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.