From: Duggan et'al' Nature Genetics 21:1014, 1999

1
Microarray-Based Assays (The Basics)
Each feature or spot represents a specific
expressed gene (mRNA).
The fluorescent intensity of each feature
correlates with the expression level of the gene
(mRNA) in the samples under investigation.
From Duggan et.al. Nature Genetics 2110-14, 1999
2
Microarray-Based Assays (The Basics)
The Key to Nucleic Acid Detection is
Sequence-Specific Affinity
GC content (base paring) generally dictates
thermodynamics of complementary binding. Tm
Melting Temperature
3
Microarray-Based Assays (The Basics)
PROBE is DNA spotted (attached) to the
solid substrate (non-fluorescent glass slide).
4
Microarray Production
384-well plate
5

• Microarray Oligonucleotide Probe Design System
• Experimental Assessment of DNA Binding Behavior
• Design and Implementation

6
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
Microarray Oligonucleotide Probes
Mismatched Base Pairs
1) AGCCCGATGATGAGCGACTCACCACGGGCCACGGCTTCTGACTCTCT
TT 0 2) AGCCCGATGATGAGCGACTCACCAGGGGCCACGGCTTCTGAC
TCTCTTT 1 3) AGCCCGATGATGAGCCACTCACCACGGGCCAGGGCTT
CTGACTCTCTTT 2 4) AGCCCGATGATCAGCGACTCACCTCGGGCCAC
GGCATCTGACTCTCTTT 3 5) AGCCCCATGATCAGCGAGTCACCTCGG
GCCACGCCTTCTGACTCTCTTT 5
Normalized Target/Probe
32?C
42?C
52?C
Microarray Oligonucleotide Probes
Kane, MD et. al. Gene Cloning and Expression
Technologies (2002) Ch. 35537-547
7
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
A.
Yick_337 Yick_884 Yick_GAP Yeio_997 Yeio_469 Yeio
_GAP Yabm_392 Yabm_552 Yabm_GAP
B.
yicK cDNA
yeiO cDNA
yabM cDNA
Kane, MD et. al. Nucleic Acids Res. (2000)
284552-4557
8
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
A.
80 Overall Homology (Cross-hybridization
detected) Yick_337 gcgtacttctgagtagttttgcttccacc
gcaaacccgcaaatgttcgcc
yeiO 335
gcgtctttcttagcagctttggctcgaccgctaacccgcaaatgtttgcc
74 Overall Homology (No cross-hybridization
detected) yicK 471 gttatcgggccaccgctcgcttatgaac
tggcaatgggatttagttttaa
Yeio_469
gtcattggcccaccgctggcttatgccttagcgatgggtttcagctttac
B.
Kane, MD et. al. Nucleic Acids Res. (2000)
284552-4557
9
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
Probe Pair 1 2 3 4 5
Overlap w/ Conserved Region 15 20 25 30 35
DAP
variable region
DAPD
conserved regions
Kane, MD et. al. Nucleic Acids Res. (2000)
284552-4557
10
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
Length of Complementary Base Pairing with DAPD
cDNA
25 30 35 20 15 25 20
15 30 35
DAPD cDNA Signal (Cy3)
DAP cDNA Signal (Cy5)
Kane, MD et. al. Nucleic Acids Res. (2000)
284552-4557
11
Assessment of Sequence-Specific
Cross-Reactivity/Cross-Hybridization
Ratio of Target/Non-Target Cross-Hybridization
120
100
80
Ratio of Target Signal (DAP) to Non-Target Signal
(DAPD)
60
40
20
0
15
20
25
30
35
Length of Continuous Complementary Base
Pairing with Non-Target (DAPD) cDNA
Kane, MD et. al. Nucleic Acids Res. (2000)
284552-4557
12

• Microarray Oligonucleotide Probe Design System
• Experimental Assessment of DNA Binding Behavior
• Design and Implementation

