DNA microarray

Total slides : 511April 8, 2023

DNA microarray

Isfahan University of Medical Science, School of Pharmacy

Department of Clinical Biochemistry

DNA MICROARRAY

DNA MICROARRAY

(An overview)(An overview)

By:

A.N. Emami Razavi


DNA microarray

Outlines Outlines

Introduction Applications Types of DNA microarray Microarrey process Microarray analysis

IntroductionIntroduction

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DNA microarray

What is a Microarray?

Microarray” has become a general term, there are many types now DNA microarrays Protein microarrays Transfection microarrays Antibody microarray Tissue microarray Chemical compound microarray …

We’ll be discussing DNA microarrays


DNA microarray

A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to a chemical matrix. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously.


DNA microarray

The affixed DNA segments are known as probes (although some sources will use different nomenclature such as reporters), thousands of which can be placed in known locations on a single DNA microarray. Microarray technology evolved from Southern blotting, whereby fragmented DNA is attached to a substrate and then probed with a known gene or fragment.


DNA microarray

DNA microarrays can be used to detect DNA (e.g., in comparative genomic hybridization); it also permits detection of RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins, which is referred to as "expression analysis" or expression profiling.


DNA microarray

Since there can be tens of thousands of distinct probes on an array, each microarray experiment can potentially accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations. The use of a collection of distinct DNAs in arrays for expression profiling was first described in 1987, and the arrayed DNAs were used to identify genes whose expression is modulated by interferon.


DNA microarray

These early gene arrays were made by spotting cDNAs onto filter paper with a pin-spotting device. The use of miniaturized microarrays for gene expression profiling was first reported in 1995, and a complete eukaryotic genome (Saccharomyces cerevisiae) on a microarray was published in 1997.


DNA microarray

Microarrays are Popular

PubMed search "microarray"= 23,559 papers (february 17,2008) // // = 28,027 papers (march,9,2008)

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ApplicationsApplications



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Gene expression profiling In different cells/tissues During the course of development Under different environmental or chemical stimuli In disease state versus healthy

Molecular diagnosis: Molecular classification of disease

Drug development Identification of new targets

Pharmacogenomics Individualized medicine


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Comparative genomic hybridization Assessing genome content in different cells or closely related

organisms.

SNP detection arrays

Identifying single nucleotide polymorphism among alleles within or between populations.

Chromatin immunoprecipitation (ChIP) studies Determining protein binding site occupancy throughout the genome,

employing ChIP-on-chip technology.


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Areas being studied with microarrays

Differential gene expression between two (or more) sample types

Similar gene expression across treatments

Tumor sub-class identification using gene expression profiles

Classification of malignancies into known classes

Identification of “marker” genes that characterize different tumor classes

Identification of genes associated with clinical outcomes (e.g. survival)


DNA microarray

mRNA levels compared in many different contexts

Different tissues, same organism (brain v. liver)

Same tissue, same organism (tumor v. non-tumor)

Same tissue, different organisms (wt v. mutant)

Time course experiments (development)

Types of DNA Microarray

Types of DNA Microarray



DNA microarray

Types of Microarrays

Spotted DNA arrays (“cDNA arrays”) Developed by Pat Brown (Stanford) PCR products (or long oligos) from known genes (~100 nt) spotted on

glass, plastic, or nylon support Customizable and off the shelf

Gene Chips Oligonucleotide arrays (Affymetrix)

Large number of 20-25mers/gene Enabled by photolithography from the computer industry Off the shelf

Ink-jet microarrays (Agilent) 25-60mers “printed” directly on glass Four cartridges: A, C, G, and T Flexible, rapid, but expensive


DNA microarray

Spotted DNA arrays

In spotted microarrays, the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs. There probes are synthesized prior to deposition on the array surface and are then "spotted" onto glass. A common approach utilizes an array of fine pins or needles controlled by a robotic arm that is dipped into wells containing DNA probes and then depositing each probe at designated locations on the array surface. The resulting "grid" of probes represents the nucleic acid profiles of the prepared probes and is ready to receive cDNA derived from experimental or clinical samples.


DNA microarray

Building a cDNA chip

Arrayed Library(96 or 384-well plates of bacterial glycerol stocks)

PCR amplification of target DNA

(cDNA or portion of genomic DNA)

Consolidate into plates

Spot as microarrayon glass slides


DNA microarray

Make Chip

Glass slide

Robot spotterMicroarray “spotters” are high-precision robots with metal pins that dip into DNA solution & tap down on glass slide (pins work like a fountain pen)


DNA microarray

This technique is used by research scientists around the world to produce "in-house" printed microarrays from their own labs. These arrays may be easily customized for each experiment, because researchers can choose the probes and printing locations on the arrays, synthesize the probes in their own lab (or collaborating facility), and spot the arrays. They can then generate their own labeled samples for hybridization, hybridize the samples to the array, and finally scan the arrays with their own equipment. This provides a relatively low-cost microarray that is customized for each study, and avoids the costs of purchasing often more expensive commercial arrays that may represent vast numbers of genes that are not of interest to the investigator.


