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-amplification of 9 dystrophin gene exons, a method attempting simultaneous analysis of DMD/BMD should offer unambiguous resolution and identification of 18 DNA fragments ranging in size from approximately 100 to 500 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 230,000, 55,000 Mn) polyacrylamides; (c) substitution of four fragments in the classical multiplex reaction (181 and 535 bp in the Beggs, 416 and 459 bp in the Chamberlain) with four new fragments of different lengths (170, 313, 154 and 88 bp, respectively). These new conditions allow resolution and unambiguous identification of all 18 PCR-amplified fragments in a single electrophoretic run. The set of 18 fragments comprises the following: 88, 113, 139, 154, 170, 196, 202, 238, 268, 271, 313, 331, 357, 360, 388, 410, 506 and 547 bp.
Gelfi
C
C
ITBA, CNR, Milano, Italy.
Orsi
A
A
Leoncini
F
F
Righetti
P G
PG
Spiga
I
I
Carrera
P
P
Ferrari
M
M
D000180:Acrylic Resins / Q000737:chemistry; D016189:Dystrophin / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005091:Exons; D006801:Humans; D007231:Infant, Newborn; D008969:Molecular Sequence Data; D009136:Muscular Dystrophies / Q000175:diagnosis / Q000235:genetics*; D015997:Neonatal Screening; D016133:Polymerase Chain Reaction / Q000379:methods*; D011296:Prenatal Diagnosis; D000099166:Polyacrylamides
D000180:Acrylic Resins; D016189:Dystrophin; D000099166:Polyacrylamides