introduction the central dogma -...
TRANSCRIPT
2011-09-26
1
Mikromacierze oligonukleotydowe i cDNA
nowe narzędzie w diagnozowaniu i terapii nowotworów
Pracownia Biologii Molekularnej i FarmakogenomikiZakład Biochemii FarmaceutycznejWydział FarmaceutycznyUM w Łodzi
prof. dr hab. n. farm. Marek Mirowski
DNA
mRNA
Transcription
IntroductionIntroduction
The Central Dogma The Central Dogma of Molecular Biologyof Molecular Biology
Cell
Polypeptide(protein)
TranslationRibosome
©1998 Timothy G. Standish
DNA makes RNA makes Protein
7
From Gene To Drug
DNAMRNASplice forms
Phosphorylation, cleavage,
glycosylation, sulfation
Gene Translation Post-translationalExpression modification
30,000-40,000 90,000-120,000 400,000-500,000 4,000-5,000genes proteins protein isoforms drug targets
Gene “Genomics”DNA: ACGT
Year 2000: Human genome sequenced
(complete gene list)
~30,000 genes/cell
Protein “Proteomics”(complete protein content)amino acids
~50,000-200,000 proteins
~5,000-10,000 per cell
mRNA
Posttranslational modification
transcription
translation
Metabolite “Metabolomics” (with in a cell) small molecules
Metabonomics(complete metabolome)
RNA microarray from http://www.mcb.arizona.eduwardlab/microarray.html
Gene
promoter Structural gene
flank flank
upstream downstream5’ 3’
•A gene is a unit of inheritance
•Carries the information for a:
-polypeptide
-structural RNA molecule
2011-09-26
2
Selective Gene Expression
• How do cells become specialized?
• Different genes are activated at different times during development.
2011-09-26
3
SUG
AR
-PHO
SPHA
TE
BA
CK
BO
NE
H
P
O
HO
O
O
CH2
HOH
P
O
O
HO
O
O
CH2
H
P
O
OH
HO
O
O
CH2
NH2
N
N
N
N
O
O
NH2N
NH
N
N
N O
NH2
N
B A
S E S
DDNNAA
OH
P
O
HO
O
O
CH2
HO
O
H 2N
NHN N
N H
H
P HO
O
O
CH2
O
O
N
O
H 2N
NH
H2O
H OH
P
O
HO
O
O
CH2
CH 3
O
O
HNN
H2O
5’Phosphate group
3’Hydroxyl group
5’Phosphategroup
3’Hydroxyl group
The Key to Nucleic Acid Detection is “Sequence-Specific Affinity”
CAGTAACGGTT
5’
3’
GTCATTGCCAA
5’
3’
Microarray-Based Assays (The Basics)
©2000 Timothy G. Standish
Denaturation Denaturation and and RenaturationRenaturation? Heating double stranded DNA can overcome the
hydrogen bonds holding it together and cause the strands to separate resulting in denaturation of the DNA
? When cooled relatively weak hydrogen bonds between bases can reform and the DNA renatures
TACTCGACATGCTAGCACATGAGCTGTACGATCGTG
Double stranded DNA
TACTCGACATGCTAGCACATGAGCTGTACGATCGTG
Double stranded DNA
Renaturation
TACTCGACATGCTAGCAC
ATGAGCTGTACGATCGTG
Denatured DNA
Denatur
ation
Single stranded DNA ©2000 Timothy G. Standish
HybridizationHybridization
DNA from source “Y”
TACTCGACAGGCTAG
CTGATGGTCATGAGCTGTCCGATCGATCAT
DNA from source “X”
TACTCGACAGGCTAG
HybridizationHybridization
http://www.nobel.se/chemistry/laureates/1993/mullis-autobio.html
Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction.
