Rapid direct sequence analysis of the dystrophin gene

Abstract

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%-65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method-currently the most widely available method of mutational analysis-detects approximately 98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed "SCAIP" (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.

Figure 1

Agarose gel analysis of primary PCR products. An aliquot of each well from the 96-well PCR amplification plate is loaded in a 96-well format onto an agarose gel. Electrophoretic separation distance for each band is ∼1.8 cm, because the wells are angled slightly in relation to the migration path. The products are from a patient with a multiexon deletion who is missing exons 20–30 and the DMD260 promoter. Products corresponding to exons 1–78 are located in sequential wells, starting left to right and top to bottom, followed by the multiple exon 79 and alternate promoter products. Note the absence of products in wells corresponding to exons 20–30 and to Dp260.

Figure 2

Average Phrap score coverage of DMD exons and promoter regions. Each block represents the length of the individual PCR products, with the exonic sequence indicated by the thick line on the top horizontal axis. The average Phrap score observed in the present study is plotted along its horizontal position, with the vertical axis ranging from Phrap score 15 to 50. Phrap scores >50 are not shown, and the portions of the plot corresponding to the exons ±100 nt are indicated in gray.