GeneticGenomic Technology Platforms Available Through the

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Genetic/Genomic Technology Platforms Available
Through the Keck Laboratory
NIDA National Institute On Drug
Abuse Neuroproteomics Center
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12 Keck Laboratory Resources

• 5 Genomic Resources
• Affymetrix Genechip
• Glass Slide Microarray
• DNA Sequencing
• Oligo Synthesis
• SNP Genotyping
• 6 Proteomic Resources
• Amino Acid Analysis
• Biophysics
• Mass Spectrometry
• Peptide Synthesis
• Small Scale (Fmoc)
• Large Scale (tBoc)
• Protein Sequencing
• Biostatistics Resource

Cluster Analysis of DNA Microarray Data
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Brief Overview of the Keck Laboratory

• Founded in 1980
• 40 Full time staff.
• 100 Major instrument systems purchased at a cost
  of gt11 million dollars.
• 10 Resources at 300 George St, Biophysics
  Resource in SHM, and Small Scale Peptide
  Synthesis Resource in Boyer Center.
• Completes gt270,000 protein and nucleic acid
  syntheses and analyses annually for 260 Yale and
  640 non-Yale investigators at 300 institutions in
  27 countries.
• gt90 of analyses are requested by Yale
  investigators
• In July, 2004 the Keck web pages received 880,000
  requests from 7,000 distinct hosts.
• (http//keck.med.yale.edu/)

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Overview and Inter-dependency of Keck Genomic
Technologies
DNA Sequencing
Oligo Synthesis
Modified, Non-modified DNA
Single tube samples
96-well plates
Crude and Gel-purified
Primer-walking
RNA
Affymetrix GeneChip
Spotted Microarrays
Genomic Expression Arrays
Genomic Expression Arrays
SNP 100K Genotyping Chips
Targeted/custom Arrays
Plasmid prep/PCR for custom arrays
Real-time PCR
SNP Genotyping
MS-based Sequenom
Biostatistics
Pyrosequencing
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Demand for Core Genomic Technologies Continues to
Increase
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Affymetrix Overview
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Affymetrix GeneChip Technology Expression
Assay Workflow
Labeled transcript
In Vitro transcription (Biotin-UTP Biotin-CTP)
L
L
L
L
Poly (A) Or Total RNA
RT
cDNA
Fragment (heat, Mg2)
L
L
Wash Stain
L
Hybridize (16 hours)
L
Scan
Labeled fragments
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GeneChip Probe Arrays
Hybridized Probe Cell
GeneChip Probe Array
Single stranded, labeled RNA target
Oligonucleotide probe
11µm
Millions of copies of a specific oligo-nucleotide
probe
1.28cm
gt1,300,000 Different complementary probes
Image of Hybridized Probe Array
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• Keck Affymetrix GeneChip Services
• mRNA Expression
• Preparation, Q/C, and fragmentation of labeled
  cRNA from RNA isolated by users
• Hybridization of fragmented, labeled target cRNA
  to test/standard arrays
• Washing, staining, and scanning of the array
• Analysis of the scanned image using Affymetrix
  Gene Chip Operating Software (GCOS)
• Generation of gene expression profiles
• Quantitative PCR
• SNP Genotyping
• Taq Man analysis of individual SNPs
• Genomic SNP analysis with 10K and 100K SNP chips

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Spotted Glass Slide Microarray Overview
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YCC/Keck Microarray Resource Arrays
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Outline of a Glass Slide Microarray Comparison of
mRNA Expression in Melanocytes vs Melanomas

• Isolate mRNA
• Normal Melanocyte resulting cDNA is labeled with
  Cy3
• Melanoma resulting cDNA is labeled with Cy5
• Combine and probe with human 16K oligo slide
• Cy5/Cy3 ratio provides a measure of the fold
  change in expression.

• From (2004) Expression Profiling Reveals Novel
  Pathways in Transformation of Melanocytes to
  Melanomas. Cancer Res. 64 5270-5282 (R. Halaban)

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Melanoma Cy5 (red)Normal Melanocyte Cy3
(green)
Cy5 Cy3 No significant change
Cy5 gt Cy3 Up-regulation in melanoma
Cy5 lt Cy3 Down-regulation in melanoma
The largest group of genes with increased
expression in melanoma cells belonged to receptor
activity. In addition, the data revealed novel
pathways and patterns of associated expression in
melanoma cells not reported previously.
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YCC/Keck Microarray Resource Genomic Services

• Generic cDNA and oligo arrays
• Custom cDNA and oligo arrays
• Labeling and hybridization
• Scanning
• Primary Data Analysis
• Complete project services

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Publications Utilizing the Keck Affymetrix and
Microarray Resources

• Affymetrix Resource 8 publications acknowledging
  use since opening in 2002
• Microarray Resource 39 publications
  acknowledging use since opening in 1999
• (Selected) 2004 publications cover a broad range
  of research
• Major Molecular Differences Between Mammalian
  Sexes are Involved in Drug Metabolism and Renal
  Function. Developmental Cell 6 791-800 (M.
  Snyder/Affymetrix)
• Aquaporin-4 is increased in the sclerotic
  hippocampus in human temporal lobe epilepsy. Acta
  Neuropathologica, in press (N. de
  Lanerolle/Affymetrix)
• Interferon alpha but not interleukin 12 activates
  STAT4 signaling in human vascular endothelial
  cells. J. Biol. Chem. 279(25)26789-96. (J.
  Pober/Microarray)
• Herpesvirus saimiri small nuclear RNAs recruit
  AU-rich element-binding proteins but do not alter
  host AU-rich element-containing mRNA levels in
  virally transformed T cells. Mol Cell Biol.
  24(10)4522-33 (J. Steitz/Microarray
  Biostatistics)