Scientific American. 262 (4) 56-65.
devised by Kary Mullis c1983
POLYMERASE CHAIN REACTION - PCR
A 'licence' to do molecular biology
A key central technique that has revolutionised molecular and consequently cell biology
COMPONENT VOLUME Final Concentration
10 X PCR Buffer 5µµµµl 1X
10 X dNTPs (2mM) 5µµµµl 200µµµµM
Forward primer (10pmols/µµµµl) 5µµµµl 1µM (50pmols/50µl)
Reverse primer (10pmols/µµµµl) 5µµµµl 1µM (50pmols/50µl)
Genomic DNA template 2µµµµl 1µµµµg
Thermostable polymerase (2U/µµµµl)
0.5µµµµl 1 unit
H2O (to 50µµµµl Final volume) 27.5µµµµl
TYPICAL REACTION MIXTURE
25 or 50µls in a micro Eppendorf (0.5ml) tube
2011-09-26
4
ANNEALING 37°C - 65°C
EXTENSION 72°C
25-35 CYCLES
DENATURATION 93°C - 95°C
DENATURATION 93°C - 95°C
PCR Agarose gel electrophoresis
The final product UV visualisation
3-4 hours
Microchip DNA analysis
2011-09-26
5
“PROBE” is DNA spotted (attached) to the solidsubstrate (non-fluorescent glass slide).
“TARGET ” is the fluorescence labeledcDNA representation of the mRNA and
is hybridized to the probe.
Microarray-Based Assays (The Basics)
... GCUACGAUUGCAACGCCCGAAUGGUUACCAAAAAAAAAAA...
dCTP
dATP
dTTP
dGTP
How does a DNA microarray detects gene activity?
Reverse Transcription makes cDNA from gene sequence…
AAAAAAAAAAAAAAAAmRNA
TTTTTTTTTTTGGTAACCCCCCC ATTGGGGTTGAATGTAG
cDNA
TTTTTTTTTTTGGTAACCCCCCC ATTGGGGTTGAATGTAG
2-Color System...
RNA from Normal Tissue RNA from Cancer or Drug Treated Tissue
dCTP dCTP
Reverse Transcription
2-Color System... Detection
2011-09-26
6
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.
2011-09-26
7
Bryant et al., The Lancet Infectious Diseases 4:100 (2004)
Leung & Cavalieri, Trends in Genetics 19:649 (2003)
Cy3 (Uninfected)
Cy5
(A
IDS
)
mRNA Expression in Lung
Gene Expression Profiling: DNA Microarrays
Genotyping: SNP MicroarrayGenotyping: SNP Microarray
� Immobilized allele-specific oligo probes
� Hybridize with labeled PCR product
� Assay multiple SNPs on a single arrayTTAGCTAGTCTGGACATTAGCCATGCGGAT
GACCTGTAATCG
Many other methodsMany other methodsFor SNP analysis have For SNP analysis have
been developedbeen developed
TTAGCTAGTCTGGACATTAGCCATGCGGAT
GACCTATAATCG
2011-09-26
8
Sequencing by Microarray Sequencing by Microarray TechnologyTechnology
Sequencing By HybridizationSequencing 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 MethodConventional Method MicroarrayMicroarray
50Kb x 1,000 = 50 Mb ofsequence. At a rate of 500bases per lane and 30sequencing lanes, you canproduce 15 Kb of sequenceper day. You need 10 yearsfor the project.
With one microarray of 1.25 x1.25 cm dimension, you canscan 50 Kb of sequence at once.You need 1,000 microarrays tocomplete task. This may becompleted in a few days.
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
2011-09-26
9
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
ApplicationsApplications
Screening for:Screening for:
� Small molecule targets
� Post-translational modifications
� Protein-protein interactions
� Protein-DNA interactions
� Enzyme assays� Epitope mapping
2011-09-26
10
2011-09-26
11
Applications of Applications of MicroarraysMicroarrays
� Gene expression patterns� Single nucleotide polymorphism (SNP) detection� Sequence by hybridization / genotyping / mutation
detection� Study protein expression (multianalyte assay) � Protein-protein interactions� Pathogen analysis� Transcriptional factors
Provides: Massive parallel information
2011-09-26
12
Commercial Microarray for Clinical Use Commercial Microarray for Clinical Use (Pharmacogenomics)(Pharmacogenomics)
Roche Product
CYP 450 Genotyping(drug metabolizing system)
FDA ConfusionClass 1 medical device? (no PMA)
Class 2 or 3 medical device?(requires pre-market approval)
From: Nature Biotechnology 2003 21:959-60
2011-09-26
13
Centrum Badań DNA (http://wwwcbdna.pl/)Poznański Park Naukowo-Techniczny
• Oferta 16 róŜnych testów (predyspozycje do raka piersi i jajnika, 88 róŜnych mutacji w 6 genach m.in. BRCA1 i BRCA2
• Panel na mukowiscydozę 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