dna micro array

8/2/2019 Dna Micro Array

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DNA Microarray Assay

Submitted By : Prosun RoyRoll No. : MS111136Session : 2010-2011Title of the Course: Advanced Pharmaceutical AnalysisCourse No. PHARM 5207Pharmacy DisciplineKhulna University, Khulna-9208


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The Central Dogma of Biology

SequencingFragment assemblyGene finding

Linkage analysisetcHomology searches

Annotation

IsolationSequencingRNA structure predictionGene expression:

microarraysetc

Protein structureprediction

Protein foldingHomology searchesFunctional pathways

Annotation

Challenges:

DNA

RNA

Protein

transcription

translation


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DNA Microarray: Introduction

Microarray

(gene chip/DNA chip/biochip)collection of microscopic DNA

spots attached to a solid surface

Array

orderly arrangement ofthe probes of interest

Microsmall size

A DNA Microarray is a multiplex technology used in molecular biology and

in medicine. It consists of an arrayed series of thousands of microscopicspots of DNA oligonucleotides, called features, each containing

picomoles (1012 moles) of a specific DNA sequence, known as probes

(or reporters).

This can be a short section of a gene or other DNA element that are usedto hybridize a cDNA or cRNA sample (called target) under high-

stringency conditions. Probe-target hybridization is usually detected and

quantified by detection of fluorophore-, silver-, or chemiluminescence-

labeled targets to determine relative abundance of nucleic acid

sequences in the target.


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Microarray: History

1975 Southern Blotting technology (Edward Southern)

1991 First high-density nylon filter arrays (Lennon, Lehrach)

1995 cDNA-Microarrays (Schena et al.)

1996 Affymetrix Genechip Technology

2002 Agilent open array technology

2003 Illumina Bead Array technology


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4 x 4 or 8x4 sectors

17...38 rows andcolumns per sector

ca. 460046000probes/array

sector: correspondsto one print-tip

A Microarray slide

Slide: 25x75 mm

Spot-to-spot: ca. 150-350 mm


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DNA Microarray: Principle

Single-stranded DNA has a natural affinity to annealto its complementary sequence of single-strandedDNA or RNA.

a sample, in appropriate buffer, will hybridize to theimmobilized complementary DNA oligo.

a fluorescent dye is either added prior to sampleaddition and hybridization or after the DNA

hybridization to the Microarray. Prior to sampleaddition, one or two fluorescent dyes can be used.

In this context, a Microarray is a high-throughputDNA or RNA hybridization platform for performinggene expression analysis

Unlike its predecessors in gene expression studies,Microarray allow for gene expression analysis ofthousands of genes, capable of covering the wholegenome from as little as 50100 ng of total RNA.

synthesize gene-specific probes onto a siliconsurface or immobilized prefabricated cDNA/oligosonto derivative glass slides using robotic printing

instruments.


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DNA Microarray Assay: Overview

microarray analysis

scanning

cDNA clones(probes)

PCR productamplificationpurification

printing

0.1nl / spotHybridize

RNA

Tumorsample

cDNA

RNA

Referencesample

cDNA

excitationredlasergreen

laser

emission

overlay images and normalise


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Microarray Steps

Experiment and Data Acquisition

Chip manufacturing

Sampling and labeling

Hybridization

Image scaling

Data acquisition Data normalization

Data analysis

Biological interpretation

Biological questionDifferentially expressed genesSample class prediction etc.

Testing

Biological verificationand interpretation

Microarray experiment

Estimation

Experimental design

Image analysis

Normalization

Clustering Discrimination

R,G

16-bit TIFF files

(Rfg, Rbg), (Gfg, Gbg)


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Types of Microarrays

Spotted DNA arrays

Laborious

Inexpensive

Moderate specificityModerate representationLow DensityCannot detect polymorphism

Affymetrix gene chipsEasyExpensiveHigh specificity

High representationHigh densityCan detect polymorphism
http://www.gene-chips.com/sample1.html


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The Microarray Technologies

Spotted Microarray Affymetrix GeneChip

cDNAs, clones, or short and long

oligonucleotides deposited onto

glass slides

Each gene (or EST) represented

by its purified PCR product

Simultaneous analysis of two

samples (treated vs untreated cells)

provides internal control.

short oligonucleotides synthesized in situ

onto glass wafers

Each gene represented multiply - using

16-20 (preferably non-overlapping)

25-mers.

