Einführung in die Genetik - Technische Universität...

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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] “Downloads” is linked to Schneitz web page 1
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Transcript of Einführung in die Genetik - Technische Universität...

  • Einführung in die Genetik

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

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

    “Downloads” is linked to Schneitz web page

    1

    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]:[email protected]:[email protected]:[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

    2

  • Genetic Recombination in Bacteria and their Viruses

    Genetics 07

    3

  • Summary• Plasmids

    • small DNA circles (1-2% of bacterial DNA), replicate autonomously in bacterial cell

    • contain additional genes (e.g, resistance genes, F genes)• Conjugation

    • directional transfer of DNA from a donor to a recipient cell, requires physical contact

    • F plasmid confers “maleness”• Hfr strains

    • copy of F plasmid integrated somewhere in bacterial chromosome

    • produces high number of recombinants in Hfr x F- crosses

    • merozygote exconjugants where multiple crossovers can occur between exo- and endogenote

    • Interrupted mating and recombination mapping• circular genetic map of E. coli

    4

  • Summary• Bacteriophages

    • bacterial viruses• Virulent phages

    • immediately lyse and kill their host bacterium• e.g., bacteriophages P1, T4

    • Temperate phage• maintained in host bacterium without immediately killing the

    host

    • e.g., bacteriophage λ• Prophage

    • phage genome that is integrated into the host chromosome• lysogenic bacterium carries a prophage

    • General transduction• Phage transfers any piece of bacterial genomic DNA between

    cells

    • Special transduction• Prophage integrated at a single, specific site in bacterial

    chromsome (e.g., λ attachment site)

    • transfers only genes located close by the attachment site5

  • Recombinant DNA Technology

    Genetics 08

    6

  • Molecular cloning - what for?

    • to sequence them (individual genes or whole genomes)

    • to put them in an order (genomes and genome fragments)

    • to do something with them (express their gene products/proteins)

    • to manipulate them (make gene fusions, introduce mutations)

    • ...

    7

  • Cloning for DNA sequencing

    • Mutations in the BREAST CANCER1 (BRCA1) geneSequencing requires the clonal amplification of the DNA fragments to be sequenced

    8

  • Cloning for genome assembly

    • Sorted fragments of an assembled genomeSequencing requires the clonal amplification of the DNA fragments to be sequenced

    9

  • Cloning for genetic engineering

    • Protein expression in a heterologous host (bacteria)Recombinant protein expression requires cloning

    10

  • Cloning for cell biology

    • Protein localization in a (plant) cell using green fluorescent proteinGene fusions can be generated by molecular cloning

    GFP-fusion protein (of phytochrome) that moves to the nucleus after detection of red light

    11

  • Molecular cloning

    DNA sequencing 1

    Examples

    Polymerase chain reaction

    Recombination-based cloning

    DNA sequencing II

    12

  • Molecular cloning

    13

  • Cloning and cloning vectors

    Restriction digest

    Ligation

    Transformation

    Clonal amplification

    DNA preparation

    Selection

    Restriction digestor sequencing

    14

  • Restriction digest

    Ligation

    Transformation

    Clonal amplification

    DNA preparation

    Selection

    Restriction digestor sequencing

    Restriction sites

    Origin of replication (ori)

    Antibiotic resistance(amp, tet)

    Cloning and cloning vectors

    15

  • restriction sites(polylinker, multiple cloning site)

    (lac promoter)

    origin of replication (ori)

    selection(for plasmid)

    (lacZ reporter)

    selection(for insert)

    Cloning and cloning vectors

    16

  • Restriction enzymes

    • there are hundreds of restriction enzymes available commercially

    • restrcition enzymes can generate different types of overhangs

    • there is a restriction enzyme basically for every DNA sequence

    • the frequency depends on the length of the recognition site (e.g. for a hexameric site the frequency is on average 4096 bp)

    17

  • 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

    18

  • • restriction enzymes are isolated from bacteria

    • bacteria have restriction enzmyes to protect themselves against foreign DNA

    • they protect their own DNA by DNA methlyation

    Restriction enzymes

    19

  • T4 DNA Ligase

    20

  • Cloning and cloning vectors

    restriction sites(polylinker, multiple cloning site)

    (lac promoter)

    origin of replication (ori)

    selection(for plasmid)

