Introduction to Gene Chips andIntroduction to Gene Chips andMicroarray Expression DataMicroarray Expression Data
Dr. Travis Doom, Assistant Professor
BIRG LabDepartment of Computer Science and Engineering
Wright State University
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OutlineOutline
DNA Microarrays– Fabrication
– Application Microarray Data
– Analysis Techniques New Technology &
Open Commentary
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Fabrication via Printing DNA sequence stuck
to glass substrate DNA solution pre-
synthesized in the lab Fabrication In Situ
Sequence “built” Photolithographic
techniques use light to release capping chemicals
365 nm light allows 20-m resolution
FabricationFabrication
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DNA MicroarraysDNA Microarrays Each probe consists of thousands of strands of identical
oglionucleotides– The DNA sequences at each probe represent important
genes (or parts of genes) Printing Systems
– Ex: HP, Corning Inc.– Printing systems can build lengths of DNA up to 60
nucleotides long– 1.28 x 1.28+ cm glass wafer
• Each “print head” has a ~100 m diameter and are separated by ~100 m. ( 5,000 – 20,000 probes)
Photolithographic Chips– Ex: Affymetix – 1.28 x 1.28 cm glass/silicon wafer
• 24 x 24 m probe site ( 500,000 probes)
– Lengths of DNA up to 25 nucleotides long– Requires a new set of masks for each new array type
GeneChip
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Practical Application of DNA MicroarraysPractical Application of DNA Microarrays
DNA Microarrays are used to study gene activity (expression)– What proteins are being actively produced by a group of cells?
• “Which genes are being expressed?”
How?– When a cell is making a protein, it translates the genes (made of DNA)
which code for the protein into RNA used in its production– The RNA present in a cell can be extracted– If a gene has been expressed in a cell
• RNA will bind to “a copy of itself” on the array• RNA with no complementary site will wash off the array
– The RNA can be “tagged” with a fluorescent dye to determine its presence
DNA microarrays provide a high throughput technique for quantifying the presence of specific RNA sequences
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The ProcessThe Process
CellsPoly-ARNA
AAAA
cDNA
L L L
L
IVT
10% Biotin-labeled UracilAntisense cRNA
L
Fragment (heat, Mg2+)
Labeledfragments
Hybridize Wash/stain Scan
L
(In-vitro Transcription)
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Hybridization and StainingHybridization and Staining
LL
GeneChip BiotinLabeled cRNA
+L
L
L
L
L
L
L
L
L
L+
SAPEStreptavidin-phycoerythrin
Hybridized Array
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The ResultThe ResultA light source scans the array, causing the dyes to fluoresce
The glow is picked up by a sensor and is used to determine the relative abundance of the RNA
This information must be processed to determine the level of activity for each expressed gene
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The GoalsThe Goals Basic Understanding
– Arrays can take a snap shot of which subset of genes in a cell is actively making proteins
– Heat shock experiments Medical diagnosis
– Microarrays can indicate where mutations lie that might be linked to a disease. Still others are used to determine if a person’s genetic profile would make him or her more or less susceptible to drug side effects
– 1999 – A genechip containing 6800 human genes was used distinguish between myeloid leukemia and lympholastic leukemia using a set of 50 genes that have different activity levels
Drug design– Pharmaceutical firms are in a rush to translate the human genome results into
new products• Potential profits are huge• First, though, they must figure out what the genes do, how they interact, and how
they relate to diseases.
– Evaluation, Specificity, Response
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The GainsThe Gains
A decade of rapid advances in biology has swept an avalanche of genetic information into scientist’s laps.
