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DNA CHIPS-Technology and Utility
Yanal Alkuddsi Ph.D Student
Dept. of Genetics and Plant BreedingUniversity of Agricultural Sciences
Dharwad, Karnataka, India, 580005
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CONTENT
1.INTRODUCTION
2.MICROARRAYS: MAKING THEM AND USING THEM
3. SEQUENCE DATA BASES FOR MICROARRAY
4. BASIC DATA ANALYSIS
5. APPLICATIONS
6.CONCLUSION
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A- INRODUCTION
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What is a Microarray ?
An arrangement of DNA sequences on a solid support
A surface (nylon, glass, or plastic).Containing hundreds to thousand pixels.Each pixel has copies of a sequence of single stranded DNA(ssDNA).
Each such sequence is called a probeEach microarray contains thousands of genes Able to simultaneously monitor gene expression levelsin all these genes
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B- Making Microarrays
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1. Manufacturing of the microarray2. Experimental design and choice of reference: what to compare
to what?
3. Target preparation (labeling) and hybridization
4. Image acquisition (scanning) and quantification (signal
intensity to numbers)
5. Database building, filtering and normalization
6. Statistical analysis and data mining
The 6 steps of a DNA microarray experiment
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Greatly reduced cross-hybridization
Uniform Tm Requires knowledge of gene
sequences
Cross-hybridization possible Non-uniform Tm No gene sequence knowledge
required
Oligonucleotide(Affymetrix)
cDNA(complete sequences )
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Different Types of DNA MicroarraysTwo types: Affymetrix or cDNA glass slide arrays
Long Sequences
Spot Unknown Sequences More variability Arrays cheaper
Short SequencesSpot Known SequencesMore reliable data Arrays typ ically more expensive How DNA sequences are laid down;
Robotic spotting How DNA sequences are laid down; In Situ synthesis
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DNA probes attachment chemistry
Covalent reaction5aliphatic amine group of
probe + chemical linkers on glass
Non Covalent reaction
phosphate group + chemicallinkers on glass
Microarrays Types cont
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Surface chemistry
DNA probes Covalent Non cove lent
Oligonucleotides
Yes No
cDNA Yes Yes
DNA probes attachment chemistry
Most oligonucleotides are smaller than cDNA4/7/2010 14DNA Chips
Microarrays Types cont
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Firstly developed at Stanford University.
A) Robotic Spotting
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1- Manufacturing of the microarray
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384 wellplateContains cDNAprobes
Glass Slide Array of bound cDNA probes
4x4 blocks = 16 print-tip groups
Print-tipgroup 6
cDNA clonesSpotted in duplicate
Print-tipgroup 1
Pins collect cDNAfrom wells
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Microarray ofthousands of
genes on aglass slide
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One spot on a microarray contains many DNA strandsof the same sequence
One spot = one gene
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B) In Situ synthesis oligonucleotide arrays
Oligonucleotides are built up base by base on the surface ofthe array by two methods:
Affymetrix
Microarrays Types cont
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Ink Jet Printing
1. Affymetrix Microarrays
This technique consist of the followingproperties:
One chip per sample
One for control One for each experimentEach probe 25 bp long22-40 probes per genePerfect Match (PM) as well as Mis
Match (MM) probes are present
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Affymetrix photolitography
Lipshutz et al ., 1999
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Ink Jet Printing
Four cartridges are loaded with the fournucleotides: A, G, C,T
As the printer head moves across the array,the nucleotides are deposited in pixels wherethey are needed.
This way (many copies of) a 20-60 base longoligo is deposited in each pixel.
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A GTC
Ink Jet Printing (Agilent)
The array is a stack ofimages in the colorsA, C, G, T.
Entirely controlled by thecomputer
High spot quality
98% of coupling efficiency
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1.Sample preparation and labeling
2.Hybridisation3.Washing
4.Image acquisition
C- Using Microarrays
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1.Preparation of Samples
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Use oligo(dT) on a separationcolumn to extract mRNA from totalcell populations.
Use oligo(dT) initiated polymeraseto reverse transcribe RNA intofluorescence labeled cDNA. RNA isunstable because of environmentRNA-digesting enzymes.
