2011-09-26
Introduction
Introduction
Mikromacierze
The Central Dogma
The Central Dogma
oligonukleotydowe i cDNA
of Molecular Biology
of Molecular Biology
nowe narzędzie w diagnozowaniu i
terapii nowotworów
Cell
DNA
Transcription
Pracownia Biologii Molekularnej i Farmakogenomiki
Zakład Biochemii Farmaceutycznej
mRNA
Wydział Farmaceutyczny
Translation
Ribosome
UM w A
odzi
Polypeptide
prof. dr hab. n. farm. Marek Mirowski
(protein)
©1998 Timothy G. Standish
From Gene To Drug
DNA makes RNA makes Protein
Phosphorylation, cleavage,
MRNA
DNA glycosylation, sulfation
Splice forms
30,000-40,000 90,000-120,000 400,000-500,000 4,000-5,000
genes proteins protein isoforms drug targets
Gene Translation Post-translational
Expression modification
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Year 2000: Human genome sequenced
DNA: ACGT
Gene
 Genomics Gene
transcription (complete gene list)
mRNA
~30,000 genes/cell
" A gene is a unit of inheritance
translation " Carries the information for a:
Posttranslational
modification
-polypeptide
RNA microarray from http://www.mcb.arizona.edu
wardlab/microarray.html
-structural RNA molecule
Protein
 Proteomics
amino acids
(complete protein content)
flank
flank
~50,000-200,000 proteins
3
5
promoter Structural gene
~5,000-10,000 per cell
upstream downstream
 Metabolomics
Metabolite
(with in a cell) small molecules
Metabonomics (complete metabolome)
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Selective Gene Expression
" How do cells
become
specialized?
" Different genes
are activated at
different times
during
development.
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5 Phosphate group
3 Hydroxyl group Microarray-Based Assays (The Basics)
OH
NH2 H OH
HO P O
D
D N The Key to Nucleic Acid Detection is  Sequence-Specific Affinity
N
O
CH2 N N O
O 5 3
CH2
O
G C
N O P HO
N
H T A
O
O H OH
H2O C G
HO P O
N A T
NH
O
T A
CH2 N N NH2 O
O CH2
A
A
T A
G C
P HO
C G
O H
NH2
H C G
H2O
HO P O
N A T
O
N A T
CH2 O
O CH2 5 Phosphate
3
5
group
P
OH H
3 Hydroxyl group
Hybridization
Hybridization
Denaturation and Renaturation
Denaturation and Renaturation
? Heating double stranded DNA can overcome the
DNA from source  X
hydrogen bonds holding it together and cause the
CTGATGGTCATGAGCTGTCCGATCGATCAT
strands to separate resulting in denaturation of
TACTCGACAGGCTAG
the DNA
? When cooled relatively weak hydrogen bonds
between bases can reform and the DNA renatures
Denatured DNA
Hybridization
ATGAGCTGTACGATCGTG Hybridization
ATGAGCTGTACGATCGTG ATGAGCTGTACGATCGTG
TACTCGACAGGCTAG
TACTCGACATGCTAGCAC TACTCGACATGCTAGCAC
DNA from source  Y
Double stranded DNA Double stranded DNA
TACTCGACATGCTAGCAC
Single stranded DNA
©2000 Timothy G. Standish ©2000 Timothy G. Standish
TYPICAL REACTION MIXTURE
POLYMERASE CHAIN REACTION - PCR
25 or 50µls in a micro Eppendorf (0.5ml) tube
A 'licence' to do molecular biology
COMPONENT VOLUME Final
A key central technique that has revolutionised molecular and Concentration
consequently cell biology 10 X PCR Buffer
1X
5µ
µl
µ
µ
10 X dNTPs (2mM)
200µ
µM
5µ µ
µl µ
µ
µ
Forward primer (10pmols/µl) 1µM (50pmols/50µl)
µ
µ
µ
5µ
µl
µ
µ
Reverse primer (10pmols/µ 1µM (50pmols/50µl)
µl)
µ
µ
5µ
µl
µ
µ
Genomic DNA template
1µ
µg
2µ µ
µl µ
µ
µ
devised by Kary Mullis c1983
Thermostable polymerase
1 unit
0.5µ
µl
µ
µ
http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html
(2U/µl)
µ
µ
µ
Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction.
H2O (to 50µ
µl Final volume)
µ 27.5µ
µ µl
µ
µ
Scientific American. 262 (4) 56-65.
3
3
H
C
S
O
U
N
G
H
N
B
A
O
R
A
-
P
H
S
N
O
2
H
S
E
N
P
N
H
O
S
A
T
O
E
O
B
A
O
H
O
N
C
N
N
K
H
N
B
O
N
2
H
O
O
N
E
O
O
H
O
H
R
n
e
o
n
i
t
a
a
t
r
u
u
r
t
a
a
t
i
n
o
e
n
D
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DENATURATION
PCR Agarose gel electrophoresis
93°C - 95°C
ANNEALING
37°C - 65°C
25-35 CYCLES 3-4 hours
DENATURATION EXTENSION
93°C - 95°C 72°C
The final product UV visualisation
Microchip DNA analysis
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Microarray-Based Assays (The Basics)
How does a DNA microarray detects gene activity?
