title: Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing.
abstract: The X-linked dystrophin gene is well known for its involvement in Duchenne/Becker muscular dystrophies and for its exceptional megabase size. This locus at Xp21 is prone to frequent random molecular changes, including large deletions and duplications, but also smaller variations. To cope with such huge sequence analysis requirements in forthcoming diagnostic applications, we employed the power of the parallel 454 GS-FLX pyrosequencer to the dystrophin locus. We enriched the genomic region of interest by the robust amplification of 62 fragments under universal conditions by the long-PCR protocol yielding 244,707 bp of sequence. Pooled PCR products were fragmented and used for library preparation and DNA sequencing. To evaluate the entire procedure we analyzed four male DNA samples for sequence coverage and accuracy in DNA sequence variation and for any potential bias. We identified 562 known variations and 55 additional variants not yet reported, among which we detected a causative Arg1844Stop mutation in one sample. Sanger sequencing confirmed all changes. Unexpectedly, only 3 x coverage was sufficient for 99.9993% accuracy. Our results show that long PCR combined to massive pyrosequencing is very reliable for the analysis of the biggest gene of the human genome and open the doors to other demanding applications in molecular diagnostics.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche (CNR-ITB), I-20090 Segrate, Italy.
authors.forename: Raoul Jean Pierre
authors.initials: RJ
authors.lastname: Bonnal
authors.forename: Marco
authors.initials: M
authors.lastname: Severgnini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Castaldi
authors.forename: Roberta
authors.initials: R
authors.lastname: Bordoni
authors.forename: Michele
authors.initials: M
authors.lastname: Iacono
authors.forename: Amelia
authors.initials: A
authors.lastname: Trimarco
authors.forename: Annalaura
authors.initials: A
authors.lastname: Torella
authors.forename: Giulio
authors.initials: G
authors.lastname: Piluso
authors.forename: Stefania
authors.initials: S
authors.lastname: Aurino
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Condorelli
authors.forename: Gianluca
authors.initials: G
authors.lastname: De Bellis
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Nigro
mesh_terms: D001483:Base Sequence; D001704:Biopolymers / Q000235:genetics; D016189:Dystrophin / Q000235:genetics*; D056426:Genetic Loci / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
chemical_list: D001704:Biopolymers; C484258:DMD protein, human; D016189:Dystrophin