ARMS-PCR, quantitative PCR, and Sanger sequencing

Standard PCR and Sanger sequencing were carried out as described previously [13,14]. For the ARMS-PCR assay, the PCR reaction (total volume, 24 μl) contained 1 μl of genomic DNA, 12 μl of AccuStart™ II GelTrack PCR SuperMix (QuantaBio, 89235-012), 1 μl of the inner mixed primers (10 μM of N3IFor and N3IRev), 1 μl of the outer mixed primers (1 μM of N3OFor1 and N3ORev1), and 9 μl of water. Amplification of genomic DNA was achieved in a T100 thermocycler (Bio-Rad) with the following program: an activation step of the DNA polymerase for 5 min at 94°C, 30 cycles of DNA amplification with each cycle including 30 sec at 94°C, 45 sec at 63°C, and 60 sec at 72°C, and a final elongation step (10 min at 72°C). The size and yield of each amplicon was resolved by standard electrophoresis on 1.5% agarose gels containing ethidium bromide. The gel pictures were taken by a ChemiDoc™ MP Imaging System (Bio-Rad). For qPCR assay, amplicons were run and analyzed by an Applied Biosystems 7,500 real-time PCR system and the dedicated software provided by the thermocycler manufacturer (Thermo Fisher). The qPCR reaction (total volume, 20 μl) contained 1 μl of genomic DNA, 10 μl 2X Power SYBR Green PCR Master Mix (Fisher, 4367659), 1 μl of the mixed primers (10 μM of N3MuP3F, and N3MuP3R), and 8 μl of water. We used the β-actin gene (primers seen in Table 1) as an internal control for qPCR assay. We used a pair of primers (seen in Table 1) to amplify genomic DNA containing the Notch3 mutation. Sanger direct DNA sequencing was carried out by Eurofins Genomics.