Data Microaarray and applications

7/31/2019 Data Microaarray and applications

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DNA MICROARRAY AND ITS APPLICATION

Ananta K. Ghosh

Department of BiotechnologyIndian Institute of Technology, Kharagpur


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WHAT IS AN ARRAY ?

An array is an orderly arrangement of samples. It provides a

medium for matching known and unknown DNA samples based onbase-pairing rules and automating the process of identifying the

unknowns.

An array experiment can make use of common assay systems such asmicroplates or standard blotting membranes, and can be created by

hand or make use of robotics to deposit the sample.

Arrays can be of two types: macroarrays or microarrays.the

difference being the size of the sample spots.

Macroarrays contain sample spot sizes of about 300 microns or

larger and can be easily imaged by existing gel and blot scanners.

The sample spot sizes in microarray are typically less than 200

microns in diameter and these arrays usually contains thousands ofspots.


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DNA MICROARRAY

It consists of large number of known cDNA or oligonucleotides

spotted onto a carrier in a logical and orderly fashion.

DNA microarray, or DNA chips are fabricated by high-speed

robotics, generally on glass but sometimes on nylon substrates, for

which probes with known identity are used to determinecomplementary binding, thus allowing massively parallel gene

expression and gene discovery studies.

An experiment with a single DNA chip can provide researchers

information on thousands of genes simultaneously - a dramatic

increase in throughput.


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APPLICATION OF DNA MICROARRAY

Two major application forms for the DNA microarray technology:

Determination of expression level (abundance) of genes.

2) Identification of sequence (gene / gene mutation)


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GENE EXPRESSION

Gene expression is the term used to described the transcription of

the information contained within the DNA-the repository of genetic

information-into messenger RNA molecules that are then translated

into the proteins that perform most of the critical functions of cells.

Gene expression is a highly complex and tightly regulated processthat allows a cell to respond dynamically both to environmental

stimuli and to its own changing needs.

This mechanism acts as both an on/off switch to control which

genes are expressed in a cell as well as a volume control that

increases or decreases the level of expression of particular genes asnecessary.


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ANALYSIS OF GENE EXPRESSION

Thousands of genes and their products (i.e., RNA and proteins) in a

given living organism function in a complicated and orchestrated

way that creates the mystery of life.

However, traditional methods in molecular biology (northern

hybridization or Biochemical approaches) generally work on a "onegene in one experiment" basis, which means that the throughput is

very limited and the "whole picture" of gene function is hard to

obtain

DNA sequencing programme unravels the complete genome

sequences of many organisms and identifies many new genes. But

only after the full functions of the new genes are discovered the fullimpact of these genome project can be realized.


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ANALYSIS OF GENE EXPRESSION

DNA sequencing programme unravels the complete genomesequences of many organisms and identifies many new genes. But

only after the full functions of the new genes are discovered the full

impact of these genome project can be realized. And scientists are

looking for different technologie. to address this issue

DNA Microarray is one such technology which allows thesimultaneous analysis of thousands of genes and their functions

This technology promises to monitor the whole genome on a single

chip so that researchers can have a better picture of the interactions

among thousands of genes simultaneously.


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TECHNOLOGY FOR MICROARRAY

Two major technologies are currently used for DNA microarray

A. cDNA microarray : It was developed by Brown and Schena at

Stanford University and comprises a large number of cDNA(500 5000 bases long) spotted on to a glass slide, in a high

density pattern and exposed to a set of targets either separately

or in a mixture.

B Oligonucleotide array or chips: It was developed by the leading

manufacturer, Affymatrix, and is made from oligonucleotides(20-80 nucleotides) array synthesized in situ on the glass surface

by photolithiography or by conventional synthesis followed by on

chip immobilization. The array is exposed to labeled sampleDNA, hybridized, and the identity/abundance of complementary

se uences are determined.

