Analysis of DNA polymerase activity in vitro using non-radioactive primer extension assay in an automated DNA sequencer

D. O. Lopes, C. G. Regis-da-Silva, A. Machado-Silva, A. M. Macedo, G. R. Franco, J. S. Hoffmann, C. Cazaux, S. D.J. Pena, S. M.R. Teixeira, C. R. Machado

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Although different DNA polymerases have distinct functions and substrate affinities, their general mechanism of action is similar. Thus, they can all be studied using the same technical principle, the primer extension assay employing radioactive tags. Even though fluorescence has been used routinely for many years for DNA sequencing, it has not been used in the in vitro primer extension assay. The use of fluorescence labels has obvious advantages over radioactivity, including safety, speed and ease of manipulation. In the present study, we demonstrated the potential of non-radioactive in vitro primer extension for DNA polymerase studies. By using an M13 tag in the substrate, we can use the same fluorescent M13 primer to study different substrate sequences. This technique allows quantification of the DNA polymerase activity of the Klenow fragment using different templates and under different conditions with similar sensitivity to the radioactive assay.

Original languageEnglish
Pages (from-to)250-255
Number of pages6
JournalGenetics and Molecular Research
Volume6
Issue number2
StatePublished - 2007
Externally publishedYes

Keywords

  • DNA polymeras
  • DNA synthesis in vitro
  • Non-radioactive assay

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