Multiplex ligation-dependent probe amplification (MLPA) assay was performed with the SALSA MLPA P034/P035 DMD kit (MRC Holland, Amsterdam, The Netherlands), according to the manufacturer’s recommendations. The SALSA MLPA P095 Aneuploidy kit (MRC Holland, Amsterdam, The Netherlands) was used in patient II-3 to exclude the presence of an extra X-chromosome.
Following denaturation of Genomic DNA, MLPA mix and the probes are briefly added to the samples for overnight hybridization. Ligation reaction and multiplex PCR reaction were performed with a Ligase-65 master mix and polymerase master mix, respectively. The PCR products were then separated on an ABI 3130xL automatic DNA sequencer (Life Technologies, Carlsbad, CA, USA). Coffalyser.Net package (MRC-Holland, Amsterdam, The Netherlands) was performed for MLPA data analysis.
Following denaturation of Genomic DNA, MLPA mix and the probes are briefly added to the samples for overnight hybridization. Ligation reaction and multiplex PCR reaction were performed with a Ligase-65 master mix and polymerase master mix, respectively. The PCR products were then separated on an ABI 3130xL automatic DNA sequencer (Life Technologies, Carlsbad, CA, USA). Coffalyser.Net package (MRC-Holland, Amsterdam, The Netherlands) was performed for MLPA data analysis.