Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis

Grade, K; Grunewald, Ingrid; Graupner, I; Behrens, F; Coutelle, Charles

doi:10.1007/bf00202861

Cited by 15 publications

(9 citation statements)

References 16 publications

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“…Many times the size of the gene and the great number of possible mutations makes it a laborious task. An alternative to sequencing the whole gene are screening techniques, such as Single Strand Conformational Polymorphisms (SSCP) [1,2] denaturing High Performance Liquid Chromatography (dHPLC) [3,4] or more recently, the High Resolution Melting (HRM) technique [5]. All these screening methods are pre-sequencing because detect deviations from normal pattern due to the mutation.…”

Section: Introductionmentioning

confidence: 99%

Screening of Specific Gene Mutations Associated with Cystic Fibrosis

et al. 2014

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A selective screening method for detection of gene mutations in highly mutated genes associated with cystic fibrosis is described. The method relies on a disposable DNA electrochemical biosensor based on the use of a selective pentaammine [3‐(2‐phenanthren‐9‐yl‐vinyl)‐pyridine] ruthenium complex as electrochemical indicator. This complex incorporates dual functionalities: the Ru center provides a redox probe and the ligand endows the complex with an intercalative character making it capable of binding preferently to double‐stranded DNA. The approach allows the simultaneous detection of different mutations and the determination of homozygous and heterozygous directly in large PCR amplicons extracted from blood cells. The results agree well to those obtained by High Resolution Melting sequencing suggesting that the proposed biosensing procedure is a robust and sensitive technique for screening mutations.

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Section: Introductionmentioning

confidence: 99%

Screening of Specific Gene Mutations Associated with Cystic Fibrosis

et al. 2014

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“…SSCP analysis of PCR-amplified exons was performed by electrophoresis of 0.1-0.5~1 of the PCR mixture which was denatured with a 10-fold excess of formamide/ 15mM EDTA/SO mM NaCl for 5 min at 95°C followed by 5 min cooling on ice. Conditions for horizontal electrophoresis and silver staining as well as the equipment used were the same as indicated by Grade et al (1994). Single-stranded DNA was obtained by alkali denaturation of biotinylated PCR products and separation with streptavidin-coated magnetic beads (Dynal, Oslo, Norway) as recommended by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

BRCA1 mutations in German breast-cancer families

et al. 1996

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We analyzed germline mutations of the BRCA1 gene in 20 German breast/ovarian-cancer families. BRCA1 mutations co-segregating with breast-cancer susceptibility were identified in 3 of these families. All mutations were found to generate a premature stop codon leading to the synthesis of truncated BRCA1 proteins of different length. Nine polymorphisms were detected in BRCA1, 4 of which have not been described previously. Analysis of familial tumors for LOH revealed that only the disease-related allele of BRCA1 was present.

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“…CF mutations are currently identified by a variety of molecular biology approaches, including polymerase-chain reaction (PCR) [Saiki et al, 1985] and hybridization (ASO) [Ballabio et al, 1990;Ng et al, 1991;Richards et al, 1993], heteroduplex formation [Rommens et al, 1990], single-stranded conformation polymorphism [Orita et al, 1989;Grade et al, 1994], denaturing gradient gel electrophoresis [Ferec et al, 1992], reverse dot-blot hybridization [Chehab and Wall, 1992], and PCR sequencing [Chehab and Wall, 1992;Shoshani et al, 1992]. It should be pointed out that these methods have been highly informative and successfully applied in basic as well as epidemiology studies; however, most of them are tedious, in some cases technically complex, and difficult to be routinely employed in a clinical context when quality of services, speed, accuracy, and low costs are the most important requirements [Lundemberg et al, 1991;Jenkins, 1994].…”

Section: Introductionmentioning

confidence: 99%

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR

et al. 2001

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In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.

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Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis

Cited by 15 publications

References 16 publications

Screening of Specific Gene Mutations Associated with Cystic Fibrosis

Screening of Specific Gene Mutations Associated with Cystic Fibrosis

BRCA1 mutations in German breast-cancer families

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR

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