Properties of cold-active uracil-DNA glycosylase from Photobacterium aplysiae GMD509, and its PCR application for carryover contamination control

Abstract

Owing to its selective uracil-excision property, uracil-DNA glycosylase (UDG) has been widely utilized in diagnostic PCR applications as an effective decontamination method. Since mesophilic UDGs in PCR has been shown to degrade not just contaminant DNA but also target amplicon, there has been an increase in demand for cold-active UDGs. We characterized UDG from Photobacterium aplysiae GMD509 (Pop GMD509 UDG) expressed in Escherichia coli BL21 (DE3). The optimal temperature range of the enzyme was 25-30 degrees C, which is considerably lower than any other reported UDG. and the half-life of the enzyme at 40 degrees C and 50 degrees C was approximately 77 s and 33 s. respectively. These results clearly demonstrate the fragility of this enzyme upon heating. In addition, we compared the carryover contamination control property of Pap GMD509 UDG with other commercialized UDGs. The results indicate that Pap GMD509 UDG is capable of degrading contaminant DNA without a preincubation step before the main PCR reaction. These attributes imply that the Pop GMD509 UDG is a highly adequate enzyme to prevent carryover contamination during PCR. (C) 2008 Elsevier Inc. All rights reserved.