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Yale Microarray Database (YMD)

• YMD is an Oracle database that stores
• Raw microarray images
• Corresponding image quantitative data produced by
  Axons GenePixPro
• Descriptions of the microarray experiments
• Supplementary data for publications
• Data stored in YMD can be
• Queried for primary data analysis
• Easily submitted to the NCBI Gene Expression
  Omnibus (GEO) database
• Easily analyzed by several statistical tools
  linked to YMD

http//info.med.yale.edu/microarray/
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YMD Tool Keck ARray Manager Annnotator (KARMA)

• Provides a Web interface to annotate and compare
  array platforms in and between a single or
  multiple organisms.
• http//biryani.med.yale.edu/karma/cgi-bin/mysql/ka
  rma.pl
• The annotation and comparison is based on
  integration of genome annotation from multiple
  sources (e.g., GO, SwissProt, Unigenes, LocusLink
  and Homologene)
• The integrated information is stored in an Oracle
  database and is updated periodically. This is
  automated by a set of Perl programs some of which
  were adapted from Stanfords SOURCE project.
• Research that led to KARMA was published in the
  2004 NAR special issue on server applications
• Cheung, K.H., Hager, J., Pan, D., Srivastava,
  R., Mane, S., Li, Y., Miller, P., Williams, K.
  (2004) KARMA A web server application for
  comparing and annotating heterogeneous microarray
  platforms. Nucleic Acids Research 32(Web Server
  issue)W441-W444.

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KARMA Analysis of Overlap Between Mouse
Affymetrix Chip versus Keck (NIA) cDNA and
(Operon) 70-mer Glass Slide Arrays
AFFY U74AV2 25-mer 12,488
7,041 Genes are shared between this Affymetrix
chip and glass slide microarray
Overlap is based on number of genes sharing the
same Unigene cluster ID.
7,041
4,635
3811
OMM 16K 70-mer 16,655
NIA 15K cDNA 15,245
5,685
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SNP Genotyping Technologies
Affymetrtix SNP chip is designed for whole genome
association or linkage studies.  The other three
technologies are for finer linkage or association
with custom SNPs  and genome regions.
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Affymetrix Human 100K SNP GeneChip Mapping

• The 100K Mapping Set is composed of 2 arrays
  each representing gt50,000 SNPs
• One array uses DNA that is digested with the XbaI
  restriction enzyme while the other uses HindIII
• Median intermarker distance 8.5 kb
• Expected call rate gt95
• Major steps with this technology
• Genomic DNA digested with a restriction enzyme
• Resulting fragments are ligated to an adapter
  sequence
• Generic primer that recognizes the adapter
  sequence is used to PCR amplify adapter-ligated
  fragments
• Amplified DNA is fragmented, labeled, and
  hybridized to a 50K SNP chip which is then scanned

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Affymetrix GeneChip SNP Genotyping
250 ng Genomic DNA
Restriction enzyme digestion
Xba
Xba
Xba
Adapter ligation
Fragmentation and Labeling
Hyb Scan on Standard Hardware
Adapted from Affymetrix
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Sequenom SNP Genotyping One or Two Base
Extension With MALDI-MS Detection
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SNP Genotyping by Pyrosequencing
Each of four dNTPs is added one at a time to a
primertemplate complex, if incorporated by DNA
polymerase, a luciferase-coupled reaction
produces light.
Light Intensity
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Keck Biostatistics Resource Services

• Microarray Data Analysis
• Basic data pre-processing
• Identification of differentially expressed genes
• Clustering analysis
• Classification analysis
• Pathway analysis
• Bioinformatics analysis
• Literature searches (e.g., PubMatrix might be
  used to search a list of genes onto the PubMed
  database for relevant terms of interest to
  researchers)

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Hierarchical Clustering of an Affymetrix Study of
Drug-induced Vascular Injury
Red Higher expression with drug
Clustered High Dose Subjects
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New Center for High Performance Computation In
Biomedicine Will Support Advanced Analysis of
Genomic Data

• The Supercomputer for this Center was recently
  funded by an NCRR High End Instrumentation Grant
• DC 1.6 million PI Ken Williams
• Grant application was supported by 31 key
  personnel from Yale, Rutgers, Rockefeller, and
  Wesleyan Universities.
• A new Keck High Performance Computing Resource
  will provide technical programming support
• Yale Academic Media Technology will provide
  systems administration support
• Genomics research supported by the Center will
  include
• Complex disease gene mapping
• Processing large microarray data sets
• Mapping relationships between gene expression
  patterns and cis-regulatory elements

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Additional Information is Available from the
Directors and Managers of Keck Genomics Resources
and the Keck Web Pages
19 Highly Dedicated and Experienced Staff in
these 6 Keck Resources Stand Ready to Bring
State-of-the-Art Genomic Technologies to Bear on
Your Research http//keck.med.yale.edu/