Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we describe the use of a DNA amplification technique in which modified primers introduce a base substitution adjacent to the codon of interest and create an artificial restriction site for the detection of mutations which do not produce or modify a naturally occurring restriction site (restriction site generating-polymerase chain reaction, RG-PCR). RG-PCR was developed and applied to the screening in an Italian population sample of several relatively common cystic fibrosis mutations which are not amenable to analysis with a known restriction endonuclease: G542X, 2869insG, Y913C, N1303K, and 1717-1GA. This method, which allows the identification of virtually any single base change by restriction enzyme analysis and without the need for molecular probes, is rapid and easy to perform. The combined use of RG-PCR for several different CF mutations in multiplex tests further expands the advantages of this approach.
 ...
PMID:Restriction site generating-polymerase chain reaction (RG-PCR) for the probeless detection of hidden genetic variation: application to the study of some common cystic fibrosis mutations. 134 44

DNA diagnostic tests often utilize restriction endonuclease digestion of PCR-amplified portions of genes under analysis. When partial digestion occurs, the resulting patterns may lead to error in diagnosis. To overcome such potential errors in cystic fibrosis testing, we have developed internal markers that can increase the precision and reliability of genotype assignments.
 ...
PMID:Utility of internal markers to improve the accuracy of cystic fibrosis genotype analysis. 138 68

Detection of small alterations or abnormalities in genomic DNA (eg, point mutations or small deletions) has become increasingly important in the diagnosis of genetic disease and polymorphism. When a mutation or polymorphism creates a new restriction endonuclease site, it can easily be identified by polymerase chain reaction (PCR) amplification of the DNA region of interest, followed by digestion with the restriction endonuclease. However, useful restriction sites are the exception, and a variety of specialised techniques have been developed to identify subtle DNA abnormalities. We have shown that where a DNA mutation does not create a useful novel restriction site, such a site can be introduced by PCR and specially chosen primers. The approach is simple and inexpensive and should be broadly applicable in the diagnosis of genetic polymorphism and mutation. The technique is illustrated here by the three base-pair deletion responsible for most cases of cystic fibrosis and by detection of the point mutation in the rhodopsin gene that has been associated with some cases of autosomal dominant retinitis pigmentosa.
 ...
PMID:Diagnosis of genetic disease by primer-specified restriction map modification, with application to cystic fibrosis and retinitis pigmentosa. 167 37

Polymorphism in the restriction fragments length of human DNA sequences linked to mucoviscidosis locus was studied in the healthy control group and in the families affected by mucoviscidosis. The plasmid clones metH, pJ3.11,XV-2c and pKM.19 were used as hybridization probes. The allelic frequencies of the polymorphic loci were determined for total population and for affected families. The linkage disequilibrium between the disease locus and linked polymorphic loci detectable with XV-2c (TaqI endonuclease) and pKM.19 (PstI endonuclease) was demonstrated. The high informational value of DNA-diagnosis of mucoviscidosis in the family studies with the use of four DNA probes combination has been demonstrated.
 ...
PMID:[Polymorphism of nucleotide sequences of human genomic DNA linked to a mucoviscidosis locus]. 197 61

The haploid nucleus of a human cell contains 3 X 10(9) base pairs. Organized in linear duplex, this DNA would stretch out to a length of some 90 cm. Thus, organization of chromosomes has been a major subject for pioneer cytogenetists. Long lasting controversies on the strandedness of chromosomes, together with newly developed banding techniques, led us to molecular cytogenetics. Next, the discovery of reverse transcriptase, restriction endonucleases, and other recombinant DNA methods have enabled us to isolate and characterize genes from any organism and to determine the DNA sequences and any encoded protein sequences. These new technologies have already helped us to understand many inherited diseases at a molecular level. In sickle cell anemia, thalassemia and in other mendelian disorders we can know their molecular defects by examining the DNA from peripheral leukocytes, without the need for complex biochemical assays or biopsies. Southern blot analysis using restriction endonuclease and a probe is a basic tool for molecular diagnosis. cDNA or DNA fragments are used as probes. Recently, synthesized oligonucleotide probes are available, if the DNA sequence of a gene is determined. In addition, restriction fragment length polymorphisms (RFLPs), play a very important role in the molecular diagnosis. Linkage analysis using RFLPs linked to the gene locus of a certain disease also permits the detection of the patients and carriers within families with genetic diseases of unknown cause. Starting with the genetic map and physical map, genes for cystic fibrosis and Duchenne muscular dystrophy have recently been isolated and cloned.
 ...
PMID:[Recent advances in human molecular genetics]. 197 24

