Linked Life Data

20670611

Source:http://linkedlifedata.com/resource/pubmed/id/20670611

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2010-9-6
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
1096-0309
pubmed:author
pubmed:copyrightInfo
Copyright 2010 Elsevier Inc. All rights reserved.
pubmed:issnType
Electronic
pubmed:day
15
pubmed:volume
406
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
176-84
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Reliable resequencing of the human dystrophin locus by universal long polymerase chain reaction and massive pyrosequencing.
pubmed:affiliation
Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche (CNR-ITB), I-20090 Segrate, Italy.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't