Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

Roberts, Roland G.; Coffey, Alison J.; Bobrow, Martin; Bentley, David

doi:10.1016/0888-7543(92)90005-d

Cited by 84 publications

(38 citation statements)

References 27 publications

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“…Finally, we have defined the transcriptional start site and sequenced the 5' upstream region of this gene as well as that of the adult isoform, ~1. Vectorette PCR has already been shown to be a useful technique in determining exon-intron boundaries (Roberts et al 1992). …”

Section: Discussionmentioning

confidence: 99%

“…To define the structure of genes lying in these contigs, vectorette polymerase chain reaction (PCR) can be used to define the exon-intron boundaries without the need for additional cloning steps. This has the advantage of being simple and rapid and is especially useful in defining the organization of large genes such as dystrophin (Roberts et al 1992). However, YACs often contain deletions and lack critical exons.…”

Section: U77724-u777321mentioning

confidence: 99%

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Structure of the human alpha 2 subunit gene of the glycine receptor--use of vectorette and Alu-exon PCR.

Monani¹,

Burghes²

1996

Genome Res.

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The alpha subunit of the glycine receptor is encoded by multiple genes that display developmental and tissue-specific expression. The alpha 1 subunit gene is expressed predominantly in the adult brain stem and spinal cord, whereas the alpha 2 subunit gene is expressed in fetal brain and spinal cord. We wished to determine the genomic organization of the human alpha 2 subunit gene as well as to define the 5' ends of the alpha 1 and alpha 2 subunit genes. Gene structure can be defined rapidly from yeast artificial chromosome (YAC) DNA sources by the use of vectorette-exon polymerase chain reaction (PCR). However, YACs frequently contain small deletions that complicate the determination of the complete exon-intron structure of a gene, and this often necessitates the isolation of additional clones. In this study we have used vectorette-exon PCR from YAC DNA to define exons of the glycine receptor alpha 2 subunit gene. To define those exons that were absent in the isolated YACs, we used Alu-exon PCR on genomic DNA, using nested primers to obtain specificity in the PCR reactions. The alpha 2 subunit gene was found to contain nine exons varying in size from 68 bp (exons 3A and 3B) to 581 bp (exon l). All of the intron-exon boundary sequences conform to consensus splice donor and acceptor sites. In addition, we have defined the 5' end of this gene as well as that of the alpha 1 subunit gene by RACE-PCR. The structures of the alpha subunit glycine receptor genes in humans are very similar to each other and to the alpha subunit genes in mice.

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Section: Discussionmentioning

confidence: 99%

Section: U77724-u777321mentioning

confidence: 99%

Structure of the human alpha 2 subunit gene of the glycine receptor--use of vectorette and Alu-exon PCR.

Monani¹,

Burghes²

1996

Genome Res.

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“…Dystrophin is comprised of an N-terminal actin-binding domain, 24 spectrin-like repeat units interspersed by four hinge regions, a cysteine-rich domain and a C-terminal domain. It is involved in the linkage between the cytoskeletal actin and the extracellular matrix (Blake et al, 2012;Ehmsen et al, 2013, Roberts et al, 2014and Hoffman et al, 2015.…”

Section: Introductionmentioning

confidence: 99%

In vitro antifungal potential of rhizospheric isolates against Fusarium oxysporum causing Fusarium wilt of Bt-cotton

Raut¹,

Hamde²

2016

Biosci. Biotech. Res. Comm

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The Duchenne muscular dystrophy (DMD) gene is located in the short arm of the X chromosome (Xp21). It spans 2.4 Mb of the human genomic DNA and is composed of 79 exons. Mutations in the dystrophin gene results in DMD and Beckers muscular dystrophy. In this study, the effi ciency of MLPA over multiplex PCR assays in an Iranian population was investigated.Multiplex PCR assays and multiplex ligation-dependent probe amplifi cation (MLPA) analysis were carried out in 65 patients affected with DMD. Multiplex PCR detected deletions in 51% of the patients with DMD. MLPA analysis could determine all the deletions detected by the multiplex PCR. Additionally, MLPA was able to identify one more deletion and duplication in patients without detectable mutations by multiplex PCR. Moreover, MLPA precisely determined the exact size of the deletions.Although MLPA analysis is more sensitive for detection of deletions and duplications in the dystrophin gene, multiplex PCR might be used for the initial analysis of the boys affected with DMD in the Iranian population as it was able to detect 95% of the rearrangements in patients with DMD.

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“…The dystrophin gene is an enormous gene which spreads over more than 2300 Kb, consisting of 79 exons 1,2 and encodes the protein dystrophin (427 kDa) localised in the inner surface of the sarcolemma of muscle fibres. 3,4 Defects of the gene cause the allelic neuromuscular disorders Duchenne and Becker muscular dystrophies.…”

Section: Introductionmentioning

confidence: 99%

Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients

et al. 1999

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The distal part of the human dystrophin gene is characterised by particular features and seems to play an important functional role. Additionally in recent years several data have implicated minor mutations in this gene region in some patients with mental retardation (MR). In order to screen for pathogenic mutations at the distal part of the human dystrophin gene we have used single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) in 35 unrelated male Greek DMD/BMD patients with no detectable deletions. Seven patients also had severe mental retardation. Direct sequencing of samples demonstrating a shift of SSCA mobility revealed six different and pathogenic minor changes, five in DMD and one in a BMD patient. Four of the mutations were found in DMD patients with severe MR. Three of these mutations were localised in exon 66, which presents an interesting similarity with part of the 3' end of the genome of eastern equine encephalomyelitis virus (EEEV). The present data from Greek DMD/BMD patients give further information about the phenotypic effects consequent on mutations in exons at the distal part of the human dystrophin gene.

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Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

Cited by 84 publications

References 27 publications

Structure of the human alpha 2 subunit gene of the glycine receptor--use of vectorette and Alu-exon PCR.

Structure of the human alpha 2 subunit gene of the glycine receptor--use of vectorette and Alu-exon PCR.

In vitro antifungal potential of rhizospheric isolates against Fusarium oxysporum causing Fusarium wilt of Bt-cotton

Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients

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