microarray

DNA Microarrays

Ms.ruchi yadavlecturer

amity institute of biotechnologyamity university

lucknow(up)

Gene expression

• A human organism has over 250 different cell types (e.g., muscle, skin, bone, neuron), most of which have identical genomes, yet they look different and do different jobs

• It is believed that less than 20% of the genes are‘expressed’ (i.e., making RNA) in a typical cell type

• Apparently the differences in gene expression is what makes the cells different

Gene Expression

Patrick Schmid 6

Uses and Applications

Microarrays:Universal Biochemistry Platforms

PeptidesPeptides ProteinsProteins

Carbohydrates

LipidsLipids

Small moleculesSmall molecules

DNADNA

Some questions for the golden age of genomics

• How gene expression differs in different cell types?

• How gene expression differs in a normal and diseased (e.g., cancerous) cell?

• How gene expression changes when a cell is treated by a drug?

• How gene expression changes when the organism develops and cells are differentiating?

• How gene expression is regulated – which genes regulate which and how?

What is a DNA Microarray? (cont.)

Cheung et al. 1999

• Biological Samples in 2D Arrays on Membranes or Glass Slides

What is a DNA Microarray?

• Also known as DNA Chip• Allows simultaneous measurement of the

level of transcription for every gene in a genome (gene expression)

• Microarray detects mRNA, or rather the more stable cDNA

MICROARRAY TYPES

The Colours of a Microarray

• GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA.

• RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA.

• YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA.

• BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA.

Microarray Steps

• Experiment and Data Acquisition– Sample preparation and labelling

– Hybridisation

– Washing

– Image acquisition

• Data normalization

• Data analysis

• Biological interpretation

I. Target and probe preparation

There are many ways to obtain a labeled target sample.

...GGCUUAAUGAGCCUUAAAAAA...AmRNA

TTTTTT...T

viral enzyme reverse transcriptaserecognizes poly-T bound to poly-Aand begins to add complementaryDNA nucleotides. The C nucleotidesare dyed.

AAA GGCTCTTAAGCC...poly-A tail

poly-T primer

cDNA target

Hybridization and Data Analysis

Spotted Array Synthesis.

Microarray Experiment

How do we manufacture a microarray?

• Start with individual genes, e.g. the ~6,200 genes of the yeast genome

• Amplify all of them using polymerase chain reaction (PCR)

• “Spot” them on a medium, e.g. an ordinary glass microscope slide

• Each spot is about 100 µm in diameter• Spotting is done by a robot• Complex and potentially expensive task

Robotic spotting

DNA Samples on 96 well plates

The PixSys 5500 Arraying Robot (Cartesian Technologies)

Vacuum wash station

The print head holds up to 32 pinsin a 8x4 format

Vacuum hold-down platform (50 slide capacity)

Robotic arm

Contact Printing

Non Contact Printing

InkJet (HP/Canon) technology

• 1 drop = 100 picolitres

Spotting the Probes on the Microarray 8 X 4 Print Headmicroarray slide

plate with wells holding probes in solution

All spots of the same color are made at the same time.

All spots in the same sector are made by the same pin.

Using cDNA Microarrays to Measure mRNA Levels

ACCTG...GACCTG...GACCTG...G

TTCTG...ATTCTG...ATTCTG...A

GGCTT...CGGCTT...CGGCTT...C

ATCTA...AATCTA...AATCTA...A

ACGGG...TACGGG...TACGGG...T

CGATA...GCGATA...GCGATA...G

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Sample 1

Sample 2

Microarray Slide

Spots(Probes)

UnknownmRNASequences(Target)

Extract mRNA

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Sample 1

Sample 2

Convert to cDNA and Label with Fluorescent Dyes

Sample 1

Sample 2

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Sample 1

Sample 2

Mix Labeled cDNA

Sample 1

Sample 2??????????

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Sample 1

Sample 2

Hybridize cDNA to the Slide

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Sample 1

Sample 2

Excite Dyes with Laser

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Sample 1

Sample 2

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Quantify Signals

ACCTG...G

76527652138138

TTCTG...A

5708570843884388

GGCTT...C

85668566765765

ATCTA...A

120812081344213442

ACGGG...T

6784678497629762

CGATA...G

6767239239

Sample 1

Sample 2

Patrick Schmid 37

Oligonucleotide Microarray

• Gene chip (DNA chip, Affymetrix chip):

• Oligonucleotide (20~80-mer oligos) is synthesized either in situ (on-chip)

• Developed at Affymetrix, Inc. , under the GeneChip® trademark

Affymetrix Chip

• Each gene has 16 – 20 pairs of probes synthesized on the chip

• Each pairs of probes have two oligonucleotide

–Perfect match (PM, reference seq) ATG…C…TGC

(20-25 bases)

–Mismatch (MM, one base change) ATG…T…TGC

• A MM oligo is identical to a PM oligo except that the middle nucleotide (13th of 25) is intentionally replaced by its complementary nucleotide.

