Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing

Raoul Jean Pierre Bonnal, Marco Severgnini, Alessandra Castaldi, Roberta Bordoni, Michele Iacono, Amelia Trimarco, Annalaura Torella, Giulio Piluso, Stefania Aurino, Gianluigi Condorelli, Gianluca De Bellis, Vincenzo Nigro

Research output: Contribution to journalArticle

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× 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.

Original languageEnglish
Pages (from-to)176-184
Number of pages9
JournalAnalytical Biochemistry
Volume406
Issue number2
DOIs
Publication statusPublished - Nov 2010

Keywords

  • 454 sequencing
  • Dystrophin gene
  • Long PCR
  • Resequencing

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Bonnal, R. J. P., Severgnini, M., Castaldi, A., Bordoni, R., Iacono, M., Trimarco, A., Torella, A., Piluso, G., Aurino, S., Condorelli, G., De Bellis, G., & Nigro, V. (2010). Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing. Analytical Biochemistry, 406(2), 176-184. https://doi.org/10.1016/j.ab.2010.07.022