Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase
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RESEARCH PAPER
Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase Jinying Liu 1 & Jiangyan Zhang 1
&
Meiqi Chen 1 & Dehui Qiu 1 & Xuechong Lv 1 & Qi Jiang 1 & Yongqiang Cheng 1
Received: 29 July 2020 / Revised: 30 September 2020 / Accepted: 7 October 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10−4 to 5 U/mL with four orders of magnitude, and the detection limit is 2.7 × 10−4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids postcontamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes. Keywords Uracil-DNA glycosylase . Real-time detection . Magnetic separation . Ligation chain reaction . Fluorescence detection
Introduction DNA damage occurs continuously in cells, both during normal metabolic processes and due to external environmental factors, resulting in genome instability, disruption of normal cell physiology, and high likelihood of disease [1, 2]. Base excision repair is a DNA damage repair pathway for removal of the damaged bases by a DNA-repair enzyme [3–5]. UracilElectronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02997-8) contains supplementary material, which is available to authorized users. * Jiangyan Zhang [email protected] * Yongqiang Cheng [email protected] 1
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Institute of Life Science and Green Development, Hebei University, Baoding 071002, China
DNA glycosylase (UDG), as an important DNA-repair enzyme for the repair
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