter of symbol [help]
ABCDEFGHIJKLMNOPQRSTUVWXYZ
Page 1 of 4: Cadherins
Symbol
CDH1
CDH2
CDH3
CDH4
CDH5
CDH6
CDH7
CDH8
CDH9
CDH10
CDH11
CDH12
CDH12P
CDH13
CDH15
CDH16
CDH17
CDH18
CDH19
CDH20
CDH21
CDH23
CDH24
Full Name
Cytogenetic Location
cadherin 1, type 1, E-cadherin (epithelial) 16q22.1
cadherin 2, type 1, N-cadherin (neuronal) 18q12.1
cadherin 3, type 1, P-cadherin (placental) 16q22.1
cadherin 4, type 1, R-cadherin (retinal)
20q13.3
cadherin 5, type 2, VE-cadherin
reserved
cadherin 6, type 2, K-cadherin (fetal kidney) 5p14-p15.1
cadherin 7, type 2
18q22-q23
cadherin 8, type 2
16q22.1
cadherin 9, type 2 (T1-cadherin)
reserved
cadherin 10, type 2 (T2-cadherin)
5p13-p14
cadherin 11, type 2, (osteoblast)
16q22.1
cadherin 12, type 2 (N-cadherin 2)
5p14-p13
cadherin 12 (N-cadherin 2) pseudogene
5q13
cadherin 13, H-cadherin (heart)
16q24.2
cadherin 15, M-cadherin (myotubule)
16q24.3
cadherin 16, KSP-cadherin
16q21-q22
cadherin 17, LI cadherin (liver-intestine)
8q22.2-q22.3
cadherin 18, type 2
5p15.1-p15.2
cadherin 19, type 2
18q22-q23
cadherin 20, type 2
18q22-q23
cadherin 21
reserved
cadherin related 23
10q21-q22
cadherin-like 24
reserved
PubMed ID
9925936
2384753
1427864
10191097
2059658
7743525
9615235
9615235
2059658
2059658
9615235
7731968
7731968
8673923
1427864
9721215
9615235
9030594
11090341
PubMed Reference for Cadherin 2
N-cadherin gene maps to human chromosome 18 and is
not linked to the E-cadherin gene.
Walsh FS, Barton CH, Putt W, Moore SE, Kelsell D, Spurr N, Goodfellow PN.
Department of Experimental Pathology, UMDS, Guy's Hospital, London, England.
cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening
with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the
predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs
over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated
in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp
of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb
in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of
5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be
mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have
evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.
http://www.gdb.org/
Search
Genomic Segments
All Biological Data
People
Citations
by
Name/GDB ID
Keyword
DNA Sequence ID
Submit
Gene List - Accession #, Map Coordinates
Gene Map of Chromosome 8q13.3-8q24.22
Displayed by MapView 2.4
Custom Microarray Printing
• Glass slide substrate
• 500-10,000 spots/slide
• Robotic spotting device
• cDNA clones
• PCR products
• Oligonucleotides
(50-70mers)
The Mguide: A complete guide to building your own microarrayer.
http://cmgm.stanford.edu/pbrown/mguide
Oligonucleotides (50-70mers)
•
•
•
•
are sequence verified
eliminate clone library banking
eliminate PCR and PCR contamination issues
are inexpensive (500ng of purified 75mer will
print 12,000 arrays each with 3 replicate spots)
• can be directed at specific exons to detect
splice variants
• can be designed to distinguish closely related
genes
Array-based genotyping vs.
gene expression analysis
Gene Expression
Genotyping
• 1-40 markers/sample
• multiple samples/chip
• 100-10,000 genes / chip
• sample tracking
• automated sample
and reagent handling
• 2-4 color fluorescence
detection
•
•
•
•
• 1-2 samples/chip
library of array elements
high speed/high density printing
data interpretation
database management
:::::::::::::::
:::::::::::::::::::::::::
:::::::::::::::
:::::::::::::::::::::::::
:::::::::::::::
:::::::::::::::::::::::::
:::::::::::::::
:::::::::::::::::::::::::
:::::::::::::::::::::::::
Solid-phase primer extension assay:
“Mini-sequencing”, “Genetic Bit Analysis (GBA)”
1. Hybridization of template DNA to solid-phase primer
5’
X=C/T polymorphism
3’
Hybridized
template
TACGTGACTGATGCTTAGCTTCAATGAGXTGATGTAG
|||||||||||||||||||||
GACTACGAATCGAAGTTACTC
Solid-phase oligonucleotide
Glass slide
3mm
A Microarray Strategy
Gene Discovery
(large arrays, limited
numbers of samples)
Custom DNA microarrays
(small arrays, large
numbers of samples)
Genotyping (SNPs)
The challenges for chip technology I.
• Incredible amounts of “data”
•
•
•
•
An incomplete database
Technical issues involving multiple technologies
Relatively primitive tools for handling data
Major issues for statistical methodologies
Normalization
Replicates
Significant changes
The challenges for chip technology II.
• Major challenge to scientific thought and method
Big science or “organismal” biology
Hypothesis-driven vs. “Fishing Expedition”
• How is the transcriptosome regulating the proteome?
Clinical Specimens
Laser Capture Microscopy
RNA/Signal Amplification