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C Coutelle

Publications and source records attributed to C Coutelle.

At least 109 records · Page 6Linked to original sources
PubMed1990

Deletion analysis of DMD/BMD families from the German Democratic Republic and selected regions of Czechoslovakia and Hungary.

Over the last two years we have screened 183 DMD/BMD families requesting prenatal diagnosis. Using cDNA probes cf56a,b we have detected exon deletions in 72 of them. In 62 cases the deletion was also detectable with currently available PCR primers. Deletion analysis for exons 8, 17, and 19, using either PCR or Southern blotting techniques, was performed for 65 of the 111 families which showed no deletions with cf56a,b. Eight of them were deleted for one or more of these exons. PCR offers new possibilities for deletion analysis in families without a living patient using either Guthrie papers or histologically conserved material from the dead patient. In 20 of 25 patients, we observed concordance between the clinical picture and the molecular deletion analysis in accordance with the open reading frame hypothesis. Five patients, however, presented with DMD in spite of our analysis showing an in frame deletion. Carrier determination in families in which DMD is caused by a deletion using linkage, dosage, or breakpoint analysis is discussed.

Blotting, Southern↗
PubMed1990

Automated sequencing of fluorescently labelled DNA by chemical degradation.

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.

Automation↗
PubMed1989

Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis.

Gene sequences in human oocytes were studied to investigate the possibility of diagnosing inherited or sporadic genetic disease before implantation after in vitro fertilisation. By specific amplification the possibility of analysing the DNA from single human oocytes for a specific gene was shown, and genotypes for markers closely linked to cystic fibrosis and Duchenne muscular dystrophy were determined. Single oocytes were used to approximate the total amount of DNA present in a single cell taken for biopsy from a 4-16 cell blastocyst. With a new technique for specific DNA amplification, the polymerase chain reaction, these data can be obtained within several hours of cell isolation. Extreme care must be taken to avoid any contamination of the sample with DNA from other sources. With this technique genotyping for single gene disorders is feasible with an accuracy and on a time scale that would allow implantation of the zygote after in vitro fertilisation without freezing.

Base Sequence↗
PubMed1989

RFLP-discordance within the human phenylalanine hydroxylase locus.

A search for crossover events was performed in seven families with two phenylketonuria-affected children applying seven enzymes for the detection of eight restriction fragment length polymorphisms (RFLPs) by hybridisation with a human phenylalanine hydroxylase (PAH) cDNA probe. A discordance was detected within the PvuIIb-RFLP pattern of two affected sibs. This observation is most likely the result of a cross-over event in the PAH locus. Alternative explanations are discussed. Non-paternity could be excluded by DNA fingerprinting using the oligonucleotide probe (GTG)5.

Crossing Over, Genetic↗
PubMed1989

A new neomycin phosphotransferase II solid phase assay in combination with polyacrylamide sodium dodecylsulphate gel electrophoresis.

A new general method for the determination of neomycin phosphotransferase (NPT) II (EC 2.7.1.95) activity in cell extracts after separation in SDS-polyacrylamide gels is described. The enzymatic activity of NPT II is restored after SDS-polyacrylamide gel electrophoresis by incubating the gel for 3 h (20 mM Tris-HCl buffer, pH 7.4). The enzymatic activity is determined by in situ phosphorylation of aminoglycoside antibiotics bound to solid supports and brought into direct contact with the gel surface. A novel, mechanically stable, negatively charged matrix was synthesized for use in this solid phase enzyme assay and compared to phosphocellulose and carboxymethylcellulose paper. This new method allows the easy and exact determination of the molecular weight of any fusion protein with NPT II by assaying the position of the enzymatic activity in the gel and a consecutive immunological reaction following protein transfer onto nitrocellulose membranes.

Aminoglycosides↗
PubMed1989

[The development of molecular human genetics and its significance for perspectives of modern medicine].

The introduction of molecular human genetics has become a paradigma for the application of genetic engineering in medicine. The main principles of this technology are the isolation of molecular probes, their application in hybridization reactions, specific gene-amplification by the polymerase chain reaction, and DNA sequencing reactions. These methods are used for the analysis of monogenic diseases by linkage studies and the elucidation of the molecular defect causing these conditions, respectively. They are also the basis for genomic diagnosis of monogenic diseases, introduced into the health care system of the GDR by a national project on Duchenne/Becker muscular dystrophy, Cystic Fibrosis and Phenylketonuria. The rapid development of basic research on the molecular analysis of the human genome and genomic diagnosis indicates, that human molecular genetics is becoming a decisive basic discipline of modern medicine.

