Anno accademico 2002-2003
Canale A: Prof. Malavasi
Lezione 1: La Genetica Umana sul web
Il Progetto Genoma Umano
http://www.ncbi.nlm.nih.gov/omim/
- OMIM: online mendelian inheritance in man
- Creato da Victor McKusick
- Continuamente aggiornato
- Punto chiave per acquisire informazione sui caratteri mendeliani umani,
patologici e non
- A ogni carattere viene attribuito un numero a 6 cifre MIM
http://www.ncbi.nlm.nih.gov/disease/
http://www.marchofdimes.com/index.asp
http://www.faseb.org/genetics/
http://sigu.univr.it/
http://www.ncbi.nlm.nih.gov/
http://www.ncbi.nlm.nih.gov/prow/
http://www.genome.gov/
Human Genome Project
- 1970: possibilità di manipolare il DNA
- 1980: costruzione di parti della Human Genome
Map
- 1988: inizio della collaborazione scientifica
internazionale attraverso HUGO
- 1990: programmi di ricerca sul genoma iniziati in
Italia, Inghilterra, Francia, Europa, Giappone e
Canada
- 1990: l’NIH e il US Dept of Energy hanno dato il
via a un progetto congiunto di 5 anni
- prerequisiti:
History
Bilancio del Progetto Genoma Umano
Studi sull’uomo
Costi di funzionamento
Altri organismi
3 milioni di paia di basi
30-35.000 geni al massimo
Sequence
Hybridization of nucleic acids is based on complementarity
A DNA or RNA “probe” is a single strand nucleid acid whose
nucleotide sequence allows it to hybridize uniquely to its
complementary nucleic acid
In classical techniques such as Southern or Northern blotting
The labeled probe
DNA or RNA are
separated on a gel
The gel is transfered
to a filter
finds complementary
targets
Hybridization to solid-state probe array allows detection of
multiple sequences in the same complex sample
Individual probes are
spotted to a filter
…and hybridized
to the filter
RNA or DNA are
extracted from tissues
… labeled during RT
DNA microarrays
DNA chips: thee are currently two major types available:
cDNA microarrays
Oligonucleotide arrays
10,000 - 20,000 probes / cm2 are
spotted on glass slides using an
automated microarrayer
Probes are cDNA or PCR products
representing known genes or
simply EST
Up to 250,000 oligonucleotides / cm2
are sythesized directly on the chip
surface, using a photolitographic
technique.
These 20-25nt long oligonucleotides
represent sequences of known genes
or EST
Highly parallel probe
arrays: “DNA chips”
Dot diameter: 200m
Pitch: 400 m
Fluorochrome labelling of DNA and RNA
RNA from sample and from reference are
labeled by introducing two different
fluorochromes.
This allows co-hybridization of the two
samples to the same chip, providing
direct comparison by two-color analysis
Use of microarrays for Gene
Expression Profiling
“Test” sample (tumor tissue, stimulated cells)
RNA Extraction,
cDNA Synthesis
and labelling
Hybridization
“reference” sample (normal tissue, unstimulated cells)
Transformation to
false-color code
->6-fold
-3-6-fold
-1-3-fold
equal to median
+1-3-fold
+3-6-fold
+>6-fold
Representation
of results
Gene Id.
Sample X
Laser scanning of
individual chips
HNF3a
KDR/Flk1
ERa
Keratin 17
Troponin I
Integrin b4
GATA bp3
AP-2a
…….
……
…..
….
...
Cluster analysis 1
HMEC
HUVEC
MDAMB231
BT549
SKBR3
BT474
MCF7
T47D
->6-fold
-3-6-fold
-1-3-fold
equal to median
+1-3-fold
+3-6-fold
+>6-fold
HNF3a
KDR/Flk1
ERa
Keratin 17
Troponin I
Integrin b4
GATA bp3
AP-2a
Cluster analysis 2
HMEC
HUVEC
MDAMB231
BT549
SKBR3
BT474
MCF7
T47D
->6-fold
-3-6-fold
-1-3-fold
equal to median
+1-3-fold
+3-6-fold
+>6-fold
KDR/Flk1
AP-2a
Troponin I
Keratin 17
Integrin b4
HNF3a
GATA bp3
ERa