{"title":"Characterization of Heat-labile Uracil-DNA Glycosylase from <i>Oncorhynchus mykiss</i> and its Application for Carry-over Contamination Control in RT-qPCR.","authors":"Qingyuan Huang, Yaqi Zhang, Wenhao Hu, Keqi Chen, Jian Zhang, Zhidan Luo, Chen Lu","doi":"10.2174/0109298665283737240122105923","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Objective: </strong>This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression.</p><p><strong>Methods: </strong>The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout <i>(Oncorhynchus mykiss)</i> and expressed in <i>Escherichia coli</i> 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.</p><p><strong>Results: </strong>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<sup>+ </sup> and K<sup>+</sup> 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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":20736,"journal":{"name":"Protein and Peptide Letters","volume":null,"pages":null},"PeriodicalIF":1.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein and Peptide Letters","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2174/0109298665283737240122105923","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍：
Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations.
Protein & Peptide Letters focuses on:
Structure Studies
Advances in Recombinant Expression
Drug Design
Chemical Synthesis
Function
Pharmacology
Enzymology
Conformational Analysis
Immunology
Biotechnology
Protein Engineering
Protein Folding
Sequencing
Molecular Recognition
Purification and Analysis
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