Measurements of DNA-loop formation via Cre-mediated recombination.

Nucleic acids research (2012-05-17)
Massa J Shoura, Alexandre A Vetcher, Stefan M Giovan, Farah Bardai, Anusha Bharadwaj, Matthew R Kesinger, Stephen D Levene
ABSTRACT

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 594 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Supelco
Atto 594, suitable for fluorescence, ≥90.0% (HPCE)
Supelco
Atto 594 maleimide, BioReagent, suitable for fluorescence, ≥80% (coupling rate)
Sigma-Aldrich
Atto 594 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 594 iodoacetamide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 594-Biotin, ≥90% (HPCE)

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