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 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 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

GENOMICS NITROGENOMICS PERSONAL GENOMICS METAGENOMICS

WHOLE GENOME SEQUENCING BAC to BAC Sequencing Whole Genome Shotgun Sequencing

Whole genome sequencing BAC to BAC Sequencing Shotgun Sequencing 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. 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.

 

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 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 Theproteome This will vary with 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 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

Protein Chemistry Assay Techniques Gel Electrophoresis Isoelectric point Molecular weight Liquid Chromatography Fluorescence Staining Affinity capture Phosphorylation Protein Binding Receptors Complexes Mass Spectrometry Accurate molecular weight

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 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 Protein separation Molecular weight (Mw) Isoelectric point (pI) Staining Birds-eye view of protein abundance

PROTOMAP 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).

Mass Spectrometer Ionizer Sample Mass Analyzer Detector MALDI + _ Mass Analyzer Detector MALDI Electro-Spray Ionization (ESI) Time-Of-Flight (TOF) Quadrapole Ion-Trap Electron Multiplier (EM)

LC/MS for Peptide Abundance Enzymatic Digest and Fractionation

LC/MS for Peptide Abundance Liquid Chromatography Mass Spectrometry LC/MS: 1 MS spectrum every 1-2 seconds

The NMR Spectrometer =>

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.

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

TECHNIQUES GC MS NMR HPLC RHPLC

APPLICATIONS Toxicology Nutrition Medical profiling Pharmacology Diagnostics……….

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.