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Introduction Basic Genetic Mechanisms Eukaryotic Gene Regulation The Human Genome Project Test 1 PowerPoint Presentation
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Introduction Basic Genetic Mechanisms Eukaryotic Gene Regulation The Human Genome Project Test 1

Introduction Basic Genetic Mechanisms Eukaryotic Gene Regulation The Human Genome Project Test 1

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Introduction Basic Genetic Mechanisms Eukaryotic Gene Regulation The Human Genome Project Test 1

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  1. Molecular Genetics The Human Genome: Biology and Medicine • Introduction • Basic Genetic Mechanisms • Eukaryotic Gene Regulation • The Human Genome Project • Test 1 • Genome I - Genes • Genome II – Repetitive DNA • Genome III - Variation • Test 2 • Monogenic and Complex Diseases • Finding ‘Disease’ Genes • Pharmacogenomics • Test 3 • Your Presentations • Your Presentations • Happy New Year!  http://priede.bf.lu.lv/ Studiju materiāli / MolekularasBioloģijas / MolGen / EN

  2. J. Watson (~ 1988) about the Human Genome project's goal: "to find out what being human is."

  3. The Human Genome Project (HGP) 1984 - idea of sequencing the entire human genome • Robert Sinsheimer • discussionto be or not to be? • the sequence of mitochondrial genome was already determined (1981) 1988 - US National Research Council endorses the concept 1990 - the 15-year project officially begins • NIH (National Institutes of Health) un DOE (Department of Energy) • James Watson (till 1992), Francis Collins (up to the finish line) 20 genome centres - USA, UK,France, Japan, Germany, China International Human Genome Sequencing Consortium (IHGSC)

  4. HGP aims extended far beyond obtaining the full DNA sequence of the human genome

  5. The project went for a phased approach 2003 “Genome race”

  6. How to determine the sequence of DNA? • amplify • make DNA single-stranded • perform sequencing • read the sequence • 500 - 700 bp

  7. DNA can be amplified in two ways

  8. Molecular Biology of the Gene, 5th Edition Reminder on DNA synthesis (replication) in cells

  9. Sanger (1975-77) dideoxy or chain termination method

  10. The dideoxy or chain termination (also known as enzymatic) method (or a labeled nucleotide)

  11. Fluorescent dyes (1986 - ...) capillary electrophoresis computer screen Genomes, 2nd Edition

  12. Automated sequencers (1986 - 1989- 1998)

  13. Automated production lines for sample preparation

  14. Cheaper and quicker – the human genome sequence came within reach

  15. How to determine the sequence of a genome? • brake the genome into fragments, make a library • determine the sequence of fragments, i.e., clones • find overlapping sequences (computer) • assemble the master sequence (computer) • close the gaps - “finishing” • up to 5 million bp, i.e., bacterial genomes SHOTGUN method

  16. Construction of a genomic library

  17. Principle of the shotgun approach (1981) to SEQUENCE ASSEMBLY Genomes, 2nd Edition

  18. Principle of the shotgun approach (1981) to SEQUENCE ASSEMBLY Contig – contiguous block of sequence Recombinant DNA, 3rd Edition

  19. Assembly of the sequence blocks Scaffold - sets of contigs joined by paired reads from both ends of plasmid inserts Molecular Biology of the Gene, 5th Edition

  20. Different parts of the genome are not equally covered by clones and sequences Genomes, 2nd Edition • Clone /sequence coverage – the number of independent clones/sequences that are needed to ensure having a complete sequence. • A 1x sequence coverage - means that the number of base pairs determined equals the number of base pairs in the genome: • 1x coverage produces only ~ 63% of the genome’s sequence • 6x – 99.75% • 10x – 99.995% Cloned DNA for sequencing must represent the entire genome and be sequenced multiple times

  21. The first whole-genome shotgun (1995) Genomes, 2nd Edition A human pathogen that causes upper respiratory and middle ear infections, and even meningitis. 1.8 million bp 19 687 clones 28 643 sequencing reactions (16% failures); a total of 11 631 485 bp determined (6x coverage) 140 contigs

