Genomic & Postgenomic Technologies

Genomic & Postgenomic Technologies

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

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

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　　

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

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

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.

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

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…

What is the difference between human & ape?Genomic research

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　　　　　

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

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　　　　　

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

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

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

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!

Allele specific hybridization

Mini-sequencing

Ligation assay

Ligation with enzyme

Ligation with enzyme

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

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)

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

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

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

Sniper assay

DNA sample containing SNP site

＋

Padlockprobe

Cyclization

Non-cyclization

Molecularbeacon

Circular PCR

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

Pyro-sequencing

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

SNP typing using MS PINPOIN assay

PROBE AssaySNP typing using MS

VSET assaySNP typing using MS

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