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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kinetoplast DNA (kDNA), the mitochondrial DNA in kinetoplastids, is a network containing several thousand topologically interlocked minicircles. We investigated cell cycle-dependent changes in the localization of kDNA replication enzymes by combining immunofluorescence with either hydroxyurea synchronization or incorporation of fluorescein-dUTP into the endogenous gaps of newly replicated minicircles. We found that while both topoisomerase II and DNA polymerase beta colocalize in two antipodal sites flanking the kDNA during replication, they behave differently at other times. Polymerase beta is not detected by immunofluorescence either during cell division or G1, but is abruptly detected in the antipodal sites at the onset of kDNA replication. In contrast, topoisomerase II is localized to sites at the network edge at all cell cycle stages; usually it is found in two antipodal sites, but during cytokinesis each postscission daughter network is associated with only a single site. During the subsequent G1, topoisomerase accumulates in a second localization site, forming the characteristic antipodal pattern. These data suggest that these sites at the network periphery are permanent components of the mitochondrial architecture that function in kDNA replication.
...
PMID:Changes in organization of Crithidia fasciculata kinetoplast DNA replication proteins during the cell cycle. 981 50

A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a rare mutant allele based solely upon its altered nucleotide sequence. ACB-PCR genotypic selection operates through the preferential PCR amplification of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele. In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutant allele than the wild-type allele is incorporated to reduce the background and increase sensitivity. Using ACB-PCR, the CAA-->AAA base substitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10(-5). To accurately quantify the occurrence of this particular mutation, an internal amplification standard (AS) DNA was constructed. The H-ras and AS DNAs were subject to the same genotypic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of various components of the ACB-PCR. The concentration of dNTPs, blocker primer and Perfect Match Polymerase Enhancer, as well as the choice of thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAA-->AAA mutation in the H-ras and AS DNAs. Using the identified conditions, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 10 to 1 in 10(5)). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.
...
PMID:Detection of a mouse H-ras codon 61 mutation using a modified allele-specific competitive blocker PCR genotypic selection method. 986 88

We report here a novel DQB1 allele (DQB1*0616) identified during sequence-based HLA typing. Polymerase chain reaction with proofreading Pwo DNA polymerase and pfu DNA polymerase and subsequent sequencing yielded identical results as that with Taq DNA polymerase. Molecular cloning and sequencing confirmed that the new DQB1 allele is identical to DQB1*0602 at exon 2 except for a single nucleotide substitution (TAC-->AAC), changing codon 60 from Tyr to Asn. This is the first report of polymorphism of DQB1 alleles at codon 60.
...
PMID:Identification of a novel DQB1 allele DBQ1*0616. 1032 44

Polymerase chain reaction (PCR) has been used with increasing frequency to diagnose infectious and genetic diseases. In this study, the effects of heparin on PCR were investigated, because heparinized blood may sometimes be used in PCR studies. HLA-DQA1 gene amplification was used as a model. PCR was clearly interfered with when heparinized blood was used as a source of template DNA, and the degree of interference was affected by the following three factors; (1) type of Taq DNA polymerase; (2) leukocyte count in blood; and (3) concentration of heparin contained. When additional tests were conducted with additions of definite heparin concentrations to a PCR reaction mixture, specimens with large amounts of DNA tended to exhibit less interference by heparin. The addition of > or = 0.1 to 0.0016 U of heparin per reaction mixture (50 microl) suppressed DNA amplification in a dose-dependent fashion. We therefore concluded that much care should be taken when heparinized blood is used as a PCR material.
...
PMID:Effects of heparin on polymerase chain reaction for blood white cells. 1032 79

A new method is described to enrich genomic libraries for clones containing microsatellite repeats. The method involves selection on completed M13 genomic libraries rather than on genomic DNA before library construction. It uses two reactions, in which microsatellite oligonucleotides prime strand extension. The first reaction uses a biotinylated primer allowing vectors with microsatellite-containing inserts to be selected with streptavidin-coated magnetic beads. This reaction may be dependent on the strand displacement activity of the Klenow fragment of DNA Polymerase I. The second strand extension reaction is included to improve the relative transformation efficiency of microsatellite-containing clones. In control experiments starting with 0.7% microsatellite-containing clones, enrichment averaged 99.5%. The method was tested empirically on antechinus and abalone genomic libraries in which enrichment for (CA)n microsatellites was efficient enough that clones could be sequenced without further screening. This protocol is technically straightforward and permits the isolation of a large number of microsatellite markers in less time than is required to execute traditional protocols involving rounds of filter hybridization.
...
PMID:Microsatellites obtained using strand extension: an enrichment protocol. 1034 7

