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.
Protein Pept Lett. 2024;31(3):169-177. doi: 10.2174/0109298665283737240122105923.
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.
This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression.
The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout and expressed in 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.
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.
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.
热不稳定尿嘧啶-DNA 糖基化酶（HL-UDG）常用于消除 DNA 扩增过程中的携带污染。然而，目前常用的 HL-UDG 在 50°C 时会明显失活，因此不适合在最佳温度为 42°C 的特定一步法 RT-qPCR 方案中使用逆转录酶。
本研究旨在探索具有更低失活温度的新型 HL-UDG 并进行重组表达。
从冷水鱼类虹鳟鱼中克隆编码 HL-UDG 的基因，并在大肠杆菌中进行高产量表达。彻底研究了该酶的热稳定性和其他酶学特性。然后将新型 HL-UDG 用于控制一步法 RT-qPCR 中的携带污染。
本研究表达的虹鳟截断型 HL-UDG（OmUDG）与重组大西洋 cod UDG（rcUDG）具有很高的氨基酸相似性（84.1%的同一性），容易在 40°C 时变性。OmUDG 的最适 pH 为 8.0，最适 Na 和 K 浓度均为 10mM。由于其失活温度低于 rcUDG，因此 OmUDG 可用于在适度逆转录温度下的一步法 RT-qPCR 中消除携带污染。
我们成功地鉴定并重组表达了一种新型的 HL-UDG，其失活温度为 40°C。它适用于消除一步法 RT-qPCR 中的携带污染。
