Taq pcr master mix

For verification of the WES results, the related MSX1 (NM_002448) fragments were sequenced using Sanger sequencing. Genomic DNA from these family members was isolated according to the procedure described above. The primers for two exons of MSX1, MSX1-1F (5’-CTGGCCTCGCCTTATTAGC-3’) and MSX1-1R (5’-GCCTGGGTTCTGGCTACTC-3’) and MSX1-2F (5’-TGGCGGCACTCAATATCTGG-3’) and MSX1-2R (5’-CAGATCTGTCGTGGGTGTTCA-3’), were specifically designed to detect variants. The exonic region of the MSX1 gene of five families was amplified using PCR with Taq PCR Master Mix (BioTek, Beijing, China). The PCR products were sequenced by Tsingke Biological Technology (Beijing, China). Once the frameshift variant was detected, the PCR product harbouring this variant was cloned into the pClone007 Simple Vector (Tsingke, Beijing, China) to identify the exact status of the variant.

From the WES results, we identified a novel variant of BMPR2 (RefSeq NM_001204), which was validated via Sanger sequencing and familial co-segregation. Subsequently, to further confirm the genetic role of BMPR2 in oligodontia, gene screening was performed via Sanger sequencing using samples of 130 unrelated individuals with nonsyndromic oligodontia with undefined etiology in our clinical database. Polymerase chain reaction (PCR) was performed to amplify the coding region of BMPR2 (primers and amplification conditions can be provided) using Taq PCR Master Mix (BioTek, Beijing, China). The PCR products were sequenced by Tsingke Biological Technology (Beijing, China).

To verify the WES results, the related EDA and WNT10A fragments were sequenced using Sanger sequencing. Genomic DNA from the brothers was isolated according to the procedure using a the TIANamp Blood DNA Midi Kit (Tiangen, Beijing, China) according to the manufacturer’s procedure. The primers used were specifically designed to detect the variations (Table 1). The coding sequences of the EDA and WNT10A genes were amplified using PCR with Taq PCR Master Mix (BioTek, Beijing, China). The PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Beijing). The results were compared with the reference sequences for each gene (EDA, NM_001399; WNT10A, NM_025216) (UCSC, http://genome.ucsc.edu/) to verify the results of WES.
Table 1

Gene variations and primer sequences

Variation	Forward primer	Reverse primer
EDA c.878 T > G (p.L293R)	5′-AAGTTTGGCCTTCTAGGCTACC-3′	5′-CCTGCACCGGATCTGCATTC-3′
WNT10A c.511C > T (p.R171C)	5′-CGCTTTTGCCTACGCCATC-3′	5′-AACTCGGTTGTTGTGAAGCC-3′
WNT10A c.637G > A (p.G213S)	5′-CGCTTTTGCCTACGCCATC-3′	5′-AACTCGGTTGTTGTGAAGCC-3′