Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Polymerase chain reaction (PCR)-based diagnosis was carried out in 62 patients (57 probands) with Duchenne or Becker muscular dystrophy (DMD or BMD) and 226 members in 57 families. The PCR studies were also performed for carrier detection in 57 mothers and 58 sisters, and prenatal diagnosis of 4 fetuses at risk of DMD. The PCR with 7 sets of primers, which amplify 7 different exon-sequences of the dystrophin gene, detected gene deletion of at least one exon in 49% of the probands. The PCR with the other 4 primer sets, which amplify 3 intragenic loci, and subsequent endonuclease digestion detected in 84% of the mothers a heterozygous pattern in at least one such locus/segment. Using the same primer sets, carrier detection was successful in 5 sisters of familial DMD cases, while recombination between the ERT87 and the 3' end intragenic loci was observed in 11% of family members studied. Prenatal diagnosis was made in all the 4 fetuses; two males were affected, one male fetus non-affected, and the remaining one female fetus a carrier. Thus, the PCR study and the primers used in the present study are useful and convincing for rapid diagnosis of DMD and/or BMD.
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PMID:Gene-deletion and carrier detections, and prenatal diagnosis of Duchenne muscular dystrophy by analysis of the dystrophin gene amplified by polymerase chain reaction. 181 Oct 98

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.
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PMID:[Recent advances in human molecular genetics]. 197 24

A DNA deletion in a patient with Becker muscular dystrophy (BMD) has been delineated by restriction endonuclease mapping. The deletion is unusually small, removing six kilobases (kb) of DNA distal to pERT 87-1 (DXS164). This region has previously been shown to contain an exon of a candidate gene which, when defective, causes Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy. Removal of this exon and surrounding DNA is apparently sufficient, in this case, to cause a BMD phenotype. The occurrence of this deletion in DXS164 would appear to confirm that this region is part of the BMD locus. Many DMD patients have deletions in and around this region, adding further evidence for the allelic nature of the two disorders. This fortuitous deletion may identify a functionally important domain of the protein product in terms of the severity of phenotype manifested.
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PMID:A small deletion in the Duchenne/Becker muscular dystrophy locus--a functionally important region? 304 May 77

A method is proposed to calculate the maximum likelihood estimate of gene frequency and linkage disequilibrium from disease-codominant marker conditional data. The method is illustrated using data on sickle-cell anemia and Duchenne muscular dystrophy and linked polymorphic restriction endonuclease cleavage sites.
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PMID:Estimation of the marker gene frequency and linkage disequilibrium from conditional marker data. 632 Jun 41

Construction of recombinant adenovirus, which contain human microdystrophin, and then transfection into mesenchymal cells( MSCs) of mdx mice were done, and genetically-corrected isogenic MSCs were acquired; the MSCs transplantation into the mdx mice was then done to treat the Duchenne muscular dystrophy( DMD). Microdystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I ; the production was inserted directionally into pShuttle-CMV. The plasmid of pShuttle-CMV-MICRO was digested by Pme I , the fragment containing microdystrophin was reclaimed and transfected into E. coli BJ5183 with plasmid pAdeasy-1. After screening by selected media, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by observing the CPE of cells and by the PCR method. Finally, MSCs of mdx mice were infected with the culture media containing recombinant adenovirus, and the expression of microdystrophin was detected by RT-PCR and immunocytochemistry. Recombinant adenovirus including microdystrophin was constructed successfully and the titer of recombinant adenovirus was about 5.58 x 10(12) vp/mL. The recombinant adenovirus could infect MSC of mdx mice and microdystrophin could be expressed in the MSC of mdx mice. Recombinant adenovirus including microdystrophin was constructed successfully, and the microdystrophin was expressed in the MSC of mdx mice. This lays the foundation for the further study of microdystrophin as a target gene to correct the dystrophin-defected MSC for stem cell transplantation to cure DMD.
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PMID:[Construction of recombinant adenovirus including microdystrophin and expression in the mesenchymal cells of mdx mice]. 1736 84

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.
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PMID:Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol. 1795 67

Non-autonomous retrotransposon-mediated mobilizations of the Alu family are known pathogenic mechanisms of human disease. Here, we report a pathogenic, contemporary, non-autonomous retrotransmobilization of part of a novel non-coding gene into the dystrophin gene. In a Japanese Duchenne muscular dystrophy patient, a 330-bp-long de novo insertion was identified in exon 67 of dystrophin. The insertion induced exon 67-skipping in the dystrophin mRNA, creating a premature stop codon. The sequence of the insertion had certain characteristics of retrotransposons: an antisense polyadenylation signal accompanied by a poly(T) sequence and a target site duplication. The insertion site matched the consensus recognition sequence for the L1 endonuclease, indicating a retrotransposon-mediated event, although the inserted sequence did not match any known retrotransposons. The origin of the inserted sequence was mapped to a gene-poor region of chromosome 11. The inserted fragment was expressed in multiple human tissue RNAs, indicating that it is a novel transcript. The full length of the transcript was cloned and showed no meaningful protein coding ability.
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PMID:Contemporary retrotransposition of a novel non-coding gene induces exon-skipping in dystrophin mRNA. 2082 76

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD), and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations, which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases where other techniques have failed.
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PMID:Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol. 2393 97

Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.
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PMID:Quantification of designer nuclease induced mutation rates: a direct comparison of different methods. 2741 95

CRISPR was first observed in 1987 in bacteria and archaea and was later confirmed as part of bacterial adaptive immunity against the attacking phage. The CRISPR/Cas restriction system involves a restriction endonuclease enzyme guided by a hybrid strand of RNA consisting of CRISPR RNA and trans-activating RNA, which results in gene knockout or knockin followed by nonhomologous end joining and homology-directed repair. Owing to its efficiency, specificity, and reproducibility, the CRISPR/Cas restriction system was said to be a breakthrough in the field of biotechnology. Apart from its application in biotechnology, CRISPR/Cas has been explored for its therapeutic potential in several diseases including cancer, Alzheimer's disease, sickle cell disease, Duchenne muscular dystrophy, neurologic disorders, etc., wherein CRISPR/Cas components such as Cas9/single guide RNA (sgRNA) ribonucleoprotein, sgRNA/mRNA, and plasmid were delivered. However, limitations including immunogenicity, low transfection, limited payload, instability, and off-target binding pose hurdles in its therapeutic use. Nonviral vectors (including cationic polymers, lipids, etc.), classically used as carriers for therapeutic genes, were used to deliver CRISPR/Cas components and showed interesting results. Herein, we discuss the CRISPR/Cas system and its brief history and classification, followed by its therapeutic applications using current nonviral delivery strategies.
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PMID:CRISPR/Cas System for Genome Editing: Progress and Prospects as a Therapeutic Tool. 3112 33


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