DNA CHIPSTechnology and Utility

Yanal Alkuddsi Ph.D Student Dept. of Genetics and Plant Breeding University of Agricultural Sciences Dharwad, Karnataka, India, 580005

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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Analysis of gene expression at the single gene level.

Methods for measuring a single gene: Northern Blots
Measure RNA levels by hybridization of a labeled probe to total RNA.

Reporter Genes
Use of an enzyme to measure the amount of transcription from a promoter.

Quantitative real-time RT-PCR.

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Assaying the regulation of thousands of genes in a single experiment

DNA microarrays
DNA molecules printed at high density used to determine the level of RNA or DNA in a sample. Can be thought of a reverse Northern blots

Other technologies
SAGE Microbeads
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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 probe Each microarray contains thousands of genes Able to simultaneously monitor gene expression levels in all these genes

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Definitions:
Target - the nucleic acid (cDNA) sample whos identity and quantity are being measured. Probe an attached nucleic acid with a known sequence (the DNA chip). Fluorophore usually green and red labels attached to the target to enable visualizing expression. Array a set of DNA reagents for measuring the amount of sequence counterparts among the mRNA of the sample.
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B- Making Microarrays

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The 6 steps of a DNA microarray experiment

1. Manufacturing of the microarray 2. 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
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Workflow

Biological sample of some sort

Extract mRNA

Amplify

Label and Fragment

Analyze down to one number per gene 4/7/2010

Find features in scan Scan chip DNA Chips Hybridise to a chip 11

Different Types of DNA Microarrays

Two types: Affymetrix or cDNA glass slide arrays

Oligonucleotide
(Affymetrix) Greatly reduced crosshybridization Uniform Tm Requires knowledge of gene sequences

cDNA(complete sequences)
Cross-hybridization possible Non-uniform Tm No gene sequence knowledge required
Long Sequences Spot Unknown Sequences More variability Arrays cheaper

Short Sequences Spot Known Sequences More reliable data Arrays typically more expensive

How DNA sequences are laid down;

In 4/7/2010 Situ synthesis

How DNA sequences are laid down; Robotic spotting

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Microarrays Types cont

DNA probes attachment chemistry

Covalent reaction 5aliphatic amine group of probe + chemical linkers on glass Non Covalent reaction phosphate group + chemical linkers on glass

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Microarrays Types cont

DNA probes attachment chemistry Surface chemistry DNA probes Oligonucleot ides cDNA Covalent Yes Yes Non cove lent No Yes

Most oligonucleotides are smaller than cDNA

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http://www.gene-chips.com/GeneChips.html#What DNA Chips 15

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1- Manufacturing of the microarray

A) Robotic Spotting

Firstly developed at Stanford University.

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Pins collect cDNA from wells

384 well plate

Print-tip group 1 Contains cDNA probes

Glass Slide
Array of bound cDNA probes 4x4 blocks = 16 print-tip groups

cDNA clones
Spotted in duplicate

Print-tip group 6
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Microarray of thousands of genes on a glass slide

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One spot on a microarray contains many DNA strands of the same sequence

One spot = one gene

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http://bioinformatics.picr.man.ac.uk/mbcf/overview_ma.jsp

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Microarrays Types cont

B) In Situ synthesis oligonucleotide arrays Oligonucleotides are built up base by base on the surface of the array by two methods:
Affymetrix Ink Jet Printing 1. Affymetrix Microarrays
This technique consist of the following properties: One chip per sample One for control One for each experiment Each probe 25 bp long 22-40 probes per gene Perfect Match (PM) as well as Mis Match (MM) probes are present 4/7/2010 DNA Chips

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Affymetrix photolitography

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Lipshutz et al., 1999 22

Why do we have mismatch probes?

Mismatch probes (MM) are trying to detect background. The mismatch probes are supposed to detect things that are close but not an exact match. It is assumed that these things also bind to the perfect match (PM), erroneously.

