einführung in die genetik - developmental biology · 11 veränderungen der chromosomen 08. 01. 13...
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Einführung in die Genetik
Prof. Dr. Kay Schneitz (EBio Pflanzen)http://plantdev.bio.wzw.tum.deschneitz@wzw.tum.de
Prof. Dr. Claus Schwechheimer (PlaSysBiol)http://wzw.tum.de/sysbiolclaus.schwechheimer@wzw.tum.de
“Downloads” is linked to Schneitz web page
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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
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Genetic Recombination in Bacteria and their Viruses
Genetics 07
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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. coli4
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
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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)
• ...
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Cloning for DNA sequencing
• Mutations in the BREAST CANCER1 (BRCA1) gene
Sequencing requires the clonal amplification of the DNA fragments to be sequenced
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Cloning for genome assembly
• Sorted fragments of an assembled genome
Sequencing requires the clonal amplification of the DNA fragments to be sequenced
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Cloning for genetic engineering
• Protein expression in a heterologous host (bacteria)
Recombinant protein expression requires cloning10
Cloning for cell biology
• Protein localization in a (plant) cell using green fluorescent protein
Gene fusions can be generated by molecular cloning
GFP-fusion protein (of phytochrome) that moves to the nucleus after detection of red light
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Molecular cloning
DNA sequencing 1
Examples
Polymerase chain reaction
Recombination-based cloning
DNA sequencing II
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Molecular cloning
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Cloning and cloning vectors
Restriction digest
Ligation
Transformation
Clonal amplification
DNA preparation
Selection
Restriction digestor sequencing
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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
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restriction sites(polylinker, multiple cloning site)
(lac promoter)
origin of replication (ori)
selection(for plasmid)
(lacZ reporter)
selection(for insert)
Cloning and cloning vectors
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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)
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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
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• 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
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T4 DNA Ligase
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Cloning and cloning vectors
restriction sites(polylinker, multiple cloning site)
(lac promoter)
origin of replication (ori)
selection(for plasmid)
(lacZ reporter)
selection(for insert)
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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)
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Transformation
Plasmid after ligation
Heat shock transformation(Alternative: electro shock) Selection
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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)
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DNA preparation
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DNA sequencing
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The dideoxy sequencing method (1)
Nobel prize in 1980Walter Gilbert and Frederick Sanger(Sanger sequencing)
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The dideoxy sequencing method (2)
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(Fluorescent) dye terminator technology
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Examples
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Cloning vectors for sequencing
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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 information
Product 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-1!T
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
calI 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
glutathione S-transferase
Amp r
Acrylamide protein gel (SDS-PAGE)
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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)
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Polymerase Chain Reaction (1)
Melting of DNA 95 °C
Primer annealing ca. 55 °C
Primer extension 72 °C
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Primer extension72 °C
Melting of DNA95 °C
Primer annealing ca. 55 °C
Primer extension72 °C
Polymerase Chain Reaction (2)
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Primer extension72 °C
Melting of DNA 95 °C
Primer annealing ca. 55 °C
Primer extension72 °C
...and so forth
Polymerase Chain Reaction (3)
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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
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Primer (oligonucleotide) orders
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Taq polymerase - a heat stable polymerase
Yellowstone National Park
Thermus aquaticus
Taq polymerase
Karry MullisNobel prize 1993
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First generation PCR machinesthree waterbaths - three temperatures
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PCR machines - the real thing
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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)
• ...
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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)
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Recombination-based cloning
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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
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DNA sequencing II
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Next generation sequencing
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Third generation sequencing
http://www.youtube.com/watch?v=q8gK4P7jg_s49
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)
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The end
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