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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 21:10-14, 1999
Microarray-Based Assays (The Basics)
The Key to Nucleic Acid Detection is “Sequence-Specific Affinity”
5’ 3’
G
T
C
A
T
T
G
C
C
A
A
C
A
G
T
A
A
C
G
G
T
T
3’ 5’
“GC” content (base paring) generally dictates thermodynamics of complementary binding. Tm = Melting Temperature
Microarray-Based Assays (The Basics)
“TARGET” is the fluorescence labeled
cDNA representation of the mRNA and
is hybridized to the probe.
“PROBE” is DNA spotted (attached) to the solid
substrate (non-fluorescent glass slide).
Microarray Production
384-well plate
Biological
Samples
Microarray Hybridization
Microarray Imaging
Microarray Raw Data Analysis
Microarray Statistical Data Analysis
Microarray Oligonucleotide Probe Design System
• Experimental Assessment of DNA Binding Behavior
• Design and Implementation
Assessment of Sequence-Specific Cross-Reactivity/Cross-Hybridization
Microarray Oligonucleotide Probes
Mismatched Base Pairs
1) AGCCCGATGATGAGCGACTCACCACGGGCCACGGCTTCTGACTCTCTTT
2) AGCCCGATGATGAGCGACTCACCAGGGGCCACGGCTTCTGACTCTCTTT
3) AGCCCGATGATGAGCCACTCACCACGGGCCAGGGCTTCTGACTCTCTTT
4) AGCCCGATGATCAGCGACTCACCTCGGGCCACGGCATCTGACTCTCTTT
5) AGCCCCATGATCAGCGAGTCACCTCGGGCCACGCCTTCTGACTCTCTTT
1.00
Normalized Target/Probe
0.90
0.80
0.70
0.60
0.50
32C
0.40
0.30
0.20
42C
0.10
52C
0.00
1
2
3
4
5
Microarray Oligonucleotide Probes
Kane, MD et. al. Gene Cloning and Expression Technologies (2002) Ch. 35:537-547
0
1
2
3
5
Assessment of Sequence-Specific Cross-Reactivity/Cross-Hybridization
Yabm_392
Yabm_552
Yabm_GAP
Yeio_997
Yeio_469
Yeio_GAP
Oligo Name
Sequence
yick_337
gcgtacttctgagtagttttgcttccaccgcaaacccgcaaatgttcgcc
yick_884
aatgtgtttttacgccagcgtactcatggcgacgactccggcggttgagc
yeio_997
ctgatgcccggtcaggcgggttcagccaccacgctctataccaacacttc
yeio_469
gtcattggcccaccgctggcttatgccttagcgatgggtttcagctttac
yabm_392 aatatgcggataactcggcgcgagaagtggtgatgtttagctcggtgatg
yabm_552
cagtctggtattgattgcatttatgcttccgtctgtggcgcgggtagaac
Yick_337
Yick_884
Yick_GAP
A.
B.
yicK cDNA
yeiO cDNA
yabM cDNA
Kane, MD et. al. Nucleic Acids Res. (2000) 28:4552-4557
yick
64%
74%
54%
30%
% Homology
yeio yabm
80% 56%
60% 40%
64%
68%
66%
36%
Assessment of Sequence-Specific Cross-Reactivity/Cross-Hybridization
14 Nucleotides; 6 (42%) GC
A.