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Lipshutz et al., 1999

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2. Inkjet Printed Microarrays

Inkjet head
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Ink Jet Printing

Four cartridges are loaded with the four nucleotides: A, G, C,T As the printer head moves across the array, the nucleotides are deposited in pixels where they are needed. This way (many copies of) a 20-60 base long oligo is deposited in each pixel.

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Ink Jet Printing (Agilent)

The array is a stack of images in the colors A, C, G, T. Entirely controlled by the computer High spot quality 98% of coupling efficiency

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C- Using Microarrays
1.Sample preparation and labeling 2.Hybridisation 3.Washing 4.Image acquisition

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1.Preparation of Samples
Use oligo(dT) on a separation column to extract mRNA from total cell populations. Use oligo(dT) initiated polymerase to reverse transcribe RNA into fluorescence labeled cDNA. RNA is unstable because of environment RNA-digesting enzymes. Alternatively use random priming for this purpose, generating a population of transcript subsequences

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How does a DNA microarray detects gene activity? Reverse Transcription makes cDNA from gene sequence
mRNA
AAAAAAAAAAAAAAAA

...GCUACGAUUGCAACGCCCGAAUGGUUACCAAAAAAAAAAA... CGATG CTAACGTTGCG GGCTTACCA ATGGTTTTTTTTTTT cDNA dTTP dCTP CGATG CTAACGTTGCG GGCTTACCA ATGGTTTTTTTTTTT dGTP dATP
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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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Labeled DNA copies of all mRNA from one sample (i.e. from a tumor) is hybridized to array x Dye

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http://bioinformatics.picr.man.ac.uk/mbcf/overview_ma.jsp

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2.Hybridization

Hybridization chamber
3XSSC HYB CHAMBER ARRAY LIFTER SLIP SLIDE
LABEL

SLIDE LABEL

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Temperature 45-65 Oc Time 12-24 hr Formamide (Lowers the Tm) Na+- improve stringency 34 Rept.DNA- block cross.hyb

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
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Microarray-Based Assays

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).
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3.Washing
To remove the excess of solution left on the array after hybridization process To improve the stringency of the experiments

Targets (fluorescently tagged)

probe sets (oligos) Non-bound ones are washed away

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4. Image acquisition ( Scanning )

Detector

Image

Duplicate spots
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Shine laser light on array and labeled DNA fragments glow

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Sample 1 -red dye Sample2 green dye

Spot Red Green Yellow Black Gene expression Sample 1 Sample2 Both No

Coding scheme:
Green = repressed (less mRNA) gene in experiment Red = induced (more mRNA) gene in experiment Black = no change (1:1 ratio)
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Total process

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D- Sequence data bases for Microarray

What resources could we use to design our own custom array? Microarray to particular disease, tissue or organism Will need to identify the genes that might be expressed.

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Data Bases
Primary gene sequence databases (Gene Bank, EMBL, DDGJ) holds all published sequences and are basis for all other data bases Secondary gene sequence databases (Uni Gene, TIGR, RefSeq) are excellent resources for designing Microarrays Genomic databases (TIGR, SGD ) are excellent resources for designing arrays for small organisms and can also be used for more complex organisms

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Designing of oligonucleotide probes

An oligonucleotide probe is short piece of single stranded DNA complimentary to the target gene whose expression is measured on the microarray by that probe. Usually probes for microarray are designed within several hundred bases of 3 end of target sequences. Good oligonucleotide properties: 1. Sensitive 2. Specific
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3. Isothermal

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1.Sensitive probe Is one that returns strong signal. When the complimentary sequence 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. repetitive sequences

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Probes secondary structures

The probe dont self hybridise, either oligonucleotide could form a stem loop structure or dimerise with neighbouring identical oligonucleotide on the surface of the array. (exclusion of palindrome sequences)

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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 genes by Homology search algorithms.
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BLAST FASTA Smith-Wterman algorithm

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3.Isothermal probe
Probes behave similarly under the hybridisation conditions of microarray experiment temperature, salt concentration and formamide concentration Base staking model is used in determining the melting temperature. It consider both base composition and order of bases in the sequence Mfold software
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Experimental Error
Sample contamination Poor quality/insufficient mRNA Reverse transcription bias Fluorescent labeling bias

