Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR

• Published in: Protein & Peptide Letters
• Main Authors: Huang, Qingyuan, Zhang, Yaqi, Hu, Wenhao, Chen, Keqi, Zhang, Jian, Luo, Zhidan, Lu, Chen
• Other Authors: Lianyungang Science and Technology Project
• Format: Article in Journal/Newspaper
• Language: English
• Published: Bentham Science Publishers Ltd. 2024
• Subjects: atlantic cod
• Online Access: http://dx.doi.org/10.2174/0109298665283737240122105923; https://www.eurekaselect.com/article/download?doi=10.2174/0109298665283737240122105923; https://www.eurekaselect.com/226814/article

Background: Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. Objective: This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. Methods: The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. Results: This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. Conclusion: We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.