DNA MICROARRAY

DNA MICROARRAY: 

DNA MICROARRAY Mrs. Rani Ashok Asst. Prof. of Zoology, Lady Doak College, Madurai – 2 Email: eaarani@rediffmail.com Genome Chip

Enabling Technologies: 

Enabling Technologies MICROARRAYS: CHIPPING AWAY AT THE MYSTERIES OF SCIENCE AND MEDICINE Microarrays allow scientists to analyze expression of many genes in a single experiment quickly and efficiently. They represent a major methodological advance and illustrate how the advent of new technologies provides powerful tools for researchers. Scientists are using microarray technology to try to understand fundamental aspects of growth and development as well as to explore the underlying genetic causes of many human diseases.

DNA Microarray: 

DNA Microarray Monitor the whole genome on a single chip Researchers can have a better picture of the interactions among thousands of genes simultaneously

DNA Microarray: 

DNA Microarray Orderly arrangement of samples provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identif contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scannersying the unknowns

DNA Microarray: 

DNA Microarray sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots require specialized robotics and imaging equipment that generally are not commercially available as a complete system.

FEATURES: 

FEATURES Parallelism Thousands of genes simultaneously – Miniaturization Small chip size – Multiplexing Multiple samples at the same time – Automation Chip manufacturing Reagents

Slide 7: 

DNA microarrays can be used To detect differences in the levels gene expression in different populations of cells on a genome-wide level.

Applications of DNA microarray analysis: 

Applications of DNA microarray analysis Differing expression of genes over time, between tissues, and disease states Identification of complex genetic diseases Drug discovery and toxicology studies Mutation/polymorphism detection (SNP’s) Pathogen analysis

Application – diagnosis of the disease: 

Application – diagnosis of the disease Identification of sequence (gene / gene mutation) Determination of expression level (abundance) of genes Identify the genome expression for hereditary diseases Map out which genome expression would directly related to certain hereditary diseases. Once the sequence for different hereditary diseases was clarified, the commercially available diagnostic biochip for the hereditary diseases would become possible. During the viral infection, the viral genome would upregulate the protein expression in the host level so that it would help to produce the DNA polymerase for viral DNA systhesis . As the virus start to duplicate the virus particle, the viral genome would show significant abnormal expression. By using the DNA array, it could also be detect while the infection is in the early stage.

MICROARRAY STEPS: 

MICROARRAY STEPS Experiment and Data Acquisition Chip manufacturing Sampling and labeling Hybridization Image scaling Data acquisition Data normalization Data analysis Biological interpretation

PRINCIPLE: 

PRINCIPLE Similar to Northern Major differences from Northern Base-Pairing, hybridization between nucleic Acids Detects thousands of genes simultaneously /individual Probes fixation on glass slide / nylon membrane Target samples labeling with fluorescent/radioactive dNTP

TECHNOLOGIES: 

TECHNOLOGIES Bead Method Photolitography ( Affymetrix , Febit , Nimblegen ) Inkjet (Agilent, Canon) Robot spotting (many providers)

The Illumina Beadarray Technology: 

The Illumina Beadarray Technology Highly redundant ~50 copies of a bead 60mer oligos Absolute expression Each array is deconvoluted using a colour coding tag system Human, Mouse, Rat, Custom

Slide 15: 

Kenneth Kuhn et al. Genome Res. 2004; 14: 2347-2356 Design of a randomly assembled gene-specific probe array x,y array coordinate

Affymetrix photolitography: 

Affymetrix photolitography Each probe 25 bp long 22-40 probes per gene Perfect Match (PM) as well as MisMatch (MM) probes

Febit/NimbleGen photolitography: 

Febit / NimbleGen photolitography

Robot Spotting: 

Robot Spotting

InkJet (HP/Canon) technology: 

InkJet (HP/Canon) technology

ARRAY CHIP TYPE: 

ARRAY CHIP TYPE cDNA chip (DNA microarray, two-channel array) Gene chip (DNA chip, Affymetrix chip) Probe cDNA (500~5,000 bases long) is immobilized to a solid surface such as glass Using robot spotting Traditionally called DNA microarray Firstly developed at Stanford University. Oligonucleotide (20~80-mer oligos ) is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization Historically called DNA chips Developed at Affymetrix , Inc. , under the GeneChip ® trademark Many companies are manufacturing oligonucleotide based chips using alternative technologies

Affymetrix Chip: 

Affymetrix Chip Each gene has 16 – 20 pairs of probes synthesized on the chip Each pairs of probes have two oligonucleotides Perfect match (PM, reference seq ) ATG…C…TGC (20-25 bases) Mismatch (MM, one base change) ATG…T…TGC The scanned result for a given gene is the average differences between PM and MM signals, over probes (MAS5 algorithm)

Affymetrix Microarray Experiment: 

Affymetrix Microarray Experiment Sample RNA labeling Fragmentation First-strand cDNA synthesis Reverse transcriptase Second-strand synthesis • DNA polymerase cDNA purification In Vitro transcription to synthesize biotin-labeled RNA T7 enzyme Use heat and Mg++ Reduce RNA to 25-200 bp fragment Facilitate efficient and reproducible hybridization

Affymetrix Microarray Experiment: 

Affymetrix Microarray Experiment Hybridization Wash and Stain Preheat hybrid mix solution (99 C) Affy chip in hybrid solution 5min Add probe and hybridization for 16 hours Wash buffer Stain with a fluorescent molecule ( streptavidin-phycoerythrin ) that binds to biotin A signal amplification step that employs anti- Streptavidin antibody (goat) and biotinylated goat IgG antibody

Affymetrix Microarray Experiment: 

Affymetrix Microarray Experiment Scan Data Acquisition Affymetrix scanner and follow the menu . dat image file . cel tab delimited file .CHP data file Affymetrix Microarray Suite GCOS ( Genechip Operating System) Need chip description file (CDF) For probe location

cDNA Microarray Experiment: 

cDNA Microarray Experiment Array fabrication Probe preparation DNA clones Unigene EST clustering PCR amplification of clones Array printing RNA extraction (control, test) RNA labeling Incorporate fluorescently labeled deoxyribonucleotides First strand cDNA Cyanine5 labels Test sample RNA Cyanine3 labels Control sample RNA Mix the labeled two RNAs

Affymetrix Microarray Experiment: 

Affymetrix Microarray Experiment Hybridization Slide scanning Prehybridize slide 42C 45 min Hybridize preheated probes 16-20 hours C3 16-bit TIFF image file C5 16-bit TIFF image file

Affymetrix Microarray Experiment: 

Affymetrix Microarray Experiment Data Acquisition Image GenePix Quantarray Need chip description file (CDF) For probe location

EXPERIMENT: 

EXPERIMENT DNA MICROARRAY

Experiment & data acquisition: 

Experiment & data acquisition Mechanism

Genome-wide data sets: considerations and complications: 

Genome-wide data sets: considerations and complications Factors to consider when analysing genome-wide data sets Complications when comparing genome-wide data sets How were the data generated? What method was used? Are there any technical limitations? On how many repeats is the data set based? How reproducible are the data? Has the error rate been estimated? If so, how high is it? Can known and trusted examples be confirmed? How complete is the data set? Which genes or proteins are missing? How was the data analysed ? Do genes or proteins of interest also appear in other data sets? Different isolates and experimental conditions have been used Databases are not interconnected There is no unifying data format (. GCT format is close…..) Keywords for database searches are not standardized Some data are not readily accessible or in the public domain

Slide 33: 

Thank You