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.