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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: 200m 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
