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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

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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

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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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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

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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

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“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

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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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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

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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

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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

ApplicationsApplications

Screening for:Screening for:

� Small molecule targets

� Post-translational modifications

� Protein-protein interactions

� Protein-DNA interactions

� Enzyme assays� Epitope mapping

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support@sabiosciences.com

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

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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 ConfusionClass 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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Microarray analysis