Heterochronous multiplex real-time PCR with intercalating dye using uracil-DNA N-glycosylase (UNG) and multiple primer pairs to revaluate post PCR product.
| Autor: | Mizumoto-Teramura Y; Department of Otolaryngology and Head and Neck Surgery, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan., Kamogashira T; Department of Otolaryngology and Head and Neck Surgery, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan., Kondo K; Department of Otolaryngology and Head and Neck Surgery, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan., Yamasoba T; Department of Otolaryngology, Tokyo Teishin Hospital, Japan. |
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| Jazyk: | angličtina |
| Zdroj: | MethodsX [MethodsX] 2024 Jun 22; Vol. 13, pp. 102818. Date of Electronic Publication: 2024 Jun 22 (Print Publication: 2024). |
| DOI: | 10.1016/j.mex.2024.102818 |
| Abstrakt: | Real-time PCR with intercalating dyes can only be performed once. The expensive fluorescent hydrolysis probes are target specific and are suitable to detect multiplex targets. Uracil-DNA N-glycosylase (UNG), which specifically hydrolyzes and degrades any uracil-containing PCR products, is often applied before PCR to reduce carryover contamination. We developed an optimized protocol for recovering DNA from PCR products and revaluating by real-time PCR with intercalating dye using UNG processing, which is particularly useful when the sample volume is very small and insufficient for multiple assays of real-time PCR.•A real-time PCR master mix with dUTP instead of dTTP was used.•UNG at 1 % and 10 % concentrations of PCR product volumes were used for the first and second processing.•The second real-time PCR was performed with different primer pairs than the first real-time PCR. Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (© 2024 The Author(s).) |
| Databáze: | MEDLINE |
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