Vorlesung „Grundlagen der Genetik“
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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) - subitem: Lehre - subitem: Skripte - password: 4VLAG16 - 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 - to give a survey of enzymes/techniques used in molecular biology - to introduce tools of recombinant DNA-technology - explain how cells can be used as a protein-factory - 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