Reverse Transcription makes cDNA from gene sequence&
 TARGET is the fluorescence labeled
cDNA representation of the mRNA and
mRNA AAAAAAAAAAAAAAAA
is hybridized to the probe.
...GCUACGAUUGCAACGCCCGAAUGGUUACCAAAAAAAAAAA...
CGATGCTAACGTTGCGGGCTTACCAATGGTTTTTTTTTTT
 PROBE is DNA spotted (attached) to the solid cDNA
substrate (non-fluorescent glass slide).
dTTPATGCTAACGTTGCGGGCTTACCAATGGTTTTTTTTTTT
dCTP
CG
dGTP dATP
2-Color System... Detection
2-Color System...
RNA from Normal Tissue RNA from Cancer or Drug Treated Tissue
dCTP dCTP
Reverse Transcription
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2-Color Laser Scanner
Figure 3b. Genes distinguishing ALL from AML. The 50 genes most highly correlated with the ALL/AML class
distinction are shown. Each row corresponds to a gene, with the columns corresponding to expression levels in
different samples. Expression levels for each gene are normalized across the samples such that the mean is 0 and the
standard deviation is 1. Expression levels greater than the mean are shaded in red, and those below the mean are
shaded in blue. The scale indicates standard deviations above or below the mean. The top panel shows genes highly
expressed in ALL, the bottom panel shows genes more highly expressed in AML. Note that while these genes as a
group appear correlated with class, no single gene is uniformly expressed across the class, illustrating the value of a
multi-gene prediction method.
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Gene Expression Profiling: DNA Microarrays
Leung & Cavalieri, Trends in Genetics 19:649 (2003)
mRNA Expression in Lung
Cy3 (Uninfected)
Bryant et al., The Lancet Infectious Diseases 4:100 (2004)
Genotyping: SNP Microarray
Genotyping: SNP Microarray
Immobilized allele-specific oligo probes
Hybridize with labeled PCR product
Assay multiple SNPs on a single array
TTAGCTAGTCTGGACATTAGCCATGCGGAT
GACCTGTAATCG
TTAGCTAGTCTGGACATTAGCCATGCGGAT
Many other methods
Many other methods
GACCTATAATCG
For SNP analysis have
For SNP analysis have
been developed
been developed
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Cy5 (AIDS)
2011-09-26
Sequencing by Microarray
Sequencing by Microarray
Technology
Technology
Sequencing By Hybridization
Sequencing By Hybridization
Address the need for high-speed, low-cost
sequencing of large sequences in parallel.
Example:
Consider examining 50Kb of sequence for 1,000
individuals.
Conventional Method Microarray
Conventional Method Microarray
With one microarray of 1.25 x
50Kb x 1,000 = 50 Mb of
1.25 cm dimension, you can
sequence. At a rate of 500
scan 50 Kb of sequence at once.
bases per lane and 30
You need 1,000 microarrays to
sequencing lanes, you can
complete task. This may be
produce 15 Kb of sequence
completed in a few days.
per day. You need 10 years
for the project.
Conferences in Research and Practice in Information
Technology Series; Vol. 33 archive
Proceedings of the First Asia-Pacific bioinformatics
conference on Bioinformatics 2003 - Volume 19
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Proc Natl Acad Sci U S A. 2003 September 2; 100(18): 10393 10398.
Published online 2003 August 13. doi: 10.1073/pnas.1732912100.
Copyright © 2003, The National Academy of Sciences
Applications
Applications
Screening for:
Screening for:
Small molecule
targets
Post-translational
modifications
Protein-protein
interactions
Protein-DNA
interactions
Enzyme assays
Epitope mapping
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Applications of
Applications of
Microarrays
Microarrays
Gene expression patterns
Single nucleotide polymorphism (SNP) detection
Sequence by hybridization / genotyping / mutation
[e-mail]
detection
Study protein expression (multianalyte assay)
Protein-protein interactions
Pathogen analysis
Transcriptional factors
Provides: Massive parallel information
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Commercial Microarray for Clinical Use
Commercial Microarray for Clinical Use
(Pharmacogenomics)
(Pharmacogenomics)
Roche Product
CYP 450 Genotyping
(drug metabolizing
system)
FDA Confusion
Class 1 medical device? (no PMA)
Class 2 or 3 medical device?
(requires pre-market approval)
From: Nature Biotechnology 2003 21:959-60
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Centrum Badań DNA (http://wwwcbdna.pl/)
Poznański Park Naukowo-Techniczny
" Oferta 16 ró\
nych testów (predyspozycje do raka piersi i
jajnika, 88 ró\
nych mutacji w 6 genach m.in. BRCA1 i
BRCA2
" Panel na mukowiscydozÄ™ 254 mutacje
" Talasemia
" Retinopatia
" Diagnostyka zapalenia opon mózgowo-rdzeniowych
" Mutacje genu K-ras
" Choroby odkleszczowe
" Diagnostyka sepsy
" Diagnostyka infekcji dróg oddechowych, moczowo-
płciowych
Microarray analysis
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