Initiation of the microgene polymerization reaction with non-repetitive homo-duplexes

Mark Itsko, Arieh Zaritsky, Avinoam Rabinovitch, Eitan Ben-Dov

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Microgene Polymerization Reaction (MPR) is used as an experimental system to artificially simulate evolution of short, non-repetitive homo-duplex DNA into multiply-repetitive products that can code for functional proteins. Blunt-end ligation by DNA polymerase is crucial in expansion of homo-duplexes (HDs) into head-to-tail multiple repeats in MPR. The propagation mechanism is known, but formation of the initial doublet (ID) by juxtaposing two HDs and polymerization through the gap has been ambiguous. Initiation events with pairs of HDs using Real-Time PCR were more frequent at higher HD concentrations and slightly below the melting temperature. A process molecularity of about 3.1, calculated from the amplification efficiency and the difference in PCR cycles at which propagation was detected at varying HD concentrations, led to a simple mechanism for ID formation: the gap between two HDs is bridged by a third. Considering thermodynamic aspects of the presumed intermediate "nucleation complex" can predict relative propensity for the process with other HDs.

Original languageEnglish
Pages (from-to)606-613
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume368
Issue number3
DOIs
StatePublished - 11 Apr 2008

Keywords

  • Blunt-end ligation by DNA polymerase
  • Head-to-tail primer polymerization
  • Molecularity
  • PCR artifacts
  • Repeated motifs

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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