1. Chapter 14 Genomes and Genomics

2. Sequencing DNA dideoxy (Sanger) method ddGTP ddATP ddTTP ddCTP 5’TAATGTACG TAATGTAC TAATGTA TAATGT TAATG TAAT TAA TA T Fred Sanger, Nobel prize 1980

3. Sequencing DNA dideoxy (Sanger) method Leroy Hood, Caltech Fluorescence based sequencing Norm Dovici – Capillary electrophoresis

4. Sequencing DNA dideoxy (Sanger) method

5. Genomics era: High-throughput DNA sequencing  The first high-throughput genomics technology was automated DNA sequencing in the early 1990.  In September 1999, Celera Genomics completed the sequencing of the Drosophila genome.  Baker’s yeast, Saccharomyces cerevisiae (15 million bp), was the first eukaryotic genome to be sequenced.  TIGR (The Institute for Genomics Research) 1995 – first whole genome sequence, H. influenza

6. Genomics: Completed genomes as 2002  Currently the genome of over 600 organisms are sequenced:  This generates large amounts of information to be handled by individual computers. http://www.genomesonline.org/

7. Cloning/libraries BAC, YAC and ESTs BAC = bacterial artificial chromosome –150 kb, replicate in E.coli YAC = yeast artificial chromosome –150 kb -1.5 Mb, replicate in yeast

8. Assembling contigs

9. Ordered-clone Sequencing Clones ordered by restriction enzyme sites

10. Annotation ORF – open reading frame EST- Expressed sequence tag –Based on mRNA Comparative genomics

11. The trend of data growth  21 st century is a century of biotechnology:  Microarray : Global expression analysis: RNA levels of every gene in the genome analyzed in parallel.  Proteomics : Global protein analysis generates by large mass spectra libraries.  Metabolomics: Global metabolite analysis: 25,000 secondary metabolites characterized  Genomics : New sequence information is being produced at increasing rates. ( The contents of GenBank double every year )  Glycomics: Global sugar metabolism analysis

12. How to handle the large amount of information? Drew Sheneman, New Jersey--The Newark Star Ledger Answer: bioinformatics and Internet

13. Bioinformatics history IBM 7090 computer  In1960s: the birth of bioinformatics  Margaret Oakley Dayhoff created:  The first protein database  The first program for sequence assembly  There is a need for computers and algorithms that allow: Access, processing, storing, sharing, retrieving, visualizing, annotating…

14. DNA (nucleotide sequences) databases  They are big databases and searching either one should produce similar results because they exchange information routinely. -GenBank (NCBI): www.ncbi.nlm.nih.gov -Arabidopsis: ( TAIR) www.arabidopsis.org  Specialized databases:Tissues, species… -ESTs (Expressed Sequence Tags) ~at NCBI ~at TIGR -...many more!

15. Comparative genomics BLAST – basic local alignment and search tool (http://www.ncbi.nlm.nih.gov/) Homologs orthologs paralogs

16. Question You are a researcher who has tentatively identified a human homolog of a yeast gene. You determine the DNA sequence of cDNAs of both your yeast gene and the human gene and decide to compare the gene sequences, as well as the predicted protein sequence of each, using alignment software. You would expect the greatest sequence identity from comparisons of the: a. cDNA sequences b. Protein sequences c. Genomic DNA sequences d. Both (a) and (b) will give you equivalent sequence similarity e. All will give equivalent sequence similarity

17. What is a microarray?

18. Types of Arrays Expression Arrays –cDNA –Genome Affymetrix (GeneChip®) Agilent Tiling arrays

19. Overview of Microarrays

20. Transcription Profiling of a mutant WT mutant

21. A “good” microarray plate Red = only in treatment Green = only in normal Yellow = found in both Black = found in neither

22. Results 100’s of genes identified, those turned on, those turned off

23. Expression map red = up regulated green= down regulated

24. Question Microarray technology directly involves: a.PCR b.DNA sequencing c.Hybridization d.RFLP detection e.None of the above

25. Protein – protein interactions ChIP (chomatin immunoprecipitation) Yeast two hybrid Bi Molecular Fluorescence Complementation (BMFC)

26. ChIP and ChIP- chip

27. Yeast two hybrid

28. Citovsky et al., 2006 Bi Molecular Fluorescence Complementation (BMFC)

29. Reverse genetics Gene knockouts RNAi Overexpression Altered expression

30. Summary DNA Sequencing and the rise of genomics Annotation of genome sequence –Comparative genomics –Functional genomics Protein-protein interactions ESTs Reverse genetics