2011-09-26

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Mikromacierze

oligonukleotydowe i cDNA

nowe narzędzie w diagnozowaniu i

terapii nowotworów

Pracownia Biologii Molekularnej i Farmakogenomiki
Zakład Biochemii Farmaceutycznej
Wydział Farmaceutyczny
UM w Łodzi

prof. dr hab. n. farm. Marek Mirowski

DNA

mRNA

Transcription

Introduction

Introduction

The Central Dogma

The Central Dogma

of Molecular Biology

of Molecular Biology

Cell

Polypeptide
(protein)

Translation

Ribosome

©1998 Timothy G. Standish

DNA makes RNA makes Protein

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From Gene To Drug

DNA

MRNA
Splice forms

Phosphorylation, cleavage,

glycosylation, sulfation

Gene Translation Post-translational

Expression

modification

30,000-40,000 90,000-120,000 400,000-500,000 4,000-5,000
genes 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.edu
wardlab/microarray.html

Gene

promoter

Structural gene

flank

flank

upstream

downstream

5’

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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3

S

U

G

A

R

-P

H

O

S

P

H

A

T

E

B

A

C

K

B

O

N

E

H

P

O

HO

O

O

CH

2

H

OH

P

O

O

HO

O

O

CH

2

H

P

O

OH

HO

O

O

CH

2

NH

2

N

N

N

N

O

O

NH

2

N

NH

N

N

N

O

NH

2

N

B

A

S

E

S

D

D

N

N

A

A

O

H

P

O

HO

O

O

CH

2

HO

O

H

2

N

N

HN

N

N

H

H

P

HO

O

O

CH

2

O

O

N

O

H

2

N

N

H

H

2

O

H

OH

P

O

HO

O

O

CH

2

C

H

3

O

O

H

N

N

H

2

O

5’Phosphate group

3’Hydroxyl group

5’Phosphate

group

3’Hydroxyl group

The Key to Nucleic Acid Detection is “Sequence-Specific Affinity”

C

A

G

T

A

A

C

G

G

T

T

5’

3’

G

T

C

A

T

T

G

C

C

A

A

5’

3’

Microarray-Based Assays (The Basics)

©2000 Timothy G. Standish

Denaturation

Denaturation

and

and

Renaturation

Renaturation

?

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

T

A

C

T

C

G

A

C

A

T

G

C

T

A

GC

A

C

A

T

G

A

G

C

T

G

T

A

C

G

A

T

C

G

T

G

Double stranded DNA

T

A

C

T

C

G

A

C

A

T

G

C

T

A

GC

A

C

A

T

G

A

G

C

T

G

T

A

C

G

A

T

C

G

T

G

Double stranded DNA

Ren

atu

ratio

n

T

A

C

T

C

G

A

C

A

T

G

C

T

A

GC

A

C

A

T

G

A

G

C

T

G

T

A

C

G

A

T

C

G

T

G

Denatured DNA

De

na

tur

ati

on

Single stranded DNA

©2000 Timothy G. Standish

Hybridization

Hybridization

DNA from source “Y”

T

A

C

T

C

G

A

C

A

GG

C

T

A

G

C

T

G

A

T

GG

T

C

A

T

G

A

G

C

T

G

T

CC

G

A

T

C

G

A

T

C

A

T

DNA from source “X”

T

A

C

T

C

G

A

C

A

GG

C

T

A

G

Hybridization

Hybridization

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

5

µµµµ

l

1X

10 X dNTPs (2mM)

5

µµµµ

l

200

µµµµ

M

Forward primer

(10pmols/

µµµµ

l)

5

µµµµ

l

1

µ

M (50pmols/50

µ

l)

Reverse primer

(10pmols/

µµµµ

l)

5

µµµµ

l

1

µ

M (50pmols/50

µ

l)

Genomic DNA template

2

µµµµ

l

1

µµµµ

g

Thermostable polymerase
(2U/

µµµµ

l)

0.5

µµµµ

l

1 unit

H

2

O (to 50

µµµµ

l Final volume)

27.5

µµµµ

l

TYPICAL REACTION MIXTURE

25 or 50

µ

ls 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 solid

substrate (non-fluorescent glass slide).

“TARGET” is the fluorescence labeled

cDNA representation of the mRNA and

is hybridized to the probe.

Microarray-Based Assays (The Basics)

...

GCUACGAUUGCAACGCCCGAAUGGUUACC

AAAAAAAAAAA

...

dCTP

dATP

dTTP

dGTP

How does a DNA microarray detects gene activity?

Reverse Transcription makes cDNA from gene sequence…

AAAAAAAAAAAAAAAA

mRNA

TTTTTTTTTTT

G

G

T

A

A

C

C

C

C

C

C

C

A

T

T

G

G

G

G

T

T

G

A

A

T

G

T

A

G

cDNA

TTTTTTTTTTT

G

G

T

A

A

C

C

C

C

C

C

C

A

T

T

G

G

G

G

T

T

G

A

A

T

G

T

A

G

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)

C

y

5

(

A

ID

S

)

mRNA Expression in Lung

Gene Expression Profiling: DNA Microarrays

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

Many other methods

Many other methods

For SNP analysis have

For SNP analysis have

been developed

been developed

TTAGCTAGTCTGGACATTAGCCATGCGGAT

GACCTATAATCG

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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

Conventional Method

Microarray

Microarray

50Kb x 1,000 = 50 Mb of
sequence. At a rate of 500
bases

per

lane

and

30

sequencing lanes, you can
produce 15 Kb of sequence
per day. You need 10 years
for the project.

With one microarray of 1.25 x
1.25 cm dimension, you can
scan 50 Kb of sequence at once.
You need 1,000 microarrays to
complete task. This may be
completed 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

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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[e-mail]

Applications of

Applications of
Microarrays

Microarrays

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 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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