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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Letter Article

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

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

Volume 31, Issue 3, 2024

Published on: 09 February, 2024

Page: [169 - 177] Pages: 9

DOI: 10.2174/0109298665283737240122105923

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.

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Graphical Abstract

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