The Experts below are selected from a list of 87 Experts worldwide ranked by ideXlab platform
Nicola Longo - One of the best experts on this subject based on the ideXlab platform.
-
validation of Dye Binding high resolution thermal denaturation for the identification of mutations in the slc22a5 gene
Human Mutation, 2005Co-Authors: Steven F. Dobrowolski, Jason T. Mckinney, Cristina Amat Di San Filippo, K. G. Sim, Bridget Wilcken, Nicola LongoAbstract:Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate Dye-Binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA Binding Dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate Dye-Binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. Hum Mutat 25:306–313, 2005. © 2005 Wiley-Liss, Inc.
-
Validation of Dye-Binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene
Human mutation, 2005Co-Authors: Steven F. Dobrowolski, Jason T. Mckinney, Cristina Amat Di San Filippo, K. G. Sim, Bridget Wilcken, Nicola LongoAbstract:Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate Dye-Binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA Binding Dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate Dye-Binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. Hum Mutat 25:306–313, 2005. © 2005 Wiley-Liss, Inc.
Luis M Hernández - One of the best experts on this subject based on the ideXlab platform.
-
Identification of low-Dye-Binding (ldb) mutants of Saccharomyces cerevisiae.
FEMS yeast research, 2004Co-Authors: Isaac Corbacho, Isabel Olivero, Luis M HernándezAbstract:We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-Dye-Binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.
-
Identification of low‐Dye‐Binding (ldb) mutants of Saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Isaac Corbacho, Isabel Olivero, Luis M HernándezAbstract:Abstract We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-Dye-Binding) mutants. This was done by complementation of the mutant’s phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.
Steven F. Dobrowolski - One of the best experts on this subject based on the ideXlab platform.
-
validation of Dye Binding high resolution thermal denaturation for the identification of mutations in the slc22a5 gene
Human Mutation, 2005Co-Authors: Steven F. Dobrowolski, Jason T. Mckinney, Cristina Amat Di San Filippo, K. G. Sim, Bridget Wilcken, Nicola LongoAbstract:Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate Dye-Binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA Binding Dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate Dye-Binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. Hum Mutat 25:306–313, 2005. © 2005 Wiley-Liss, Inc.
-
Validation of Dye-Binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene
Human mutation, 2005Co-Authors: Steven F. Dobrowolski, Jason T. Mckinney, Cristina Amat Di San Filippo, K. G. Sim, Bridget Wilcken, Nicola LongoAbstract:Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate Dye-Binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA Binding Dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high-resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G>T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate Dye-Binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. Hum Mutat 25:306–313, 2005. © 2005 Wiley-Liss, Inc.
Dolores Pérez-bendito - One of the best experts on this subject based on the ideXlab platform.
-
Quantitation of fusidane antibiotics in pharmaceuticals using the surfactant–Dye Binding degree method
Analytica Chimica Acta, 2005Co-Authors: Esther María Costi, María Dolores Sicilia, Soledad Rubio, Dolores Pérez-benditoAbstract:Abstract The determination of fusidic acid (FA)/sodium fusidate (SF) in dosage forms based on their modification of the Binding degree of the cationic surfactant didodecyldimethylammonium bromide (DDABr) to the anionic Dye Coomassie Brilliant Blue G (CBBG) was proposed. The formation of mixed DDABr–CBBG aggregates was monitored from changes in the spectral features of the Dye. Addition of fusidane antibiotic to DDABr–CBBG aqueous mixtures caused a decrease in the surfactant–Dye Binding degree as a result of the formation of DDABr–drug aggregates. At the working pH (7.0), FA and SF were as fusidate anion; therefore, both hydrophobic and attractive electrostatic interactions were responsible for the formation of these mixed surfactant–drug aggregates. Based on the mathematical expression previously derived for determining surfactants, a linear calibration in the fusidane antibiotic concentration range 1.9–70 mg L−1 was obtained. The surfactant to Dye Binding degree (SDBD) method offered important advantages over conventional method used for quality control of fusidane antibiotics in terms of sensitivity (detection limit = 0.6 mg L−1), selectivity, which permitted a minimum sample treatment (only dissolution of dosage forms), precision [relative standard deviations for the whole analytical process = 1.5–2.0% (n = 6)], rapidity, because of the speed of both sample treatment and determination step, required instrumentation (a photometric titrator) and cost. Pharmaceutical samples analyzed included tablets, pomades and creams.
-
Surfactant to Dye Binding Degree-Based Methodology for the Determination of Ionic Amphiphilic Compounds
Analytical Chemistry, 2003Co-Authors: Rafael Fabios, María Dolores Sicilia, Soledad Rubio, Dolores Pérez-benditoAbstract:A new analytical measurement parameter based on the effect of amphiphilic substances on the degree of Binding of a surfactant to Dye molecules, which induce the formation of surfactant premicellar aggregates, is presented. The theory for Dye−surfactant intermolecular interactions in mixed surfactant systems, which assumes a mononuclear model for the formation of Dye-induced premicellar aggregates, has been used to derive an expression that provides linear calibrations for the determination of amphiphilic compounds. The Dye−surfactant interactions involved have been investigated, and the variables affecting the measurement analytical parameter have been discussed. The analytical applicability of the surfactant−Dye Binding degree method is demonstrated by quantifying major anionic surfactants at the nanograms-per-milliliter level and determining the total concentration of these amphiphilic substances in sewage samples (average recoveries ranged from 98 to 102%).
Isaac Corbacho - One of the best experts on this subject based on the ideXlab platform.
-
Identification of low-Dye-Binding (ldb) mutants of Saccharomyces cerevisiae.
FEMS yeast research, 2004Co-Authors: Isaac Corbacho, Isabel Olivero, Luis M HernándezAbstract:We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-Dye-Binding) mutants. This was done by complementation of the mutant's phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.
-
Identification of low‐Dye‐Binding (ldb) mutants of Saccharomyces cerevisiae
Fems Yeast Research, 2003Co-Authors: Isaac Corbacho, Isabel Olivero, Luis M HernándezAbstract:Abstract We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-Dye-Binding) mutants. This was done by complementation of the mutant’s phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.
