Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.
Protein Pept Lett
; 31(3): 169-177, 2024.
Article
em En
| MEDLINE
| ID: mdl-38343045
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.Palavras-chave
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oncorhynchus mykiss
/
Uracila-DNA Glicosidase
/
Temperatura Alta
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Protein Pept Lett
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China