Each oligonucleotide has single-base

mismatch partner for internal control of

hybridization specifity.

relative gene expressions absolute gene expressions


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Spotted MicroarrayProcess

CTRL

TEST


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Affymetrix GeneChipProbe Arrays

24m

Each probe cell or feature containmillions of copies of a specificoligonucleotide probe

Image of Hybridized Probe Array

Over 250,000 different probescomplementary to geneticinformation of interest

Single stranded, fluorescentlylabeled DNA target

Oligonucleotide probe

**

**

*

1.28cm

GeneChip Probe Array Hybridized Probe Cell

BGT108_DukeUniv


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Microarray Experiment: Materials Needed

1. RNA (isolation from a biologicalsample).

2. Microarray chip (preferably commercialplatforms).

3. In vitro transcription/RNA amplification

kit4. Labeling kit (often specific and

optimized to the Microarray platform ofinterest).

5. Hybridization station and chambers

6. Scanner for image capture

7. Software for data analysis.


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RNA Extraction & Hybridization

Hybridize

RNA

Tumorsample

cDNA

RNA

Referencesample

cDNA


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Spotted Array Technology

Printing Slides

Pi ll DNA


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384 wellplate

Contains cDNA

probes

Glass Slide

Array of bound cDNA probes

4x4 blocks = 16 print-tip groups

Print-tipgroup 6

cDNA clones

Spotted in duplicate

Print-tipgroup 1

Pins collect cDNAfrom wells


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Scanning & Image Analysis


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200 10000 50.00 5.64

4800 4800 1.00 0.00

9000 300 0.03 -4.91

Cy3 Cy5Cy5Cy3

Cy5Cy3log2

Genes

Experiments 8

4

2

fold

2

4

8

Underexpressed

Overexpressed

Image Analysis & Data Visualization


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Normalization

Biased towards the green channel & Intensity dependent artifacts


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Affymetrix Data Mining Tool

Affymetrix NetAffx

Biomax Gene Expression Analysis Suite

GeneData Expressionist

Informax Xpression

Invitrogen Corp. ResGen Pathways

Rosetta Resolver Gene Expression System

Silicon Genetics GeneSpring Spotfire

Data Analysis

Software Package


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Applications of Microarray

Analysis of patterns of gene expression

Functional relationship between genes

Expression in coregulatory gene group Monitoring changes in genomic DNA

Cellular pathways affected by mutation Changes in expression profiles of mutants

Simultaneous detection of many genes

Gene discovery

Pathway analysis Molecular basis of disease progression Molecular signatures of pathogens

Comparative genomic studies of pathogens Virulence difference

Pathogen genetics and manifestation

Life cycle Replication, translational control

Host-parasite interaction Pathogen establishment Host cell recognition Host cell response

Parasite response to host immune response


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Applications of Microarray


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Constraints

Complex system of eukaryotes & multicellularorganisms

Transcriptome analysis

Developing technology

Many stages Design of experiment

Array quality

Highly variable data

Analysis of data

Published experiments

Cost


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The Future of DNA Microarray

Gene discovery. DNA Microarraytechnology helps in theidentification of new genes, know about their functioning andexpression levels .

Disease diagnosis. classify the types of cancer on the basis of thepatterns of gene activity in the tumor cells.

Pharmacogenomics.is the study of correlations betweentherapeutic responses to drugs and the genetic profiles of thepatients.

Toxicogenomics. microarray technology allows us to research the

impact of toxins on cells. Some toxins can change the geneticprofiles of cells, which can be passed on to cell progeny.

Personalized Medicine. Personalized treatment plans can bedeveloped for each individual patient.

dna micro array

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