REQUIREMENT OF MICROARRAY EXPERIMENT


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REQUIREMENT OF MICROARRAY EXPERIMENT

a) The chip itself with its special surface.

b) The device for producing microarrayas by spotting the nucleicacids onto the chip or for their in situ synthesis.

c) A fluidic system for hybridization of labelled probes to target

DNA

d) A scanner to read the hibridization signals on the chip.

e) Sophisticated software programs to quantify and interpret the

results

FABRICATION OF MICROARRAY


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FABRICATION OF MICROARRAY

Sourcing DNA /clones: What to spot ?

The first step in creating a microarray is to find the source of genesor DNA that will be arrayed on to a glass slide.

a) One can use publicly available clones such as those from the

IMAGE consortium or Research Genetics where relatively large

number of clones can be obtained relatively inexpensively.

b) Generates clones in house which may be time consuming and

expensive.

P ti d ifi ti f DNA


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Preparation and purification of DNA

Microarray can be made by printing plasmids, or most commonly, a

section of the plasmid. The relevant part of the plasmid DNA are

amplified by PCR, product purity and amount is checked, and then

spotted down in concentrated form. Usually a 96 well format is used

for this purpose. PCR product should be of similar concentration

(approximately 500ng/ul) and sizes (0.2-1.2 kb)

Selection of controls

As with any experimental system, the inclusion of relevant controls

is essential for the meaningful interpretation of data downstream.

Some researcher use housekeeping genes which are expressed

constitutively and whose level of expression is thought to be stable.

D iti f DNA


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Deposition of DNA

Deposition of the PCR product are done on glass, nylon or othersupports using a robot on Poly-L-Lysine coated glass slides. Typically

0.5-10 nl of DNA is deposited in a spot 100-150 um in diameter and a

distance of 200-250 um from neighboring spots.

Spotting is done in humidity controlled chamber because humiditydetermines the rate of evaporation of water from the arrayed spots.

Rapidly dried spots may be uneven, with most of the DNA in the cent

whereas slow drying might result in creeping, and spot spreading. Sp

should be perfect circular shape with an even density of DNA.

Post deposition processing

After printing, the slides are dried for 24 hours at room temperatureThe deposited DNA is then immobilized, usually by UV irradiation


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EXPERIMENTS USING MICROARRAY


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EXPERIMENTS USING MICROARRAY

1. RNA preparation:

High quality total RNA are extracted by guanidium isothiocyanate method andmRNA purified by oligo dT cellulose chromatography. Quality and quantity of

RNA should be checked properly (Bioanalyzer)

2. Sample labelling:

A single round of reverse transcription reaction is used to generate a labelled

cDNA probe from the sample. Usually 2-5 ug of mRNA is used for labelling withFluorophores.

Dual label hybridization is a technique often used to compensate for differences in

spotted genes. Two samples (normal and experimental are labeled with paired

fluorophores (Cy3 and Cy5) that are competitively co-hybridized to the same

arary..


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3 Hybridization of sample to microarray


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3. Hybridization of sample to microarray

Glass microarrays are hybriidized by spotting a small volume of sample (20-25 ul,prepared in hybridization solution) on the microaray and then carefully dropping

a coverslip onto it to spread the solution over the entire slide and eliminating the

air. The slides are then placed in a humidified (to prevent evaporation)

hybridization chamber at 42C (20-25C below the Tm ) for 12-16 hours.

4. Post hybridization processing:

After hybridization, the microarray are subjected to a series of washes to remove

unbound, labeled probe and non-specifically bound sequences. During washingstringent conditions are applied by increasing the temperature or lowering the

ionic strength of the washing buffer.


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5. Image capture :


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5. Image capture :

After hybridization, a digital image of the array is made by ascanner. Most scanner are equipped with confocal laser microscope

and a CCD camera.

In practice, the combination of 532 nm (green) and 633nm (red)

lasers is used most frequently with Cy3 and Cy5 fluorochromes.


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A typicalmicroarray slide

after scanning


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6. Image Analysis and Data handling:


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g y g

Once a digital image of the array is obtained, it must be analyzed. The first step inthe analysis of data is log transformation of the data. This is because the value

from instruments are often biased to small values, with few high values. For each

spot, the red to green fluorescence ratio is calculated.