DNA microarray

Oligonucleotide arrays

In oligonucleotide microarrays, the probes are short sequences designed to match parts of the sequence of known or predicted open reading frames. Although oligonucleotide probes are often used in "spotted" microarrays, the term "oligonucleotide array" most often refers to a specific technique of manufacturing. Oligonucleotide arrays are produced by printing short oligonucleotide sequences designed to represent a single gene by synthesizing this sequence directly onto the array surface instead of depositing intact sequences.


DNA microarray

Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer probes produced by Affymetrix) depending on the desired purpose; longer probes are more specific to individual target genes, shorter probes may be spotted in higher density across the array and are cheaper to manufacture.

One technique used to produce oligonucleotide arrays include photolithographic synthesis (Agilent and Affymetrix) on a silica substrate where light and light-sensitive masking agents are used to "build" a sequence one nucleotide at a time across the entire array.


DNA microarray


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Spotted Vs. Oligonucleotide array

Spotted Arrays

Relative cheap to make (~$10 slide)

Flexible - spot anything you want

Cheap so can repeat experiments many times

Highly variable spot deposition

Usually have to make your own

Affy Gene Chips

Expensive ($500 or more) Limited types avail, no chance

of specialized chips Fewer repeated experiments

usually More uniform DNA feaures

Can buy off the shelf

DNA MicroarrayProcess

DNA MicroarrayProcess



DNA microarray

Overview of the processQuestion

Sample Preparation

Data Analysis

Microarray

Hybridization

Microarray Detection


DNA microarray

Gene Expression Patterns

Genes are expressed when they are copied into mRNA or RNA (transcription)

Differential gene expression: which genes are expressed in which cells or tissues at a given point in time or in the life of the organism.

Total RNA can be isolated from cells or tissues under different experimental conditions and the relative amounts of transcribed RNA can be measured

The change in expression pattern in response to an experimental condition, environmental change, drug treatment, etc. sheds light into the dynamic functioning of a cell


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Experiment process

1. Collect tissue

2. Isolate RNA

3. Isolate mRNA

4. Make labeled DNA copy

5. Apply DNA

6. Scan microarray

7. Analyze data


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Equipments


DNA microarray

Collect tissue


DNA microarray

Isolate RNA


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Isolate mRNA


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Make labeled DNA copy


DNA microarray

Apply DNA


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Scan microarray


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Analyze data


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QuantificationCy5 at 635 Cy3 at 532

Overlay images


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Expression profiling with DNA microarraysData acquistion

HYBRIDIZATION

Spot #1

Spot #3

Spot #2

Spot #4

Spot #6

Spot #5

Relative intensity

Relative intensity

cDNA “control”Cy3 labeled

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Output Output

cDNA “treament”Cy5 labeled

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Image Analysis & DataData Visualization

200 10000 50.00 5.644800 4800 1.00 0.009000 300 0.03 -4.91

Cy3 Cy5Cy5Cy3

Cy5Cy3log2

Gen

es

Experiments842

fold248

Underexpressed

Overexpressed


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Sample Data


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Data storage and retrieval

Filtering

Normalization

Analysis


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Analysis of Microarray Data Clustering

Idea: Groups of genes that share similar function have similar expression patterns Hierarchical clustering k-means Bayesian approaches Projection techniques

Principal Component Analysis Independent Component Analysis

Classification Idea: A cell can be in one of several states

(Diseased vs. Healthy, Cancer X vs. Cancer Y vs. Normal) Can we train an algorithm to use the gene expression patterns to determine which state a

cell is in? Support Vector Machines Decision Trees Neural Networks K-Nearest Neighbors

Thank youThank you

Questions ? Questions ?


DNA microarray

Two-color vs. one-color detection Two-Color microarrays are typically hybridized with cDNA

prepared from two samples to be compared (e.g. diseased tissue versus healthy tissue) and that are labeled with two different fluorophores. Fluorescent dyes commonly used for cDNA labelling include Cy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and Cy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). The two Cy-labelled cDNA samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores after excitation with a laser beam of a defined wavelength. Relative intensities of each fluorophore may then be used in ratio-based analysis to identify up-regulated and down-regulated genes.

DNA microarray

Education

expression

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pcr products

thearray surface

fluorescenceemission

light spectrum

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