Experimental and computational analysis of DNA unwinding and polymerization kinetics

Manjula Pandey, Mikhail K. Levin, Smita S. Patel

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Citations (Scopus)

Abstract

DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state and single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants. Using the hexameric helicase and the DNA polymerase from bacteriophage T7 as model systems, we provide stepwise procedures for measuring the kinetics of the reactions they catalyze based on radioactivity and fluorescence. We also describe analysis of the experimental measurements using publicly available models and software gfit ( http://gfit.sf.net ).

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages57-83
Number of pages27
Volume587
DOIs
StatePublished - 2010

Publication series

NameMethods in Molecular Biology
Volume587
ISSN (Print)10643745

Fingerprint

Polymerization
DNA
Bacteriophage T7
DNA-Directed DNA Polymerase
Radioactivity
Software
Fluorescence

Keywords

  • DNA polymerase
  • DNA synthesis
  • DNA unwinding
  • gfit
  • global regression analysis
  • Hexameric helicase
  • primer extension
  • replication
  • strand displacement
  • T7 bacteriophage

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

Pandey, M., Levin, M. K., & Patel, S. S. (2010). Experimental and computational analysis of DNA unwinding and polymerization kinetics. In Methods in Molecular Biology (Vol. 587, pp. 57-83). (Methods in Molecular Biology; Vol. 587). https://doi.org/10.1007/978-1-60327-355-8_5

Experimental and computational analysis of DNA unwinding and polymerization kinetics. / Pandey, Manjula; Levin, Mikhail K.; Patel, Smita S.

Methods in Molecular Biology. Vol. 587 2010. p. 57-83 (Methods in Molecular Biology; Vol. 587).

Research output: Chapter in Book/Report/Conference proceedingChapter

Pandey, M, Levin, MK & Patel, SS 2010, Experimental and computational analysis of DNA unwinding and polymerization kinetics. in Methods in Molecular Biology. vol. 587, Methods in Molecular Biology, vol. 587, pp. 57-83. https://doi.org/10.1007/978-1-60327-355-8_5
Pandey M, Levin MK, Patel SS. Experimental and computational analysis of DNA unwinding and polymerization kinetics. In Methods in Molecular Biology. Vol. 587. 2010. p. 57-83. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-60327-355-8_5
Pandey, Manjula ; Levin, Mikhail K. ; Patel, Smita S. / Experimental and computational analysis of DNA unwinding and polymerization kinetics. Methods in Molecular Biology. Vol. 587 2010. pp. 57-83 (Methods in Molecular Biology).
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