Alternatively use random priming
for this purpose, generating apopulation of transcriptsubsequences
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Labeling the samples
Affymetrix arrays
Prepare biotin labeled cRNA for hybridizing the chip
Protocol is prepared by affymetrix, so every affymetrix
lab perform same steps.It make more easier to compare the results
Other arrays
Fluorescently labeled cDNA is used.
Cy3 dye excited by green laser
Cy5 dye excited by red laser
Two samples labelled with different dye.
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2-Color System...
RNA from Normal Tissue RNA from Cancer or Drug Treated Tissue
dCTP dCTP
Reverse Transcription
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http://bioinformatics.picr.man.ac.uk/mbcf/overview_ma.jsp
Labeled DNA copies of all mRNA from one sample (i.e. from a tumor) is hybridized to array
Dye
x
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LABEL
3XSSC
HYB CHAMBER
ARRAY
SLIDELIFTER SLIP
SLIDE LABEL
Temperature 45-65 Oc Time 12-24 hr Formamide (Lowers the Tm) Na+- improve stringency
Rept.DNA - block cross.hyb
Hybridization chamber
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2.Hybridization
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The Key to Nucleic Acid Detection is Sequence-Specific Affinity
C
A
G
T
A
A
C
G
G
TT
5
3
G
T
C
A
T
T
G
C
C
A A
5
3
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PROBE is DNA spotted (attached) to the solidsubstrate (non-fluorescent glass slide).
TARGET is the fluorescence labeledcDNA representation of the mRNA and
is hybridized to the probe.
Microarray-Based Assays
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4. Image acquisition ( Scanning )
Detector
Image
Duplicate spots
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Sample 1 -red dye
Sample2 green dye
Spot Geneexpression
Red Sample 1
Green Sample2
Yellow BothBlack No
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Coding scheme:Green = repressed (less mRNA) gene
in experimentRed = induced (more mRNA) gene in
experimentBlack = no change (1:1 ratio)
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Total process
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Data Bases
Primary gene sequence databases (Gene Bank, EMBL,DDGJ) holds all published sequences and are basis forall other data bases
Secondary gene sequence databases (Uni Gene, TIGR,RefSeq) are excellent resources for designingMicroarrays
Genomic databases (TIGR, SGD ) are excellent
resources for designing arrays for small organisms andcan also be used for more complex organisms
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Designing of oligonucleotide probes
An oligonucleotide probe is short piece of singlestranded DNA complimentary to the target gene whoseexpression is measured on the microarray by that probe.
Usually probes for microarray are designed withinseveral hundred bases of 3 end of target sequences.
Good oligonucleotide properties:
1. Sensitive
2. Specific
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1.Sensitive probe
Is one that returns strong signal. When the complimentarysequence present on the target
The probes dont have internal secondary structure
Example:
Designing probes for gene Homo sapience alcohol dehydrogenase beta 2 sub unit (ADH2)
These probes have low complexity sequences i.e. repetitivesequences
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2.Specific probe Is one that returns weak signals when
complimentary sequence of target is absent.
It doesn't cross hybridise to other targets
Prediction of cross hybridisation to related genesby Homology search algorithms.
BLAST FASTA
Smith-Wterman algorithm4/7/2010 48DNA Chips
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3.Isothermal probe
Probes behave similarly under the hybridisationconditions of microarray experimenttemperature, salt concentration and formamide
concentration Base staking model is used in determining the
melting temperature. It consider both basecomposition and order of bases in the sequence
Mfoldsoftware
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E i t l E
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Poor quality/insufficient mRNA
Reverse transcription bias
Fluorescent labeling bias
Sample contamination
Hybridization bias
Cross-linking of DNA (double strands )
Defective chips (scratches, degradation)
Poor probe design (cross-hybridization)
Background from non-specific hybridization
Experimental Error
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Examples of spot imperfections. A. donut shape; B. oval or pear shape; C. holey heterogeneous interior; D. high-intensity artifact;
E. sickle shape; F. scratches.4/7/2010 53DNA Chips
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Image Analysis
1. Gridding: identify spots(automatic, semiautomatic,manual)
2. Segmentation: separate spots
from background. (A), Fixedcircle (B), Adaptive circle(C), Adaptive shape (D),Histogram
3. Intensity extraction: mean ormedian of pixels in spot
4. Background correction:local or global
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Normalization
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Reject AcceptTrue Type I error
False Type II error
Null hypothesis
Analysis of differentially expressed genes
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Example1-
Samples are taken from 20 breast cancer patients, before and after 16 weekcourse of doxorubicin chemotherapy, and analyzed using microarrays. To wishto identify gene (acetyl-coenzyme Aacetyltransferase 2 ( ACAT2 )) that are up ordown regulated in breast cancer following that treatment
tcal = 3.22
t tab (at 19 df) = 2.39significant cal >tabConclude that this gene hasbeen sig. down regulatedfollowing chemotherapy atthe 1%lvel .
