Introduction of DNA microarrays

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Introduction of DNA microarrays
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• cDNA clone
• Plasmids
• PCR products
• Oligos

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How microarrays are made spotted microarrays

• DNA mechanically placed on glass slide
• Need to deliver nanoliter to picoliter volumes
• Too small for normal pipetting devices
• Robot prints, or spots, DNA in specific places

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Robotic printing or in situ synthesis

• Contact printing
• Non-contact printing
• In situ synthesis --- Affymetrix

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DNA spotting I

• DNA spotting usually uses multiple pins
• DNA in microtiter plate
• DNA usually PCR amplified
• Oligonucleotides can also be spotted

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Commercial DNA spotter
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How microarrays are madeAffymetrix GeneChips

• Oligonucleotides synthesized on silicon chip
• One base at a time
• Uses process of photolithography
• Developed for printing computer circuits

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Affymetrix GeneChips

• Oligonucleotides
• Usually 2025 bases in length
• 1020 different oligonucleotides for each gene
• Oligonucleotides for each gene selected by
  computer program to be the following
• Unique in genome
• Nonoverlapping
• Composition based on design rules
• Empirically derived

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Photolithography

• Light-activated chemical reaction
• For addition of bases to growing oligonucleotide
• Custom masks
• Prevent light from reaching spots where bases not
  wanted
• Mirrors also used
• NimbleGen uses this approach

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Example building oligonucleotides by
photolithography

• Want to add nucleotide G
• Mask all other spots on chip
• Light shines only where addition of G is desired
• G added and reacts
• Now G is on subset of oligonucleotides

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Example adding a second base

• Want to add T
• New mask covers spots where T not wanted
• Light shines on mask
• T added
• Continue for all four bases
• Need 80 masks for total
• 20-mer oligonucleotide

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Ink-jet printer microarrays

• Ink-jet printhead draws up DNA
• Printhead moves to specific location on solid
  support
• DNA ejected through small hole
• Used to spot DNA or synthesize oligonucleotides
  directly on glass slide
• Use pioneered by Agilent Technologies, Inc.

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Comparisons of microarrays
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Microarray hybridization

• Usually comparative
• Ratio between two samples
• Examples
• Tumor vs. normal tissue
• Drug treatment vs. no treatment
• Embryo vs. adult

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Comparison of microarray hybridization

• Spotted microarrays
• Competitive hybridization
• Two labeled cDNAs hybridized to same slide
• Affymetrix GeneChips
• One labeled RNA population per chip
• Comparison made between hybridization intensities
  of same oligonucleotides on different chips

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Target labeling fluorescent cDNA

• cDNA made using reverse transcriptase
• Fluorescently labeled nucleotides added
• Labeled nucleotides incorporated into cDNA

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Target labeling cRNA biotin

• cDNA made with reverse transcriptase
• Linker added with T7 RNA polymerase recognition
  site
• T7 polymerase added and biotin labeled RNA bases
• Biotin label incorporated into cRNA

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Labels

• Cy3 and Cy5
• Fluoresce at different wavelengths
• Used for competitive hybridization
• Biotin
• Binds to fluorescently labeled avidin
• Used with Affymetrix GeneChips

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Spotted-microarray hybridization

• Control and experimental cDNA labeled
• One sample labeled with Cy3
• Other sample labeled with Cy5
• Both samples hybridized together to microarray
• Relative intensity determined using confocal
  laser scanner

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Scanning of microarrays
laser

• Confocal laser scanning microscopy
• Laser beam excites each spot of DNA
• Amount of fluorescence detected
• Different lasers used for different wavelengths
• Cy3
• Cy5

detection
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Analysis of hybridization

• Results given as ratios
• Images use colors
• Cy3 Green
• Cy5 red
• Yellow
• Yellow is equal intensity or no change in
  expression

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Example of spotted microarray

• RNA from irradiated cells (red)
• Compare with untreated cells (green)
• Most genes have little change (yellow)
• Gene CDKN1A red increase in expression
• Gene Myc green decrease in expression

CDKNIA
MYC
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Analysis of cell-cycle regulation

• Yeast cells stopped at different stages of cell
  cycle
• G1, S, G2, and M
• RNA extracted from each stage
• Control RNA from unsynchronized culture

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Results of cell-cycle analysis

• 800 genes identified whose expression changes
  during cell cycle
• Grouped by peak expression
• M/G1, G1, S, G2, and M
• Four different treatments used to synchronize
  cells
• All gave similar results
• Results from Spellman et al., 1998 Cho et al.,
  1998

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Cell-cycle regulated genes

• Each gene is a line on the longitudinal axis
• Treatments in different panels
• Cell-cycle stages are color coded at top
• Vertical axis groups genes by stage in which
  expression peaks

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Affymetrix GeneChip experiment

• RNA from different types of brain tumors
  extracted
• Extracted RNA hybridized to GeneChips containing
  approximately 6,800 human genes
• Identified gene expression profiles specific to
  each type of tumor

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Profiling tumors

• Image portrays gene expression profiles showing
  differences between different tumors
• Tumors
• MD (medulloblastoma)
• Mglio (malignant glioma)
• Rhab (rhabdoid)
• PNET (primitive neuroectodermal tumor)
• Ncer normal cerebella

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Cancer diagnosis by microarray

• Gene expression differences for medulloblastoma
  correlated with response to chemotherapy
• Those who failed to respond had a different
  profile from survivors
• Can use this approach to determine treatment

60 different samples
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Analysis of microarray results

• Inherent variability need for repetition
• Biological and technical replicates
• Analysis algorithms
• Based on statistical models
• Means of generating hypotheses that need to be
  tested

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Hz-1 Functional Genomics
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Purpose

• 1. Find Hz-1 viral conserve promoter in clustered
  genes during
• productive infection.
• 2. Analysis of Hz-1 viral gene expression
  profiles during persistent
• infection.

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• 1. Template 20 ng total DNA (Viral TN368
  cell)
• primer pairs 155 specific primers
• PCR program 94? 5 mins, 53 ? 30 s, 72 ?
  30 s (35cycles)
• 2. Re-amplify failure PCR and control gene with
  appropriate Tm value.

PCR products
Cleanup procedure
PCR products
100 bp
Final concentration 200-250 ng/ul
Control gene
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CMT-GAPSII coated slide
(Gamma Amino Propyl silane)
Subarray II
Subarray I
Repeat region A
Repeat region A
Repeat region B
Repeat region B
Repeat region A
The same target DNA
Repeat region B
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Subarray II
Subarray I
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7 spot
ORF
6 spot
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Raw data
Detect image
GenePix Pro 4.1.1.31
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Microarray analysis for scent

• Chip prepare
• Probe prepare
• Scent and scentless
• Different stage in P. violacea
• Day and night
• Hybridization
• Slide screen and data analysis

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??cDNA?????????????????
T7 primer
T3 primer
2000 ???????cDNA
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Microarray analysis
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Scent gene analysis
77 genes (22)
270 genes (77)
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thanks