1. Expressed sequence tags (ESTs)
Luis Paez

2. 1. Theory 2. Applications 3. Example

3. What is a expressed sequence tag?
“ESTs are partial sequences from the extremities of cDNA resulting from a single pass sequencing of clones from cDNA libraries, and different ESTs can be obtained from one gene" (Hatey et al)

4. What is a expressed sequence tag?
“ESTs are partial sequences from the extremities of cDNA resulting from a single pass sequencing of clones from cDNA libraries, and different ESTs can be obtained from one gene" (Hatey et al)

5. Typical Workflow
But…
Many mRNA can be obtained from a single genomic DNA
Many cDNA can be obtained from a single mRNA
Many ESTs can be obtained from a single cDNA

6. Multiple mRNA from genomic DNA
Alternative promoter
Alternative splicing
Alternative polyadenylation
Blueprint and building analogy.
Proteins and RNAs determine the traits of an organism.
Highlight importance of molecular machines and transition to DNA synthesis.

7. Multiple cDNA from mRNA
mRNA secondary structures leading to early termination
mRNA secondary structures leading to truncated cDNA
Random primers that start sequence at different sites

8. Multiple ESTs from cDNA
Both extremities (5’ and 3’ ends) are sequenced
Overlapping enables construction of consensus sequences

9. Applications Identify transcriptome (mRNA) of a tissue
Identification of conserved genes and protein structures across species by comparing their ESTs
Identification of allele of a gene for a particular species population and the specificities of that gene (SNPs)
Obtained full length sequence of cDNAs
“Tagging of most genes faster and at a lower cost than complete genomic sequencing” (Adams et al, 1991)

10. Jhon Craig Venter Biochemist and biotechnologist from UC San Diego
Contributions to the human genome project.
JCVI institute
JCVI-syn.30 (mycoplasma with minimal bacterial genome)

11. 1991:Complementary DNA Sequencing
Found 337 new genes on brain tissue
48 of these genes are orthologs to other species.
46 ESTs mapped to chromosomes
By 1995, Venter group had:
Sequence of 174,472 human tags
Used 300cDNA libraries
Represented 37 different tissues

12. References
Adams, M. et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–1656 (1991).
Hatey, F. & Mulsant, P. Expressed sequence tags for genes: 21
Strausberg, R. L. & Riggins, G. J. Navigating the human transcriptome. Proceedings of the National Academy of Sciences 98, 11837–11838 (2001).
Khan, A. S. et al. Single pass sequencing and physical and genetic mapping of human brain cDNAs. Nat Genet 2, 180–185 (1992).

13. Questions