We report here a novel DQA1 allele (DQA1*0106) identified during sequence-based HLA-DQA1 typing. Polymerase chain reaction with proofreading pfu DNA polymerase and subsequent sequencing yielded identical results as that with Taq DNA polymerase. Molecular cloning and sequencing confirmed that the new DQA1 allele is identical to DQA1*01021/2 at exon 2 except for a single nucleotide substitution (ACT-->GCT), changing codon 44 from Thr to Ala. This is the first report of polymorphism at codon 44 of HLA-DQA1 alleles.
...
PMID:Identification of a novel HLA-DQA1 allele (DQA1*0106) by sequence-based DQA1 typing. 1039 13

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex. Moreover, slippage (at the proximal or 3'-end) is subject to positive and negative effects from the 5'-end (distal) of the same strand. Growing T and G strands generate T.A:T and G-G:C motif fold-back structures at the distal end that hamper slippage at the proximal end. On the other hand, growing tails at the distal end upon annealing with excess complementary template accentuates proximal slippage several-fold.
...
PMID:Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions. 1048 Oct 24

The peripheral blood cells from a patient with a B-cell lymphoma were established in long-term tissue culture. Two years after establishment of the cells in culture they were infected with herpes simplex virus type 2 and the productivity and duration of viral persistence investigated. One week after infection the lymphoblastoid cells were productively infected and have remained so for a period of over 3 years. Expression of a viral glycoprotein antigen was evaluated by using a fluorescein-labeled monoclonal anti-herpes simplex virus type 2 antibody and revealed a spectrum of staining reactions grading from a lightly stippled to very intense pattern. Polymerase chain reaction analysis of the infected cells revealed the presence of the herpes simplex virus type 2 DNA polymerase gene in the infected cells that was absent from the uninfected lymphoblastoid cells. These results taken together with the long-term growth characteristics of both the infected and uninfected lymphoblastoid cells suggest that this cell line may be a good model system for studying viral infection, viral replication, viral latency, and clinical application for the isolation of human herpes virus.
...
PMID:Persistent infection of a lymphoma cell line by herpes simplex virus. 1050 3

The Polymerase Chain Reaction (PCR) has revolutionized the analysis of DNA from a variety of sources. With its sensitivity and ability to amplify degraded DNAs and small quantities of samples, coupled with fast turn-around-time, PCR is often the analytical method of choice for DNA profiling in forensic laboratories. RFLP methods, while requiring larger amounts of high molecular weight DNA and needing approximately 6-8 weeks of analytical time, still provide a higher power of discrimination per locus than that achieved using the loci currently available for PCR. The combination of both RFLP and PCR would be advantageous for some applications. A new technique, Long PCR, allows for the effective amplification of long DNA targets from approximately 0.5 kb to > 20 kb of genomic DNA. Currently, several Long PCR systems are commercially available. Using a Taq/Pyrococcus DNA polymerase enzyme system and DNA isolated from bloodstains, we have successfully amplified 1-20 ng of Chelex-extracted DNA, an amount commonly used in Amp-FLP technology. The robustness of Long PCR in comparison to RFLP was also examined through the use of partially degraded blood samples. Long PCR was then used to amplify both D2S44 and D5S110 RFLP loci. Although all D2 and D5 alleles were detected, the larger alleles were amplified at significantly lower levels than the smaller alleles.
...
PMID:Long PCR for VNTR analysis. 1058 57

The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore gene in the pathogenesis of HBV infection are unclear. Various methods have been used to detect nucleotide (nt) 1896 precore mutants. We established a new primer-extension assay to facilitate the detection of these mutants. This assay is based upon the fact that there is no adenine in the distal precore region of wild-type HBV. Polymerase chain reaction (PCR)-amplified template DNA was denatured and annealed to the [gamma-32P]-labelled primer. During primer extension in the presence of DNA polymerase and dCTP, dGTP, dTTP and ddATP, the reaction terminates if there is a nucleotide A. When mixtures of different ratios of wild-type and nt 1896 precore mutants were analysed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r2=0. 9669). When the primer-extension assay and direct sequencing were compared in hepatitis B e antigen (HBeAg)-positive and -negative chronic active hepatitis B patients, the primer-extension assay detected a greater number of nt 1896 precore mutants than direct sequencing and thus most HBV infections were found to be mixed infections. In conclusion, the primer-extension assay is a reliable and sensitive method for the detection of nt 1896 precore mutants.
...
PMID:A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA. 1060 45


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---