Strains of Pseudomonas aeruginosa from patients with cystic fibrosis (CF) are unusual. The majority have a rough lipopolysaccharide (LPS) which renders them nontypeable by conventional typing systems based on a serological reaction with the O polysaccharide of smooth LPS. We developed a new typing scheme using a pilin gene probe as a marker for hybridization with endonuclease-digested genomic DNA from P. aeruginosa. Twenty-one different restriction fragment length polymorphism (RFLP) types were found among 249 isolates. RFLP type 7 was recovered only from patients with thermal burns (9 of 14 isolates) in both Vancouver, British Columbia, and Edmonton, Alberta, Canada. None of the other RFLP types showed a clear predilection for disease state or environmental niche. Multiple morphologically different isolates from individual patients with CF were studied; each isolate in 33 of 40 sputum samples had an identical RFLP type, despite considerable LPS serotype heterogeneity. Sequential isolates from 23 patients were studied; in 10 isolates there was a clear change in both the RFLP and the LPS serotype. We conclude that patients with CF usually harbor a single P. aeruginosa RFLP type in their sputa, but that one strain can replace another as the predominant colonizing type.
 ...
PMID:Use of a pilin gene probe to study molecular epidemiology of Pseudomonas aeruginosa. 257 4

Cosmid libraries have been constructed from DNA of somatic cell hybrid cell lines, each containing a fragment of human chromosome seven and including sequences closely linked to cystic fibrosis (CF). Cosmids containing human DNA as insert were isolated from the library. Three cosmids, when used as probes to total genomic DNA, detected polymorphic loci, each of which was shown to be in strong linkage disequilibrium with CF. Restriction endonuclease digestion of cosmid clones and use of a new, rapid method of chromosome walking based on competitive hybridisation of cosmid inserts has allowed identification of several groups of overlapping cosmids ("contigs") from the vicinity of CF.
 ...
PMID:Physical and genetic analysis of cosmids from the vicinity of the cystic fibrosis locus. 347 44

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.
 ...
PMID:Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. 633 52

Polymerase chain reaction (PCR) ribotyping detects differences in the intergenic spacer between the 16S and 23SrRNA genes. This method was applied to Burkholderia cepacia isolates from 16 Welsh cystic fibrosis (CF) patients attending three different clinics. Amplification of the intergenic spacer followed by an additional digestion step with TaqI restriction endonuclease identified seven distinct electrophoretic patterns among the patient isolates. Each of the seven patterns was distinct from that of the so called "epidemic strain" commonly isolated from patients attending clinics elsewhere in the UK. Two environmental isolates from the hospital clinics and four NCTC reference strains gave different patterns. The simplicity of the method lends itself to use in a general microbiological laboratory.
 ...
PMID:Characterisation of Burkholderia cepacia from cystic fibrosis patients living in Wales by PCR ribotyping. 747 77

W1282X (W) and delta F508 (delta) are the two most common mutations of the cystic fibrosis Israeli population. Patients who are homozygotes (WW and delta delta) as well as compound heterozygotes (W delta) present a severe phenotype of the disease. In the present study, we have developed a polymerase chain reaction (PCR)-based method for the detection of both mutations simultaneously in a single blastomere. Unfertilized human oocytes and single polyspermic blastomeres were subjected to a two-round PCR amplification: a first round of multiplex PCR followed by a second round of nested PCR, done separately at each locus. Clear signals at both loci were obtained in 51% (47/65) of oocytes and 69% (24/35) of blastomeres. The genotype of the single cell analysed was determined by endonuclease digestion of the W products and by heteroduplex formation of the delta F products. This diagnostic system will allow the identification of affected embryos (WW, delta delta, W delta) as well as phenotypically normal carriers (W+, +delta), and therefore may be used for cystic fibrosis preimplantation diagnosis in families who carry either or both mutations.
 ...
PMID:Preimplantation diagnosis of cystic fibrosis by simultaneous detection of the W1282X and delta F508 mutations. 753 Jul 26


1 2 3 Next >>