LEZIONE 3 Anno Accademico 2010/11

1. BIOTECNOLOGIE FARMACOLOGICHE CORSO DI LAUREA SPECIALISTICA IN BIOTECNOLOGIE DEL FARMACO LEZIONE 3 Anno Accademico 2010/11

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 • studigeneticidi ‘linkage’ • use of sequence tagget site (STS)

4. MITOSIS profase metafase anafase telofase • The chromosomes decondense • The nuclear envelope forms • Cytokinesis reaches completion, creating two daughter cells • 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 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

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. Mappe genetiche 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. .

9. Analisi di linkage • 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: • Establish a pedigree • Make a number of estimates of recombination frequency • Calculate a LOD score for each estimate • The estimate with the highest LOD score will be considered the best estimate • The LOD score is calculated as follows:

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

11. 1 /8 ricombina: distanzatra A e gene malattia: 0.125 cM 1 genitore – 0.125 = 0.437 2 tipi diricomb Assoc con malattia Se due eventi non sonolegati la probabilitàdiereditarli e ¼ = 0.25 La probabilitàdiunasequenzaallanascita è ilprodotto del valorediognieventoindipendente

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 Frammentazione del DNA genomico con enzimidirestrizione Separazionedeiframmenti per elettroforesisuagarosio Immobilizzazione DNA per trasferimentosumembrana Ibridazione con sondaopportunamentemarcata Identificazionedi 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. 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. 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. They are used in shotgun sequencing, specifically to aid sequence assembly.

16. SEQUENZIAMENTO DEL GENOMA UMANO

17. Progetto GENOMA UMANO • Scopi: • Sequenziamentodeigenomidiinteressebiologico o farmacologico • Studidifunzionegenica http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml

18. Progetto GENOMA UMANO • Strategie: • Generazione sistematica di mappe fisiche • Sequenziamentodi Expressed Sequence Tag (EST) • Miglioramentodelletecnologiedisequenziamento http://www.ornl.gov/sci/techresources/Human_Genome/project/about.shtml

19. Strategie di sequenziamento del genoma

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

21. 1990: Launch of the Human Genome Project1990: ELSI Founded (Ethical, Legal and Social Implications )1990: Research on BACs1991: ESTs, Fragments of Genes (expressed-sequence tag )1992: Second-generation Genetic Map of Human Genome1992: Data Release Guidelines Established1993: NEW HGP Five-year Plan1994: FLAVR SAVR Tomato (Calgene, Inc. of Davis, California )1994: Detailed Human Genetic Map1994: Microbial Genome Project (The microbes DOE chose do not cause disease but are important for their environmental, energy, and commercial roles 1990-1994 1995: Ban on Genetic Discrimination in Workplace1995: Two Microbial Genomes Sequenced1995: Physical Map of Human Genome Comp leted1996: International Strategy Meeting on Human Genome Sequencing1996: Mouse Genetic Map Completed1996: Yeast Genome Sequenced1996: Archaea Genome Sequenced1996: Health Insurance Discrimination Banned1996: 280,000 Expressed Sequence Tags (ESTs)1996: Human Gene Map Created1996: Human DNA Sequence Begins (large-scale sequencing) 1995-1996 1997: Bermuda Meeting Affirms Principle of Data Release1997: E. coli Genome Sequenced1997: Recommendations on Genetic Testing1998: Private Company Announces Sequencing Plan1998: M. Tuberculosis Bacterium Sequenced1998: Committee on Genetic Testing (Service’s Advisory Committee on Genetic Testing 1998: HGP Map Includes 30,000 Human Genes1998: New HGP Goals for 20031998: SNP Initiative Begins (single nucleotide polymorphism, multigene disorders)1998: Genome of Roundworm C. elegans Sequenced1999: Full-scale Human Genome Sequencing1999: Chromosome 22 1997-1999

22. 2000: Free Access to Genomic Information2000: Chromosome 212000: Working Draft2000: Drosophila and Arabidopsis genomes sequenced2000: Executive Order Bans Genetic Descrimination in the Federal Workplace2000: Yeast Interactome Published2000: Fly Model of Parkinson's Disease Reported2001: First Draft of the Human Genome Sequence Released2001: RNAi Shuts Off Mammalian Genes2001: FDA Approves Genetics-based Drug to Treat Leukemia Gleevec to treat patients with chronic myeloid leukemia (CML). 2000-2001 2002: Mouse Genome Sequenced2002: Researchers Find Genetic Variation Associatedwith Prostate Cancer2002: Rice Genome Sequenced2002: The International HapMap Project is Announced2002: The Genomes to Life Program is Launched2002: Researchers Identify Gene Linked to Bipolar Disorder2003: Human Genome Project Completed2003: Fiftieth Anniversary of Watson and Crick's Description of the Double Helix2003: The First National DNA Day Celebrated2003: ENCODE Program Begins2003: Premature Aging Gene Identified 2002-2003

23. 2004-The Future 2004: Rat and Chicken Genomes Sequenced2004: FDA Approves First Microarray2004: Refined Analysis of Complete Human Genome Sequence2004: Surgeon General Stresses Importance of Family History2005: Chimpanzee Genomes Sequenced2005: HapMap Project Completed2005: Trypanosomatid Genomes Sequenced2005: Dog Genomes Sequenced2006: The Cancer Genome Atlas (TCGA) Project Started2006: Second Non-human Primate Genome is Sequenced2006: Initiatives to Establish the Genetic & Environmental Causesof Common Diseases Launched

24. Alcuni risultati dell’analisi della sequenza del genoma umano • n. geni 26*103 • arrangiamentodeigeni non casuale (in cluster) • esoni 1.1% del genoma • introni 24% • sequenzeintergeniche 75% • > 1.4 milionidisitipolimorfici (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 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. 2003 NIH ENCODE Project ENCyclopdia Of DNA Elements (ENCODE). 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.

43. use gene targeting to make the resource of null alleles, marked with a high utility reporter, preferably in C57BL/6; • support a repository to house the products of this resource; • 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; • implement a data coordination center which will make the status and relevant data of the production effort available to the research community.

44. NIH 2006 Genes and Environment Initiative (GEI). 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.

45. HapMap Project 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. 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