Transcript Proteome

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
 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
Step of
period
The number of Candidates in the past 5 years in Japan
development
Basic
563, 589 compounds
research 2~ 3 years Preparation & selection of new compounds
Preclinical
research
of pharmacologic action,
metabolic pathway,
3 ~ 5 years Examination
1/2790
202 compounds
and safety of the selected compounds
Devil’s river
Clinical
research
Phase I : Confirmation of the safety for healthy persons
3 ~ 7 years Phase83IIcompounds
: Research of safe administration
1/6790for patients
Phase III: Research of effectiveness & safety or
comparison Death
with other
drugs
valley
approval
1 ~ 2 years
postcommercializing
research
During a
sales
period
35 compounds
1/16103
Research of safety, effectiveness and
quality of!
1/21677
commercialized
drug
and
promoting
the appropriate use
26 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 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
Current situation of genomic drug discovery
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?
Elucidation of the functions in diseases will be the key!
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.
Current genomic researches
have tried pulling out of all
nails on the chest.
However, the number of the
nails may be infiinite…
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.
Key to ope the chast
(Post genomic technology)
Getting treasure (new drugs) !!
Genomic
research
What is the difference
between
human & ape?
Progress of Research
genome
Genomic sequence・Polymorphism(SNP etc)
Transcriptome
Proteome
Gene transcription profile
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
Technologies in each categories
Genome
・DNA chip
・Invader assay
・Sniper assay
・PROBE assay
・Luminex
・PCR-SSCP
・PCR-RFLP etc
Structure analysis(sequence)
Polymorphism analysis
Transcriptome
・cDNA chip etc
・Differential expression analysis
Proteome
Identification
Protein function
Protein interaction
Ligand interaction
Post translational modification
・protein chip, peptide chip
・Y2H
・SELEX
・Phage display
・STABLE assay etc
Progress of research
genome
Genomic sequence・Polymorphism(SNP etc)
Transcriptome
Proteome
Gene transcription profile
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
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
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
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
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.
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
G
G
A
A
( A -A llele)
( G -A llele)
A m plified D N A frag m ent
( A -A llele)
A m plified D N A fragm ent
( G -A llele)
F luorescein-labeled O D N
C
R O X -labeled O D N
U
F lu orescein-labeled O D N
C
T A M R A -labeled O D N
U
ROX-d d C
TAMRA- d d U
P C R prim ers
G
FRET
FRET
C
G
A
U
A
FRET
R O X positive
C
G
T A M R A positive
R O X p ositive
FRET
Endogenious SNPs typing using FRET
a) TDI assay, b) DOL assay
U
G
T A M R A p ositive
b)
a)
c)
G
G
A m p lified G -A llele R N A
A m p lified G -A llele
G
h yb rid iz a tio n
h yb rid iz a tion
A m p lified G -A llele
p r i me r
p r ob e DNA
p r i me r
Ta g
C
TG
CG
G
f l u or e s c e i n - d d CTP
T
p rim er array
p rim er array
b i ot i n - d d TTP
G
C
sing le b ase ex tention
C
f l uor e s c e i n- d d CTP
T
b i ot i n -d d TTP
C
f l u or e s c e i n - d CTP
d NTP
sing le b ase ex tention
C
c Ta g
CG
olig o T ag -array
TG
CG
p rim er ex tention
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
FRET
b)
a)
flu o re p ho re
T
C
FRET
prim er
C
G
q u encher
com p lem entry seq u nece
to the tem plate inclu d ing S N P
M olecu lar B eacon
T a q -polym e ra se
P C R reaction
G
P C R reaction
C
C
f l uor es c ence
C
G
C
G
G
C
G
F R E T probe is decom posed
w ith the endonuclease activity
F lu op rescence is increased w ith the P C R reaction
SNPs typing using kinetic –PCR strategy
a) Taq-Man PCR, b) Allele-specific molecular beacon
Invader Assay
Flap
Flap
Inveder
probes
EndoflapNNuclease
Reporter
probes
C
Cleavage
G
C
N
G
Fluorophore 1
Quencher
Cleavage
N
T
Cleavage
T
N
A
A
Fluorophore 2
Quencher
Invader assay
Flap
Flap
Invader probe
cleavage
Reporter probe
C
N
G
Fluorophore
cleavage
Quencher
cleavage
Invader probe
T
N
A
Fluorophore
Reporter probe
Quencher
cleavage
Advantage: PCR is unnecessary
Drawback: Quite large amount of sample is required
Background reaction exists
Sniper assay
Circular PCR
+
DNA sample
containing SNP site
Molecular
beacon
Cyclization
Padlock
probe
Non-cyclization
Luminex Assay
PCR amplified DNA
Zip code
Capture
probe
ligation
Reporter
probe
Cell sorter
C15~C18
Fluorescent
bead
c-Zip code
25~20base
Linker sequence
25~20base
Pyro-sequencing
SNP typing using Mass Spectrometry
RFLP
:restriction fragment length polymorphysm
AATGATG
TTACTAC
AATGCTG
TTACGAC
AATG
TTA
SSOP
AATGATG
TTACTAC
CTG
CGAC
:sequence specific oligonucleotide probe
AATGCT
TTACGA
AATGCT
TTACGA
Biotin
PCR amplified DNA
Magnetic bead
modified with
streptavidin
SNP typing using MS
PINPOIN assay
SNP typing using MS
PROBE Assay
SNP typing using MS
VSET assay
Survivor assay
G
+
A
A m p lif ied D N A re g io n
in c lu d in g th e S N P s ite
p rim e r h y b rid iz atio n
G
A
ddA TP, ddGT P
ddC TP, ddTTP
e x ten tio n
r ea ctio n
G
C
A
T
A
G
C T
h etero z y g o te
m /e
E S I- M S
Schematic outline of Survivor assay
The figure shows the case of heteroxygote.