1. Genome sequencing of disease causing microorganism Romana Siddique

2. DNA Sequencing DNA seuencing refers to sequencing methods for detremining the order of the nucleotide bases- adenine, guanine, cytosine and thymine-in a molecule of DNA. The advent of DNA sequencing has significantly accelerated biological research and discovery. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the human genome project.

3. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequence of many animal, plant and microbial genomes (M. tuberculosis, V.cholerae, H.pylori,E.coli, Jute genome)

4. Genome sequencing of microorganism With the advent of high throughput technology the genome of different microorganisms have been seuenced These include among others: 1.Mycobacterium tuberculosis 2.Helicobacter pylori 3.V.holerae O1 4.Salmonella typhi /paratyphi 5.E.coli 6.Bacillus anthracis and fungal pathogem Candida albicans

5. In vivo induced antigen technology (IVIAT) pathogens, which induce or repress specific genes to optimize growth within the host environment during infection. Genes induced specifically during in vivo growth may contribute to the overall pathogenicity of an organism and may be virulence determinants

6. Several techniques to identify in vivo induced gene.  IVET(in vivo expressionTechnology)  signature-tagged mutagenesis (STM)  differential fluorescence induction(DFI)  transcriptional and proteomic profiling

7. IVET IVET is a promoter trap strategy in which random DNA is linked to a reporter gene required for growth in an animal model of infection. This system rests on positive selection to identify genes turned on during in vivo growth. Limitations: the timing and level of in vivo –induced gene expression is critical to identification of genes by IVET.

8. STM STM is a negative selection technique in which a pool of sequence-tagged mutant bacteria is administered to an animal in an appropriate model. Organisms are recovered from a normally sterile site within the animal (e.g., the spleen). Mutant bacteria present in the initial pool but not recovered from the host are lacking in a gene required for in vivo growth and survival.

9. Limitations of STM:  Need for an adequate model that facilitates recovery of bacteria from an infected host.

10. DFI(differential fluorescence induction) DFI requires the segregation of bacterial populations by fluorescence-activated cell sorting. The gene encoding green fluorescent protein (GFP) is placed downstream of a promoter library constructed from a pathogen of interest. Clones containing an active promoter–GFP fusion are separated from clones that lack expression of GFP. Sequentially sorting cells from in vivo and in vitro environments yields cells fluorescent in vivo but not in vitro.

11. A common feature of all of these strategies is that they require an animal model, which may not adequately replicate events during a natural human infection.

12. Transcriptional and proteomic profiling Transcriptional and proteomic profiling may identify genes and gene products expressed in vivo during human infection, recovery of appropriate and sufficient organisms may be problematic for many infections.

13. in vivo induced antigen technology (IVIAT)

17. Limitations of IVIAT Reactivity of identified clones may be secondary to cross-reactivity. Rests solely on the use of convalescent serum; does not detect cellular immune responses. Construction of genomic expression library in E. coli may lead to variable expression resulting from codon bias, restriction site limitations (if library generated using restriction digestion) and toxicity of overexpressed antigens.

18. Advantages of IVIAT