genomic & postgenomic technologies
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Genomic & Postgenomic Technologies. 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 - PowerPoint PPT PresentationTRANSCRIPT
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Genomic & Postgenomic Technologies
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ContentsIntroduction 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
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http://www.chem.kyushu-u.ac.jp/~katayama/
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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
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Process of drug discovery
Basic research
Preclinical research
Clinical research
approval
postcomm-ercializing research
Step of development period The 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 personsPhase II : Research of safe administration for patientsPhase 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 ~ $ 1 billion (3) Success rate : 1/21,677 the number of approved medicines in past 5 years in japan: 26
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Characteristics if Biotechnology1: 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 developmentIdeas & Speed!
Whole industrial system will changed by using genomic information
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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 genomePossible to find & use of disease-associated genes
・ Most of drug target genes are still unknown.
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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.
Key point
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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…
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What is the difference between human & ape?Genomic research
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Progress of Researchgenome
Genomic sequence ・ Polymorphism( SNP etc )
Transcriptome
Gene transcription profile
Proteome Expression profile of proteins
Functional proteomeMetabolome
Genetic functionPost translational modificationProtein interation etc.
Time consuming and enormous costElucidation of functional network of cellular
molecules
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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 interactionLigand interactionPost translational modification
・ protein chip, peptide chip・Y2H・SELEX・Phage display・STABLE assay etc
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Progress of researchgenome
Genomic sequence ・ Polymorphism( SNP etc )
Transcriptome
Gene transcription profile
Proteome Expression profile of proteins
Functional proteomeMetabolome
Genetic functionPost translational modificationProtein interation etc.
Time consuming and enormous costElucidation of functional network of cellular
molecules
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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
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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
Analysis of gene polymorphism
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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: $ 84 billion
Cohort study of SNP mapping
Technical issuesCost: 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
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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!
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Allele specific hybridization
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Mini-sequencing
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Ligation assay
Ligation with enzyme
Ligation with enzyme
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Amplified DNA fragment ( G-Allele)
GAmplified DNA fragment ( A-Allele)
A
Fluorescein-labeled ODNC U
ROX- ddC TAMRA- ddU
G AC
FRET
U
FRET
ROX positive TAMRA positive
( G-Allele)G
( A-Allele)A
Fluorescein-labeled ODN
UROX-labeled ODN
TAMRA-labeled ODN
G
FRET
FRET
ROX positive
TAMRA positive
C
PCR primers
A
G
G
C
U
Endogenious SNPs typing using FRET a) TDI assay, b) DOL assay
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cTag
GAmplified G-Allele
Tagprobe DNA
C
T
f l uorescei n-ddCTP
bi oti n- ddTTP
GC
C
single base extention
oligo Tag-array
Gpri mer
primer array
GAmplified G-Allele
C
T
fl uorescei n- ddCTP
bi oti n- ddTTP
single base extention
hybridization
GC
G
G
pri mer
C
primer array
G
G
T
Amplified G-Allele RNA
C fl uorescei n- dCTP
hybridization
dNTP
C GT
primer extention
SNPs typing using primer extention on a chipa)Oligo-Tag array, b) Primer array with single-base extentionc)Primer array with multi-base extenton
a) b) c)
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GC
T
FRET
FRET
PCR reactionTaq-polymerase
primer
G
Cfl uorescence
FRET probe is decomposed with the endonuclease activity
C
a)fluorephore
quencher complementry sequneceto the template including SNP
Molecular Beacon
PCR reaction
G
G
GC
C
C
Fluoprescence is increased with the PCR reaction
G
C
b)
SNPs typing using kinetic –PCR strategy a) Taq-Man PCR, b) Allele-specific molecular beacon
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T
N
C
G A
Reporter probes
Flap FlapInveder probesN
C T
GN N
A
Cleavage Cleavage
Endoflap Nuclease
Fluorophore 1 Fluorophore 2Quencher Quencher
C
Cleavage
T
Invader Assay
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Invader assay
Reporter probe
Fluorophore Quenchercleavage
GNC
cleavage
Flap
Reporter probeInvader probe
Fluorophorecleavage
NT
cleavage
Flap
Invader probe
A
Quencher
Advantage: PCR is unnecessaryDrawback: Quite large amount of sample is required Background reaction exists
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Sniper assay
DNA sample containing SNP site
+
Padlockprobe
Cyclization
Non-cyclization
Molecularbeacon
Circular PCR
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Luminex Assay
Fluorescent bead
C15 ~ C18
Linker sequence25 ~ 20base
c-Zip code25 ~ 20base
PCR amplified DNA
Zip code Capture
probe Reporter probe
ligation
Cell sorter
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Pyro-sequencing
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SNP typing using Mass Spectrometry
RFLP
TTACGACAATGCTG
AATG CTG
TTACTACAATGATG
TTACTACAATGATG
: restriction fragment length polymorphysm
TTA CGAC
SSOP
Magnetic bead modified with streptavidin
Biotin
TTACGA
PCR amplified DNA
AATGCTTTACGA
AATGCT
: sequence specific oligonucleotide probe
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SNP typing using MS PINPOIN assay
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PROBE AssaySNP typing using MS
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VSET assaySNP typing using MS
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AA m p lif ie d D N A re g io nin c lu d in g th e S N P s i te
d d A T P , d d G T Pd d C T P , d d T T P
p r im e r h y b r id i z a t io n
A
e x t e n t i o nr e a c t io n
AT
G+
GC
A G
m /eE S I- M S
h e t e ro z y g o te
G
TCSchematic outline of Survivor assayThe figure shows the case of heteroxygote.
Survivor assay