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2 Mappare i geni: Storicamente attraverso la costruzione di mappe fisiche (identificare e ordinare marcatori lungo il cromosoma) Attualmente con il sequenziamento

3 mappe fisiche costruite con: restriction mapping FISH ( Fluorescent in situ hybridization ) mapping studi genetici di ‘linkage’ use of sequence tagget site (STS)

4 profasemetafaseanafasetelofase The chromosomes condense and become visible The centrioles form and move toward opposite ends of the cell ("the poles") The nuclear membrane dissolves The mitotic spindle forms (from the centrioles in animal cells) Spindle fibers from each centriole attach to each sister chromatid at the kinetochore The Centrioles complete their migration to the poles The chromosomes line up in the middle of the cell ("the equator") Spindles attached to kinetochores begin to shorten. This exerts a force on the sister chromatids that pulls them apart. Spindle fibers continue to shorten, pulling chromatids to opposite poles. This ensures that each daughter cell gets identical sets of chromosomes The chromosomes decondense The nuclear envelope forms Cytokinesis reaches completion, creating two daughter cells MITOSIS

5 MEIOSIS Meiosis: a special type of cell division that occurs in sexually reproducing organisms Meiosis reduces the chromosome number by half Meiosis of diploid cells produces haploid daughter cells, which may function as gametes. Gametes undergo fertilization, restoring the diploid number of chromosomes in the zygote Meiosis and fertilization introduce genetic variation in three ways: Crossing over between homologous chromosomes at prophase I. Independent assortment of homologous pairs at metaphase I: Each homologous pair can orient in either of two ways at the plane of cell division. The total number of possible outcomes = 2n (n = number of haploid chromosomes). Random chance fertilization between any one female gamete with any other male gamete.



8 Il centimorgan (cM) è l’unità di misura della distanza genetica tra 2 loci. La distanza tra due loci che presentano una frequenza di ricombinazione dell'1% è 1 cM. Le misurazioni della distanza tra i diversi loci permettono di generare mappe genetiche o mappe cromosomiche che quindi sono il risultato di un calcolo basato sulla osservazione di frequenze di ricombinazione.. Mappe genetiche

9 The LOD score (logarithm (base 10) of odds) is a statistical test often used for linkage analysis in human populations, and also in animal and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD score favor the presence of linkage, whereas negative LOD scores indicate that linkage is less likely. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers). The method is described in greater detail by Strachan and Read. Briefly, it works as follows: 1. Establish a pedigree 2. Make a number of estimates of recombination frequency 3. Calculate a LOD score for each estimate 4.The estimate with the highest LOD score will be considered the best estimate The LOD score is calculated as follows: Analisi di linkage

10 EREDITA’ DI MARCATORI RFLP Restriction Fragment Length Polymorphism Single Nucleotide Polymorphism

11 Assoc con malattia 1 /8 ricombina: distanza tra A e gene malattia: 0.125 cM 1 genitore – 0.125 2 tipi di ricomb = 0.437 Se due eventi non sono legati la probabilità di ereditarli e ¼ = 0.25 La probabilità di una sequenza alla nascita è il prodotto del valore di ogni evento indipendente

12 Poichè la probabilità di una sequenza alla nascita misurata è 0.125, la frequenza di ricombinazione per gli otto figli sarebbe uguale a (0.4375) 7 (0.0625) 1 = 0.0001917 La probabilità di una sequenza senza linkage sarebbe: (0.25) 8 = 0.0000153 Quindi : LOD SCORE: log 12.566 = 1.099

13 RESTRICTION MAPPING 1.Frammentazione del DNA genomico con enzimi di restrizione 2.Separazione dei frammenti per elettroforesi su agarosio 3.Immobilizzazione DNA per trasferimento su membrana 4.Ibridazione con sonda opportunamente marcata 5.Identificazione di RLFP

14 restriction mapping sequence tagget site

15 A sequence-tagged site (or STS) is a short (200 to 500 base pair) DNA sequence that has a single occurrence in the genome and whose location and base sequence are known.DNAgenome STSs can be easily detected by the polymerase chain reaction (PCR) using specific primers. For this reason they are useful for constructing genetic and physical maps from sequence data reported from many different laboratories. They serve as landmarks on the developing physical map of a genome.polymerase chain reactionprimersphysical map When STS loci contain genetic polymorphisms (e.g. simple sequence length polymorphisms, SSLPs, single nucleotide polymorphisms), they become valuable genetic markers, i.e. loci which can be used to distinguish individuals.loci They are used in shotgun sequencing, specifically to aid sequence assembly.shotgun sequencingsequence assembly


17 Progetto GENOMA UMANO Scopi: Sequenziamento dei genomi di interesse biologico o farmacologico Studi di funzione genica

