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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
mRNA Analysis Methods
Northern Blot (Single Gene analysis)
Microarray Technology (Genome Wide Experiment)
•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)
Number of Genes in Different Organisms
Yeast ~ 6200 genes E. coli ~ 4200 genes
Human ~ 30,000 genes Mouse ~ 30,000 genes
Phage T4 ~ 200 genes Influenza ~ 12 genes
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
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
Number of Genes in Different Organisms
Yeast ~ 6200 genes E. coli ~ 4200 genes
Human ~ 30,000 genes Mouse ~ 30,000 genes
Phage T4 ~ 200 genes Influenza ~ 12 genes
Spotted microarrays
www.biorobotics.com
Robotic Printing
Liquid HandlingResuspension of oligos
www.qiageninstruments.com
Overview of fabrication of spotted microarrays
Introduction
Need to achieve two things:
(i)Immobilize thousands of probes specific for individual genes
(ii)Label mRNA populations
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
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=cy5yellow cy3=cy5
red cy5>cy3red cy5>cy3
green cy3>cy5green cy3>cy5
EGFP KDEGFP
cDNA “Two Color Chips”
Tar
get p
repa
rati
on
Arr
ay p
repa
rati
on
summary
This is the kind of thing you will see in YOUR microarray experiments
What’s happening at each spot?
cDNA Chip vs. Northern Blot
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!!
ABH
ABH2
ABH3
ADPRT
ADPRTL2
ADPRTL3
APEX
APEXL2
ATM
ATR
BID
BLM
BRCA1
BRCA2
BTG1
CASP2
CASP3
CASP8
CASP9
CCNH
CDK2
CDK4
CDK6
CDK7
CDK8
CETN2
DCLRE1A
DDB1
DDB2
DMC1
DUT
EGFP
ENDOG
ERCC1
ERCC2
ERCC3
ERCC4
ERCC5
EXO1
FANCA
FANCC
FANCE
FANCF
FEN1
FOS
ERCC6
G22P1
GADD45A
GADD45B
GADD45G
GTF2H4
HAP1
HCNP
HSU24186
HUS1
JUN
LIG1
LIG3
LIG4
MAD2L2
POLQ
PRKDC
PRSS25
RAD1
RAD17
RAD18
RAD23A
RAD23B
RAD50
RAD51
RAD51C
RAD51L1
RAD51L3
RAD52
RAD54B
RAD54L
RAD9
RECQL4
REV1L
alkB homolog
Breast cancer 1
Excision repair
Growth arrest and DNA-damage-inducible
No homology toHuman sequences
Alien DNA
28S
Liver
18S
28S
6 kb
4 kb
2 kb
1 kb
0.5 kb
0.2 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