Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Vorlesung „Grundlagen der Genetik“
Lecture „ Basics in Genetics“
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Schedule (prospective), Room E33 Date
Time
Subject
09.04. 09:20
DNA-Metabolism
16.04. 09:20
DNA-Metabolism
23.04. 09:20
Vectors, Transformation
30.04. 09:20
Gene-library, Gene cloning
07.05. 09:20
Gene expression
14.05. 09:20
Protein purification, Tags
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Scripts of lectures can be found at: -
Homepage of AG Prof. Rödel (http://tudresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachricht ung_biologie/genetik/allgemeine_genetik/index_html)
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subitem: Lehre
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subitem: Skripte
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password: 4VLAG16
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subitem: Graduiertenkolleg
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Aims -
to give an overview on DNA-metabolism
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to give a survey of enzymes/techniques used in molecular biology
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to introduce tools of recombinant DNA-technology
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explain how cells can be used as a protein-factory
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to introduce protein-purification in molecular biology
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Genetic information is stored on DNA
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Genetic information is stored/coded on DNA DNA is composed of 4 nucleotides (letters) DNA has a 5‘-phosphate- and 3‘-hydroxyl-end
(Watson et al., Molecular Biology of the Gene, sixth edition, Pearson International)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Base-pairing is performed via specific hydrogen-bonds DNA-strands form a double-helix
(Watson et al., Molecular Biology of the Gene, sixth edition, Pearson International)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Base-pairing in single-stranded (ss) nucleic acids leads to the formation of stem-loop structures (e.g. in tRNA or mRNA)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
DNA is stored on chromosomes Prokaryotes, often circular
Eukaryotes, classical chromsomes and DNA of mitochondria
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Sizes of genomes Very roughly: the more complex an organism is, the larger is it‘s genome
http://www.cbs.dtu.dk/staff/dave/roanoke/figure09_21.jpg
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Genetic DNA-information is translated to proteins
DNA is replicated to pass it through generations
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Polymerases synthesize DNA/RNA only in 5‘→3‘-direction
(Sambrook and Russel, Molecular cloning: a laborytory manual, CSHL press, 2001)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Types of Polymerases
DNA-dependent DNA-Polymerase: substrate DNA, produces DNA, used for replication and repair, e.g. E. coli Pol.I or Pol.III, thermostable enzymes for PCR (e.g. Taq-polymerase) DNA-dependent RNA-Polymerase: substrate DNA, produces RNA, used for transcription, e.g. Pol.II of E. coli RNA-dependent DNA-Polymerase: substrate RNA, produces DNA, found in Retroviruses, used for cDNAlibrary in vitro
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Replication via a leading and a lagging strand (challenge: only 5‘→3‘-direction of polymerases)
(Sambrook and Russel, Molecular cloning: a laborytory manual, CSHL press, 2001)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Concerted action of proteins at the „replication fork“
Protein: action - Gyrase, helicase: unwinding of DNA - ssbP: binds to/stabilizes ssDNA - Primase: synthesizes RNA-Primer - DNA-Pol.III: synthesizes DNA (fast) - DNA-Pol.I: degrades RNA-Primer, syntesizes DNA - DNA Ligase: ligates „nicks“, produces phospodiester-bonds
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Enzymatic activities of Polymerases I and III of E. coli: 5‘→3‘ polymerase activity: Pol. I, Pol. III (fast) 5‘→3‘ exonuclease activity: Pol. I (removing RNA-primer)
3‘→5‘ exonuclease activity: Pol. I, proof reading
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
E. coli Polymerase I activities
(Sambrook and Russel, Molecular cloning: a laborytory manual, CSHL press, 2001)
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Polymerase Chain Reaction exploits 5‘→3‘ polymerase activity for the amplification of target DNA
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
PCR-Cycles consist of: 1. Denaturation (Melt, about 95°C) 2. Annealing (Primer dependent, about 50°C – 60°C) 3. Polymerization (Extension, about 72°C thermostable enzymes)
Temperature profile of a PCR
Vorlesung „Grundlagen der Genetik“ Lecture „ Basics in Genetics“ Institut für Genetik, Prof. Dr. G. Rödel, Dr. K. Ostermann
Polymerase Chain Reaction leads to an exponential increase of target-DNA
Watson, Gilman, Witkowski und Zoller, Rekombinierte DNA, 2. Auflage, Spektrum Verlag