80% Overall Homology (Cross-hybridization detected);
Yick_337 gcgtacttctgagtagttttgcttccaccgcaaacccgcaaatgttcgcc
|||| |||| || || |||| || ||||| |||||||||||||| |||
yeiO 335 gcgtctttcttagcagctttggctcgaccgctaacccgcaaatgtttgcc
74% Overall Homology (No cross-hybridization detected);
B.
yicK 471 gttatcgggccaccgctcgcttatgaactggcaatgggatttagttttaa
|| || || |||||||| |||||||
| || ||||| || || ||||
Yeio_469 gtcattggcccaccgctggcttatgccttagcgatgggtttcagctttac
8 Nucleotides; 6 (75%) GC
Kane, MD et. al. Nucleic Acids Res. (2000) 28:4552-4557
Microarray Oligonucleotide Probe Design System
• Experimental Assessment of DNA Binding Behavior
• Design and Implementation
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
Obtain
Sequence
Info
Genomic
Sequence
Database
Sequence
QC
Remove Redundant Entries
Confirm RNA vs. Genomic DNA
Confirm Species Selection on Each Gene
Identify Longest mRNA
Identify Splice/Isoform Variants (Segments)
Contigs Used to Increase EST Sequence Quality
Microarray Oligonucleotide Probe Design System
Target
Gene
List
Obtain
Sequence
Info
Sequence
QC
Probe
Design
Genomic
Sequence
Database
Oligonucleotide Probe Length = 46-54 bases
AGAAGAAGCCGATGATGACGAGGACGATGAGGATGGTGATGAGGTAGAGGAAGAGGCTGAGGAACCCTAC…
Microarray Oligonucleotide Probe Design System
1)
2)
3)
4)
5)
6)
7)
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
Obtain
Sequence
Info
Genomic
Sequence
Database
Sequence
QC
Probe
Design
Primary
Probe
Evaluation
AGAGCACGCGGAGGAGCGTGCGCGGGGGCCCCGGGAGACGGCGGCGGTGGCGGCGCGGGCAGAGCAAGGA…
82.6% GC (failure)
>4 cont. G or C (failure)
46 bases
AGAAGAAGCCGATGATGACGAGGACGATGAGGATGGTGATGAGGTAGAGGAAGAGGCTGAGGAACCCTAC…
54.3% GC
46 bases
Microarray Oligonucleotide Probe Design System
1)
2)
3)
4)
5)
6)
7)
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
Obtain
Sequence
Info
Sequence
QC
Probe
Design
Primary
Probe
Eval
Secondary
Probe
Evaluation
Basic Local Alignment Search
Tool (BLAST) against published
sequences in target species.
Parse BLAST Output
Genomic
Sequence
Database
Accept/Reject Candidate Probe
Microarray Oligonucleotide Probe Design System
Target
Gene
List
Obtain
Sequence
Info
Sequence
QC
Genomic
Sequence
Database
High
Quality
mRNA
Primary
Probe
Evaluation
Probe
Design
DNA Sequences
Redundant Entries
Wrong Species
Optimal Probes:
Nearest to Desired Tm
Probe Annotated: GInumber_start_length
GC Rich/Poor
2ndary Structure
Known Splice Site
Secondary
Probe
Evaluation
Rank
Acceptable
Probes
Cross-Hybridization Potential
Microarray Oligonucleotide Probe Design System
Target
Gene
List
Obtain
Sequence
Info
Sequence
QC
Probe
Design
Primary
Probe
Evaluation
Secondary
Probe
Evaluation
Rank
Acceptable
Probes
Genomic
Sequence
Database
SYSTEM PERFORMANCE
Desk Top PC
500 mhz
512 meg RAM
File-Based
Communication
Dedicated PC
1 ghz
1 gig RAM
X  50 genes/hour generating 2
probes for each target gene (mRNA).
Oligonucleotide
Probes
Number of Spots
Spot Size
Replicates
Solid Substrate (slide)
Functional Surface Chemistry
Synthesis
Purification
Design
DNA Probes
MICROARRAY SPOTTER
Record File
(.gal file)
cDNA (PCR)
Probes
Amplification
Purification
Clones
DNA Microarrays
Processing Chemistry
Control Sample
Storage
Delivery
Sample
RT (Fluorescence)
Labeling
RNA
Isolation
Test Sample
Pre-Hybridization Chemistry
HYBRIDIZATION
Time, Temp, Buffers, Wash
IMAGING
STUDY DESIGN
and OBJECTIVES
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