Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.
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PMID:Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA. 137 52

A new method has been developed that permits the rapid amplification of unknown DNA flanking a known site so that one can walk into an uncharacterized region of DNA. This method eliminates the steps and sequence artifacts associated with cloning and permits genome walking into unclonable regions of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to the 3' end of a 5'-phosphorylated oligonucleotide using a short bridging oligonucleotide using a short bridging oligonucleotide as a splint. The phosphorylated oligonucleotide is designed to create 5'-end extensions that are complementary to the known sequence. Following denaturation and reannealing under dilute conditions that promote intra-strand annealing and under high stringency, only those DNA strands that contain the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end. This stem-loop renders a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide that anneals to the known sequence immediately adjacent to the phosphorylated oligonucleotide high-stringency annealing site. The oligonucleotide appended to the phosphorylated oligonucleotide by the heat-stable ligase can, when present in its free, non-ligated form, prime DNA polymerase-mediated amplification of those strands modified by site-specific ligation to this same oligonucleotide. This is followed by one or two nested DNA amplifications, with the final amplification primed by the phosphorylated oligonucleotide in its free, non-ligated form. We successfully applied this method to the specific amplification of 2.2 kb of DNA flanking the 5' end of the cystic fibrosis transmembrane conductance regulator cDNA using primers that anneal to the cDNA sequence and to the specific amplification of 2.2 kb of human genomic beta-globin DNA flanking the primer annealing sites.
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PMID:A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification. 750 1

The results obtained using deoxyribonucleic acid (DNA) amplification-based tests must be accurate and reproducible. One such test that simultaneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in this report. An investigation was conducted into how changes of primer, DNA template and Taq DNA polymerase concentrations and of polymerase chain reaction annealing temperatures affect the test. A total of 383 DNA samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate results are obtained even if the test is performed under suboptimal conditions. Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remaining 1.6% of samples were not mistyped, rather they were not scored owing to failure to detect control DNA sequences. These were also archival DNA preparations rather than freshly prepared samples from venous blood. Careful primer design and optimization of reaction conditions are important in the development of multiplex deoxyribonucleic acid amplification-based diagnostic tests. Providing the recommended protocols are followed, the test described here is simple to carry out, gives accurate results and works well if performed within defined operational windows for each reaction variable.
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PMID:Development and validation of a screening test for 12 common mutations of the cystic fibrosis CFTR gene. 972 5

An approach is described for in situ polymerase chain reaction (ISPCR) based on cycling primed in situ synthesis (PRINS) conditions defined for alpha-satellite DNA. Using blood cell preparations subjected to limited fixation with paraformaldehyde, ISPCR cycling resulted in a gradual buildup of amplicon at the site of synthesis, as judged by the characteristic presence of paired nuclear spots corresponding to specific centromeres. Using longer cycling regimens, primers for single copy genes also generated paired nuclear spots in a primer-pair--specific manner. In this context, the amplification refractory mutation system (ARMS) was evaluated for in situ applications. In ARMS, allele-specific primers are used in such a manner that PCR proceeds only when an exact 3' match between annealed primer and template is recognized by DNA polymerase. Using normal and mutant primers for the delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a model system, it was not possible to reliably differentiate between ARMS reactions by accumulation of direct labeled reaction product in cells, because of ARMS-independent nonspecific labeling. However, by DNA extraction and reamplification with ARMS primers, it was shown that amplicon accumulates in cells in the expected primer/template-dependent manner crucial to mutation detection by ARMS. It was also shown that nonspecific signal is due to primer dimer formation, especially in the absence of true template DNA. The impact of primer dimer formation in generating a false-positive signal is discussed. The method described here enables a cell population to be analyzed for a given point mutation.
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PMID:Adapting in situ polymerase chain reaction for genotyping of cells in suspension. 999 Apr 81

Recombinant adeno-associated serotype 2-based vectors (rAAV2) possess a number of theoretical advantages for cystic fibrosis (CF) gene therapy because they elicit little or no inflammatory response and generally result in stable expression. rAAV2 vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have previously been shown to mediate stable correction of the CF defect in CF bronchial epithelial cells and stable expression of CFTR in rabbit and nonhuman primate models. Here we report the results of the first trial initiated with rAAV in humans, a phase I study in 25 adult and adolescent CF patients with mild to moderate lung disease. Doses of the rAAV-CFTR vector (tgAAVCF) ranging from 3 x 10(1) to 1 x 10(9) replication units (RU), which is equivalent to approximately 6 x 10(4) to 2 x 10(12) DNase resistant particles (DRP), were administered to one side of the nose and to the superior segment of the lower lobe of the right lung. Several adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of the bronchoscopic procedures. Only one of the serious events was judged to be possibly vector-related (based on temporal association), and this event was a pulmonary exacerbation very similar to several others experienced by the same subject in the three months preceding vector delivery. Vector shedding was minimal throughout the study, and serum-neutralizing antibodies were detected after vector delivery to subjects in the highest dosage cohorts. Gene transfer as measured by DNA polymerase chain reaction (PCR) was not observed until cohort 10 in nasal and bronchial epithelia. Sporadic low-level copy numbers suggested gene transfer of anywhere from 0.002 copies per cell up to 0.5 copies per cell was possible; however, DNA PCR was positive in lungs prior to direct dosing suggesting aspiration from the nasal dosing. These data indicate the need for continued evaluation of rAAV-CFTR vectors in additional clinical trials.
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PMID:Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study. 1288 47