Mass analysis of the vast set of biologic data is impractical without high-throughput techniques
DNA microarrays (aka Gene chips, biochips) allow researchers to look for the presence, productivity, or sequence of thousand of genes simultaneously
Advantages: – Speed
– Feasibility
– Sensitivity
– Reproducibility
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OutlineOutline
DNA Microarrays– Fabrication
– Application Microarray Data
– Analysis Techniques New Technology &
Open Commentary
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Microarray DataMicroarray Data
First, the Problems:1. The fabrication process is not
error free2. Probes have a maximum
length 25-60 nucleotides3. Biologic processes such as
hybridization are stochastic4. Background light may skew
the fluorescence 5. How do we decide if/how
strongly a particular gene is being expressed?
Solutions to these problems are still in their infancy
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FeaturesFeatures
Problem #1: The fabrication process is not error free
Solution: Each probe does not represent a unique DNA sequence.
Probe set: A set of probes each containing the same DNA sequence (the Feature)
Remove outermost rows and columns to avoid fabrication-based error
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Feature ValueFeature Value
83 112 96 32
47 382 165 87
55 246 140 93
104 552 187 65
Remove outermost rows and columns
Find 75th percentile of remaining values
This value is taken as representative of this feature
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How Features Are ChosenHow Features Are Chosen
Multipleoligo probes
25-mers
Features
5’ 3’Gene Sequence
Problem #2: Probes have a maximum length 25-60 nucleotides:– Solution: Use multiple features per gene
– Affymetrix claims that this redundancy actually improves detection and quantification of the target gene
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Feature MismatchesFeature Mismatches
Multipleoligo probes
25-mers
Perfect MatchMismatch
5’ 3’Gene Sequence
Problem #3: Biologic processes such as hybridization are stochastic– Solution: Include a “control” for each probe – a DNA sequence which differs
only slightly from the feature
– In a 25-mer, the mismatch sequence differs in the 13th position (A-T or G-C)
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Background Noise RemovalBackground Noise Removal
Problem #4: Background light may skew the fluorescence “Measure of non-specific fluorescence attributed to hybridization
conditions and sample” = Noise Solution: Estimate background noise and subtract intensity
The array is divided into equal sectors (16 is standard) For each sector
– Find the lowest feature intensities (2%)
– Average these
– Subtract this average from the intensity value of all features in the sector
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Average Difference IntensityAverage Difference Intensity
Problem #5: How do we decide if / how strongly a particular gene is being expressed?
For a given gene– For each feature match/mismatch pair for the given gene
• Calculate the difference PM-MM
– Calculate , for this set
– Remove outliers from set• Ex: abs( (PM – MM) - ) 3
– The average (PM – MM) difference over the set (minus outliers) is the average difference intensity
– This value can be used to compare expression levels for the gene which the features represent
avgin pairsavgin pairs#
1
iii MMPMAvgDiff
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Positive & Negative Probe PairsPositive & Negative Probe Pairs
If both true, mark probe pair as positive evidence
If both true, mark as probe pair as negative evidence
PM/MM SRT
PM-MM SDT
MM/PM SRT MM-PM SDT
Problem #5: How do we decide if / how strongly a particular gene is being expressed?
For each perfect match/mismatch probe pair in the feature, perform a standard difference and ratio test
Example SRT and SDT thresholds:– SRT 1.5– SDT a multiple of intensity or
Otherwise, mark probe pair as inconclusive
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Voting Methods for Absolute CallVoting Methods for Absolute Call Problem #5: How do we decide if / how strongly a particular gene is
being expressed?– Solution: Use decision matrix to make absolute call
Positive/negative ratio PNR = # pos. calls / # neg. calls Positive fraction PF = # pos. calls / # probe pairs Log average ratio LA = 10 x avg. ( log (PM/MM) )
Absent Marginal Present
PNR 3.00 4.00
PF 0.33 0.43
LA 0.90 1.30
VOTE!
