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Search Results: 1 - 10 of 9796 matches for " BDT cycle sequencing kit v3.1 "
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Optimization of the Sample Preparation Method for DNA Sequencing
Gazi N.N. Sultana,Amir H. Khan
Journal of Biological Sciences , 2007,
Abstract: This study reports on the modification and optimization of the sample preparation method for analyzing nucleotide sequence in plant species by capillary electrophoresis. The experimental study was aimed at developing possible ways of increasing the throughput of the method. The optimization conditions modified were purification of the PCR product, concentration adjustment of cycle sequencing kit BDT v3.1 from half to quarter reactions for economic analysis. Concentrations of the tamplet, primer and program of thermocycler were also optimized. Finally the volume of Hi-Di form amide was adjusted for the required sequencing. The accuracy of analysis was greater than 99.99%. The modified method enables quality sequencing results with higher average signal intensities compared to the standard recommended method. With the optimized electrophoresis conditions reported here, greater than 700 base pairs can be analyzed within 75 min using POP7. It was found that deviation from the recommended method consistently delivers higher signal-to-noise ratios and signal intensities and longer Phred20 read lengths. It is more reproducible than that of other methods and enables rapid, more cost effective robustness for nucleotide sequencing in the samples of plant origin received from research laboratory sources.
Computer-assisted anti-AIDS drug development: cyclophilin B against the HIV-1 subtype A V3 loop  [PDF]
Alexander M. Andrianov, Ivan V. Anishchenko
Health (Health) , 2010, DOI: 10.4236/health.2010.27100
Abstract: Aim: The objects of this study originated from the experimental observations, whereby the HIV -1 gp120 V3 loop is a high-affinity ligand for immunophilins, and consisted in generating the structural complex of cyclophilin (Cyc) B belonging to immunophilins family with the virus subtype A V3 loop (SA-V3 loop) as well as in specifying the Cyc B segment forming the binding site for V3 synthetic copy of which, on the assumption of keeping the 3D peptide structure in the free state, may present a forwardlooking basic structure for anti-AIDS drug development. Methods: To reach the objects of view, molecular docking of the HIV-1 SA-V3 loop structure determined previously with the X-ray conformation of Cyc B was put into practice by Hex 4.5 program (http://www.loria.fr/~ritchied/ hex/) and the immunophilin stretch responsible for binding to V3 (Cyc B peptide) was identified followed by examination of its 3D structure and dynamic behavior in the unbound status. To design the Cyc B peptide, the X-ray conformation for the identical site of the native protein was involved in the calculations as a starting model to find its best energy structural variant. The search for this most preferable structure was carried out by consecutive use of the molecular mechanics and simulated annealing methods. The molecular dynamics computations were implemented for the Cyc B peptide by the GROMACS computer package (http:// www.gromacs.org/). Results: The overmolecular structure of Cyc B with V3 was built by computer modeling tools and the immunophilinderived peptide able to mask effectively the structurally invariant V3 segments embracing the functionally crucial amino acids of the HIV-1 gp120 envelope protein was constructed and analyzed. Conclusions: Starting from the joint analysis of the results derived with those of the literature, the generated peptide was suggested to offer a promising basic structure for making a reality of the protein engineering projects aimed at developing the anti-AIDS drugs able to stop the HIV’s spread.
V3 peptide binding pattern and HIV-1 transmission route in Rio de Janeiro
Pinto, Monica E.;Schechter, Mauro;
Memórias do Instituto Oswaldo Cruz , 1995, DOI: 10.1590/S0074-02761995000600004
Abstract: to characterize antibody binding to a panel of v3 loop peptides representing diverse hiv-1 neutralization epitopes, 149 hiv-1 infected individuals from rio de janeiro (rj) were investigated. results were analyzed with respect to risk factors for infection and other epidemiological and clinical data. peptide reactivity was not associated with sex, clinical status, cd4 counts, antigenemia or ?2-microglobulin serum level. a segregation of peptide reactivity according to route of infection was encountered. this finding suggests that more then one viral strain may be circulating in rj, in subjects with different risk factors for hiv-1 infection. an investigation of prevalent hiv-1 genotypes, serotypes and immunotypes may be of importance for the design and selection of potential vaccines to be used in brazil as well as for the selection of populations to be included in future vaccine efficacy trials.
Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity
Casseb, J.;Katzenstein, D.;Winters, M.;Brigido, L.F.M.;Duarte, A.J.S.;Hendry, R.M.;
Brazilian Journal of Medical and Biological Research , 2002, DOI: 10.1590/S0100-879X2002000300013
Abstract: the main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the v3 region of hiv-1 gp120, which is the main target for neutralizing antibodies. results from an enzyme immunoassay (eia) employing a panel of synthetic peptides of hiv-1 subtypes and using urea washes to detect high avidity antibodies (aav3) were compared with those obtained by the heteroduplex mobility assay and dna sequencing. the eia correctly typed 100% of subtype b (sensitivity = 1.0; specificity = 0.95), 100% of hiv-1 e samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype c specimens (sensitivity = 0.95; specificity = 0.94). in contrast, only 50% of subtype a (sensitivity = 0.5; specificity = 0.95), 60% of subtype d (sensitivity = 0.6; specificity = 1.0), and 28% of subtype f samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. this approach was also able to discriminate in a few samples antibodies from patients infected with b variants circulating in brazil and thailand that reacted specifically. the assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. moreover, the classification in subtypes (genotypes) may overestimate hiv-1 diversity and a classification into serotypes, based on antigenic v3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.
Relexification in a Northern Norwegian dialect?
Hilde Sollid
Nordlyd : Troms? University Working Papers on Language & Linguistics / Institutt for Spr?k og Litteratur, Universitetet i Troms? , 2003,
Abstract: This paper explores how the process of relexification can contribute to the understanding of the genesis of the new Norwegian dialect of Sappen in Nordreisa. The dialect has emerged in the context of language shift from Finnish to Norwegian, and the dialect syntax has features that might be regarded as products of relexification. One example is declarative main clauses with the finite verb in the third position (V3). The discussion adheres to a more general discussion of approaches to language genesis, where substratist and universalist (and also superstratist) theories often are regarded as contrary to each other. I argue that different theories can contribute to the understanding of different aspects of the same question.
A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C
Kumar Rajesh,Andrabi Raiees,Tiwari Ashutosh,Prakash Somi Sankaran
BMC Biotechnology , 2012, DOI: 10.1186/1472-6750-12-87
Abstract: Background Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope. Results An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner. Conclusions This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.
Serotyping HIV-1 with V3 peptides: detection of high avidity antibodies presenting clade-specific reactivity
Casseb J.,Katzenstein D.,Winters M.,Brigido L.F.M.
Brazilian Journal of Medical and Biological Research , 2002,
Abstract: The main objective of the present study was to assess the specificity and sensitivity of a modified assay using short synthetic peptides of the V3 region of HIV-1 gp120, which is the main target for neutralizing antibodies. Results from an enzyme immunoassay (EIA) employing a panel of synthetic peptides of HIV-1 subtypes and using urea washes to detect high avidity antibodies (AAV3) were compared with those obtained by the heteroduplex mobility assay and DNA sequencing. The EIA correctly typed 100% of subtype B (sensitivity = 1.0; specificity = 0.95), 100% of HIV-1 E samples (sensitivity = 1.0; specificity = 1.0), and 95% of subtype C specimens (sensitivity = 0.95; specificity = 0.94). In contrast, only 50% of subtype A (sensitivity = 0.5; specificity = 0.95), 60% of subtype D (sensitivity = 0.6; specificity = 1.0), and 28% of subtype F samples (sensitivity = 0.28; specificity = 0.95) were correctly identified. This approach was also able to discriminate in a few samples antibodies from patients infected with B variants circulating in Brazil and Thailand that reacted specifically. The assays described in this study are relatively rapid and simple to perform compared to molecular approaches and can be used to screen large numbers of serum or plasma samples. Moreover, the classification in subtypes (genotypes) may overestimate HIV-1 diversity and a classification into serotypes, based on antigenic V3 diversity or another principal neutralization domain, may be more helpful for vaccine development and identification of variants.
