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By Gunter Mertes, Thomas Schafer, Thomas A. Schild, Gerald Schmidt, Dagmar Schuster, Jorg vom Stein

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4). 3 Die PCR als Grundlage in der molekularen DNA-Analyse 27 (6) Magnesiumchlorid Die Tuq-DNA-Polymerase benotigt freies Magnesium als Kofaktor zum Einbau von Nukleotiden an das 3'-Ende der wachsenden Kette. Die Magnesiumionenkonzentration muB deshalb uber der dNTP-Konzentration liegen, da deren negative Ladungen Mg2'-Ionen binden. Ublicherweise liegt die Konzentration des freien Magnesiums etwa um 0,5-2,5 mM uber der dNTP-Konzentration: Befinden sich z. B. 0,8 mM (0,2 mM fur jedes dNTP) dNTPs im Ansatz, so ist eine MgKonzentration von ca.

Das Gen der Tuq-DNA-Polymerase wurde bereits 1989 in E. coli kloniert und exprimiert [19], so daB sich dieses Enzym mittlerweile relativ leicht und kostengiinstig in groBen Mengen herstellen lafit. Um auch bakterielle Sequenzen problemlos amplifizieren zu konnen, gibt es eine speziell aufgereinigte Form des rekombinanten Enzyms, die AmpliTaq@ DNA-Polymerase LD (Low DNA). Bei ihrer Verwendung konnen mogliche Kontaminationen mit bakterieller DNA aus dem HerstellungsprozeB praktisch ausgeschlossen werden (Perkin-Elmer).

By Alberghina, L. , 359-362. 18 Dagmar Schuster [4] Olsvik, 0. , ,,Magnetic separation techniques in diagnostic microbiology" Clinical Mikrobiology Reviews 7 (1994), 43-54. , ,,In vitro packaging of Lambda Dam Vectors and their use in cloning DNA fragments" Methods in Enzymology 68 (1980), 281-298. [6] Bolivar, F. , ,,Construction and characterization of new cloning vehicles. 11. A multipurpose cloning system" Gene 2 (1977), 95-113. , ,,Bacteriophage M13" Methods Enzymol. 153 ( 1987), 3-11. , ,,Plasmid DNA for DNA-Sequencing" DNA 4 (1985), 165-170.

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