DNA Microarrays Ms.ruchi yadav lecturer amity institute of biotechnology amity university lucknow(up)
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
Uses and Applications
Microarrays: Universal Biochemistry Platforms Peptides Proteins Carbohydrates Lipids Small molecules DNA
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.)
Biological Samples in 2D Arrays on Membranes or Glass Slides
Cheung et al. 1999
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
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.
Experiment and Data Acquisition
Sample preparation and labelling
I. Target and probe preparation
There are many ways to obtain a labeled target sample. ...GGCUUAAUGAGCCUUAAAAAA...A mRNA TTTTTT...T viral enzyme reverse transcriptase recognizes poly-T bound to poly-A and begins to add complementary DNA nucleotides. The C nucleotides are dyed. A A A G G C T C T T A A G C C ... poly-A tail cDNA target poly-T primer
Hybridization and Data Analysis
Spotted Array Synthesis.
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
DNA Samples on 96 well plates
The PixSys 5500 Arraying Robot (Cartesian Technologies) Vacuum wash station The print head holds up to 32 pins in a 8x4 format Vacuum hold-down platform (50 slide capacity) Robotic arm
Non Contact Printing InkJet (HP/Canon) technology • 1 drop = 100 picolitres
Spotting the Probes on the Microarray 8 X 4 Print Head microarray 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.
Oligonucleotide (20~80-mer oligos) is synthesized either in situ (on-chip)
Developed at Affymetrix, Inc. , under the GeneChip® trademark
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
– Mismatch (MM, one base change) ATG… T …TGC
A MM oligo is identical to a PM oligo except that the middle nucleotide (13 th 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 gene and have good hybridization characteristics.
A Probe Set for Measuring Expression Level of a Particular Gene probe pair gene sequence ...TGCAATGGGTCAGAA G GACTCCTATGTGCCT... AATGGGTCAGAA G GACTCCTATGTG AATGGGTCAGAA C GACTCCTATGTG perfect match sequence mismatch sequence probe set probe cell
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
In-situ synthesis of oligonucleotide
Patrick Schmid Affymetrix Arrays
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
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
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