18 Progetto GENOMA UMANO Strategie: Generazione sistematica di mappe fisiche Sequenziamento di Expressed Sequence Tag (EST) Miglioramento delle tecnologie di sequenziamento

19 Strategie di sequenziamento del genoma

20 Durata del sequenziamento del genoma umano con il metodo ‘Whole genome shot-gun’ 8 settembre 1999 17 giugno 2000 Investimento NIH nel progetto 3 miliardi di dollari /13 anni Termine del progetto Genoma Umano 2003 Craig Venter Francis Collin

21 1990: Launch of the Human Genome Project 1990: ELSI Founded ( Ethical, Legal and Social Implications ) 1990: Research on BACs 1991: ESTs, Fragments of Genes ( expressed-sequence tag ) 1992: Second-generation Genetic Map of Human Genome 1992: Data Release Guidelines Established 1993: NEW HGP Five-year Plan 1994: FLAVR SAVR Tomato ( Calgene, Inc. of Davis, California ) 1994: Detailed Human Genetic Map 1994: Microbial Genome Project ( The microbes DOE chose do not cause disease but are important for their environmental, energy, and commercial rolesLaunch of the Human Genome ProjectELSI FoundedResearch on BACsESTs, Fragments of GenesSecond-generation Genetic Map of Human GenomeData Release Guidelines EstablishedNEW HGP Five-year PlanFLAVR SAVR TomatoDetailed Human Genetic MapMicrobial Genome Project 1990-1994 1995: Ban on Genetic Discrimination in Workplace 1995: Two Microbial Genomes Sequenced 1995: Physical Map of Human Genome Comp leted 1996: International Strategy Meeting on Human Genome Sequencing 1996: Mouse Genetic Map Completed 1996: Yeast Genome Sequenced 1996: Archaea Genome Sequenced 1996: Health Insurance Discrimination Banned 1996: 280,000 Expressed Sequence Tags (ESTs) 1996: Human Gene Map Created 1996: Human DNA Sequence Begins ( large-scale sequencing)Ban on Genetic Discrimination in WorkplaceTwo Microbial Genomes SequencedPhysical Map of Human Genome Comp letedInternational Strategy Meeting on Human Genome SequencingMouse Genetic Map CompletedYeast Genome SequencedArchaea Genome SequencedHealth Insurance Discrimination Banned280,000 Expressed Sequence Tags (ESTs)Human Gene Map CreatedHuman DNA Sequence Begins 1995-1996 1997: Bermuda Meeting Affirms Principle of Data Release 1997: E. coli Genome Sequenced 1997: Recommendations on Genetic Testing 1998: Private Company Announces Sequencing Plan 1998: M. Tuberculosis Bacterium Sequenced 1998: Committee on Genetic Testing ( Service’s Advisory Committee on Genetic Testing 1998: HGP Map Includes 30,000 Human Genes 1998: New HGP Goals for 2003 1998: SNP Initiative Begins ( single nucleotide polymorphism, multigene disorders) 1998: Genome of Roundworm C. elegans Sequenced 1999: Full-scale Human Genome Sequencing 1999: Chromosome 22Bermuda Meeting Affirms Principle of Data ReleaseE. coli Genome SequencedRecommendations on Genetic TestingPrivate Company Announces Sequencing PlanM. Tuberculosis Bacterium SequencedCommittee on Genetic TestingHGP Map Includes 30,000 Human GenesNew HGP Goals for 2003SNP Initiative BeginsGenome of Roundworm C. elegans SequencedFull-scale Human Genome SequencingChromosome 22 1997-1999

22 2000: Free Access to Genomic Information 2000: Chromosome 21 2000: Working Draft 2000: Drosophila and Arabidopsis genomes sequenced 2000: Executive Order Bans Genetic Descrimination in the Federal Workplace 2000: Yeast Interactome Published 2000: Fly Model of Parkinson's Disease Reported 2001: First Draft of the Human Genome Sequence Released 2001: RNAi Shuts Off Mammalian Genes 2001: FDA Approves Genetics-based Drug to Treat Leukemia Gleevec to treat patients with chronic myeloid leukemia (CML).Free Access to Genomic InformationChromosome 21Working DraftDrosophila and Arabidopsis genomes sequencedExecutive Order Bans Genetic Descrimination in the Federal WorkplaceYeast Interactome PublishedFly Model of Parkinson's Disease ReportedFirst Draft of the Human Genome Sequence ReleasedRNAi Shuts Off Mammalian GenesFDA Approves Genetics-based Drug to Treat Leukemia 2000-2001 2002: Mouse Genome Sequenced 2002: Researchers Find Genetic Variation Associated with Prostate Cancer 2002: Rice Genome Sequenced 2002: The International HapMap Project is Announced 2002: The Genomes to Life Program is Launched 2002: Researchers Identify Gene Linked to Bipolar Disorder 2003: Human Genome Project Completed 2003: Fiftieth Anniversary of Watson and Crick's Description of the Double Helix 2003: The First National DNA Day Celebrated 2003: ENCODE Program Begins 2003: Premature Aging Gene IdentifiedMouse Genome SequencedResearchers Find Genetic Variation Associated with Prostate CancerRice Genome SequencedThe International HapMap Project is AnnouncedThe Genomes to Life Program is LaunchedResearchers Identify Gene Linked to Bipolar DisorderHuman Genome Project CompletedFiftieth Anniversary of Watson and Crick's Description of the Double HelixThe First National DNA Day CelebratedENCODE Program BeginsPremature Aging Gene Identified 2002-2003

