scholarly journals Mycobacterium smegmatis porin A (MspA) nanopore–based DNA sequencing using phage DNA polymerase and blocking oligomers

2012 ◽  
Vol 5 (14) ◽  
pp. 375-375
Keyword(s):  
Dna Sequencing ◽  
Dna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Phage Dna

scholarly journals Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers

2017 ◽  
Vol 114 (12) ◽  
pp. E2310-E2318 ◽  
Author(s):  
Bin Zhu ◽  
Longfei Wang ◽  
Hitoshi Mitsunobu ◽  
Xueling Lu ◽  
Alfredo J. Hernandez ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deep Sea ◽  
Phage Dna

scholarly journals UV- and MMS-induced mutagenesis of lambdaO(am)8 phage under nonpermissive conditions for phage DNA replication.

2003 ◽  
Vol 50 (4) ◽  
pp. 921-939 ◽  
Author(s):  
Joanna Krwawicz ◽  
Anna Czajkowska ◽  
Magdalena Felczak ◽  
Irena Pietrzykowska
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Protein S ◽  
Dna Lesions ◽  
E Coli ◽  
Phage Dna ◽  
Induced Mutagenesis ◽  
C Proteins ◽  
Inducible Protein

Mutagenesis in Escherichia coli, a subject of many years of study is considered to be related to DNA replication. DNA lesions nonrepaired by the error-free nucleotide excision repair (NER), base excision repair (BER) and recombination repair (RR), stop replication at the fork. Reinitiation needs translesion synthesis (TLS) by DNA polymerase V (UmuC), which in the presence of accessory proteins, UmuD', RecA and ssDNA-binding protein (SSB), has an ability to bypass the lesion with high mutagenicity. This enables reinitiation and extension of DNA replication by DNA polymerase III (Pol III). We studied UV- and MMS-induced mutagenesis of lambdaO(am)8 phage in E. coli 594 sup+ host, unable to replicate the phage DNA, as a possible model for mutagenesis induced in nondividing cells (e.g. somatic cells). We show that in E. coli 594 sup+ cells UV- and MMS-induced mutagenesis of lambdaO(am)8 phage may occur. This mutagenic process requires both the UmuD' and C proteins, albeit a high level of UmuD' and low level of UmuC seem to be necessary and sufficient. We compared UV-induced mutagenesis of lambdaO(am)8 in nonpermissive (594 sup+) and permissive (C600 supE) conditions for phage DNA replication. It appeared that while the mutagenesis of lambdaO(am)8 in 594 sup+ requires the UmuD' and C proteins, which can not be replaced by other SOS-inducible protein(s), in C600 supE their functions may be replaced by other inducible protein(s), possibly DNA polymerase IV (DinB). Mutations induced under nonpermissive conditions for phage DNA replication are resistant to mismatch repair (MMR), while among those induced under permissive conditions, only about 40% are resistant.

scholarly journals Cycle Sequencing of Filamentous Phage DNA Using a Biotinylated Primer and ΔTaq DNA Polymerase

BioTechniques ◽  
1997 ◽  
Vol 23 (1) ◽  
pp. 125-127 ◽  
Author(s):  
Jianping Qiu ◽  
Huanying Zhou ◽  
Joseph F. Aceto ◽  
Thomas Kieber-Emmons
Keyword(s):  
Dna Polymerase ◽  
Filamentous Phage ◽  
Cycle Sequencing ◽  
Phage Dna

Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase

DNA Sequence ◽  
1993 ◽  
Vol 4 (2) ◽  
pp. 79-85 ◽  
Author(s):  
James J. Earley ◽  
Helena Kuivaniemi ◽  
Darwin J. Prockop ◽  
Gerard Tromp
Keyword(s):  
Dna Sequencing ◽  
Dna Polymerase ◽  
Microtiter Plates

Targeted Ultra-Deep High Accuracy Sequencing of Pre-Treatment AML Reveals a Diversity of Mutational Phenotypes and Evidence of Preexisting Relapse-Associated Subclones

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2372-2372
Author(s):  
Marc J. Prindle ◽  
Lawrence A. Loeb ◽  
Elihu H. Estey ◽  
Pamela S. Becker
Keyword(s):  
Drug Resistance ◽  
Dna Sequencing ◽  
Dna Polymerase ◽  
Conflicts Of Interest ◽  
Dna Polymerases ◽  
Next Generation ◽  
Clinical Recurrence ◽  
Next Generation Dna Sequencing ◽  
Synonymous Mutations ◽  
Treatment Naïve