  22. Some pieces of DNA sequence are always missing Genomes, 2nd Edition

  23. Closing the gaps (“finishing”) Genomes, 2nd Edition

  24. 1743 predicted coding regions Recombinant DNA, 3rd Edition

  25. The data analysis becomes disproportionately more complex as the number of fragments increases. The shotgun method can lead to errors when repetitive regions of genome are analysed: Genomes, 2nd Edition Shotgun cannot be used on its own to sequence eukaryotic genome

  26. How to determine the sequence of a eukaryotic genome? • construct a genome map • make libraries of different size inserts • apply a variant of the shotgun approach • any genome can be sequenced in this way

  27. Physical map contain markers that aid the sequence assembly • genetic • physical Genomes, 2nd Edition

  28. The principle of using a physical map Genomes, 2nd Edition STS – sequence tagged site – a short DNA sequence (100-500bp), that is easily recognizable and occurs only once in the chromosome or genome being studied.

  29. There are two alternative strategies of eukaryotic genome sequencing Hierarchical shotgun Genomes, 2nd Edition

  30. The Human Genome Project employed the Hierarchical shotgun

  31. Large-insert cloning systems used in the project Recombinant DNA, 3rd Edition

  32. Construction of large-insert clone map Recombinant DNA, 3rd Edition

  33. Construction of large-insert clone map Genomes, 2nd Edition

  34. Human Genome Project Celera Genomics (C.Venter) “Genome race” • 29 298 mapped large-insert clones • 1246 clone contigs • 23 000 000 000 bp; 7.5x coverage • 35% of the genome in “finished form” • 27 000 000 sequences of 543 bp • 5.1 x coverage • Combined with HGP – 12.6 x

  35. Draft sequences (~90% of euchromatic part;~ 150 000 gaps) - 2001 Human Genome Project Celera Genomics (C.Venter)

  36. Finishing the sequence of the human genome (HGP) Recombinant DNA, 3rd Edition

  37. >99% euchromatic sequence with accuracy > 99.99%

  38. Recombinant DNA, 3rd Edition

  39. Recombinant DNA, 3rd Edition

  40. The sequence content of the human genome

  41. Next-generation technology brings sequencing closer to routine 2010 Goal $ 100 000 $ 1000

  42. Principle of this 2nd generation technology • Fragments are bound to beads, • the beads are captured in droplets of oil emulsion • and PCR amplification occurs in each droplet 2 • DNA is isolated, fragmented, ligated to adapters • and separated into single strands 1 Smaller beads carrying immobilized enzymes required for sequencing are deposited into wells • Emulsion is broken, DNA strands are denatured, • and beads carrying ssDNA are deposited • into wells of a fibre optic slide 4 3 Nature (2005) 437, 376 – 380. Standard technology – cloning and dideoxy-sequencing of individual clones New technology – in vitro and simultaneous sequencing of 100 000 fragments pirosequencing 5

  43. Determination of DNA sequence with PIROSEQUENCING method www.pyrosequencing.com APS: adenosine 5´ phosphosulfate

  44. 1953: F. Crick un J. Watson decipher the structure of DNA 2008: J. Watson receives a digital version of his deciphered genome

  45. What about translation of Watson’s genomic information to clinical practice? Bentley DR (2004) Genomes for medicine. Nature, 429, 440-445. Individual sequence of James Watson

  46. The era of personalized genomic medicine for Dr. Watson and everybody else has not come yet, although he is carrier of a handful disease causing mutations and disease risk variants that might catch a genetic counsellor’s interest. (HGDM – Human Gene Mutation Database)

  47. The era of personalized genomic medicine for Dr. Watson and everybody else has not come yet, although he is carrier of a handful disease causing mutations and disease risk variants that might catch a genetic counsellor’s interest. “It was so profound, how little we were actually able to say about that... To me, it really proved that this is the beginning, not the end.” (conclusion after a counselling session)

  48. (2002) 3, 44 However, other new technologies can be of practical use now or in the nearest future Genomics + genotyping Transcriptomics Proteomics Animal models + Bioinformatics

  49. DNA microchips (microarrays)

  50. Principle of mRNA analysis