23 2004-The Future 2004: Rat and Chicken Genomes Sequenced 2004: FDA Approves First Microarray 2004: Refined Analysis of Complete Human Genome Sequence 2004: Surgeon General Stresses Importance of Family History 2005: Chimpanzee Genomes Sequenced 2005: HapMap Project Completed 2005: Trypanosomatid Genomes Sequenced 2005: Dog Genomes Sequenced 2006: The Cancer Genome Atlas (TCGA) Project Started 2006: Second Non-human Primate Genome is Sequenced 2006: Initiatives to Establish the Genetic & Environmental Causes of Common Diseases LaunchedRat and Chicken Genomes SequencedFDA Approves First MicroarrayRefined Analysis of Complete Human Genome SequenceSurgeon General Stresses Importance of Family HistoryChimpanzee Genomes SequencedHapMap Project CompletedTrypanosomatid Genomes SequencedDog Genomes SequencedThe Cancer Genome Atlas (TCGA) Project StartedSecond Non-human Primate Genome is SequencedInitiatives to Establish the Genetic & Environmental Causes of Common Diseases Launched

24 Alcuni risultati dell’analisi della sequenza del genoma umano n. geni 26*10 3 arrangiamento dei geni non casuale (in cluster) esoni 1.1% del genoma introni 24% sequenze intergeniche 75% > 1.4 milioni di siti polimorfici (SNPs)


26 NATURE Human Genome Collection It is now more than 15 years since work began sequencing the 2.85 billion nucleotides of the human genome. While the draft sequence was published in Nature in 2001, researchers at the Human Genome Project continued to fill the gaps and subject individual chromosomes to ever more detailed analyses. Nature is proud to present here the complete and comprehensive DNA sequence of the human genome as a freely available resource.


28 Risultati del Progetto GENOMA UMANO



31 The Cancer Genome Atlas (TCGA) is a comprehensive and coordinated effort to accelerate our understanding of the genetics of cancer using innovative genome analysis technologies. The overarching goal of The Cancer Genome Atlas (TCGA) is to improve our ability to diagnose, treat and prevent cancer


33 The National Institutes of Health announced in 2009 the expansion of TCGA project. After a rigorous review process, the scope of the TCGA Research Network has expanded to include more than 20 tumor types and thousands of samples over the next five years. Each cancer will undergo comprehensive genomic characterization that incorporates powerful bioinformatic and data analysis components. The expansion of TCGA is expected to lead to the most comprehensive understanding of cancer genomes and will enable researchers to further mine the data generated by TCGA to improve prevention, diagnosis and treatment of cancer.

34 To identify and locate all of the genome’s protein- coding genes, non-protein coding genes, and other sequence-based functional elements. When completed, the collection of elements identified by the ENCODE program will help scientists better understand how the genome influences our health. 2003 NIH ENCODE Project ENCyclopdia Of DNA Elements (ENCODE).









43 1)use gene targeting to make the resource of null alleles, marked with a high utility reporter, preferably in C57BL/6; 2)support a repository to house the products of this resource; 3)develop improved C57BL/6 ES cells that show robust germline transmission, so that they may be used in a high throughput pipeline in generating this resource; 4)implement a data coordination center which will make the status and relevant data of the production effort available to the research community.

44 GEI has two components: the analysis of genetic variation among people with specific diseases an effort to develop technology that will find new ways to monitor environmental exposures that interact with genetic variations leading to disease. The specific diseases that GEI will focus on will be decided by peer review. NIH 2006 Genes and Environment Initiative (GEI).

45 The International HapMap Project is a multi-country effort to identify and catalog genetic similarities and differences in human beings. Using the information in the HapMap, researchers will be able to find genes that affect health, disease, and individual responses to medications and environmental factors. HapMap Project The goal of the International HapMap Project is to compare the genetic sequences of different individuals to identify chromosomal regions where genetic variants are shared















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