Oncorhynchus mykiss 的热嗜性尿嘧啶-DNA 糖基化酶的特征及其在 RT-qPCR 中携带污染控制的应用。

IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein and Peptide Letters Pub Date : 2024-01-01 DOI:10.2174/0109298665283737240122105923
Qingyuan Huang, Yaqi Zhang, Wenhao Hu, Keqi Chen, Jian Zhang, Zhidan Luo, Chen Lu
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引用次数: 0

摘要

背景:通常使用热敏性尿嘧啶-DNA 糖基化酶(HL-UDG)来消除 DNA 扩增中的携带污染。然而,常用的 HL-UDG 在 50°C 时会明显失活,因此不适合在 42°C 的最佳温度下使用反转录酶的特定一步 RT-qPCR 方案:本研究旨在探索失活温度更低且可重组表达的新型 HL-UDG:方法:从冷水性鱼类虹鳟鱼(Oncorhynchus mykiss)中克隆了编码HL-UDG的基因,并在大肠杆菌中进行了高产率表达。对该酶的热稳定性和其他酶特性进行了深入研究。随后,新型 HL-UDG 被用于控制一步 RT-qPCR 中的携带污染:结果:重组表达的虹鳟截短HL-UDG(OmUDG)与重组大西洋鳕鱼UDG(rcUDG)的氨基酸相似度高(84.1%),且在40°C时容易变性。OmUDG 的最佳 pH 值为 8.0,Na+ 和 K+ 的最佳浓度均为 10 mM。由于 OmUDG 的失活温度低于 rcUDG,因此可用于消除一步式 RT-qPCR 中的携带污染,反转录温度适中:结论:我们成功鉴定并重组表达了一种失活温度为 40°C 的新型 HL-UDG。结论:我们成功鉴定并重组表达了灭活温度为 40°C 的新型 HL-UDG,它适用于消除一步式 RT-qPCR 中的携带污染。
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Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

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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来源期刊
Protein and Peptide Letters
Protein and Peptide Letters 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
98
审稿时长
2 months
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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