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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. 300-500,000 Proteins. What is a gene?.

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Dna microarrays for biomedical research wonders and cautions

DNA Microarrays for Biomedical Research:

Wonders and Cautions

Daniel R. Salomon, M.D.

Department of Molecular and Experimental Medicine

The Scripps Research Institute


Dna microarrays for biomedical research wonders and cautions

If you don’t understand anything

today - it is my fault.

This is just technology.


Dna microarrays for biomedical research wonders and cautions

300-500,000

Proteins

What is a gene?

30-35,000

Open Reading Frames

(ORFs)

Genome

Transcriptosome

Proteosome


Dna microarrays for biomedical research wonders and cautions

Environment: ECM, Ischemia, Stress

Growth Factors

Cytokines

Development/Differentiation Programs

Baseline/Constitutive

Activated

What happens immediately, what happens next?


Dna microarrays for biomedical research wonders and cautions

Tissues are complex mixtures

of different cell types.

Infiltrating

Inflammatory

Cells

Endothelium

Epithelium

Mesenchymal Elements


Dna microarrays for biomedical research wonders and cautions

How are signal pathways

regulated by transcription?


Gene discovery vs custom arrays
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



Dna microarrays for biomedical research wonders and cautions

Affymetrix: Photolithography-based Gene Chip Printing


Dna microarrays for biomedical research wonders and cautions

Affymetrix Chip System

Chip

Hybe/Wash

Station

Array

Scanner

Informatics

Work Station


Dna microarrays for biomedical research wonders and cautions

Processing Microarray Image Data - Step 1

Scanning Image

Red - Cy5

Green - Cy3

Yellow - Both



Dna microarrays for biomedical research wonders and cautions

Processing Microarray Image Data - Step 3

Analyzing probe intensity at a single spot


Dna microarrays for biomedical research wonders and cautions

Processing Microarray Image Data - Step 4

Comparing multiple time points or conditions


Image analysis
Image Analysis

  • ImaGene (Biodiscovery)

  • QuantArray (GSI Lumonics)

  • Affymetrix GeneChip software


Data analysis
Data Analysis

  • Excel

  • GeneSpring (Silicon Genetics)

  • Affymetrix GeneChip software

  • Cluster/Treeview (Stanford)

  • NFUEGO, Promoter Cruncher, Chip Annotation databases


Dna microarrays for biomedical research wonders and cautions

Image files

(from Affymetrix or

Custom slide arrays)

Microarray specs

Patient/Clinical data

To

Web

Database

Server

Annotation

Database

Promoter

Cruncher

User Workstations

ImaGene, GeneSpring, Cluster/Treeview

GeneChip, NFUEGO


Dna microarrays for biomedical research wonders and cautions

A

C

B



Dna microarrays for biomedical research wonders and cautions

Incyte Genomics: Human UniGene I Array

(8393 unique genes or ESTs)

