Definition (1)- Hieter & Boguski
The development & application of global
• Genome-wide or
• System-wide experimental approaches to assess gene function by making
use of the information & reagents provides by structural genomics.
It is characterized by high-throughput or large scale
• Combined with statistical or computational analysis of the results.
Definition (2) – UC Davis Genome
A means of assessing phenotype
differs from more classical approaches primarily with respect to
The scale & automation of biological investigations
A classical investigation works only fxal genomics can examine
On a single gene. Over 1k-10k genes
Which genes are differentially expressed?
Which genes are co-expressed?
Which genes interact
Which genes show alternative splicing?
Which cancer has Patient X?
Is my drug safe for PatientY?
Which treatment is the most efficient?
An integrated system is needed for
DNA : Protein
Protein : Protein
High Throughput Screening
Loss / Gain of Function
Quantitative Trait Loci
Database backed storage.
Specialized signal processing algorithms.
Dedicated analysis environment.
Additional knowledge about genes and transcripts
PCR based methods.
DNA microarray analysis.
Polymerase Chain Reaction
• Developed in 1983 by Kary Mullis ,a
biochemical technology in
molecular biology used to amplify a
single or a few copies of a piece of
DNA across several orders of
magnitude, generating thousands
to millions of copies of a particular
• DNA cloning for sequencing.
• DNA-based phylogeny, or functional analysis of genes.
• Diagnosis of hereditary diseases.
• Identification of genetic fingerprints.
• PCR can be extensively modified to perform a wide array of
genetic manipulations & hence may replace gene cloning
• A DNA microarray (aka DNA chip or biochip) is a
collection of microscopic DNA spots attached to a
• can be used to detect DNA or RNA that may or
may not be translated into proteins.
• Expression Analysis/expression profiling which is a
process of measuring gene expression via cDNA
• internationally adopted standard for the
Minimal Information About a Microarray Experiment.
• The result of an MGED (www.mged.org) driven effort to
codify the description of a microarray experiment.
• Ultimately, it tries to specify the collection of information
that would be needed to allow somebody to completely
reproduce an experiment that was performed elsewhere.
Six Parts of MIAME
1. Experimental design: the set of hybridization
experiments as a whole
2. Array design: each array used and each element
(spot, feature) on the array
3. Samples: samples used, extract preparation and
4. Hybridizations: procedures and parameters
5. Measurements: images, quantification and
6. Normalization controls: types, values and
• hybridization between two DNA strands.
• A high number of complementary base pairs in a nucleotide sequence
means tighter non-covalent bonding between the two strands.
• Fluorescently labelled target sequences are used that bind to a probe
sequence generate a signal that depends on the hybridization
conditions, and washing after hybridization.
• Total strength of the signal, from a spot, depends upon the amount of
target sample binding to the probes present on that spot.
• Microarrays use relative quantitation in which the intensity of a feature
is compared to the intensity of the same feature under a different
condition, and the identity of the feature is known by its position.
• In standard microarrays, the probes
are synthesized and then attached
via surface engineering to a solid
surface by a covalent bond to a
• The solid surface can be glass or a
silicon chip, in which case they are
colloquially known as an Affy-chip
when an Affymetrix chip is used.
• Other microarray platforms, such as
Illumina, use microscopic beads,
instead of the large solid support.
Scanning of arrays
• Laser scanners
• Excellent spatial resolution
• Good sensitivity, but can bleach
• Although slow
• Charged couple device
• Spatial resolution can be a problem
• Sensitivity easily adjustable
• Faster and cheaper than lasers
• Cell growth in different environments,
• Isolate RNA cDNAs
• Measure expression using array technology
• Create database of expression information
• Data Analysis
• Display information in an easy to-use format
• Show ratio of expression under
• Different conditions Affymetrix® food chip
• microRNA detection
• Comparative Genomic Hybridization (CGH)
detects deletions or amplifications of genomic sequence
• ChIP on chip
• Single Nucleotide Polymorphism screening (SNP)
measures an individual’s genotype at known sites of variance
• Cell Arrays
• Protein Arrays
• Tissue Arrays
Other microarray-based assays
• Image analysis..
