Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses

Dobson‐Stone, Carol; Cox, Roger D.; Lonie, Lorne; Southam, Lorraine; Fraser, Maria; Wise, Carol A.; Bernier, François P.; Hodgson, Shirley; Porter, Daniel; Simpson, A. H. R. W.; Monaco, Anthony P.

doi:10.1038/sj.ejhg.5200409

Cited by 85 publications

(87 citation statements)

References 24 publications

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“…It is now possible that labeling PCR products with fluorescent and adapted SSCP and HA to automated DNA sequencing machines either slab gel electrophoresis [24][25][26][27][28] or capillary electrophoresis (CE) [29][30][31][32][33]. PCR products can be labeled by fluorescent primers [24][25][26] or fluorescent deoxynucleotides either during PCR [27] or after PCR [34,35].…”

Section: Introductionmentioning

confidence: 99%

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Tsuji

Niida

2008

Electrophoresis

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Efficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity, eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% polyacrylamide gel electrophoresis and rapid silver staining can detect all types of mutations and achieved 100% sensitivity.

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Section: Introductionmentioning

confidence: 99%

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Tsuji

Niida

2008

Electrophoresis

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“…This mixture was then annealed and analysed by denaturing high-performance liquid chromatography (DHPLC), as described previously. 15 PCR products showing a variant DHPLC pattern were purified using a QIAquick PCR purification kit (QIAGEN, Surrey, UK). These were then sequenced in both directions by means of the BigDye Terminator Cycle Sequencing Ready Reaction kit (PE-Applied Biosystems) and were run on an ABI PRISM 377 DNA Sequencer (PE-Applied Biosystems).…”

Section: Mutation Analysismentioning

confidence: 99%

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis

Dobson‐Stone

Danek

Rampoldi³

et al. 2002

Eur J Hum Genet

145

123

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Chorea-acanthocytosis (ChAc) is an autosomal recessive neurological disorder whose characteristic features include hyperkinetic movements and abnormal red blood cell morphology. Mutations in the CHAC gene on 9q21 were recently found to cause chorea-acanthocytosis. CHAC encodes a large, novel protein with a yeast homologue implicated in protein sorting. In this study, all 73 exons plus flanking intronic sequence in CHAC were screened for mutations by denaturing high-performance liquid chromatography in 43 probands with ChAc. We identified 57 different mutations, 54 of which have not previously been reported, in 39 probands. The novel mutations comprise 15 nonsense, 22 insertion/ deletion, 15 splice-site and two missense mutations and are distributed throughout the CHAC gene. Three mutations were found in multiple families within this or our previous study. The preponderance of mutations that are predicted to cause absence of gene product is consistent with the recessive inheritance of this disease. The high proportion of splice-site mutations found is probably a reflection of the large number of exons that comprise the CHAC gene. The CHAC protein product, chorein, appears to have a certain tolerance to amino-acid substitutions since only two out of nine substitutions described here appear to be pathogenic.

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“…38 The patients were selected from affected sib-pair families on the basis of sharing either one or two alleles identical by descent (IBD) in the region surrounding the candidate genes, based on flanking microsatellite marker analysis. Primers were designed to cover all exons and intron-exon boundaries of the four genes, as well as the putative promoter sequences of PEG1/MEST, COPG2, and CPA1.…”

Section: Mutation Screeningmentioning

confidence: 99%

Mutation screening and imprinting analysis of four candidate genes for autism in the 7q32 region

Bonora

Bacchelli

et al. 2002

Mol Psychiatry

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Genetic studies indicate that chromosome 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate gene approach to identify the relevant gene and report the analysis of four adjacent genes localised to a 800 kb region in 7q32 that contains an imprinted domain: PEG1/MEST, COPG2, CPA1 and CPA5-a previously uncharacterised member of the carboxypeptidase gene family. Screening these genes for DNA changes and association analysis using intragenic single nucleotide polymorphisms (SNPs) provided no evidence for an etiological role in IMGSAC families. We also searched for imprinting mutations potentially implicated in autism: analysis of both DNA methylation and replication timing indicated a normal imprinting regulation of the PEG1/COPG2 domain in blood lymphocytes of all patients tested. The analysis of these four genes strongly suggests that they do not play a major role in autism aetiology, and delineates our strategy to screen additional candidate genes in the AUTS1 locus.

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Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chromatography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses

Cited by 85 publications

References 24 publications

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Mutational spectrum of the CHAC gene in patients with chorea-acanthocytosis

Mutation screening and imprinting analysis of four candidate genes for autism in the 7q32 region

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