T E C H N I K W I S S E N L E I D E N S C H A F T. Mol Gentechnik.

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1 1 W I S S E N T E C H N I K L E I D E N S C H A F T Mol Gentechnik

2 2 Grundlagen der Gentechnik

3 3 Lehrbücher Brown T.A. Gentechnologie für Einsteiger Elsevier GmbH Spektrum Akademischer Verlag, Heidelberg 6. Auflage 2011 Müllhardt Cornel Der Experimentator Molekularbiologie/Genomics Spektrum Akademischer Verlag, 7. Auflage 2013 Nicholl Desmond S.T. An Introduction to Genetic Engineering Cambridge University Press, 3. Auflage 2008 Reece J. Richard Analysis of Genes and Genomes John Wiley & Sons, Ltd USA, Auflage 2005 Howe Christopher Gene Cloning and Manipulation Cambridge University Press, 2. Auflage 2007

4 4 Komplexer Werkzeugsatz an Methoden Gene - DNA Identifizieren Genetisch Biologisch Biochemisch Chemisch Physikalisch Gentechnik Lokalisieren Isolieren Charakterisieren Synthetisieren Modifizieren Kombinieren Transferieren

5 5 Gene Aquisition Basic Cloning and Modification Work Vector Development For Target Host E. coli Expression in Final Host

6 6 Herstellen von rekombinanten DNA Molekülen (Klonieren) DNA Fragmente Schneiden mit Restriktionsenzym ori Ligation mit DNA Ligase isolierte Vektor DNA Amp r Einbringen in lebende Zellen ori rekombinantes DNA Molekül Amp r ori geschnittene Vektor DNA Amp r

7 7 Wesentliche Arbeitsschritte für Standard Klonierexperimente Isolierung / Gewinnung von DNA Fragmenten Präparation der Vektoren Schneiden von Fragmenten und Vektoren mit geeigneten Restriktionsendonukleasen Ligation Transfer in lebende Zellen Selektion von Transformanten Analyse der rekombinanten Klone

8 8 Gewinnung von DNA Fragmenten Isolierung aus Organismen gesamte genomische DNA DNA aus Organellen Metagenomische DNA cdna (über RNA) PCR Polymerase Kettenreaktion spezifische Gene homologe Familien (degenerierte Primer) Gensynthese Oligonukleotide synthetische Gene

9 9 DNA RNA Proteine Lipide Polysaccharide Metabolite DNA Isolierung Zellaufschluss Präparation von Organellen Nucleus Mitochondria Chloroplasts Extraktion der DNA z.b.phenol Proteasen Chaotrope Agentien Detergentien Destruktion von Membranstrukturen Verdau von Proteinen Reinigung Chromatographie

10 10 Chemical DNA Synthesis Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

11 Chemical DNA Synthesis 11 DMT: Dimethoxytrityl Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

12 12 Step 1 - De-blocking (detritylation) Removal of protection group Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

13 Step 2 - Base condensation (coupling) 13 A nucleoside phosphoramidite (or a mixture of several phosphoramidites) is activated by an acidic azolide catalyst, tetrazole, Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

14 14 Step 3: Capping acetylation of the unreacted 5'-hydroxy groups using a mixture of acetic anhydride and dimethylaminopyridine. Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

15 15 Step 4 - Oxidation treatment of the support-bound material with iodine and water Step 5 Deprotection and release from carrier Result: 5 non- Phosphorylated Oligo Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

16 16 Chemical DNA Synthesis Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

17 Gene synthesis 17 Overlapping Oligonuceotides Annealing, DNA Ligase Partially overlapping Oligonucleotides Annealing, DNA Polymerase DNA Ligase PCR

18 Gene synthesis 18 Overlapping Oligonuceotides Annealing, DNA Ligase Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

19 Gene synthesis 19 Partially overlapping Oligonucleotides Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

20 20 Overlap Extension PCR Gene synthesis Annealing, DNA Polymerase Denaturation & Annealing Primerless PCR PCR with outer primers

