Express sequence tags
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ESTs are short sequences of DNA derived from cDNA clones that represent gene expression in particular cells or tissues. They provide a simple and inexpensive way to discover new genes and map their positions in genomes. To create an EST, mRNA is converted to cDNA and then sequenced, yielding short expressed DNA sequences. ESTs are deposited in public databases like NCBI's dbEST and can help identify genes, construct genome maps, and characterize expressed genes through clustering, assembly, and mapping to genomic sequences. However, isolating mRNA from some tissues can be difficult and ESTs alone do not indicate the genes they were derived from.
Express sequence tags
- 1. EXPRESS SEQUENCE TAG A Tool In Molecular Biology Dhananjay Desai Student MSc II Dept. of Microbiology N. A. C. & Sc. College, Ahmednagar [email protected]
- 2. Content Expressed sequence tag ?? Use of EST. Tag preparation. cDNA to EST. EST and NCBI. Gene mapping tool. EST format on NCBI site. Limitation. Collection.
- 3. Express Sequence Tag (EST) ??? • ESTs are small pieces of DNA sequence. • Short, single pass sequence read from mRNA. • Bits of DNA sequence , Represent gene express in single cells, tissues or organ from different organisms.
- 4. Use of EST Identify unknown gene and map their position in a genome. Provide simple and inexpensive path for discovering new gene. Genome map construction. Characterization of expressed gene.
- 5. Tag Preparation • It is a conversion of m RNA in to cDNA. • Stability of cDNA. • Expressed DNA sequences only.
- 6. cDNA ------ESTs 5’ EST- sequencing only beginning portion of cDNA. Code for protein. These region tend to conserved and not change within a gene family. 3’ EST- sequencing only ending portion of cDNA. less conservation because it is un translated region.
- 7. Gene mapping tool ETS – Genome marker. • Unique to a particular. • Easily recognizable and occur only once in genome or chromosome. Resource of gene discovery. • Tool for detection of hereditary disease involving gene. • Generation of ETS is rapid and inexpensive.
- 8. ESTs and NCBI In 1992, scientist at NCBI developed a new database designed to serve as a collection point for ESTs. Once EST that was submitted to GenBank had been screen and annotated, it was then deposited in this new database, called dbEST.
- 10. EST Format
- 11. EST Format
- 12. EST Format
- 13. EST Limitation • Very difficult to isolate mRNA from some tissue and cell types. • No indication as to the gene from which it is derived.
- 14. How to organize EST collection? Clustering :associate individual EST sequence with unique transcripts or gene. Assembling: derive consensus sequence from overlapping ESTs belonging to same cluster. Mapping: Associate ESTs with exons in genomic sequences. Interpreting: Find correct coding region.
- 15. Private EST database SANBI- South Africa MIPS- Munich and SIB TIGEM- Italy DOTS- Pennsylvania