2.3 Mutation screening
The genomic DNA extracts of the proband and parents were used for screening the DMD gene by the WES analysis. The detected variants were further evaluated by the Polyphen and SIFT software to determine pathogenicity.
2.4 Nested polymerase chain reaction (PCR) of pcMINI-gene and pcMINI-C gene
The nested PCR was initially conducted using two sets of primers (4705-DMD-F/7551-DMD-R and 5063-DMD-F/7188-DMD-R listed in Table 1) with the genomic DNA used as template. A second round of nested PCR was followed to obtain the amplicons of DMD gene. Subsequently, the PCR with the primer set of pcMINI-DMD-KpnI-F and pcMINI-DMD-EcoRI-R was conducted to generate wild-type (wt) amplicon (840bp) using the PCR product from the second round of nested PCR as template. By the same protocol, the mutant fragment 1 was amplified with the primer set of pcMINI-DMD-KpnI-F and DMD-MUT-R, whilst the mutant fragment 2 was amplified with the primer set of DMD-MUT-F and pcMINI-DMD-EcoRI-R. The amplicons of mutant fragment 1 and mutant fragment 2 were mixed with ratio of 1:1. The primer set of pcMINI-DMD-KpnI-F and pcMINI-DMD-EcoRI-R were used for amplifying pcMINI mutant type (mut) fragment (840bp). The sizes of pcMINI-C (wt/mut) amplicons are the same. The primer sequences are listed in Table 1.