Stoffel fragment

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As Stoffel fragment is in the biochemistry altered DNA polymerase and thus a DNA -generating enzyme referred to. The Stoffel fragment is based on a Taq polymerase and is, inter alia, in the polymerase chain reaction for copying used by DNA. The name of the fragment is derived from the name of the technical assistant Susanne Stoffel, who developed it from 1985 together with the biologist David Gelfand at Cetus Corporation (now Roche Molecular Systems).

properties

The Stoffel fragment is a Taq polymerase modified by protein design and thus belongs to the thermostable DNA polymerases . The Stoffel fragment is a Taq polymerase in which the 5'-3 ' exonuclease function was deleted in the gene . The first 289 amino acids are missing from the Stoffel fragment . The removal of the exonuclease function contained in the first 289 amino acids doubles the thermal stability . Due to the significantly lower exonuclease activity, the generated DNA strands are longer, which is why the Stoffel fragment is occasionally used in a polymerase chain reaction to synthesize longer DNA sequences (from two kilobases ). Furthermore, the Stoffel fragment is used in qPCR when hydrolysis of the probe is undesirable (e.g. LightCycler probes). Most of the time, the Stoffel fragment is produced recombinantly in Escherichia coli . By insertion of the helix-hairpin-helix sequences, the stability can be increased to high salt concentrations.

Individual evidence

  1. ^ A b F. C. Lawyer, S. Stoffel, RK Saiki, K. Myambo, R. Drummond, DH Gelfand: Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus. In: The Journal of biological chemistry. Volume 264, Number 11, April 1989, pp. 6427-6437, PMID 2649500 .
  2. ↑ S. Dabrowski, J. Kur: Recombinant His-tagged DNA polymerase. II. Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment). In: Acta biochimica Polonica. Volume 45, Number 3, 1998, pp. 661-667, PMID 9918492 .
  3. ^ J. Wilhelm, A. Pingoud, M. Hahn: Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes. In: BioTechniques. Volume 30, Number 5, May 2001, pp. 1052-6, 1058, 1060 passim, PMID 11355341 .
  4. ↑ Z. Yang, Y. Ding, Y. Zhang, F. Liu: Rapid purification of truncated Taq DNA polymerase Stoffel fragment by boiling lysis of bacterial expression cultures. In: Biotechnology and applied biochemistry. Volume 50, Pt 2 June 2008, pp. 71-75, doi : 10.1042 / BA20070114 , PMID 18184110 .
  5. ^ AR Pavlov, GI Belova, SA Kozyavkin, AI Slesarev: Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases. In: Proceedings of the National Academy of Sciences . Volume 99, number 21, October 2002, pp. 13510-13515, doi : 10.1073 / pnas.202127199 , PMID 12368475 , PMC 129704 (free full text).