    (lacZ reporter)

    selection(for insert)

    21

  • Cloning and cloning vectors

    Restriction digest

    Ligation

    Transformation

    Clonal amplification

    DNA preparation

    Selection

    Restriction digestor sequencing

    Restriction sites

    Origin of replication (ori)

    Antibiotic resistance(amp, tet)

    22

  • Transformation

    Plasmid after ligation

    Heat shock transformation(Alternative: electro shock) Selection

    23

  • Cloning and cloning vectors

    Restriction digest

    Ligation

    Transformation

    Clonal amplification

    DNA preparation

    Selection

    Restriction digestor sequencing

    Restriction sites

    Origin of replication (ori)

    Antibiotic resistance(amp, tet)

    24

  • DNA preparation

    25

  • DNA sequencing

    26

  • The dideoxy sequencing method (1)

    Nobel prize in 1980Walter Gilbert and Frederick Sanger(Sanger sequencing)

    27

  • The dideoxy sequencing method (2)

    28

  • (Fluorescent) dye terminator technology

    29

  • Examples

    30

  • Cloning vectors for sequencing

    31

  • Cloning GST-fusion constructs

    • Expression and purification of a GST-tagged protein

    • GST, glutathione S-transferase (protein)

    • glutathione, small molecule that can be bound to a resin

    GE Healthcare

    pGEX vectors, GST Gene Fusion System

    imagination at work

    Map of the glutathione S-transferase fusion vectors showing reading frames and main features. Even though stop codons in all three frames are not depicted in this map, all thirteen vectors have stop codons in all three frames downstream from the multiple cloning site.

    Ordering informationProduct Quantity Code no.pGEX-‐1λT EcoRI/BAP 5 µg 28-‐9546-‐56

    pGEX-‐2T 25 µg 28-‐9546-‐53

    pGEX-‐2TK 25 µg 28-‐9546-‐46

    pGEX-‐3X 25 µg 28-‐9546-‐54

    pGEX-‐4T-‐1 25 µg 28-‐9545-‐49

    pGEX-‐4T-‐2 25 µg 28-‐9545-‐50

    pGEX-‐4T-‐3 25 µg 28-‐9545-‐52

    pGEX-‐5X-‐1 25 µg 28-‐9545-‐53

    pGEX-‐5X-‐2 25 µg 28-‐9545-‐54

    pGEX-‐5X-‐3 25 µg 28-‐9545-‐55

    pGEX-‐6P-‐1 25 µg 28-‐9546-‐48

    pGEX-‐6P-‐2 25 µg 28-‐9546-‐50

    pGEX-‐6P-‐3 25 µg 28-‐9546-‐51

    Do you want to learn more? Read the GST Gene Fusion System Handbook (18-‐1142-‐75). Please contact your local GE Healthcare representative for a printed copy.

    pGEX-1λT

    pGEX-6P-1

    EcoRI SmaI SalI XhoI NotIBamHI

    PreScission™ Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro HisCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT

    pGEX-6P-2

    BamHI

    PreScission Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG

    EcoRI SmaI SalI XhoI NotI

    pGEX-6P-3

    BamHI

    PreScission Protease

    Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly ArgCTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC

    EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    BamHI EcoRI SmaI SalI XhoI NotI

    EcoRI

    CTG GTT CCG CGT GGA TCC CCG GAA TTC ATC GTG ACT GAC TGA CGA

    BamHI

    Leu Val Pro Arg Gly Ser Pro Glu Phe Ile Val Thr Asp

    Thrombin

    Stop codons

    pGEX~4900 bp

    pBR322ori

    BalI

    BspMI

    Ptac

    cal I q

    NarI

    EcoRV

    BssHII

    BstEII MluI

    ApaI

    Tth111I AatII

    PstI

    p4.5AlwNI

    pSj10 Bam7Stop7∆

    pGEX-4T-2

    pGEX-5X-1

    pGEX-5X-2

    pGEX-5X-3

    pGEX-4T-1

    pGEX-4T-3

    pGEX-3X

    pGEX-2TK

    Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerCTG GTT CCG CGT GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

    Stop codon

    Ile Glu Gly Arg Gly Ile Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspATC GAA GGT CGT GGG ATC CCC GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

