einführung in die genetik - tum...segmentally duplicated regions in the arabidopsis genome. the...

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Einführung in die Genetik Prof. Dr. Kay Schneitz (EBio Pflanzen) http://plantdev.bio.wzw.tum.de [email protected] Prof. Dr. Claus Schwechheimer (PlaSysBiol) http://wzw.tum.de/sysbiol [email protected]

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  • Einführung in die Genetik

    Prof. Dr. Kay Schneitz (EBio Pflanzen)http://[email protected]

    Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/[email protected]

    http://plantdev.bio.wzw.tum.dehttp://plantdev.bio.wzw.tum.dehttp://plantdev.bio.wzw.tum.dehttp://plantdev.bio.wzw.tum.demailto:[email protected]:[email protected]

  • Einführung in die Genetik - InhalteEinführung in die Genetik - InhalteEinführung in die Genetik - Inhalte1 Einführung 16. 10. 12 KS2 Struktur von Genen und Chromosomen 23. 10. 12 KS3 Genfunktion 30. 10. 12 KS4 Transmission der DNA während der Zellteilung 06. 11. 12 KS5 Vererbung von Einzelgenveränderungen 13. 11. 12 KS6 Genetische Rekombination (Eukaryonten) 20. 11. 12 KS7 Genetische Rekombination (Bakterien/Viren) 27. 11. 12 KS8 Rekombinante DNA-Technologie 04. 12. 12 CS9 Kartierung/Charakterisierung ganzer Genome 11. 12. 12 CS

    10 Genmutationen: Ursache und Reparatur 18. 12. 12 CS11 Veränderungen der Chromosomen 08. 01. 13 CS12 Genetische Analyse biologischer Prozesse 15. 01. 13 CS13 Transposons bei Eukaryonten 22. 01. 13 CS14 Regulation der Genexpression 29. 01. 13 KS15 Regulation der Zellzahl - Onkogene 05. 02. 13 CS

  • Recombinant DNA Technology

    Genetics 08

    Based on Chapter 11 (Griffiths; 10th ed.)

  • Summary • Plasmids and vectors• origin of replication (ori)• selection markers (AmpR, TetR etc.)• polylinker = multiple cloning site• restriction sites• blue-white selection (LacZ)

    • Cloning, recombination technology, genetic engineering• Restriction enzymes

    • sticky and blunt ends• 5’ overhangs, 3’ overhangs• methylation of DNA in E. coli

    • T4 Ligases and ligation• Topoisomerase-based cloning• Recombination-based cloning (e.g. Gateway system)

  • • Polymerase chain reaction• Melting, annealing, extension• Taq polymerase• primer, oligo(-nucleotide)

    • Transformation of ligation product to E. coli• Heat shock• Electroshock

    • DNA Preparation using alkaline lysis• DNA sequencing

    • Sanger sequencing (dideoxy sequencing, chain termination sequencing)

    • Next generation sequencing• Third generation sequencing

    Summary

  • E.coli strains (examples)

    XL1 Blue - for cloning

    Rosetta(DE3)pLysS - for protein expression

  • Mapping and characterization of entire genomes

    Genetics 09

    Based on Chapter 15 (Griffiths; 10th ed.)

  • How are genome sequences obtained?

    How is this information deciphered?

    How can comparing genomes help to understand life and evolution?

    The advances through next generation sequencing

    How does the availability of genome sequences affect biological analyses?

  • How are genome sequences obtained?

  • Restriction enzymes

    Digest of genomic DNAM size marker

    1,3 undigested

    2,4 digested

    Digest of plasmid DNAM size marker

    1,3 undigested

    2,4 digested

    Agarose gel Agarose gel

  • Genome sequencing strategy

    Arabidopsis125 000 000 bp

    Large fragmentsx 100 000 bp

    Orderd large fragments(minimal tiling path)

    Make small fragments from large fragmentsx 500 - 1000 bp

    Sequence, align and overlap reads (contig)

    Sequencing length 500-1000 bp/run

    Assemble chromosomes and genome

  • Vectors for large inserts

    BACs100 - 300 kb

    YACs50 - 2000 kb

    Phage Lambda35 - 45 kb

    BAC, bacterial artificial chromosomeYAC, yeast artificial chromosome

  • Vectors for small inserts

    Plasmid vectors

  • Genome sequencing strategyArabidopsis125 000 000 bp

    Large fragmentsx 100 000 bp

    Orderd large fragments(minimal tiling path)

    Make small fragments from large fragmentsx 500 - 1000 bp

    Sequence, align and overlap reads (contig)

    Assemble chromosomes and genome

  • Generating physical maps

  • Generating a minimal tiling path

  • From paired end reads to a contig

  • Filling contig gaps

  • How is genome sequence deciphered?

