Primer competition PCR

Haizpea Lasa-Fernandez; Laura Mosqueira-Martín; Ainhoa Alzualde; Jaione Lasa-Elgarresta; Ainara Vallejo-Illarramendi

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Primer competition PCR

HL Haizpea Lasa-Fernandez

LM Laura Mosqueira-Martín

AA Ainhoa Alzualde

JL Jaione Lasa-Elgarresta

AV Ainara Vallejo-Illarramendi

This method is extracted from research article: Sci Rep, Oct 2020

A genotyping method combining primer competition PCR with HRM analysis to identify point mutations in Duchenne animal models

DOI: 10.1038/s41598-020-74173-y

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Primer competition PCR (pcPCR) was carried out with Precision Melt Supermix (Bio-Rad) with a hot-start Taq DNA polymerase (iTaq), 200 nM of each custom-designed primer (one forward and two reverse primers), and 5% of DNA solution. The PCR protocol comprised an initial denaturation step at 95 °C for 3 min, followed by 34 cycles of 95 °C for 10 s and 65 °C for 1 min. Primer sequences and expected PCR products for WT and mutant samples are detailed in Table Table11 for the zebrafish model, and in Fig. 5 for the mouse model. HRM analysis was performed as described above, and PCR products were subsequently resolved by 3% agarose gel electrophoresis. Repeatability of the pcPCR-HRM analysis in zebrafish samples was assessed by determination of the intra- and inter-assay CV of the Tm of three runs carried out on different days.

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