Genomic & Postgenomic Technologies

1. Genomic & Postgenomic Technologies

2. Contents • Introduction • Gene diagnostics • Transcriptome and its future direction • Proteomics technologies • Technologies for assessing protein-interaction • Technologies for protein labeling • Protein array and peptide array • Mass spectroscopy & proteomics • Monitoring of protein kinases • In vivo imaging • DDS & gene delivery

3. http://www.chem.kyushu-u.ac.jp/~katayama/

4. Why do we need ‘Bio-Technologies’? 1. It makes great innovation & progress in our lives. Keeping good QOL and solving the issue of aging: Genomic drug discovery; Genomic diagnostics; New therapies; Regenerative Medicines Solving the issue of food supply: Improvement of food self-sufficiency Solving environmental issues and energy supply: Bio-process, bio-mass, bio-energy technologies 2. Growing Market in Bio-technologies Biotechnologies can make a new industrial field in worldwide. Market size：$ 2.3 trillion in 2010. Ex) Pharmaceutical industry

5. Process of drug discovery Step of development period The number of Candidates in the past 5 years in Japan 563, 589compounds postcomm-ercializingresearch Preclinical research Basic research Clinical research approval 2～ 3 years Preparation & selection of new compounds 1／2790 202compounds Examination of pharmacologic action, metabolic pathway, and safety of the selected compounds 3 ～ 5 years Devil’s river Phase I : Confirmation of the safety for healthy persons Phase II : Research of safe administration for patients Phase III: Research of effectiveness & safety or comparison with other drugs 1／6790 83compounds 3 ～ 7 years Death valley 1／16103 1～ 2 years 35compounds During a sales period 1／21677　！ Research of safety, effectiveness and quality of commercialized drug and promoting the appropriate use 26compounds (1) R&D : 9～17 years (2) Cost : about $ 6,000 million～$ 1 billion (3) Success rate : 1/21,677 the number of approved medicines in past 5 years in japan: 26

6. Characteristics if Biotechnology 1: Basic researches directly linked to applied researches All technologies are in developing Industry-academia cooperation is crucial. 2:Long incubation time Leading time is so long for practical application Product life time in drugs: 16 years to 9 years in each drug in this decade Leading time: 9 years to 13 years Development time is longer than the product lifetime! Industry-Academia relationship to acceleration of development Ideas & Speed! Whole industrial system will changed by using genomic information

7. Current situation of genomic drug discovery Elucidation of the functions in diseases will be the key! Decoding of human genome Possible to find & use of disease-associated genes Although sequences of all human genes have been elucidated…. ・Most of drug target genes are still unknown. What is important to dicover drug target genes?

8. Key point Important thing is to establish technologies that can evaluate the pathological roles of genes screened by medicinal research. From mechanism-oriented to disease-oriented Key issue that we have to establish will be… How we can validate the function of gene quickly? Establishment of new validation system of genetic function in vtro & in vivo.

9. Even if we get the treasure chest (target gene), we can’t open it (because we can’t access to its function in disease.) We have got the map (genomic sequence) to find treasure so that we can get treasure chest. Getting treasure (new drugs) !! Key to ope the chast （Post genomic technology) Current genomic researches have tried pulling out of all nails on the chest. However, the number of the nails may be infiinite…

10. Genomic research What is the difference between human & ape?

11. Progress of Research genome Genomic sequence・Polymorphism（SNP etc） Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome Genetic function Post translational modification Protein interation etc. Time consuming and enormous cost Elucidation of functional network of cellular molecules

12. Technologies in each categories Genome • ・DNAchip　・Invader assay • Sniper assay　 ・PROBEassay • Luminex　　　 ・PCR-SSCP • PCR-RFLP etc Structure analysis(sequence) Polymorphism analysis ・cDNAchipetc Transcriptome ・Differential expression analysis Proteome • ・proteinchip, peptide chip • Y2H • SELEX • Phage display • STABLEassayetc Identification Protein function Protein interaction Ligand interaction Post translational modification

