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Genomic & Postgenomic Technologies. Contents Introduction  Gene diagnostics  Transcriptome and its future direction  Proteomics technologies Technologies.

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Presentation on theme: "Genomic & Postgenomic Technologies. Contents Introduction  Gene diagnostics  Transcriptome and its future direction  Proteomics technologies Technologies."— Presentation transcript:

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 Basic research Preclinical research Clinical research approval postcomm- ercializing research Step of development periodThe number of Candidates in the past 5 years in Japan 2 ~ 3 years 3 ~ 5 years 3 ~ 7 years 1 ~ 2 years During a sales period Preparation & selection of new compounds Examination of pharmacologic action, metabolic pathway, and safety of the selected compounds 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 Research of safety, effectiveness and quality of commercialized drug and promoting the appropriate use 202 compounds 1 / 2790 83 compounds 1 / 6790 35 compounds 1 / 16103 26 compounds 1 / 21677 ! Devil’s river Death valley 563, 589 compounds (1) R&D : 9 ~ 17 years (2) Cost : about $ 6,000 million ~ (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 Although sequences of all human genes have been elucidated…. What is important to dicover drug target genes? Elucidation of the functions in diseases will be the key! Decoding of human genome Possible to find & use of disease-associated genes ・ Most of drug target genes are still unknown.

8 Important thing is to establish technologies that can evaluate the pathological roles of genes screened by medicinal research. From mechanism-oriented to disease-oriented 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… 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. Key issue that we have to establish will be… 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. Key point

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

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

11 Progress of Research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome 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 Structure analysis(sequence) Polymorphism analysis ・ DNA chip ・ Invader assay ・ Sniper assay ・ PROBE assay ・ Luminex ・ PCR-SSCP ・ PCR-RFLP etc Transcriptome ・ Differential expression analysis ・ cDNA chip etc Proteome Identification Protein function Protein interaction Ligand interaction Post translational modification ・ protein chip, peptide chip ・ Y2H ・ SELEX ・ Phage display ・ STABLE assay etc

13 Progress of research genome Genomic sequence ・ Polymorphism ( SNP etc ) Transcriptome Gene transcription profile Proteome Expression profile of proteins Functional proteome Metabolome 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 1: Menteinance of genetic information : repairing 2: Transmission of genetic information : replication 2: Transmission of genetic information : replication 3: Use of genetic information : transcription & translation 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 Polymorphism marker : Difference of DNA sequence on the genome 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 Mini-satellite : Repeat of several to tens of base sequence Micro-satellite : Repeat of 1 to 4 base sequence Micro-satellite : Repeat of 1 to 4 base sequence Base insertion and deletion : Insertion /Deletion of 1-tens of 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, Single base polymorphism ( SNP ) : 1 /1000 bases, 3-10 millions SNA on human genome 3-10 millions SNA on human genome Analysis of gene polymorphism

16 Why gene typing is needed? If gene type is elucidated, effectiveness or adverse effect of particular drug can be validated. 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: $ 84 billion Cohort study of SNP mapping 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. Social issues: Informed consent, Handling of data to protect personal information Intellectual property Intellectual property

17 SNP analysis Identification & Mapping of SNPs Ability to find many SNPs from small number of genomic samples. SNPs Map SNPs Typing Ability to typing of particular (small amount of) SNPs by using a large number of genomoc samples 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

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

22 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 a) b)c)

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

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

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

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

27 Luminex Assay Fluorescent bead C 15 ~ C 18 Linker sequence 25 ~ 20base c-Zip code 25 ~ 20base PCR amplified DNA Zip code Capture probe Reporter probe ligation Cell sorter

28 Pyro-sequencing

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

30 SNP typing using MS PINPOIN assay

31 PROBE Assay SNP typing using MS

32 VSET assay SNP typing using MS

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


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