Mizumoto-Teramura Yui, Kamogashira Teru, Kondo Kenji, Yamasoba Tatsuya
Department of Otolaryngology and Head and Neck Surgery, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.
Department of Otolaryngology, Tokyo Teishin Hospital, Japan.
MethodsX. 2024 Jun 22;13:102818. doi: 10.1016/j.mex.2024.102818. eCollection 2024 Dec.
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.
使用嵌入染料的实时PCR只能进行一次。昂贵的荧光水解探针具有靶标特异性，适用于检测多重靶标。尿嘧啶-DNA N-糖基化酶（UNG）能特异性水解和降解任何含尿嘧啶的PCR产物，常用于PCR之前以减少残留污染。我们开发了一种优化方案，用于从PCR产物中回收DNA，并使用UNG处理通过嵌入染料的实时PCR进行重新评估，当样本量非常小且不足以进行多次实时PCR检测时，该方案特别有用。
• 使用含dUTP而非dTTP的实时PCR预混液。
• 分别以PCR产物体积的1%和10%浓度的UNG用于第一次和第二次处理。
• 第二次实时PCR使用与第一次实时PCR不同的引物对进行。