• The scanned result for a given gene is the average differences between PM and MM signals, over probes

Different Probe Pairs Represent Different Parts of the Same Gene

gene sequence

Probes are selected to be specific to the target geneand have good hybridization characteristics.

A Probe Set for Measuring Expression Level of a Particular Gene

probepair

probecell

gene sequence...TGCAATGGGTCAGAAGGACTCCTATGTGCCT...AATGGGTCAGAAGGACTCCTATGTGAATGGGTCAGAACGACTCCTATGTG

perfect match sequencemismatch sequence

probe set

Affymetrix Chip

The photolithographic method

• Treat substrate with chemically protected “linker” molecules, creating rectangular array

• Selectively expose array sites to light deprotects exposed molecules, activating further synthesis

• Flush chip surface with solution of protected A,C,G,T

• Binding occurs at previously deprotected sites• Repeat steps 2&3 until desired probes are

synthesized

Photolithography

The mask only allows light to pass to specific features on the chip

Photolithography

Affymetrix chip• Photolithographic Approach

• In-situ synthesis of oligonucleotide

Photolithographic Approach

Patrick Schmid 50

Affymetrix Arrays

Affymetrix GeneChips

The black features represent no intensity (no RNA hybridized to the respective probe in the feature).

The intensity level from lowest to highest by color is: Dark blue -> Blue -> Light Blue -> Green -> Yellow -> Orange -> Red - > White.

More intensity means more RNA bound to a specific feature, which basically means the gene was expressed at a higher level.

Affymetrix GeneChip experiment

• labeled cRNA randomly fragmented in to pieces anywhere from 30 to 400 base pairs in length

• The fragmented, Biotin-labeled cRNA is added to the array

• Anywhere on the array where a RNA fragment and a probe are complimentary, the RNA hybridizes to the probes in the feature.

• The array is then washed to remove any RNA that is not stuck to an array then stained with the fluorescent molecule that sticks to Biotin (Cy5 conjugated to streptavidin)

• Lastly, the entire array is scanned with a laser and the information is kept in a computer for quantitative analysis of what genes were expressed and at what approximate level

in-situ synthesised arrays• The different methods for deprotection lead to the

three main technologies for making in-situ synthesised arrays:

• Photodeprotection using masks: this is the basis of the Affymetrix® technology.

• Photodeprotection without masks: this is the method used by Nimblegen and Febit.

• Chemical deprotection with synthesis via inkjet technology: this is the method used by Rosetta, Agilent and Oxford Gene Technology.

Photodeprotection without masks

Maskless Array Synthesis

NimbleGen Arrays

Microarray Experiment

Cancer and Microarray

* Measuring levels of gene expression

* Creating diagnostic tests to predict whether a patient has a genetic predisposition to obesity

* Designing Drugs

Gene expression and obesity

Reading an array (cont.)

Block Column Row Gene Name Red Green Red:Green Ratio

1 1 1 tub1 2,345 2,467 0.95

1 1 2 tub2 3,589 2,158 1.66

1 1 3 sec1 4,109 1,469 2.80

1 1 4 sec2 1,500 3,589 0.42

1 1 5 sec3 1,246 1,258 0.99

1 1 6 act1 1,937 2,104 0.92

1 1 7 act2 2,561 1,562 1.64

1 1 8 fus1 2,962 3,012 0.98

1 1 9 idp2 3,585 1,209 2.97

1 1 10 idp1 2,796 1,005 2.78

1 1 11 idh1 2,170 4,245 0.51

1 1 12 idh2 1,896 2,996 0.63

1 1 13 erd1 1,023 3,354 0.31

1 1 14 erd2 1,698 2,896 0.59

Campbell & Heyer, 2003

Color Coding

• Tables are difficult to read

• Data is presented with a color scale

• Coding scheme:– Green = repressed (less mRNA) gene in experiment

– Red = induced (more mRNA) gene in experiment

– Black = no change (1:1 ratio)

• Or– Green = control condition (e.g. aerobic)

– Red = experimental condition (e.g. anaerobic)

• We only use ratio

Clustering of example

Clustering of entire yeast genome

SMD Database

Microarray software

Microarray databases and tools

Microarray tools

• NetAffix Analysis center from affymetrix Array content information Probe sequences Gene annotations

• Xcluster- tool for cluster analysis

• GENECLUSTER

• TIGR Microarray MADAM-Microarray data manager SPOTFINDER-image processing tool MIDAS-Microarray data analysis system MEV-MultiExperiment Viewer

ARRAY EXPRESS (EBI)

GEO(NCBI)

microarray

g g c t c t t

g acctg

g ttctg

g sample

g cgata

t acggg

c ggctt

c atcta

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