Einführung in die Genetik

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1 Einführung in die Genetik Prof. Dr. Kay Schneitz (EBio Pflanzen) Prof. Dr. Claus Schwechheimer (PlaSysBiol) Downloads is linked to Schneitz web page 1

2 Einführung in die Genetik - Inhalte 1 Einführung KS 2 Struktur von Genen und Chromosomen KS 3 Genfunktion KS 4 Transmission der DNA während der Zellteilung KS 5 Vererbung von Einzelgenveränderungen KS 6 Genetische Rekombination (Eukaryonten) KS 7 Genetische Rekombination (Bakterien/Viren) KS 8 Rekombinante DNA-Technologie CS 9 Kartierung/Charakterisierung ganzer Genome CS 10 Genmutationen: Ursache und Reparatur CS 11 Veränderungen der Chromosomen CS 12 Genetische Analyse biologischer Prozesse CS 13 Transposons bei Eukaryonten CS 14 Regulation der Genexpression KS 15 Regulation der Zellzahl - Onkogene CS 2

3 Genetic Recombination in Bacteria and their Viruses Genetics 07 3

4 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

5 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 site 5

6 Recombinant DNA Technology Genetics 08 6

7 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

8 Cloning for DNA sequencing Mutations in the BREAST CANCER1 (BRCA1) gene Sequencing requires the clonal amplification of the DNA fragments to be sequenced 8

9 Cloning for genome assembly Sorted fragments of an assembled genome Sequencing requires the clonal amplification of the DNA fragments to be sequenced 9

10 Cloning for genetic engineering Protein expression in a heterologous host (bacteria) Recombinant protein expression requires cloning 10

11 Cloning for cell biology GFP-fusion protein (of phytochrome) that moves to the nucleus after detection of red light Protein localization in a (plant) cell using green fluorescent protein Gene fusions can be generated by molecular cloning 11

12 Molecular cloning DNA sequencing 1 Examples Polymerase chain reaction Recombination-based cloning DNA sequencing II 12

13 Molecular cloning 13

14 Cloning and cloning vectors Restriction digest Ligation Transformation Selection Clonal amplification DNA preparation Restriction digest or sequencing 14

15 Cloning and cloning vectors Restriction digest Restriction sites Ligation Transformation Selection Origin of replication (ori) Antibiotic resistance (amp, tet) Clonal amplification DNA preparation Restriction digest or sequencing 15

16 Cloning and cloning vectors restriction sites (polylinker, multiple cloning site) selection (for plasmid) (lacz reporter) (lac promoter) origin of replication (ori) selection (for insert) 16

17 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

18 Restriction enzymes Digest of genomic DNA M size marker 1,3 undigested 2,4 digested Agarose gel Digest of plasmid DNA M size marker 1,3 undigested 2,4 digested Agarose gel 18

19 Restriction enzymes restriction enzymes are isolated from bacteria bacteria have restriction enzmyes to protect themselves against foreign DNA they protect their own DNA by DNA methlyation 19

20 T4 DNA Ligase 20

21 Cloning and cloning vectors restriction sites (polylinker, multiple cloning site) selection (for plasmid) (lacz reporter) (lac promoter) origin of replication (ori) selection (for insert) 21

22 Cloning and cloning vectors Restriction digest Restriction sites Ligation Transformation Selection Origin of replication (ori) Antibiotic resistance (amp, tet) Clonal amplification DNA preparation Restriction digest or sequencing 22

23 Transformation Plasmid after ligation Heat shock transformation (Alternative: electro shock) Selection 23

24 Cloning and cloning vectors Restriction digest Restriction sites Ligation Transformation Selection Origin of replication (ori) Antibiotic resistance (amp, tet) Clonal amplification DNA preparation Restriction digest or sequencing 24

25 DNA preparation 25

26 DNA sequencing 26

27 The dideoxy sequencing method (1) Nobel prize in 1980 Walter Gilbert and Frederick Sanger (Sanger sequencing) 27

28 The dideoxy sequencing method (2) 28

29 (Fluorescent) dye terminator technology 29

30 Examples 30

31 Cloning vectors for sequencing 31

32 Cloning GST-fusion constructs pgex-6p-1 PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Glu Phe Pro Gly Arg Leu Glu Arg Pro His CTG 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 BamHI EcoRI SmaI SalI XhoI NotI PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg Ala Ala Ala Ser CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCA GGA ATT CCC GGG TCG ACT CGA GCG GCC GCA TCG pgex-6p-3 SmaI BamHI EcoRI SmaI SalI XhoI NotI PreScission Protease Leu Glu Val Leu Phe Gln Gly Pro Leu Gly Ser Pro Asn Ser Arg Val Asp Ser Ser Gly Arg CTG GAA GTT CTG TTC CAG GGG CCC CTG GGA TCC CCG AAT TCC CGG GTC GAC TCG AGC GGC CGC EcoRI XhoI SmaI SalI XhoI NotI BspMI Ptac BalI glutathione S-transferase psj10 Bam7Stop7 Tth111I AatII Amp r PstI pgex ~4900 bp NarI EcoRV lac I q p4.5 AlwNI Acrylamide protein gel (SDS-PAGE) BssHII ApaI BstEII pbr322 ori Expression and purification of a GST-tagged protein MluI GST, glutathione S-transferase (protein) glutathione, small molecule that can be bound to a resin 32

33 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 properties 33

34 Polymerase chain reaction (PCR) 34

35 Polymerase Chain Reaction (1) Melting of DNA 95 C Primer annealing ca. 55 C Primer extension 72 C 35

36 Polymerase Chain Reaction (2) Primer extension 72 C Melting of DNA 95 C Primer annealing ca. 55 C Primer extension 72 C 36

37 Polymerase Chain Reaction (3) Primer extension 72 C Melting of DNA 95 C Primer annealing ca. 55 C Primer extension 72 C...and so forth 37

38 Typical PCR Program Step 1: Melting of DNA 95 C - 4 min Step 2: Step 3: Step 4: Melting of DNA 95 C - 1 min Primer annealing 55 C - 1 min Primer extension 72 C - 1 min per kb go to Step 2-29 times Step 5: Step 6: Final primer extension 72 C - 5 min Storage 4 C forever 38

39 Primer (oligonucleotide) orders 39

40 Taq polymerase - a heat stable polymerase Yellowstone National Park Thermus aquaticus Taq polymerase Karry Mullis Nobel prize

41 First generation PCR machines three waterbaths - three temperatures 41

42 PCR machines - the real thing 42

43 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

44 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

45 Recombination-based cloning 45

46 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

47 DNA sequencing II 47

48 Next generation sequencing 48

49 Third generation sequencing 49

50 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

51 The end 51

Einführung in die Genetik

Einführung in die Genetik Prof. Dr. Kay Schneitz (EBio Pflanzen) http://plantdev.bio.wzw.tum.de schneitz@wzw.tum.de Prof. Dr. Claus Schwechheimer (PlaSysBiol) http://wzw.tum.de/sysbiol claus.schwechheimer@wzw.tum.de