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MICROAARRAY(DNA CHIP)

Presented By: Hamza KhanRegistration # FA16-RO2-019

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• Introduction

•Types of Microarray

•DNA Microarray

• Principle

Steps involved in DNA microarray

• Types of DNA Chips

cDNA - based chips

Oligonucleotide - based chips

• Applications

• Advantages and disadvantages

• SNP Array

CONTENTS

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IntroductionAn array is an orderly arrangement of samples wherematching of known and unknown DNA samples is donebased on base pairing rules.

A microarray is a pattern of ssDNA probes which areimmobilized on a surface called a chip or a slide.

Microarray technology evolved from Southern blotting

The concept of microarrays was first proposed in thelate 1980s by Augenlicht and his colleagues.

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Types of Microarray• DNA microarrays

• Protein microarrays

• Antibody microarray

• Tissue microarray

• Chemical compound microarray

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DNA Microarray• DNA microarrays are solid supports, usually of glass orsilicon, upon which DNA is attached in an organized gridfashion.

• Each spot of DNA, called a probe, represents a singlegene.

• There are several synonyms of DNA microarrays such asDNA chips, gene chips, DNA arrays, gene arrays andbiochips.

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Principle• The principle of DNA

microarrays lies on thehybridization between thenucleotide.

• Using this technology thepresence of one genomic orcDNA sequence in 1,00,000or more sequences canbe screened in a singlehybridization.

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a) A DNA chip can be

manufactured to contain

hundreds of thousands of

synthetic single-stranded DNA

sequences.

b) Unknown DNA from a patient is

separated into single strands,

enzymatically cut and labeled with

a fluorescent dye.

Steps

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c) The unknown DNA is inserted into the

chip and allowed to hybridize with the

DNA on the chip.

d) The tagged DNA will bind only to the

complementary DNA on the chip. The

bound DNA will be detected by its

fluorescent dye and analyzed by a

computer. The green light is a gene

expressed in normal cells; red is a

mutated gene expressed in tumor cells;

and yellow, in both cells.

Fig: DNA Chip Technology

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Design of a

DNA

Microarray

System

DNA Microarray Technology

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1. Sample

preparation

2. Purification

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3. Reverse

Transcription

4. Labelling

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5.Hibridization

6. Scanning

7.Normalization

and analysis

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There are 2 types of DNA Chips/Microarrays:

Types of DNA Chips

1. cDNA based microarray

2. Oligonucleotide based microaaray

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•This type of chips are prepared by using cDNA, it is calledcDNA chips or cDNA microarray or probe DNA. The cDNAsare amplified by using PCR. Then these immobilized on asolid support made up of nylon filter of glass slide.

• Small volume of this DNA preparation is spotted on solidsurface making physical contact between these two. DNA isdelivered mechanically or in a robotic manner.

•When one DNA spotting is done, the pin is washed and loaded with fresh DNA to Start the second cycle.

cDNA Based Chips

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Synthesis

Selection of the material tospot onto the microscopeglass surface.

Preparation and purificationof DNA sequencesrepresenting the gene ofinterest.

Spotting DNA solution ontochemically modified glassslides via a contact printing orinkjet printing.

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Oligonucleotide Microarray

• In oligonucleotide microarrays, the probes areshort sequences designed to match parts of thesequence of interest.

• Oligonucleotide arrays are produced by printingshort oligonucleotide sequences designed torepresent a single gene by synthesizing this sequencedirectly onto the array surface instead of depositingintact sequences.

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Synthesis

•Light is directed through a photolithographic mask tospecific areas of array surface.

•Activation of areas for chemical coupling. Attachmentof A nucleotide containingphotolabile protectinggroup X (MeNPOC).

•Addition of 2ndbuilding block T containinga photolabile protectinggroup X. This process isrepeated until the desiredproduct is obtained.

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Applications

MICROARRAY AS A GENE

EXPRESSION PROFILING

TOOL

MICROARRAY AS A

COMPARATIVE GENOMICS

TOOL

DISEASE DIAGNOSIS

DRUG DISCOVERY

TOXICOLOGICAL

RESEARCH

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• Provides data for thousands of genes.

• One experiment instead of many.

• Fast and easy to obtain results.

• Huge step closer to discovering cures for diseasesand cancer.

• Different parts of DNA can be used to study gene expression.

Advantages

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Disadvantages

• The biggest disadvantage of DNA chips is thatthey are expensive to create.

• The production of too many results at a timerequires long time for analysis, which is quitecomplex in nature.

• The DNA chips do not have very long shelf life,which proves to be another major disadvantage ofthe technology.

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• A SNP is defined as a single base change in a DNAsequence that occurs in a significant proportion (morethan 1 percent) of a large population

• SNPs are found in coding and (mostly) noncoding regions.

• Occur with a very high frequency about 1 in 1000bases to 1 in 100 to 300 bases.

• SNPs in coding regionsmay alter the protein structure made by that coding region.

SNPs (Single Nucleotide Polymorphisms)

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SNP Array

• SNP array is a type of DNA Microarray which isused to detect polymorphism within a population• The basic principles of SNP array are the same asthe DNA microarray• The three mandatory components of the SNParrays are:

• An array containing immobilized allele-specific oligonucleotide (ASO) probes.•Fragmented nucleic acid sequences of target,labeled with fluorescent dyes.•A detection system that records and interpretsthe hybridization signal.

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SNP Array

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