• Church, George M.; Porreca, Gregory J.; Shendure, Jay; Rosenbaum, Abraham Meir 2017-04-18 The present invention relates to methods for sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size, including synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region, immobilizing the concatemer to the specific region to make an immobilized concatemer, and sequencing the immobilized concatemer. • Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A 2012-01-01 Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled 'intractable' resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the 'non-contiguous finished' Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing. • Richard A Hurt Full Text Available Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled 'intractable' resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the 'non-contiguous finished' Desulfovibrio desulfuricans ND132 genome (6 intractable gaps and the Desulfovibrio africanus genome (1 intractable gap. The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. 'Electrician' is also used as the name of a role in stagecraft, where electricians are tasked primarily with hanging, focusing, and operating stage lighting. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing. • Kaufer, Benedikt B; Arndt, Sina; Trapp, Sascha; Osterrieder, Nikolaus; Jarosinski, Keith W 2011-10-01 Telomerase reverse transcriptase (TERT) and telomerase RNA (TR) represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR) on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV) as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5) by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1) that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2) that this strategy could be used to generate novel vaccine candidates against virus • Benedikt B Kaufer 2011-10-01 Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates • Nakamura, Sumiko; Tsushima, Ryosuke; Ohtsubo, Ken'ichi 2013-01-01 We developed a method for the preparation of template DNAs for polymerase chain reaction (PCR) from beer. We improved the method by (i) lyophilizing and pulverizing the beer to concentrate DNAs, (ii) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction by the use of heat-resistant amylase and proteinase K, (iii) separation of template DNA by purification using 70% EtOH extraction and isopropyl alcohol precipitation, and (iv) the use of magnetic beads to purify DNA. We developed suitable 7 STS ( sequence-tagged site) primers related to beer quality for PCR, and it proved possible to identify 16 dominant malting barley cultivars and 22 kinds of beers. To digitize the results of PCR, discriminative DNA bands were binarized as 0 (disappeared) or 1 (appeared) and subjected to multiple regression analysis. Estimation formulae for the quality of beer were developed using the above-mentioned independent variables based on the results of PCR against dependent variables related to the qualities of beer, including foam stability, bitterness, sourness and astringency. These equations showed multiple regression coefficients of 0.93, 0.82, 0.87, and 0.87 for calibration. • Uprety, Bibek; Westover, Tyler; Stoddard, Michael; Brinkerhoff, Kamron; Jensen, John; Davis, Robert C; Woolley, Adam T; Harb, John N 2017-01-24 An improved method for the metallization of DNA origami is examined in this work. DNA origami, a simple and robust method for creating a wide variety of nanostructured shapes and patterns, provides an enabling template for bottom-up fabrication of next-generation nanodevices. Selective metallization of these DNA templates is needed to make nanoelectronic devices. Here, we demonstrate a metallization process that uses gold nanorod seeds followed by anisotropic plating to provide improved morphology and greater control of the final metallized width of the structure. In our approach, gold nanorods are attached to an origami template to create a seed layer. Electroless gold deposition is then used to fill the gaps between seeds in order to create continuous, conductive nanowires. Importantly, growth during electroless deposition occurs preferentially in the length direction at a rate that is approximately 4 times the growth rate in the width direction, which enables fabrication of narrow, continuous wires. The electrical properties of 49 nanowires with widths ranging from 13 to 29 nm were characterized, and resistivity values as low as 8.9 × 10(-7) Ωm were measured. The anisotropic metallization process presented here represents important progress toward the creation of nanoelectronic devices by molecularly directed placement of functional components onto self-assembled biological templates. • Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S 2004-02-01 An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost. • Kanavarioti, Anastassia 2015-03-01 Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ss DNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. ‘Base calling’ becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ss DNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA. • Cho, S.B.; Hong, J.S.; Pak, J.H. [Korea University, Seoul (Korea); Kim, Y.M. [National Institute of Health, Seoul (Korea) 2002-02-01 One of the important roles of a DNA chip is the capability of detecting genetic diseases and mutations by analyzing DNA sequence. For a successful electrochemical genotyping, several aspects should be considered including the chemical treatment of electrode surface, DNA immobilization on electrode, hybridization, choice of an intercalator to be selectively bound to double standed DNA, and an equipment for detecting and analyzing the output singal. Au was used as the electrode material, 2-mercaptoethanol was used for linking DNA to Au electrode, and methylene blue was used as an indicator that can be bound to a double stranded DNA selectively. From the analysis of reductive current of this indicator that was bound to a double stranded DNA on an electrode, a normal double stranded DNA was able to be distinguished from a single stranded DNA in just a few seconds. Also, it was found that the peak reduction current of indicator is proportional to the concentration of target DNA to be hybridized with probe DNA. Therefore, it is possible to realize a simple and cheap DNA sensor using the electrochemical measurement for genotyping. 20 refs., 8 figs., 1 tab.
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