PCR-DGGE Analysis of the Bacterial Community of Chinese Liquor High and Medium Temperature Daqu
利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

GAO Yi-Bao,WANG Hai-Yan,XU Yan,
高亦豹
,王海燕,徐岩

微生物学通报 , 2010,
Abstract: The bacterial community structure of 5 high and medium temperature Chinese liquor Daqu were investigated using PCR-DGGE (Polymerase chain reaction-denaturing gradient gel electrophoresis) as a culture-independent method. DNA sequencing was proceeded to obtain the dominant bacterial population information. The result of DGGE profile showed that Weissella cibaria, Lactobacillus helveticus, L. fermentum and L. panis were commonly detected in all the five Daqu. Thermoactinomyces sanguinis was detected only in the Jiangqu sample. Compared with culture-dependent method, DGGE was able to detect Staphylococcus xylosus and Klebsiella oxytoca. The correlation between the craftwork of Daqu and the bacterial community structure was obvious. The Daqu bacterial Shannon-Wiener index decreased as the craftwork temperature of Daqu increasing. PCR-DGGE was proved to be a powerful tool for gaining detailed insight into the bacterial diversity of the Chinese liquor Daqu.
Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression
Bánky Balázs,Rásó-Barnett Lívia,Barbai Tamás,Tímár József
Molecular Cancer , 2012, DOI: 10.1186/1476-4598-11-83
Abstract: Background CD44 is considered as ‘a’ metastasis associated gene, despite the fact that it is an umbrella term for a group of molecules produced from a single gene by alternative splicing. However, little consideration is given to the above in the literature of colorectal carcinomas as well as other tumour types, leading to confusion and contradictory results about its possible role in tumour progression. Methods We compared the CD44 alternative splice pattern (ASP) of three genetically different human colorectal cancer cell lines (HT25, HT29, HCT116) using a series of PCR reactions and next- generation sequencing method, as well as identified a colorectal adenocarcinoma specific CD44 ASP. This ASP was further investigated in terms of its qualitative and quantitative stability in our experimental iso- and xenograft mouse models for colorectal cancer progression. A complex preclinical experimental set-up was established to separately test the different steps of tumour progression and the role of tumour microenvironment, respectively, focusing on the role of ‘CD44’ in this process. Results We managed to present a colorectal cancer-specific CD44 ASP, which remained unchanged from cell lines throughout primary tumour formation and metastatic progression. Furthermore, we report a unique roster of all expressed CD44 variant isoforms characteristic to colorectal cancer. Finally, on quantitative assessment of the variable exons v3 and v6, higher co-expression levels were found to be characteristic to metastatically potent tumour cells. Conclusion Particular CD44 variant isoforms seem to act as “metastasis genes” via tumour microenvironment-driven shifts in v3 and v6 expressions. However, this function may just affect a minority of tumour subclones. This fact and the huge potential number of different CD44 splice variants that can contain v3 and v6 domains can explain incoherence of clinical studies regarding functional asessment of CD44 variants, as well as diminish the chances of using CD44 variants for predictive purpose.
Two variants of HIV-1 B serotype are transmitted heterosexually in S?o Paulo, Brazil
Casseb, J.;Hong, M.A.;Gonsalez, C.;Brígido, L.F.;Duarte, A.J.S.;Michael-Hendry, R.;
Brazilian Journal of Medical and Biological Research , 1998, DOI: 10.1590/S0100-879X1998001000002
Abstract: hiv-1 variability may have an important impact on transmission and pathogenicity. better characterization of the hiv epidemic in brazil is necessary for the development of vaccine trials in this country. we analyzed sera from 108 hiv-1-infected volunteers from s?o paulo city to determine serotype and reactivity for v3 motifs of hiv in this population, and the relationship to transmission mode. we concluded that the hiv-1 b serotype is frequent among heterosexually infected women, even in the absence of anal sex, and that two major v3 motifs, gpgr and gwgr, had similar prevalence among women (48% and 52%, respectively) and men (56% and 44%, respectively). we also observed an equal distribution of these strains regardless of their cd4+ t cell counts, clinical status, and mode of transmission. even though v3 serology for hiv-1 subtyping is an inexpensive tool for use in developing countries, additional methods, such as heteroduplex mobility assay and direct dna sequencing, should be included to determine hiv-1 genetic diversity.
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