dna chips microarray

8/13/2019 Dna Chips Microarray

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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 )


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



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Firstly developed at Stanford University.

A) Robotic Spotting


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



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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4/7/2010 DNA Chips 22

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


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


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


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

3. Isothermal4/7/2010 45DNA Chips


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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


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 .



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 .




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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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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


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


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?


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

dna chips microarray

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