  • Genome size

    Bacteriophage fx 174 (5.3 kb, first sequenced genome 1977)Mitochrondrial DNA (human; 16.3 kb)Bacteriophage l (48.5 kb)Chloroplast DNA (Marchantia; 121 kb)Vaccinia virus (192 kb)Cytomegalovirus (CMV; 229 kb)Bacteria (Haemophilus influenzae; 1,830 kb)Bacteria (Escherichia coli; 4,600 kb)Yeasts (Saccharomyces cerevisiae; 12,100 kb)Insects (Drosophila; 130,000 kb)Plant (Arabidopsis; 157,000 kb)Man (3,200,000 kb)Plant (Wheat; 17,000,000 kb)Fish (Protopteros aethiopicus; 130,000,000 kb) = largest genome

  • Chromosome numbers

  • Segmentally duplicated regions in the Arabidopsis genome.The Arabidopsis genome initiative, Nature 408, 796-815 (2000)

    DNA sequence comparison

    SyntenyArabidopsis ChromosomeNOR, nucleolus organizing region

  • Elements and sites be recognized by more or less conserved DNA sequence elements, can therefore be predicted by bioinformatics

    Exon/intron structure particularly important because it allows to predict the sequence of a protein

    Structure of a eukaryotic gene

  • cDNA = complementary DNA of mRNA

    EST = expressed sequence tag, sequenced cloned mRNA/cDNA

    Predicting and confirming genesfrom a genomic sequence and cDNA/ESTs

  • Translating genomic information into protein

  • Making gene predictions based on genome sequence

  • No correlation between genome size and gene numbers

    Number of genes Genome size (Mb)

  • How comparing genomes can help to understand life and evolution

  • Segmentally duplicated regions in the Arabidopsis genome.The Arabidopsis genome initiative, Nature 408, 796-815 (2000)

    Synteny

    SyntenyArabidopsis ChromosomeNOR, nucleolus organizing region

  • Genome evolution

    Synteny

  • The advances through next generation sequencing

  • Next generation sequencing

  • Shearing of the DNA!to 300 - 800 bp fragments!

    Adaptor ligation!B Primer is biotinylated!

    Streptavidin beads capture biotinylated B primed fragments -> Emulsion PCR is used to amplify fragment on the beads!

    Distribution of beads to a fibre-optic PicoTiterDevice!

    Million fold amplification of PCR!fragment on the beads!

    Pyrosequencing!

    Roche 454/GS FLX Sequencing Technology

  • N.b. next generation sequencing allows to obtain full genome sequences while omitting the cloning steps, thus saving time and cost.

    An assembled and related genome may be used as a scaffold for genome assembly

    The information about the full genome architecture may not be required in

    A genome sequence map

  • Genome sequencing is automated

  • ...and largely institutionalized

  • NGS genome sequencing revolution

    •  (Crop) plant genomes (published)

    rice (2002)

    poplar (2006)

    grape (2007)

    papaya (2008)

    cucumber (2009)

    maize (2009)

    sorghum (2009)

    soybean (2010)

    apple (2010)

    strawberry (2010)

    •  Model plant genomes

    Arabidopsis (2000)

    Brachypodium (2010)

    1001 Genomes project (2011)

  • The 1000 (Human) Genomes project

  • How does the availability of genome sequences affect biological analyses?

  • Functional studies - Gene knock outs

  • Functional studies - Gene targeting

  • Functional studies - Gene targeting

  • Functional studies - Insertion mutagenesis

  • Functional studies - Insertion mutagenesis

    How to generate a random insertion mutant collectiongenerate a big population with randomly tagged linesamplify tagged locus with TAIL PCRsequence amplified locusput sequence in a databaseothers interested in the tagged gene/locus can obtain a mutant

  • Functional studies - Insertion mutants

  • Transcriptomics and gene expression profiling

    Microarrays

    Heat map

  • What you need to know and understand

    for the exam and for your life....

    ... organization of a (eukaryotic) gene

    ... vector types

    ... usefuless of genomic sequences

  • The end