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

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

Volume: 31 Issue: 3

Author(s): Qingyuan Huang, Yaqi Zhang, Wenhao Hu, Keqi Chen, Jian Zhang, Zhidan Luo and Chen Lu*

Affiliation:

• Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang, 222005, China
• Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, 222005, China
• Jiangsu Best Enzymes Biotech Co., Ltd., Lianyungang, 222005, China

Keywords: Uracil-DNA glycosylase, Oncorhynchus mykiss, recombinant expression, heat-labile, RT-qPCR, Escherichia coli.

Abstract:

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.

Cite this article as:

Huang Qingyuan, Zhang Yaqi, Hu Wenhao, Chen Keqi, Zhang Jian, Luo Zhidan and Lu Chen*, Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR, Protein & Peptide Letters 2024; 31 (3) . https://dx.doi.org/10.2174/0109298665283737240122105923