• Translate the scan into
• Check for defects.
• Quality metrics provided by
Bioinformatics of Microarrays
• Array design: choice of sequences
to be used as probes
• Analysis of scanned images
• Spot detection, normalization,
Primary analysis of hybridization
• Basic statistics, reproducibility, data scattering, etc.
• Comparison of multiple samples
• Clustering, SOMs (Self-Organizing Maps (a subtype of artificial
neural network, low-dimensional views of high-dimensional
• Unsupervised learning
• Sample tracking and data basing of results.
Benefits of using a data repository
Facilitates data sharing
Catalogued / Backed-up
Pervasive advertisement for your work
Access to data for analysis and
Improves search capabilities
Encourages development of more
capable software for annotation,
analysis and submission
• The sheer volume of data, specialized formats (such as
MIAME), and curtain efforts associated with the datasets
require specialized databases to store the data.
• Makes it easy to compare 2 Data files.
• Easy to access.
• User friendly.
• Worldwide avalabilility
Microarray Data on the Web
• Many groups have made their raw data available, but in
• Some groups have created searchable databases
• There are several initiatives to create “unified” databases
• EBI: Array Express
• NCBI: Gene Expression Omnibus
Companies are beginning to sell microarray expression data
Which software should I use??
Commercial vs. Open Source
Ease of Use
GeneSpring > maxdView > R/BioConductor
Fine tuned control
R/BioConductor > maxdView > GeneSpring
Quality Control Normalisation Analysis Presentation
Study of the proteome.
The proteome is the complete complement of proteins found
in a complete genome or specific tissue.
Proteomics and genomics are inter-dependent
Primary Protein products
Functional protein products
Determination of gene
Aims of Proteomics
• Detects the different proteins expressed by tissue,
cell culture, or organism using 2-Dimensional Gel
• Stores the information in a database
• Compares expression profiles between a healthy cell
vs a diseased cell
• The data comparison can then be used for testing
and rational drug design.
• Motion of charged molecules in an electric field.
• Polyacrylamide gel provides a porous matrix
• (PAGE – Polyacrylamide Gel Electrophoresis)
• Sample is stained with comassie blue to make it
visible in the gel.
• Sample placed in wells on the gel.
2D – Separation is based on size and
• First step is to separate based on charge or isoelectric point, called
• Then separate based on size (SDS-PAGE).
• Second Dimension.
• Separation by size.
• Run perpendicular to Isoelectric focusing.
• The only unresolved proteins after the first and
second dimensions are those proteins with the
same size and same charge – rare!
2D-PAGE Analysis Software
• 2D-PAGE technology has been in use for over 20 years,
and potentially provides a vast amount of information
about a protein sample.
• However, due to difficulties with data analysis, it remains
only partially exploited.
List of 2-D GEL DATABASES
One can find an extensive list of such databases by following these links.
We would discuss a few “Interesting ones”.
•World 2-D PAGE
•Ludwig Institute of Cancer Research
Try these links
and then Time to chill!!!
• Mass spectrometry (Mass Spec or MS) uses high energy
electrons to break a molecule into fragments.
• Separation and analysis of the fragments provides
• Molecular weight
• Mass spectrum of 2-methylpentane
• Concept of genetics –Klug & Cummings, 10th edition
• Perou, C.M., Sorlie,T., Eisen, M.B., van de Rijn, M., Jeffrey, S.S., Rees, C.A., Pollack, J.R.,
Ross, D.T., Johnsen, H., Akslen, L.A., Fluge, O., Pergamenschikov,A., Williams, C., Zhu,
S.X., Lonning, P.E., Borresen-Dale, A.L., Brown, P.O., Bolstein, D. 2000. Molecular
portraits of human breast tumors. Nature 406(6797):747-752.
• What is functional genomics?
• What are the implications of functional genomics?
• Name diff. tech. involved in transcriptome analysis?
• Explain exploitation of DNA microarray in hybridization tech.?
• How microarray database is different from microarray analysis?
• What are the different tools for microarray analysis?
• What is the need of microarray analysis?
• Define Proteomics?
• Explain various tech. involved in protein separation?