Hybridization bias Cross-linking of DNA (double strands) Poor probe design (cross-hybridization) Defective chips (scratches, degradation)

Background from non-specificDNA hybridization 4/7/2010 Chips

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E- DNA Microarrays Data Analysis

Feature extraction software is convert the image into the numerical information that quantifies the gene expression

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Problems with microarray images

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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.
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Image Analysis
Gridding: identify spots (automatic, semiautomatic, manual) 2. Segmentation: separate spots from background. (A), Fixed circle (B), Adaptive circle (C), Adaptive shape (D), Histogram 3. Intensity extraction: mean or median of pixels in spot 4. Background correction: local or global
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1.

Normalization

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Raw data

Centered

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Scaled

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Analysis of differentially expressed genes

Null hypothesis

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Reject Accept True Type I error DNA Chips False Type II error

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Analysis of differentially expressed genes

Example1Samples are taken from 20 breast cancer patients, before and after 16 week course of doxorubicin chemotherapy, and analyzed using microarrays. To wish to identify gene (acetyl-coenzyme Aacetyltransferase 2 (ACAT2)) that are up or down regulated in breast cancer following that treatment

tcal = 3.22 ttab (at 19 df) = 2.39

significant cal >tab Conclude that this gene has been sig. down regulated following chemotherapy at the 1%lvel.
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Analysis of differentially expressed genes

Example 2Bone marrow samples are taken from 27 patients suffering from acute lymphoblastic leukemia(ALL) and 11 patients suffering from acute myeloid leukemia (AML) and analysed using Affymetrix arrays. To wish to identify the gene (Metallothionein IB ) that are up or down regulated in ALL relative to AML.

tcal = 4.35 ttab (at 36 df) = 2.445

significant cal >tab Conclude that this gene has been sig. high expression in AML than ALL at the 1%lvel.
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Hierarchical Clustering
Example Samples 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 gene expression? Correlation data

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Hierarchical Clustering

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Hierarchical Clustering

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Hierarchical Clustering

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Hierarchical Clustering

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Hierarchical Clustering
Linkage analysis
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

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Biological question Differentially expressed genes Sample class prediction etc. Experimental design Microarray experiment

16-bit TIFF files

Image analysis

Normalization

R, G
Estimation Testing Clustering Discrimination

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Biological verification DNA Chips and interpretation

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Applications What problems can it solve? Differing expression of genes over time, between tissues, and disease states Identification of complex genetic diseases Drug discovery and toxicology studies Mutation/polymorphism detection Pathogen analysis
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Example:1 microarrays for disease diagnosis: Microarrays may help guide doctors in determining effective breast cancer therapy Current methods including pathology exam (loooking at cells) and molecular markers (examining few, specific genes) only give hints. Microarrays of human genome used to: detect patterns of genetic activity in a tumor Test for chance of developing metastases (cancer that spreads)

Example:2 microarrays to study biological processes

Microarrays may help discover ways to prevent or stop the spread of SARS SARS chip: New microarray with entire genome of SARS virus Contains all 29,700 base pairs of the SARS virus Could help detect differences in particular strains of the virus and study the virus evolution over time. Further studies may determine better ways to contain the spread of SARS.

U.S. National Inst. of Allergy and Infectious Diseases (NIAID), Affymetrix, Inc, through Pathogen Functional Genomics Resource Center (TIGR).

Example:3
Microarray analyses of pathogen treated vs. mock treated Solanaceae

+pathogen -pathogen mRNA from two treatments

Reverse transcription and fluorescent labeling of cDNA Hybridization to microarray

Quantification of expression levels

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References
Basic microarray analysis: grouping and feature reduction by Soumya Raychaudhuri, 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: A Benchmark by A. Ultsh, C. Vetter; FG Neuroinformatik & Kunstliche Intelligenz Research Report 0994

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