Analysis of differentially expressed genes
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A l i f diff ti ll d
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Example 2-
Bone marrow samples are taken from 27 patients suffering from acute
lymphoblastic leukemia(ALL) and 11 patients suffering from acute myeloidleukemia (AML) and analysed using Affymetrix arrays. To wish to identifythe gene ( Metallothionein IB ) that are up or down regulated in ALL relativeto AML.
tcal = 4.35
t tab (at 36 df) = 2.445significant cal >tabConclude that this gene hasbeen sig. high expression inAML than ALL at the1%lvel .
Analysis of differentially expressed genes
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Hierarchical Clustering
ExampleSamples were taken from 39 patients suffering from
diffuse large B cell lymphomas
Which genes are co regulated in this disease?
Whether the groups of patients with similar geneexpression?Correlation data
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Hierarchical Clustering
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Hierarchical Clustering
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Hierarchical Clustering
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Single Linkage Shortest link between two clusters
Complete Linkage Longest link between two clusters
Average Linkage Average of distances between all
pairs of objects
Average Group Linkage Groups once formed are
represented by their mean values,and then those are averaged
http:// www.resample.com/xlminer/help/HClst/HClst_intro.htm
Hierarchical ClusteringLinkage analysis
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Biological question
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Differentially expressed genesSample class prediction etc.
Testing
Biological verification
and interpretation
Microarray experiment
Estimation
Experimental design
Image analysis
Normalization
Clustering DiscriminationR, G
16-bit TIFF files
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Applications
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Differing expression of genes over time, betweentissues, and disease states
Identification of complex genetic diseases
Drug discovery and toxicology studies
Mutation/polymorphism detection
Pathogen analysis
What problems can it solve?
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Applications
Example:1
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pmicroarrays for disease diagnosis: Microarrays may help guidedoctors in determining effective breast cancer therapy
Current methods including pathologyexam (loooking at cells) and molecularmarkers (examining few, specific genes)only give hints.
Microarrays of human genome used to:detect patterns of genetic activity in atumorTest for chance of developing metastases(cancer that spreads)
Example:2microarrays to study biological processes
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microarrays to study biological processesMicroarrays may help discover ways to prevent or stop the spread ofSARS
SARS chip:New microarray wi th entire genome of SARS virusContains all 29,700 base pairs of the SARS virusCould help detect di fferences in particular strains
of the virus and study the virus evolution overtime.Further studies may determine better ways tocontain the spread of SARS.
U.S. National Inst . of Allergy and InfectiousDiseases (NIAID), Affymetrix, Inc, throughPathogen Functional Genomics Resource Center(TIGR).
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References
Basic microarray analysis: grouping and feature reduction by SoumyaRaychaudhuri, Patrick D. Sutphin, Jeffery T. Chang and Russ B. Altman;Trends in Biotechnology Vol. 19 No. 5 May 2001
Self Organizing Maps, Tom Germano,http://davis.wpi.edu/~matt/courses/soms
Data Analysis Tools for DNA Microarrays by Sorin Draghici; Chapman& Hall/CRC 2003
Self-Organizing-Feature-Maps versus Statistical Clustering Methods: ABenchmark by A. Ultsh, C. Vetter; FG Neuroinformatik & KunstlicheIntelligenz Research Report 0994
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