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An Introduction to DNA microarrays

Rebecca Fry, Ph.D.(and Leona Samson)

http://www.buffalo.edu/UBT/UBT

What is a DNA Microarray?

genes or gene fragments attached to a substrate (glass)

Hybridized slide

Two dyes

Image analyzed

Tens of thousands of spots/genes

=entire genome in 1 experiment

A Revolution in Biology

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mRNA Analysis Methods

Northern Blot (Single Gene analysis)

Microarray Technology (Genome Wide Experiment)

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•mRNAs separated on gel according to size•mRNAs transferred to a membrane and hybridized with small number (1-5) of radioactively labeled DNA probes. •Probe corresponds to gene of interest•Target RNA is spatially fixed and the labeled probe is in solution •Low throughput

NORTHERN BLOTS

i.e., your cloned gene(s)

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Number of Genes in Different Organisms

Yeast ~ 6200 genes E. coli ~ 4200 genes

Human ~ 30,000 genes Mouse ~ 30,000 genes

Phage T4 ~ 200 genesInfluenza ~ 12 genes

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i.e., your cloned gene(s)

We could just 35,000 Northern

to monitor expression of all

genes!!!

Northern BlotsImmobilized mRNA population hybridized with labeled probe representing one gene

DNA MicroarraysImmobilized probes hybridized with labeled mRNA population representing all expressed genes

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Need to achieve two things:

(i)Immobilize thousands of probes specific for individual genes

(ii)Label mRNA populations

EGFP ORF

1 846

ATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGCTTAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGA

Designing Oligo Probes

70 mer oligo

specific to gene

of interest

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Number of Genes in Different Organisms

Yeast ~ 6200 genes E. coli ~ 4200 genes

Human ~ 30,000 genes Mouse ~ 30,000 genes

Phage T4 ~ 200 genesInfluenza ~ 12 genes

Spotted microarrays

www.biorobotics.com

Robotic Printing

Liquid HandlingResuspension of oligos

www.qiageninstruments.com

Overview of fabrication of spotted microarrays

Introduction

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Need to achieve two things:

(i)Immobilize thousands of probes specific for individual genes

(ii)Label mRNA populations

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562 nm

cy3

cy5

www.amersham.com

cy3 cy5

Differential dye incorporationcy5 less well than cy3Light sensitivity: cy5 more easily degradedProtect your reactions from light!!

664 nm

510 nm

emission

emission

Introduction

cy3 and cy5: Commonly used dyes

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www.genetics.ucla.edu

Labeled cDNA preparation

RNA

Sample 2

Spotted MicroarrayTarget preparation

Reverse transcriptionFlourescent dyes

Introduction

EGFP KD

Combined in equal amounts

Spotted microarray target preparationDirect labeling

cy5cy3

cDNAcDNA

EGFP

Co-hybridized to array

www.genetics.ucla.edu

Labeled cDNA preparation

RNA

Sample 2

Spotted MicroarrayTarget preparation

Reverse transcriptionFlourescent dyes

Introduction

Combined in equal amounts

Spotted microarray target preparationDirect labeling

cy5cy3

cDNAcDNA

Co-hybridized to array

yellow cy3=cy5

red cy5>cy3green cy3>cy5

EGFP KDEGFP

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cDNA “Two Color Chips”

Targ

et p

repa

ratio

n

Arr

ay p

repa

ratio

nsummary

This is the kind of thing you will see in YOUR microarray experiments

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What’s happening at each spot?

cDNA Chip vs. Northern Blot

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Two Popular Microarraying Platforms

www.molgen.mpg.de

Spotted microarrays

cDNA: PCR products (500-1,000bp)synthesized oligos (70 mer)

>10,000 probes

Affymetrix“Gene Chip”500,000 probes25 mer(represents a fragment of a gene)

Introduction

Commercially available Oligo microarray

www.the-scientist.com

So..what gene probes are represented on the array you

will use??

EGFP, p53, EXO1, AAG, ATM, and ATR….

But we added in a bunch more!!

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BRCA2

BRCA1

BLM

BID

ATR

ATM

APEXL2

APEX

ADPRTL3

ADPRTL2

ADPRT

ABH3

ABH2

ABH

DDB1

DCLRE1A

CETN2

CDK8

CDK7

CDK6

CDK4

CDK2

CCNH

CASP9

CASP8

CASP3

CASP2

BTG1

EXO1

FANCA

FANCC

FANCE

FANCF

ERCC5

ERCC4

ERCC3

ERCC2

ERCC1

ENDOG

EGFP

DUT

DMC1

MAD2L2

LIG4

LIG3

LIG1

JUN

HUS1

HSU24186

HCNP

HAP1

GTF2H4

GADD45G

GADD45B

GADD45A

G22P1 POLQ

PRKDC

PRSS25

RAD1

RAD17

RAD18

RAD23A

RAD23B

RAD50

RAD51

RAD51C

RAD51L1

RAD51L3

RAD52

RAD54B

alkB homolog

Breast cancer 1

Excision repair

Growth arrest and DNA-damage-inducible

No homology toHuman sequences

Alien DNA

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28S

Liver

18S

28S

6 kb

4 kb

2 kb

1 kb0.5 kb0.2 kb

Electropherogram (28S/18S Ratio~2)

18S

28S

Fluo

resc

ence

Time (seconds)

0

10

20

30

40

50

60

70

19 24 29 34 39 44 49 54 59 64 69

Sample 1 Ladder Sample 2

RNA quality control

Lad, 1,2

Gel Image (in silico) Sharp, Clear Bands

Pre-labeling quality control:

Determine RNA Quality Agilent Bioanalyzer: 50-500 ngNo more formaldehyde gels!!

Microarray Measurements

Scanner Image Analysis .txt or .xls file

Image Analysis: Spotted arrays