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TECHNIQUES INVOVED IN PROTEOMICS,GENOMICS,TRANSCRIPTOMICS……. AIN US SABA 15. OUTLINE. BASIC PRINCIPLE OF ALL OMICs GENOMICS TRANSCRIPTOMICS PROTEOMICS METABLOMICS APPLICATIONS CONCLUSION. BASIC PRINCIPLE OF OMICS. GENOMICS.

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techniques invoved in proteomics genomics transcriptomics

TECHNIQUES INVOVED IN PROTEOMICS,GENOMICS,TRANSCRIPTOMICS…….

AIN US SABA

15

outline
OUTLINE
  • BASIC PRINCIPLE OF ALL OMICs
  • GENOMICS
  • TRANSCRIPTOMICS
  • PROTEOMICS
  • METABLOMICS
  • APPLICATIONS
  • CONCLUSION
genomics
GENOMICS
  • 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
history
HISTORY
  • 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
whole genome sequencing
Whole genome 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.

BAC to BAC Sequencing

Shotgun Sequencing

trancriptomics
Trancriptomics
  • 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)
proteomics
PROTEOMICS
  • 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.
proteome
PROTEOME
  • 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.
complexity of problem
COMPLEXITY OF PROBLEM
  • 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,Proteomicsconfirms the presence of the protein and provides a direct measure of the quantity present.
  • PTM
protein chemistry assay techniques
Protein Chemistry Assay Techniques
  • Affinity capture
    • Phosphorylation
  • Protein Binding
    • Receptors
    • Complexes
  • Mass Spectrometry
    • Accurate molecular weight
  • Gel Electrophoresis
    • Isoelectric point
    • Molecular weight
  • Liquid Chromatography
  • Fluorescence
  • Staining
techniques
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
affinity capture
AFFINITY CAPTURE
  • Antibodies are adding to the protein mixture
  • Antibodies bind to proteins that have modified
  • Proteins of interest can be separated based on the modification.
2d gel electrophoresis
2D Gel-Electrophoresis
  • Protein separation
    • Molecular weight (Mw)
    • Isoelectric point (pI)
  • Staining
  • Birds-eye view of protein abundance
protomap
PROTOMAP
  • PRoteinTOpography 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).
mass spectrometer

Sample

+

_

Detector

Ionizer

Mass Analyzer

Mass Spectrometer

ElectronMultiplier(EM)

Time-Of-Flight (TOF)

Quadrapole

Ion-Trap

MALDI

Electro-SprayIonization (ESI)

lc ms for peptide abundance1

Liquid Chromatography

LC/MS for Peptide Abundance

Mass

Spectrometry

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

metablomics
METABLOMICS
  • 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.
history1
HISTORY
  • 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
applications
APPLICATIONS
  • Toxicology
  • Nutrition
  • Medical profiling
  • Pharmacology
  • Diagnostics……….
conclusion
CONCLUSION
  • 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.