Child↗
PubMed1988

Linkage disequilibrium between RFLP haplotype 2 and the affected PAH allele in PKU families from the Berlin area of the German Democratic Republic.

Probands from 26 PKU-affected families of the Berlin area were analyzed with respect to the allele frequency distribution of six RFLPs in linkage with the normal and the PKU alleles of the phenylalanine hydroxylase gene. These investigations confirm most of the RFLP haplotypes observed by Güttler and colleagues in the Danish population and describe two additional ones. They detect no significant differences in the single RFLP or RFLP haplotype distribution on the normal chromosomes in comparison with the Danish families and confirm a prevalent association of the RFLP haplotypes 1, 4, and 7 with the normal PAH allele. In contrast to the Danish investigations, in our study the PKU allele is found most frequently linked to haplotype 2, rather then to haplotype 3. In one of our patients we found a substitution of the normal 19-kb MspI fragment by a 13.5- and a 5.5-kb fragment, reported up to now only in one other German family.

Alleles↗
PubMed1988

A protein-free blocking system for the covalently binding matrix cyanuric chloride-activated paper in immunological procedures.

A new protein-free blocking system containing 10% (w/v) sulfanilic acid/10% (v/v) triethanolamine/0.05% (v/v) Tween 20 has been used to block free binding sites of the covalently binding matrix cyanuric chloride-activated paper (CCA-paper). This method allows a reversible staining of protein blots with Coomassie brilliant blue after each step of the immunological procedure and reuse of the blots for a repeated antibody probing. Non-radioactive and radioactive detection procedures have been compared with blots on CCA-paper and nitrocellulose. The best combination is a Coomassie brilliant blue staining and immunological detection with 125I-protein A.

Cross-Linking Reagents↗
PubMed1987

Use of cyanuric chloride-activated paper for detection of subpicogram quantities of specific DNA sequences and its application to linked restriction fragment length polymorphism analysis in a Duchenne muscular dystrophy affected family.

Conditions for the optimal use of cyanuric chloride-activated (CCA) paper in Southern transfer hybridization experiments of genomic DNA were investigated. They depend critically on pH and ionic strength during transfer and on the composition of the hybridization solution. Simplified hybridization conditions using a SSC/dextran sulfate system at 65 degrees C without sodium dodecyl sulfate and the complex Denhardt's solution are applied. CCA paper allows repeated use in hybridization experiments. Under optimized conditions CCA paper allows a more sensitive detection of single-copy gene sequences in the subpicogram range than do nylon membranes. Application of these transfer and hybridization conditions with our newly developed CCA paper to carrier determination and prediction of the healthy male haplotype demonstrates its usefulness for prenatal counseling of a Duchenne muscular dystrophy family.

DNA↗
PubMed1987

Isolation of a conserved sequence deleted in Duchenne muscular dystrophy patients.

We have isolated a DNA sequence (HIP25) by subtraction- hybridisation which is deleted in a number of Duchenne muscular dystrophy (DMD) patients. HIP25 is conserved in evolution and hybridises to human fetal and adult muscle mRNA. HIP25 is absent in human fetal fibroblast mRNA. Physical mapping data localise this sequence within Xp21 between the breakpoints of X;autosome translocations found in two females suffering from the disease. HIP25 is a candidate exon sequence for the basic defect in DMD boys deleted at this locus.

Adult↗
PubMed1987

Crossovers in two German cystic fibrosis families determine probe order for MET, 7C22 and XV-2c/CS.7.

We have followed the segregation of the probes pJ3.11, 7C22, pB79a, and MET through cystic fibrosis families in the German Democratic Republic with two affected sibs. Two families with a crossover between MET and the CF phenotype were detected. In one of these families recombination was also observed between the DNA probe 7C22 and CF, and between the markers XV-2c and CF, which suggests that XV-2c, MET and 7C22 are all on the same side of CF. The other MET recombinant family is informative with XV-2c and does not recombine, which excludes the genetic order XV-2c--MET--CF if multiple recombinant events are disregarded. These two families together demonstrate that recombinations may occur in a very small genetic interval, which has important implications for prenatal diagnosis based on data from linked markers.

Crossing Over, Genetic↗