AMPLIFICATION OF 18 DYSTROPHIN GENE EXONS IN DMD BMD PATIENTS - SIMULTANEOUS RESOLUTION BY CAPILLARY ELECTROPHORESIS IN SIEVING LIQUID POLYMERS

Duchenne (DMD) and Becker (BMD) muscular dystrophies are the two most common myopathies described so far: In the late 80s, Chamberlain et al . and Beggs et al. proposed two PCR assays allowing detection of over 98% DMD/BMD deletions. Since each of them is based on specific co-ampl ification of 9 dystrophin gene exons, a method attempting simultaneous analysis of DMD/BMD should offer unambiguous resolution and identific ation of 18 DNA fragments ranging in size from approximately 100 to 50 0 bp. We have developed a novel capillary electrophoresis method that allows simultaneous analysis of the two PCR sets with full diagnostic value. It consists of (a) an ultrastable inner capillary coating based on a novel acrylamide monomer (N-acryloyl amino ethoxy ethanol); (b) a very low viscosity (barely 70 mPa) sieving polymer solution, formed by short-chain (average mol wt of 230000, 55000 M(n)) polyacrylamides; (c) substitution of four fragments in the classical multiplex reactio n (181 and 535 bp in the Beggs, 416 and 459 bp in the Chamberlain) ,vi th Sour new fragments of different lengths (170, 313, 154 and 88 bp, r espectively). These new conditions allow resolution and unambiguous id entification of all 18 PCR-amplified fragments in a single electrophor etic run. The set of 18 fragments comprises the following: 88, 113, 13 9, 154, 170, 196, 202, 238, 268, 271, 313, 331, 357, 360, 388, 410, 50 6 and 547 bp.