21 Overlap Extension PCR 21 Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

22 Overlap Extension PCR 22 Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

23 23 PCR Template-DNA DNA Polymerase Nukleotide T Denaturierung Primer-Annealing Puffer Primer DNA Synthese Thermocycler t

24 24 PCR Amplifikation von DNA Zwischen zwei Primern

25 25 PCR Amplifikation von DNA Zwischen zwei Primern

26 26 PCR Critical Factors Thermal Reactor Cycling Profile # Primer Annealing # Primer Extension Denaturation Cycle Number Final Extension Thermostable Polymerase # Type and Quality # Concentration MgCl 2 Concentration dntp Concentration Primer Sequences # Internal Secondary Structures # Balanced Base Distribution (A/T-G/C domains) # Complementarity at 3 -ends Template # Template/Primer Ratio # Purity Contamination

27 27 PCR Heat Transfer - Reproducibility

28 28 Thermostable DNA Polymerases t 1/2 (95 C) Proof-reading (Error rate) 5 3 Exonuclease ends Taq 40 min No (2 * 10-5 ) yes 3 A Tth 20 min no yes 3 A DeepVent 23 h yes no blunt Pfu > 2 h Yes (2,8 * 10-6 ) no blunt Phusion 50 min (97,5 C) Yes (4,4 * 10-7 ) blunt Q5 Yes (Fidelity >100 xtaq) blunt

29 29 Phusion High-Fidelity DNA Polymerase Phusion DNA polymerase brings together a novel Pyrococcus like enzyme with a processivity-enhancing domain (DNA binding Domain) increase in processivity shorter extension times more robust high yield amplification

30 Comparison of high-fidelity polymerases 30 NEB

31 31 PCR Strategies Touch-Down PCR stepwise lowering of annealing temperature Nested PCR 1 st PCR 2 nd PCR Anchored PCR Ligate anchor to end known sequence Asymmetric PCR ss DNA

32 32 Addition of restriction sites by PCR Howe Christopher Gene Cloning and Manipulation Cambridge University Press, 2. Auflage 2007

33 33 Hot Start PCR DNA Polymerase soll erst nach dem Erreichen hoher Temperaturen aktiviert werden - Physische Trennung durch Wachs -Thermolabile Maskierung der Polymerase Aktvierung bei hohen Temperaturen z.b. durch Antikörper oder gentechnische Modifikation

34 34 Real Time PCR -- qpcr

35 SYBR Green Incorporation into dsdna 35 Glick, Molecular Biotechnology, ASM Press, 4. Auflage, 2010

36 36 FRET Detection Systems Fluorescence resonance energy transfer (FRET) principles. Excitation of a donor dye (D) by an external light source (h) leads to longer wavelength emission by a reporter dye (R) when the two are in close proximity. Hybridization probe principles. temperature than the wild-type perfect duplex. Walter H. Koch, Nature Reviews Drug Discovery 3, (September 2004) doi: /nrd1496

37 37 TaqMan hydrolysis probe principles. The 5'- nuclease activity of thermostable polymerases used in the polymerase chain reaction (PCR) cleaves hydrolysis probes during the amplicon extension step, which separates the detectable reporter fluorophore (R) from a quencher (Q). Fluorescence emitted when excited by an external light source (hnu) at each PCR cycle is proportional to the amount of product formed. Walter H. Koch, Nature Reviews Drug Discovery 3, (September 2004) doi: /nrd1496 Molecular Beacons: The probe sequence in the loop binds spontaneously to the target RNA at physiological temperatures, separates a terminally linked pair of fluorophore and quencher, and restores the fluorescence of the quenched fluorophore. Musa M Mhlanga & Sanjay Tyagi, Nature Protocols 1, (2006) Published online: 2 November 2006 doi: /nprot