Rapid detection of deletions in the Duchenne muscular dystrophy gene by PCR amplification of deletion-prone exon sequences

Abstract

Using the polymerase chain reaction (PCR) technique, we have screened the DNA of 42 patients with Duchenne or Becker muscular dystrophy for deletions within the DMD gene. Two regions within putative deletion "hot spots" of this gene were tested, and deletions were found in 16.6% of patients. The oligonucleotide primers employed in this study initiate the amplification of exon sequences and were used to test the suitability and reliability of PCR in deletion screening and prenatal diagnosis using various numbers of cycles and artificial contamination ratios. We compared our approach with both "multiplex DNA amplification" and Southern blot analysis. A comparative evaluation of currently available techniques is presented.

The molecular genetic analysis of haemophilia A; characterization of six partial deletions in the factor VIII gene

“…The true frequency was assumed to be even higher than this, since very small deletions are likely to go undetected by Southern blotting and can only be detected by DNA sequencing. The frequency of deletion-type mutations detected in this study is however significantly lower than previously reported (about 1%; Fisher's exact (Nicholls et al 1987) and the Duchenne muscular dystrophy protein (Koenig et al 1987;Hentemann et al 1990). In some cases, Alu repetitive elements have been implicated in the deletion process, which may thus be mediated by non-reciprocal crossing-over.…”

Prenatal deletion detection in a sporadic case of Duchenne muscular dystrophy without genotype information from the affected individual

“…These results directed us to perform one single hybridization experiment, which at present seems to be more reliable than amplification experiments in prenatal diagnosis [9]. With the obtained data the sporadic case became a familial case, since the only known patient and the two fetuses had the same deletion within the limits of reasonable doubt.…”

“…Multiplex DNA amplification was performed according to Chamberlain et al [4] with modifications described elsewhere [9].…”

“…This allows the identification of the disease-causing mutation in 30%-40% of all DMD patients. Application of this method in prenatal diagnosis has been extensively discussed [9] and enabled us to perform direct diagnosis in the following case.…”

Molecular genetic analysis of factor X deficiency: gene deletion and germline mosaicism

“…Multiplex PCR analysis (e.g. Hentemann et al 1990) would have avoided the necessity to make comparisons between different amplification reactions but this was not possible since (1) the PCR amplification of different exons required different reaction conditions, (2) interference was observed between oligonucleotide primer pairs when included in the same reaction and (3) two of the resulting PCR products were similarly sized.…”