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Average Difference and Absolute CallAverage Difference and Absolute Call Problem #5: How do we decide if / how strongly a particular gene is
being expressed? Which of these do you base a decision on, for whether a gene is being
expressed? Use the absolute call for decision if a particular gene is being expressed
Use average difference to compare how strongly a gene which is present is expressed
avgin pairsavgin pairs#
1
iii MMPMAvgDiff
Absent Marginal Present
PNR 3.00 4.00
PF 0.33 0.43
LA 0.90 1.30
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Comparison AnalysisComparison Analysis Compare probe sets between two gene chips to determine whether gene
expression increased, did not change or decreased Comparison analysis has its own set of problems:
– The signals must be adjusted (if necessary) to normalize average signal levels
For each perfect match/mismatch probe pair in the feature, perform a difference and ratio test
If both true, mark probe pair as evidence of increase from base
– PM/MMexperiment – PM/MMbase Change Threshold
– (PM-MM)experiment /(PM-MM)base Percentage Change Threshold
If both true, mark probe pair as evidence of decrease from base
– PM/MMbase - PM/MMexperiment Change Threshold
– (PM-MM)base / (PM-MM)experiment Percentage Change Threshold
Otherwise mark probe pair as unchanged
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Voting Methods for Comparison CallVoting Methods for Comparison Call Increase fraction IR = # increase calls / # PP used Increase ratio DR = # increase calls / # decrease
calls Log average ratio change LAC = LAexp – Labase
If a change is called, use the average difference to measure percent change Are there better ways to extract patterns from multivariate gene
expression profiles?
No Change Marginal Increase
IF .33 .43
IR 3.0 4.0
LAC 0.90 1.30
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OutlineOutline
DNA Microarrays– Fabrication
– Application Microarray Data
– Analysis Techniques New Technology &
Open Commentary
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Does Moore’s Law apply to Gene Chips?Does Moore’s Law apply to Gene Chips? Ideally, we
would like to fit all of an organism’s genes on one chip– Current
estimates for Humans are between 30,000 – 40,000 genes
Cost
0
0.2
0.4
0.6
0.8
1
1.2
1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Years
Do
llars
/ge
ne
Density
0
50000
100000
150000
200000
1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004
Year
Ge
ne
s/c
hip
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Field-Programmable Microarrays?Field-Programmable Microarrays?
Nanogen has produced a silicon chip embedded with 100 “programmable” probe pads– 80m platinum pads (each spaced about 200um apart)
– Each pad can have apply a voltage (-1.3 to 2.0 V) Since DNA carries a negative charge, applying a positive charge on a
pad “corrals” DNA onto that spot– This is used to build custom arrays by washing the chip in a single stranded
DNA solution, biasing the desired spot on the chip, and then chemically fixing the DNA to that spot
The electric charge is also useful during the hybridization reaction– Pooling the DNA onto the charged pads increases the reaction by a factor of
1000
– Reversing the charge “shakes loose” imperfectly matched DNA leading to more accurate results
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From the Rumor-MillFrom the Rumor-Mill
Xeotron Corp: Maskless lithography– An array of micro mirrors are used to direct/block light during fabrication
Motorola: 3D microarrays– Arrays with a coating of acrylimide gel to allow “certain enzymatic
reactions” to occur that might be important to lab-on-a-chip applications Motorola: Electrical intensity measures
– Arrays contain embedded circuitry to detect hybridization through a change in conductance rather than fluorescence
Ciphergen Biosystems Inc. & Packard Instrument Co.: Protein chips– Creates microarrays of antibodies (rather than DNA) to bind and identify
proteins
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AcknowledgementsAcknowledgements
David Paoletti, Ph.D. Student, BIRG Lab, Wright State University. Berberich, S, and McGorry, M; GeneChip protocols, Wright State
University. Moore, S K; Making chips to probe genes, IEEE Spectrum, March
2001, 54-60. GeneChip Gene Expression Algorithm Training, Affymetrix.
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Questions ?Questions ?
DNA Microarrays– Fabrication
– Application Microarray Data
– Analysis Techniques New Technology &
Open Commentary
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The EndThe End
DNA Microarrays– Fabrication
– Application Microarray Data
– Analysis Techniques New Technology &
Open Commentary