To modifying the structure of a specific gene. Gene targeting vector introduced into the cell. Vector modifies the normal chromosomal gene through homologous recombination. Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy. Treating human diseases by genetic approaches – Gene Therapy. Gene Therapy – Replacing the defective gene by normal copy of the gene. Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna. EST is a short sub-sequence of a cDNA sequence. Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination. Approximately 74.2 million ESTs are available in public databases. EST results from one-short sequencing of a cloned cDNA. Low-quality fragments. Length is approximately 500 to 800 nucleotides.

1. GENE TARGETING AND
SEQUENCE TAGS
ALEN SHAJI
P1914015

2. GENE TARGETING
 To modifying the structure of a specific gene.
 Gene targeting vector introduced into the cell.
 Vector modifies the normal chromosomal gene through homologous
recombination.

3. HOMOLOGOUS RECOMBINATION
 Occurs within the chromosome.
 One piece of DNA to be exchanged for another piece.
 Cellular mechanism.
 DNA undergoing recombination be almost identical/homologous in sequence.
 Sequence on either side of the target should be identical, to promote more
efficient targeting and recombination.
 Modification of more or less any gene of interest, in more or less any way
desired.

4.  Mutating specific genes
 Knock out specific genes.
 Knock in specific genes – Introduction of novel pieces of DNA into a specific
chromosomal location – To examine the function of a gene in a variety of
ways.
 By changing single genes, it is possible to remove or alter large pieces of
chromosomes.
 Genes can be removed from a particular tissue.

7. THERAPEUTIC POTENTIAL OF GENE
TARGETING
 Useful in treating some human genetic disorders – Hemophilia, Duchenne
Muscular Dystrophy.
 Treating human diseases by genetic approaches – Gene Therapy.
 Gene Therapy – Replacing the defective gene by normal copy of the gene.

8. AWARDS
 Mario R. Capecchi, Martin. J. Evans and Oliver Smithies were awarded the
2007 Nobel Prize in Physiology and Medicine.
 Work – “Principles for introducing specific gene modifications in mice by the
use of embryonic stem cells”, or gene targeting.

9. SEQUENCE TAG
 Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp
long, of a complimentary DNA/cDNA.
 EST is a short sub-sequence of a cDNA sequence.
 Used to identify gene transcripts, and are instrumental in gene discovery and
in gene-sequence determination.
 Approximately 74.2 million ESTs are available in public databases.
 EST results from one-short sequencing of a cloned cDNA.
 Low-quality fragments.
 Length is approximately 500 to 800 nucleotides.

10.  Complementary to mRNA.
 ESTs represents portions of expressed genes.
 Represented in databases as either cDNA/mRNA sequence or as the reverse
complement of the mRNA, the template strand.
 One can map ESTs to specific chromosome locations using physical mapping
techniques – Radiation hybrid mapping, Happy mapping, or FISH.
 Alternatively, if the genome of the organism that originated the EST has been
sequenced, one can align the EST sequence to that genome using a computer.

11.  The current understanding of the human set of genes includes the existence
of thousands of genes based solely on EST evidence.
 ESTs have become a tool to refine the predicted transcripts for those genes,
which leads to the prediction of their protein products and ultimately of their
function.
 The situation in which those ESTs are obtained gives information on the
conditions in which the corresponding gene is acting.
 ESTSs contain enough information to permit the design of precise probes for
DNA microarrays that then can be used to determine gene expression profiles.

12. REFERENCE
 Joyner, A., ed. Gene Targeting; A Practical Approach, 2nd ed. New York; Oxford
University; Press, 1999.
 Torres, R.M., and R. Kuhn. Laboratory Protocols for Conditional Gene
Targeting; New York; Oxford University; Press, 1997.
 Campagne f, Skrabanek L; Bioinformatics; 2006.
 Christoffels A, van Gelder A, Greyling G, Miller R, Hide W; “stack; Sequence
Tag Alignment and Consensus Knowledgebase”; 2001.

13. THANK YOU