Dogmatic View of Gene Expression

12. DNA RNA Protein Dogmatic View of Gene Expression Post-transcriptional Control: Quantitative Control: Levels of mRNA not proportional to levels of mRNA synthesized or protein produced Qualitative Control: More than one protein from a single gene (e.g. Differential RNA Processing or RNA editing)

13. An “RNA-Centric” View of Gene Expression RNA DNA Protein

15. TRANSCRIPTOMICS

17. Identification de BAC contenant une unité de transcription

20. Expressed sequence tags

24. Three principles underlie the SAGE methodology:  1)  A short sequence tag (10-14bp) contains sufficient information to uniquely identify a transcript provided that the tag is obtained from a unique position within each transcript;  2)  Sequence tags can be linked together to form long serial molecules that can be cloned and sequenced;  3)  Quantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript

25. Restriction digest double-stranded cDNA with a 4-base cutter “anchoring enzyme” (NlaIII); bind to streptavidin coated beads Generate cDNA primed with biotin-oligo(dT) AAAA TTTT AAAA TTTT AAAA TTTT GTAC AAAA TTTT Divide pool in half & ligate to different linkers (1 or 2), both of which have a restriction site for the “tagging enzyme” (BsmFI) GTAC AAAA TTTT CATG GTAC AAAA TTTT CATG GTAC 2 1 Restriction digest with a “reach & grab” enzyme (BsmFI) which recognizes the linker sequences and cuts downstream in a sequence independent fashion; fill-in 5’ overhang to blunt ends. GGATGCATGOOOOOOOOOO CCTACGTACOOOOOOOOOO GGATGCATGXXXXXXXXXX CCTACGTACXXXXXXXXXX 2 1 Blunt end ligate pool 1 to pool 2, and PCR amplify with primers specific to linker sequences 1 and 2 Tag 1 Tag 2 GGATGCATGXXXXXXXXXXOOOOOOOOOOCATGCATCC CCTACGTACXXXXXXXXXXOOOOOOOOOOGTACGTAGG 1 2 Ditag Restriction digest with same anchoring enzyme (above); gel isolate ‘di-tags’; concatenate ditags and ligate to cloning/sequencing vector Ditag Ditag -----CATGXXXXXXXXXXOOOOOOOOOOCATGXXXXXXXXXXOOOOOOOOOOCATG---- ----GTACXXXXXXXXXXOOOOOOOOOOGTACXXXXXXXXXXOOOOOOOOOOGTAC---- Tag 1 Tag 2 Tag 3 Tag 4

26. DNA microarrays DNA microarrays rely on the hybridization properties of nucleic acids to monitor DNA or RNA abundance on a genomic scale in different types of cells. The ancestor of microarrays: the Northern blot.

28. High-Density Oligonucleotide Arrays Chee M. Assessing genetic information with high-density oligonucleotide arrays. Science 1996

29. High-Density Oligonucleotide Arrays Chee M. Assessing genetic information with high-density oligonucleotide arrays. Science 1996

40. Analyse fonctionnelle du protéome