Clone ID Accession # Gene Name

1362728 AF068236 nitric oxide synthase 2A (inducible, hepatocytes)

1363074 BF035921 lymphocyte cytosolic protein 1 (L-plastin)

1363684 NM_014823 KIAA0344 gene product

1363832 NM_006405 transmembrane 9 superfamily member 1

1364004 AA778107 Homo sapiens mRNA; cDNA DKFZp586O2124

1364225 AW136140 Homo sapiens cDNA: FLJ23053 fis, clone LNG02858

1365434 AI627624 zinc finger protein 195

1365507 BE045743 LBP protein 32

1365962 U75898 heat shock 27kD protein 2

1365975 AA595575 Homo sapiens cDNA: FLJ23516 fis, clone LNG04848

1366043 BE043061 Homo sapiens cDNA FLJ12366 fis, clone MAMMA1002411

1366085 NM_001129 AE-binding protein 1

1366602 AI416967 MUM2 protein

1366614 N68666 f-box and leucine-rich repeat protein 7

1366817 R58925 EST

1366978 AF007170 DEME-6 protein

1367201 AI754198 KIAA0076 gene product

1367516 AA813998 ESTs

1367527 AF072164 HsHomo sapiens HSFE-1 mRNA, partial cds

1367862 H39214 ESTs

1368173 Y00698 phosphofructokinase, muscle

1368319 AA348317 ESTs

1368493 AK000005 FLJ00005 protein

1368653 X57548 cadherin 2, type 1, N-cadherin (neuronal)


Dna microarrays for biomedical research wonders and cautions

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


Dna microarrays for biomedical research wonders and cautions

Page 1 of 4: Cadherins

Symbol Full Name Cytogenetic Location PubMed ID

CDH1 cadherin 1, type 1, E-cadherin (epithelial) 16q22.1 9925936

CDH2cadherin 2, type 1, N-cadherin (neuronal) 18q12.1 2384753

CDH3 cadherin 3, type 1, P-cadherin (placental) 16q22.1 1427864

CDH4 cadherin 4, type 1, R-cadherin (retinal) 20q13.3 10191097

CDH5 cadherin 5, type 2, VE-cadherin reserved 2059658

CDH6 cadherin 6, type 2, K-cadherin (fetal kidney) 5p14-p15.1 7743525

CDH7 cadherin 7, type 2 18q22-q23 9615235

CDH8 cadherin 8, type 2 16q22.1 9615235

CDH9 cadherin 9, type 2 (T1-cadherin) reserved 2059658

CDH10 cadherin 10, type 2 (T2-cadherin) 5p13-p14 2059658

CDH11 cadherin 11, type 2, (osteoblast) 16q22.1 9615235

CDH12 cadherin 12, type 2 (N-cadherin 2) 5p14-p13 7731968

CDH12P cadherin 12 (N-cadherin 2) pseudogene 5q137731968

CDH13 cadherin 13, H-cadherin (heart) 16q24.2 8673923

CDH15 cadherin 15, M-cadherin (myotubule) 16q24.3 1427864

CDH16 cadherin 16, KSP-cadherin 16q21-q22 9721215

CDH17 cadherin 17, LI cadherin (liver-intestine) 8q22.2-q22.3 9615235

CDH18 cadherin 18, type 2 5p15.1-p15.2 9030594

CDH19 cadherin 19, type 2 18q22-q23

CDH20 cadherin 20, type 2 18q22-q23

CDH21 cadherin 21 reserved

CDH23 cadherin related 23 10q21-q22 11090341

CDH24 cadherin-like 24 reserved


Dna microarrays for biomedical research wonders and cautions

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.


Dna microarrays for biomedical research wonders and cautions

http://www.gdb.org/

Search bySubmit

Genomic Segments Name/GDB ID

All Biological Data Keyword

People DNA Sequence ID

Citations

Gene List - Accession #, Map Coordinates


Dna microarrays for biomedical research wonders and cautions

Gene Map of Chromosome 8q13.3-8q24.22

Displayed by MapView 2.4


Custom microarray printing
Custom Microarray Printing

  • Glass slide substrate

  • 500-10,000 spots/slide

  • Robotic spotting device

  • cDNA clones

  • PCR products

  • Oligonucleotides

    • (50-70mers)


Dna microarrays for biomedical research wonders and cautions

The Mguide: A complete guide to building your own microarrayer.

http://cmgm.stanford.edu/pbrown/mguide


Oligonucleotides 50 70mers
Oligonucleotides (50-70mers) microarrayer.

  • 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

Genotyping microarrayer.

Gene Expression

  • 1-40 markers/sample

  • multiple samples/chip

  • sample tracking

  • automated sample and reagent handling

  • 2-4 color fluorescence

  • detection

  • 100-10,000 genes / chip

  • 1-2 samples/chip

  • library of array elements

  • high speed/high density printing

  • data interpretation

  • database management

Array-based genotyping vs.gene expression analysis

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Solid phase primer extension assay mini sequencing genetic bit analysis gba
Solid-phase primer extension assay: microarrayer.“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


Dna microarrays for biomedical research wonders and cautions

3mm microarrayer.


A microarray strategy
A Microarray Strategy microarrayer.

Gene Discovery

(large arrays, limited

numbers of samples)

Custom DNA microarrays

(small arrays, large

numbers of samples)

Genotyping (SNPs)


Dna microarrays for biomedical research wonders and cautions

The challenges for chip technology I. microarrayer.

  • 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


Dna microarrays for biomedical research wonders and cautions

The challenges for chip technology II. microarrayer.

  • Major challenge to scientific thought and method

  • Big science or “organismal” biology

  • Hypothesis-driven vs. “Fishing Expedition”

  • How is the transcriptosome regulating the proteome?


Rna signal amplification

Clinical Specimens microarrayer.

Laser Capture Microscopy

RNA/Signal Amplification