Dna isolation kit

Genomic DNA was extracted from the peripheral blood of the subjects using a DNA isolation kit (Bioteke, AU1802). Concentrations were determined on a Qubit fluorometer (Invitrogen, Q33216) using a Qubit dsDNA HS assay kit (Invitrogen, Q32851). Agarose gel (1%) electrophoresis was performed for quality control. By performing the multiplex ligation-dependent probe amplification (MLPA) technique in all patients with demyelinating and intermediate CMT, 163 index patients with PMP22 duplications or deletions were initially excluded. Between 2007 and 2012, GJB1 mutations were screened in 88 index patients by direct Sanger sequencing. After 2012, NGS gene panels covering 165 CMT and related disease genes were applied to the 154 index patients, and WES was performed in 60 index patients. All suspected variants were validated by Sanger sequencing (Figure 1). Analysis of GJB1 mutation was based on the transcript version NM_000166.

Genomic DNA was extracted from the peripheral blood of the subjects using a DNA isolation kit (Bioteke, AU1802). Concentrations were determined with a Qubit fluorometer (Invitrogen, Q33216) and a Qubit dsDNA HS assay kit (Invitrogen, Q32851). Agarose gel (1%) electrophoresis was performed for quality control. The multiplex ligation-dependent probe amplification (MLPA) technique was applied in all patients with demyelinating and intermediate CMT, and 171 index patients with PMP22 duplications were initially excluded. After performing Sanger sequencing (before 2012) or next generation sequencing (NGS) of a gene panel (after 2012) in the remaining patients (314 index patients), 78 patients with CMT2 and dHMN remained undiagnosed. Then, whole-exome sequencing (WES) was performed for 78 index patients. Sample dilution, flow cell loadings, and sequencing were performed according to the Illumina specifications. DNA libraries were sequenced on the HiSeq X10 (Illumina, San Diego, CA) as paired-end 150-bp reads. (The flow chart of genetic testing is illustrated in Figure 1). All suspected variants were validated by Sanger sequencing of SORD. All nine SORD coding exons (NM_003104) were amplified by polymerase chain reaction. The amplicons were analyzed using an ABI 3730XL DNA analyzer (Applied Biosystems, Waltham, MA) in accordance with the manufacturer's protocol.

Genomic DNA was extracted from 200 μl EDTA-anticoagulated peripheral blood sample with a DNA isolation kit (BioTeke, Peking, China) as the manufacturer's direction. DNA was stably stored at −20°C until assayed. Genotyping of the IL-31 gene polymorphism was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We designed the PCR primers with software Primer 3 (http://bioinfo.ut.ee/primer3‐0.4.0/primer3/) [27 (link)] as shown in Table 1. The 10 μl PCR reaction system was consisted of 1.0 μl DNA and 5 μl 2× Power Taq PCR Master Mix (BioTeke, Peking, China), forward and reverse primer 0.1 μl, respectively, and reserved volume was made up to 10 μl by sterilized water. The PCR condition was designed as 95°C for 4 min firstly, then 33 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally, 72°C for 10 min. Furthermore, the PCR products were digested in 37°C stable incubation by distinguished restriction enzyme MboII (New England Biolabs, Peking, China) for 30 minutes of rs4758680 and ScrFI (New England Biolabs, Peking, China) for 2 hours of rs7977932 as shown in Table 1, separately. Ultimately, the results were visually analyzed by 6% polyacrylamide gels in silver staining. To verify the genotyping results, DNA sequencing was performed in about 20% PCR-amplified DNA samples randomly.