Date post: | 11-May-2015 |
Category: |
Education |
Upload: | ruchibioinfo |
View: | 5,748 times |
Download: | 3 times |
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
2
3
4
5
Gene Expression
Patrick Schmid 6
Uses and Applications
7
Microarrays:Universal Biochemistry Platforms
8
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?
9
What is a DNA Microarray? (cont.)
10
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
11
MICROARRAY TYPES
12
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.
13
Microarray Steps
• Experiment and Data Acquisition– Sample preparation and labelling
– Hybridisation
– Washing
– Image acquisition
• Data normalization
• Data analysis
• Biological interpretation
14
I. Target and probe preparation
15
16
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
17
Spotted Array Synthesis.
18
Microarray Experiment
19
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
20
Robotic spotting
21
22
DNA Samples on 96 well plates
23
24
24
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
25
Non Contact Printing
26
InkJet (HP/Canon) technology
• 1 drop = 100 picolitres
27
28
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.
29
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
??????????
??????????
????????????????????
??????????
??????????
????
????
??
????
????
??
??????????
??????????
Sample 1
Sample 2
Microarray Slide
Spots(Probes)
UnknownmRNASequences(Target)
30
Extract mRNA
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
??????????
??????????
????????????????????
??????????
??????????
????
????
??
????
????
??
??????????
??????????
Sample 1
Sample 2
31
Convert to cDNA and Label with Fluorescent Dyes
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
Sample 1
Sample 2
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
Sample 1
Sample 2
32
Mix Labeled cDNA
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
Sample 1
Sample 2??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
33
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
Sample 1
Sample 2
Hybridize cDNA to the Slide
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
??????????
34
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
Sample 1
Sample 2
Excite Dyes with Laser
?????????? ??????????
?????????? ???????????????????? ???????????????????? ??????????
?????????? ??????????
?????????? ???????????????????? ?????????? ?????????? ??????????
?????????? ??????????
?????????? ??????????
35
ACCTG...GACCTG...GACCTG...G
TTCTG...ATTCTG...ATTCTG...A
GGCTT...CGGCTT...CGGCTT...C
ATCTA...AATCTA...AATCTA...A
ACGGG...TACGGG...TACGGG...T
CGATA...GCGATA...GCGATA...G
Sample 1
Sample 2
Scan
?????????? ??????????
?????????? ???????????????????? ???????????????????? ??????????
?????????? ??????????
?????????? ???????????????????? ?????????? ?????????? ??????????
?????????? ??????????
?????????? ??????????
36
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
38
39
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
40
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
41
42
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.
43
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
44
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
45
Photolithography
46
The mask only allows light to pass to specific features on the chip
Photolithography
47
Affymetrix chip• Photolithographic Approach
• In-situ synthesis of oligonucleotide
48
Photolithographic Approach
49
Patrick Schmid 50
Affymetrix Arrays
Affymetrix GeneChips
51
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
52
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
53
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.
54
Photodeprotection without masks
55
Maskless Array Synthesis
56
57
NimbleGen Arrays
Microarray Experiment
58
Cancer and Microarray
59
* Measuring levels of gene expression
* Creating diagnostic tests to predict whether a patient has a genetic predisposition to obesity
* Designing Drugs
60
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
61
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
62
Campbell & Heyer, 2003
Clustering of example
63
Campbell & Heyer, 2003
Clustering of entire yeast genome
64
Campbell & Heyer, 2003
SMD Database
65
Microarray software
66
Microarray databases and tools
67
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
68
ARRAY EXPRESS (EBI)
69
GEO(NCBI)
70