PURPOSE: Large rearrangements in the dystrophin gene is detected in about 65-70% of patients by multiplex PCR or Southern blot, although detection of point mutations and microlesions is currently in progress. The purpose of this study is to carry out mutation analysis of the dystrophin gene by application of PCR-related molecular diagnostic methods in Duchenne muscular dystrophy (DMD). METHODS: Five patients diagnosed as a DMD by muscle biopsy, and their first-degree relatives and mothers were enrolled in this study. The genomic DNA was extracted from the peripheral blood lymphocyte. We used a total of 28 pairs of primers including Beggs' and Chamberlain's primers. The multiplex PCR was performed in 4 groups; 5'Rxn2, 3'Rxn2, Rxn2, and CRxn2. For exon duplication analysis, multiplex PCR and gel densitometry were carried out by comparing the band intensities among individual bands. For groups with no detectable deletion, single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing method were performed with individual PCR of candidate exons. RESULTS: About 12-20microg of genomic DNA was extracted from 1mL of blood, and the size of DNA was over 50kb. Up to 9 PCR products were made from multiplex PCR using the genomic DNA. Among 5 families with DMD, No. 6 had about 240kb DNA deletion from exon 45 and 47-53, and No. 11 had about 130kb deletion from exon 47-49 and 53. No. 1 showed duplication of exon 43 when the multiplex PCR products were analyzed by a densitometer. When the deletion/duplication negative No. 3 was analyzed by SSCP method, exon 43 and 49 showed abnormal band patterns. The abnormal band pattern of exon 43 was caused by deletion mutation of A residue, which resulted in pretermination of dystrophin synthesis, meanwhile exon 49 showed transversion mutation of C G at intron 49. CONCLUSION: Based on the results of this study, the methods of multiplex PCR, SSCP and direct DNA sequencing of PCR products made it possible to analyze several types of mutation of DMD.