13
Microarray Oligonucleotide Probe Design System
Objective Computationally Design Gene-Specific
Microarray Probes
Commercial Objective Produce and Sell
Pre-Printed Oligo-Probe Microarrays
Data Flow
Target Gene List
Sequence QC
Obtain Sequence Info
Genomic Sequence Database
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Microarray Oligonucleotide Probe Design System
Target Gene List
Probe Design
Obtain Sequence Info
Sequence QC
Genomic Sequence Database
Oligonucleotide Probe Length 46-54 bases
AGAAGAAGCCGATGATGACGAGGACGATGAGGATGGTGATGAGGTAGAGG
AAGAGGCTGAGGAACCCTAC
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Microarray Oligonucleotide Probe Design System

• Accept if GC content range 40-60
• Reject 4 or more contiguous bases of G or C
  (2ndary Structure)
• Reject palindromic-hairpin sequences greater than
  7 bases (2ndary Structure)
• Reject known splice site sequences
• Accepted if Calculated Tm 52.4 ?C 4.0 ?C
• No more than 75 homology within the target
  region of non-target genes.
• No more than 14 contiguous base pairs with
  non-target genes.

Target Gene List
Primary Probe Evaluation
Obtain Sequence Info
Sequence QC
Probe Design
Genomic Sequence Database
16
Microarray Oligonucleotide Probe Design System

• Accept if GC content range 40-60
• Reject 4 or more contiguous bases of G or C
  (2ndary Structure)
• Reject palindromic-hairpin sequences greater than
  7 bases (2ndary Structure)
• Reject known splice site sequences
• Accept if Calculated Tm 52.4 ?C 4.0 ?C
• No more than 75 homology within the target
  region of non-target genes.
• No more than 14 contiguous base pairs with
  non-target genes.

Target Gene List
Basic Local Alignment Search Tool (BLAST) against
published sequences in target species. Parse
BLAST Output Accept/Reject Candidate Probe
Secondary Probe Evaluation
Obtain Sequence Info
Sequence QC
Probe Design
Primary Probe Eval
Genomic Sequence Database
17
Microarray Oligonucleotide Probe Design System
Obtain Sequence Info
Target Gene List
Genomic Sequence Database
GC Rich/Poor 2ndary Structure Known Splice Site
Primary Probe Evaluation
Sequence QC
Probe Design
Secondary Probe Evaluation
High Quality mRNA
DNA Sequences Redundant Entries Wrong Species
Rank Acceptable Probes
Cross-Hybridization Potential
Optimal Probes Nearest to Desired Tm Probe
Annotated GInumber_start_length
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Microarray Oligonucleotide Probe Design System
Target Gene List
Sequence QC
Probe Design
Primary Probe Evaluation
Secondary Probe Evaluation
Rank Acceptable Probes
Obtain Sequence Info
Genomic Sequence Database
SYSTEM PERFORMANCE X ? 50 genes/hour generating
2 probes for each target gene (mRNA).
Dedicated PC 1 ghz 1 gig RAM
Desk Top PC 500 mhz 512 meg RAM
File-Based Communication
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Synthesis Purification
Oligonucleotide Probes
Design
Number of Spots Spot Size Replicates
Solid Substrate (slide) Functional Surface
Chemistry
DNA Probes
MICROARRAY SPOTTER
cDNA (PCR) Probes
Amplification Purification
Clones
Record File (.gal file)
DNA Microarrays
Processing Chemistry
Control Sample
RNA Isolation
RT (Fluorescence) Labeling
Storage Delivery
Sample
Test Sample
Pre-Hybridization Chemistry
HYBRIDIZATION
Time, Temp, Buffers, Wash
STUDY DESIGN and OBJECTIVES
IMAGING
Resolution, Region of Interest
Raw Data Image (.tif file)
Raw Data Analysis
Spot Finding, Background Subtraction, Signal
Intensity, Normalization
Gene Specific Data
Biological/Bioinformatics Analysis