what is a dna microarray?web.mit.edu/beh.109/www/module3/lectureslides/be109... · what is a dna...
TRANSCRIPT
![Page 1: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/1.jpg)
1
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
![Page 2: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/2.jpg)
2
mRNA Analysis Methods
Northern Blot (Single Gene analysis)
Microarray Technology (Genome Wide Experiment)
![Page 3: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/3.jpg)
3
•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)
![Page 4: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/4.jpg)
4
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
![Page 5: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/5.jpg)
5
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
![Page 6: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/6.jpg)
6
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
![Page 7: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/7.jpg)
7
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
![Page 8: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/8.jpg)
8
Need to achieve two things:
(i)Immobilize thousands of probes specific for individual genes
(ii)Label mRNA populations
![Page 9: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/9.jpg)
9
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
![Page 10: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/10.jpg)
10
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
![Page 11: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/11.jpg)
11
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
![Page 12: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/12.jpg)
12
What’s happening at each spot?
cDNA Chip vs. Northern Blot
![Page 13: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/13.jpg)
13
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!!
![Page 14: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/14.jpg)
14
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
![Page 15: What is a DNA Microarray?web.mit.edu/beh.109/www/Module3/lectureslides/be109... · What is a DNA Microarray? genes or gene fragments attached to a substrate (glass) Hybridized slide](https://reader030.vdocuments.us/reader030/viewer/2022041017/5ec9bf218102227f5b779327/html5/thumbnails/15.jpg)
15
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