The second step is normalization of data. Rather than obtaining absolute values a

ratio of test to control is obtained.

Gene expression changes are often expressed as a fold difference, either greater

than two fold or less than 0.5 fold

With respect to handling and analyzing these large amount of data various

bioinformatics tools have been introduced and used.


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Steps in the design and implementation of a DNA
http://www.healthtech.com/glossaries/content/microarrays.htmhttp://www.healthtech.com/glossaries/content/microarrays.htm


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p g p

microarray experiment

1) Probe

(cDNA/oligo

with knownidentity)

2) Chip

fabrication

(Puttingprobes on

the chip)

3) Target

(fluorecently

labeledsample)

4) Assay 5) Readout

6)

Informatics

Small

oligos,

cDNAs,

chromosome

,

...

(whole

organism on

a chip?)

Photolithogr

aphy,

pipette,

drop-touch,

piezoelectric

(ink-jet),

electric, ...

RNA,

(mRNA==>)

cDNA

Hybridization,...

Fluorescence,

Image

processing,

bioinformatics, data

mining and

visualization

APPLICATION OD DNA MICROARRAY
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I. Changes in Gene Expression Levels

The immobilized DNA is cDNA derived from the mRNA of known genes, and thecontrol and sample DNA hybridized to the chip is cDNA derived from the mRNA

of normal and diseased tissue, respectively.

If a gene is overexpressed in a certain disease state, then more sample cDNA, ascompared to control cDNA, will hybridize to the spot representing that expressed

gene. In turn, the spot will fluoresce red with greater intensity than it will fluoresce

green.

Once researchers have characterized the expression patterns of various genes

involved in many diseases, cDNA derived from diseased tissue from any individual

can be hybridized to determine whether the expression pattern of the gene from

the individual matches the expression pattern of a known disease. If this is thecase, treatment appropriate for that disease can be initiated.

Expression chips can be used to develop new drugs. For instance, if a certain gene

is overexpressed in a particular form of cancer, researchers can use expression

chips to see if a new drug will reduce overexpression and force the cancer into


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II. Genomic Gains and Losses

DNA repair genes are thought to be the body's frontline defense againstmutations and, as such, play a major role in cancer. Mutations within these

genes often manifest themselves as lost or broken chromosomes.

Researchers use a technique called microarray Comparative GenomicHybridization (CGH) to look for genomic gains and losses or for a change in the

number of copies of a particular gene involved in a disease state. In microarray

CGH, large pieces of genomic DNA serve as the target DNA, and each spot of

target DNA in the array has a known chromosomal location. The hybridizationmixture will contain fluorescently labeled genomic DNA harvested from both

normal (control) and diseased (sample) tissue.

If the number of copies of a particular target gene has increased, a large amountof sample DNA will hybridize to those spots on the microarray that represent

the gene involved in that disease, whereas comparatively small amounts of

control DNA will hybridize to those same spots. As a result, those spots

containing the disease gene will fluoresce red with greater intensity than they

will fluoresce green, indicating that the number of copies of the gene involved in


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In Brief: Microarray Applications

Microarray type Application

CGHTumor classification, risk assessment, and

prognosis prediction

Expression analysis

Drug development, drug response, and therapy

development

Mutation/Polymorphism analysis

Drug development, therapy development, andtracking disease progression

Microarray Data Management


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Considering the amount of data that can potentially be generated

using a single microarray chip, many new challenges are presented

in terms of data tracking and analysis.

To support the public use and dissemination of gene expression

data, NCBI has launched the Gene Expression Omnibus, or GEO.

GEO represents NCBI's effort to build an expression data repository

and online resource for the storage and retrieval of gene expression

data from any organism or artificial source
http://www.ncbi.nlm.nih.gov/geo/http://www.ncbi.nlm.nih.gov/geo/


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Data Microaarray and applications

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