    Stop codons

    Ile Glu Gly Arg Gly Ile Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala SerATC GAA GGT CGT GGG ATC CCC GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG TGA

    Stop codon

    Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

    Stop codons

    Leu Val Pro Arg Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His Arg AspCTG GTT CCG CGT GGA TCC CCG GAA TTC CCG GGT CGA CTC GAG CGG CCG CAT CGT GAC TGA

    Stop codons

    Leu Val Pro Arg Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg Ile Val Thr AspCTG GTT CCG CGT GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC ATC GTG ACT GAC TGA

    Stop codons

    ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA CTG ACT GACIle Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser

    Stop codons

    Leu Val Pro Arg Gly Ser Arg Arg Ala Ser Val

    Kinase

    CTG GTT CCG CGT GGA TCT CGT CGT GCA TCT GTT GGA TCC CCG GGA ATT CAT CGT GAC TGAStop codons

    Thrombin

    Thrombin

    Thrombin

    Thrombin

    Factor Xa

    Factor Xa

    Factor Xa

    Factor Xa

    EcoRIBamHI SmaI

    EcoRIBamHI SmaI

    pGEX-2T

    CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA CTG ACGLeu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp

    Stop codonsEcoRI

    Thrombin

    BamHI SmaI

    glutat

    hione

    S-transfe

    rase

    Amp r

    Acrylamide protein gel (SDS-PAGE)

    32

  • Cloning GFP-fusions

    • Expression of a GFP-tagged protein for cell biological studies

    • GFP, green fluorescent protein

    • GFP can be seen based on its fluorescent properties33

  • Polymerase chain reaction (PCR)

    34

  • Polymerase Chain Reaction (1)

    Melting of DNA 95 °C

    Primer annealing ca. 55 °C

    Primer extension 72 °C

    35

  • Primer extension72 °C

    Melting of DNA95 °C

    Primer annealing ca. 55 °C

    Primer extension72 °C

    Polymerase Chain Reaction (2)

    36

  • Primer extension72 °C

    Melting of DNA 95 °C

    Primer annealing ca. 55 °C

    Primer extension72 °C

    ...and so forth

    Polymerase Chain Reaction (3)

    37

  • Typical PCR Program

    Step 1: Melting of DNA 95 °C - 4 min

    Step 2: Melting of DNA 95 °C - 1 minStep 3: Primer annealing 55 °C - 1 minStep 4: Primer extension 72 °C - 1 min per kb

    go to Step 2 - 29 times

    Step 5: Final primer extension 72 °C - 5 minStep 6: Storage 4 °C forever

    38

  • Primer (oligonucleotide) orders

    39

  • Taq polymerase - a heat stable polymerase

    Yellowstone National Park

    Thermus aquaticus

    Taq polymerase

    Karry MullisNobel prize 1993

    40

  • First generation PCR machinesthree waterbaths - three temperatures

    41

  • PCR machines - the real thing

    42

  • What is so cool about PCR?

    • highly sensitive amplification of desired gene fragment possible

    • amplification without propagation of the cloned fragments in bacteria: Good enough for sequencing and cloning! But flanking DNA sequences have to be known for primer design!

    • primers can be designed to insert restriction sites

    • primers can be designed to introduce desired sequence changes (mutations)

    • ...

    43

  • Cloning PCR products• TAQ polymerase adds an “A” to the 3’-end of its amplification

    products

    • vectors for the cloning of blunt-ended or A-tailed PCR products are available

    • use Topoisomerase for ligation (TOPO cloning)

    44

  • Recombination-based cloning

    45

  • Recombination-based cloning

    • vector system for the cloning of the same gene insert into different destination vectors (Gateway-cloning etc.)

    • gene integration by recombination with recombinases

    46

  • DNA sequencing II

    47

  • Next generation sequencing

    48

  • Third generation sequencing

    http://www.youtube.com/watch?v=q8gK4P7jg_s49

    http://www.youtube.com/watch?v=q8gK4P7jg_shttp://www.youtube.com/watch?v=q8gK4P7jg_s

  • What you need to know and understand

    for the exam and for your life....

    ... the principles of cloning

    ... elements of cloning vectors

    ... the need and purpose of cloning

    ... Sanger sequencing (dideoxy-method)

    ... polymerase chain reaction (PCR)

    50

  • The end

    51