13. Progress of research genome Genomic sequence・Polymorphism（SNP etc） Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome Genetic function Post translational modification Protein interation etc. Time consuming and enormous cost Elucidation of functional network of cellular molecules

14. Nucleic acid：DNA,RNA(mRNA, tRNA, rRNA) Missions of gene 　１：Menteinance of genetic information：repairing 　２：Transmission of genetic information：replication 　３：Use of genetic information：transcription & translation Gene: Region of genomic DNA coding protein Genome : Whole set of genes in particular species Total gene is only 3% of whole genomic DNA

15. Analysis of gene polymorphism Polymorphism marker：Difference of DNA sequence on the genome High polymorphism, but the distribution is less and heterogenious Mini-satellite：Repeat of several to tens of base sequence Micro-satellite：Repeat of 1 to 4 base sequence Base insertion and deletion： Insertion /Deletion of 1-tens of base sequence Low polymorphism, but are a lot of distributed on genomic DNA uniformly Single base polymorphism（SNP)：1 /1000 bases, 3-10 millions SNA on human genome

16. Why gene typing is needed? If gene type is elucidated, effectiveness or adverse effect of particular drug can be validated. In USA in 1994, 2 million people got extension of hospital stay and 100,000 people died due to the drug side effect. Medical expenses: $ 84billion Cohort study of SNP mapping Social issues: Informed consent, Handling of data to protect personal information Intellectual property Technical issues Cost: Current technology takes $ 40 billion for the analysis of 1000 SNPs. Profiling of large number of SNPs is required for disease diagnostics.

17. SNPanalysis Identification & Mapping of SNPs Ability to find many SNPs from small number of genomic samples. SNPsMap Ability to typing of particular (small amount of) SNPs by using a large number of genomoc samples SNPsTyping If the SNPs typing is performed genome-wide, around 100 million of SNPs have to be typed. Speed & Cost Effectiveness!

18. Allele specific hybridization

19. Mini-sequencing

20. Ligation assay Ligation with enzyme Ligation with enzyme

21. Endogenious SNPs typing using FRET a) TDI assay, b) DOL assay

22. b) c) a) SNPs typing using primer extention on a chip a)Oligo-Tag array, b) Primer array with single-base extention c)Primer array with multi-base extenton

23. C b) a) SNPs typing using kinetic –PCR strategy a) Taq-Man PCR, b) Allele-specific molecular beacon

24. Flap Flap Invader Assay C T Reporter probes Invederprobes Endoflap Nuclease A Cleavage Cleavage G Fluorophore 2 Fluorophore 1 Quencher N N T Quencher C N N A G Cleavage C T

25. Invader assay Flap Flap cleavage cleavage Reporter probe Reporter probe Invader probe T Invader probe C N N A G Fluorophore Fluorophore Quencher Quencher cleavage cleavage Advantage: PCR is unnecessary Drawback: Quite large amount of sample is required Background reaction exists

26. Sniper assay Circular PCR ＋ Cyclization DNA sample containing SNP site Padlock probe Non-cyclization Molecular beacon

27. LuminexAssay PCRamplified DNA Zipcode ligation Captureprobe Reporter probe Cell sorter c-Zip code 25～20base C15～C18 Linker sequence 25～20base Fluorescent bead

28. Pyro-sequencing

29. RFLP ：restriction fragment length polymorphysm AATGATG AATGCTG TTACTAC TTACGAC AATGATG AATG CTG TTACTAC TTA CGAC SSOP ：sequence specific oligonucleotide probe AATGCT AATGCT TTACGA TTACGA Biotin PCR amplified DNA Magnetic bead modified with streptavidin SNP typing using Mass Spectrometry

30. PINPOINassay SNP typing using MS

31. PROBEAssay SNP typing using MS

32. VSETassay SNP typing using MS

33. Survivor assay Schematic outline of Survivor assay The figure shows the case of heteroxygote.