Abi 3500xl dx genetic analyzer

Sanger sequencing was performed as previously described to verify the variants identified by targeted NGS [13 (link)]. Briefly, forward and reverse PCR primers were designed to amplify the fragments covering the variant sites. PCR products were purified with shrimp alkaline phosphatase and exonuclease and then directly sequenced on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems, Foster City, USA). Co-segregation analysis was performed in the families with identified mutations.

Four pairs of primer sequences were designed for variants in the MITF gene (NM_000248.3). (Table S1). To confirm the detected variants, standard Sanger sequencing protocols were performed on an ABI 3500xL Dx Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific). Gene mutations were analyzed using Chromas (ver. 2.6.5).

Long‐range PCR of GD‐395 was performed on 2720 Thermal Cycler (Applied Biosystems) using the TaKaRa LA Taq^® Hot Start Version (Takara Bio, Otsu, Japan). Approximately 50 ng of high quality template DNA was added to a 15 ul standard reaction. The forward primer (TMPRSS3_In2_F) and the reverse primer (TMPRSS3_Ex12_R) were added to a final concentration of 0.67 μmol/L. Thermocycling conditions were as follows: 1 cycle of 95°C for 5 min, 30 cycles of 98°C for 20 s and 68°C for 12 min, and 1 cycle of 68°C for 7 min. Gap‐PCR was designed to detect the certain CNV in single PCR amplification for GD‐395 and his family members. One reverse primer (TMPRSS3_Ex12_R) and two forward primers (TMPRSS3_In2_R and TMPRSS3_Ex11_F) were added to a single gap‐PCR amplification. Standard protocols of Sanger sequencing were followed on the ABI 3500xL Dx Genetic Analyzer (Applied Biosystems) to confirm detected variants in cases and extended families.