Label-free and sensitive detection of uracil-DNA glycosylase using exponential real-time rolling circle amplification. Issue 20 (15th May 2018)

• Record Type: Journal Article
• Authors: Xu, Yuan; Cui, Yun-Xi; Zhao, Qiu-Ge; Tang, An-Na; Kong, De-Ming
• Is Part Of: Analytical methods. Volume 10:Issue 20(2018)
• Page Start: 2405
• Page End: 2410
• Publication Date: 2018-05-15
• Subjects: Chemistry, Analytic -- Periodicals; Analytical biochemistry -- Periodicals; Chemical laboratories -- Standards -- Periodicals; 543.1905
• DOI: 10.1039/c8ay00742j
• Languages: English
• ISSNs: 1759-9660

Abstract

Abstract : Schematic representation of an exponential rolling circle amplification-based uracil-DNA glycosylase activity-sensing platform. Abstract : Sensitive evaluation of the uracil-DNA glycosylase (UDG) activity is greatly significant in both fundamental biochemical process studies and disease prognosis. In this study, a simple but sensitive UDG activity-sensing strategy was designed on the basis of UDG-triggered rolling circle amplification (RCA) reaction. In this strategy, two oligonucleotides were used. The hairpin-like structure of the oligonucleotide containing a uracil nucleotide is destroyed in the presence of UDG, and then can be employed to form the circular template of RCA and initiate the subsequent RCA reaction. The participation of a nicking endonuclease makes the RCA reaction proceed in an exponential amplification mode. The amplification product may fold into a G-quadruplex structure, which can be specifically combined with thioflavin T to generate a fluorescence signal without any extra label. This UDG activity-sensing strategy was demonstrated to work well in both end-point and real-time detection modes with high sensitivity and excellent specificity. As low as 5.5 × 10 −5 U mL −1 UDG could be detected. The advantages of simple operation, short RCA time and automatic measurement using commercial instruments make the real-time detection mode suitable for high-throughput detection with reduced risk of amplification product carryover contamination, and its application feasibility in real samples was demonstrated by UDG activity analysis of cell lysate.