TECHNIQUES INVOVED IN PROTEOMICS,GENOMICS,TR

ANSCRIPTOMICS…….AIN US SABA

15

BASIC PRINCIPLE OF ALL OMICs GENOMICS TRANSCRIPTOMICS PROTEOMICS METABLOMICS APPLICATIONS CONCLUSION

OUTLINE

BASIC PRINCIPLE OF OMICS

The actual term 'genomics' is thought to have been coined by Dr. Tom Roderick

Genomics is study of the genomes of organisms. determine

◦ the entire DNA sequence of organisms◦ fine-scale genetic mapping◦ studies of intragenomic phenomena

aim of this genetic◦ pathway◦ functional information analysis response to the entire

genome's networks

GENOMICS

The first genomes to be sequenced, a virus and a mitochondrion, and were done by Fred Sanger.

In 1972, Walter Fiers and his team were the first to determine the gene for Bacteriophage MS2 coat protein.

The first free-living organism to be sequenced was that of Haemophilus influenzae (1.8 Mb) in 1995

As of September 2007, the complete sequence was known of about ◦ 1879 viruses◦ 577 bacterial ◦ 23 eukaryote

A rough draft of the human genome was completed by the Human Genome Project in early 2001

HISTORY

GENOMICS

NITROGENOMICS

PERSONAL GENOMICS

METAGENOMICS

WHOLE GENOME SEQUENCING

BAC to BAC Sequencing Whole Genome Shotgun Sequencing

Whole genome sequencingBAC to BAC Sequencing Shotgun Sequencing

cutting the chromosomes into large pieces,physical mapping

Several copies of the genome are randomly cut into pieces

Each fragments is inserted into a BAC-a bacterial artificial chromosome.

These pieces are fingerprinted to give each piece a unique identification tag that determines the order of the fragments.

Making M13 library &sequencing These sequences are fed into PHRAP

that looks for common sequences that join two fragments together.

Multiple copies of the genome are randomly shredded into pieces

Each 2,000 and 10,000 bp fragment is inserted into a plasmid

Both the 2,000 and the 10,000 bp plasmid libraries are sequenced.

Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each chromosome.

 

The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.

Study of transcriptome is called transcriptomics.

Techniques Expression profiling DNA microarrays SAGE(serial analysis of gene expression)

Trancriptomics

The term "proteomics“ was first coined in 1997 to make an analogy with genomics "proteome" is a blend of "protein" and

"genome“ coined by Marc Wilkins in 1994 Proteomics is the large-scale study

of proteins  structures  functions.

PROTEOMICS

Theproteome ◦ is the entire complement of proteins◦  including the modifications made to a particular

set of proteins This will vary with

◦ time ◦ distinct requirements ◦ or stresses, that a cell or organism undergoes.

PROTEOME

proteomics is considered the next step in the study of biological systems.

It is much more complicated than genomics organism's genome is more or less constant  proteome differs from cell to cell and from time to time. done by mRNA analysis, correlate with protein content now known that mRNA is not always translated into

protein SO,Proteomics confirms the presence of the protein and

provides a direct measure of the quantity present. PTM

COMPLEXITY OF PROBLEM

19

Gel Electrophoresis◦ Isoelectric point◦ Molecular weight

Liquid Chromatography

Fluorescence Staining

Affinity capture◦ Phosphorylation

Protein Binding ◦ Receptors◦ Complexes

Mass Spectrometry◦ Accurate

molecular weight

Protein Chemistry Assay Techniques

develop an antibody which is specific to that modification.

For glycosylation lectins  two-dimensional gel electrophoresis“  PROTOMAP which combines  SDS-PAGE with shotgun proteomics

TECHNIQUES

AFFINITY CAPTURE

1) Antibodies are adding to the protein mixture

2) Antibodies bind to proteins that have modified

3) Proteins of interest can be separated based on the modification.

23

Protein separation◦ Molecular weight

(Mw)◦ Isoelectric point (pI)

Staining

Birds-eye view of protein abundance

2D Gel-Electrophoresis

PRotein TOpography and Migration Analysis Platform

Is combination of SDS-PAGE with shotgun proteomics is performed by

◦ resolving control and experimental samples in separate lanes of a 1D SDS-PAGE gel

◦ proteins in these bands are sequenced using shotgun proteomics. ◦ Sequence information from all of these bands

are bioinformatically integrated into a visual format called a peptograph 

◦ which plots gel-migration in the vertical dimension (high- to low-molecular weight, top to bottom)

◦ sequence coverage in the horizontal dimension (N- to C-terminus, left to right).

 PROTOMAP

27

Mass Spectrometer

Ionizer

Sample

+_

Mass Analyzer Detector

MALDIElectro-Spray

Ionization (ESI)

Time-Of-Flight (TOF)QuadrapoleIon-Trap

ElectronMultiplier(EM)

28

LC/MS for Peptide Abundance

Enzymatic DigestandFractionation

29

LC/MS for Peptide Abundance

LC/MS: 1 MS spectrum every 1-2 seconds

MassSpectrometry

Liquid Chromatography

30

The NMR Spectrometer

=>

Newborn cousin to genomics and proteomics study of chemical processes involving

metabolites. "systematic study of the unique chemical

fingerprints that specific cellular processes leave behind

The metabolome ◦ represents the collection of all metabolites in a

biological cell, tissue, organ or organism, which are the end products of cellular processes

◦ metabolic profiling can give an instantaneous snapshot of the physiology of that cell.

METABLOMICS

In 2005, first metabolite database(called METLIN) In January 2007, scientists at the University of

Alberta and the University of Calgary completed the first draft of the human metabolome.

They catalogued approximately ◦ 2500 metabolites,◦ 1200 drugs ◦ 3500 food components

This information, available at the Human Metabolome Database ◦ www.hmdb.ca

over 50,000 metabolites have been characterized from the plant kingdom

HISTORY

TECHNIQUES

GC MS NMR HPLC RHPLC

Toxicology Nutrition Medical profiling Pharmacology Diagnostics……….

APPLICATIONS

High-dimensional biology (HDB) refers to the simultaneous study in health and disease

The fundamental premise is that the evolutionary complexity of biological systems renders them difficult to comprehensively understand using only a reductionist approach.

Such complexity can become tractable with the use of "omics" research.

This term refers to the study of entities in aggregate. The two major advances that have made HDB possible are

technological breakthroughs that allow simultaneous examination of thousands of genes, transcripts, and proteins, etc., with high-throughput techniques and analytical tools to extract information.

CONCLUSION