Chemiluminescent heterogeneous and homogeneous-heterogeneous assays for determination of nicking endonuclease activity

Homogeneous-heterogeneous and heterogeneous formats of a simple and sensitive assay for the determination of nicking endonuclease (NE) Nt.Bst9I activity was developed. The duplex of two 26-membered biotinylated DNA oligonucleotides was used as a substrate of Nt.Bst9I. To improve the assay sensitivity the chemiluminescent detection system based on the use of conjugate of streptavidin and polyperoxidase and enhanced chemiluminescence reaction was used. Both proposed assay formats were constructed using microtiter plates as a solid support, allowing for easy automation of NE analysis using ELISA equipment. Varying the acidity, concentration of KCl and NaCl, and temperature of the reaction medium, favorable conditions were found. Although both formats of the proposed assay can be applied to estimate Nt.Bst9I activity, the heterogenous assay was more sensitive than the homogeneous-heterogeneous one.