Abstract Human tumors contain large numbers of clonal, subclonal and random mutations. Clonal mutations, present in 30% or more of the cells, are classified as either drivers that promote proliferation or passengers of unknown or to be determined function. Subclonal and random mutations that are present in a small subset of tumor cells, prior to chemotherapy, could serve as a reservoir for the emergence of drug resistance. Even in the presence of clinical complete remissions in AML, subclonal mutations could be present in a small number of cells that selectively expand during chemotherapy and result in relapse and death. Next-generation DNA sequencing now makes it feasible to monitor the frequency of different subclonal mutations in patients as tumors evolve over time, raising the possibility of personalizing treatment by anticipating the mutations that signal relapse in time to prevent clinical recurrence of AML. We have used Duplex DNA Sequencing (DS) to detect very low-frequency subclonal and random mutations in AML during relapse and prior to treatment. While whole genome sequencing (WGS) provides extensive data on the clonal distribution of mutations in AML, it lacks sufficient accuracy to identify subclonal mutations when they are present at frequencies less than 5%. In contrast, DS focuses on a limited number of target genes at the level of single DNA molecules. Mutations are scored only if they are present at the same position in both strands of the same DNA molecule and are complementary, resulting in sequencing accuracy that is more than 1000-fold greater than that of routine next-generation DNA sequencing. Using DNA from 12 treatment-naïve AML samples and 2 normal bone marrow samples, we first targeted the exons that encode the catalytic domains of the five major human replicative polymerases. We detected both synonymous and non-synonymous mutations in most of the targeted genes. Mutations in the two major human replicative DNA polymerases have been recently identified in adenocarcinomas of the colon, and mutations in the proof-reading domain of DNA polymerase epsilon result in the highest reported point mutation frequencies so far reported in any cancer. Until now, mutations in DNA polymerases have not been described in AML. Presumably a similar or higher subclonal mutation load exists in the coding regions of other genes in AML and in genes found in other tumors. In order to follow the course of mutation accumulation in AML after treatment and leading to relapse, we used capture hybridization that was designed to enrich for 15 genes previously reported to be mutated in AML. We identified multiple subclonal and random mutations in many of these genes. In one relapse sample, we identified a mutation in NRAS that was present in 32% of the cells. The same mutation was detected by DS in 1% of the cells from the same patient prior to treatment, which is well below the signal threshold of WGS. These initial studies demonstrate the feasibility of using DS to define the changes that occur during and after treatment, and suggest the use of DS to determine mutations that impart drug resistance. The findings from the DNA polymerase capture offers evidence that abundant non-synonymous mutations are present in treatment-naïve AML, implying that the seeds of treatment resistance may already have taken root by the time of diagnosis. Disclosures No relevant conflicts of interest to declare.

scholarly journals INFECTION OF MYCOBACTERIUM SMEGMATIS WITH D29 PHAGE DNA

1964 ◽  
Vol 119 (1) ◽  
pp. 139-149 ◽  
Author(s):  
Tohru Tokunaga ◽  
Margret I. Sellers
Keyword(s):  
Calcium Ions ◽  
Mycobacterium Smegmatis ◽  
Buoyant Density ◽  
Gc Content ◽  
Enzyme Hydrolysis ◽  
Productive Infection ◽  
Host Bacterium ◽  
Calf Thymus ◽  
Phage Dna ◽  
Dissymmetry Ratio

DNA extracted from D29 mycobacteriophage produced plaques when plated on Mycobacterium smegmatis 607. The host bacterium did not require alternation such as conversion to protoplasts; cells susceptible to infection with intact phage were susceptible to DNA. The bases found in calf thymus DNA constituted the bases of D29 DNA, adenine being paired with thymine and guanine with cytosine. The dissymmetry ratio (A + T/G + C) was 0.56, and the buoyant density in CsCl was 1.722 with a GC content of 63.77 per cent. The efficiency of plating of the DNA is very much lower than that of intact D29, and it penetrates the host at a slower rate. As does intact phage, D29 DNA requires calcium ions for productive infection of 607. D29 DNA is significantly inactivated by incubation with RNAase, but the inactivation probably results from a complexing with the DNA rather than from enzyme hydrolysis.

scholarly journals Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

2016 ◽  
Vol 113 (19) ◽  
pp. 5233-5238 ◽  
Author(s):  
Carl W. Fuller ◽  
Shiv Kumar ◽  
Mintu Porel ◽  
Minchen Chien ◽  
Arek Bibillo ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Real Time ◽  
Lipid Bilayers ◽  
Dna Polymerase ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Electrical Current ◽  
Sequencing Data ◽  
Sequencing By Synthesis ◽  
Dna Strand

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

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