scholarly journals Streamlined method for purifying single-stranded DNA from PCR products for frequent or high-throughput needs

BioTechniques ◽  
2005 ◽  
Vol 38 (5) ◽  
pp. 700-702 ◽  
Author(s):  
Tai-Chih Kuo
Keyword(s):  
High Throughput ◽  
Single Stranded Dna ◽  
Pcr Products

scholarly journals High throughput functional variant screens via in-vivo production of single-stranded DNA

2020 ◽  
Author(s):  
Max G. Schubert ◽  
Daniel B. Goodman ◽  
Timothy M. Wannier ◽  
Divjot Kaur ◽  
Fahim Farzadfard ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
High Throughput ◽  
Entire Genome ◽  
Single Stranded Dna ◽  
Functional Variant ◽  
Continuous Generation ◽  
Sources Of Variation ◽  
Targeted Deep Sequencing ◽  
High Throughput Screens

AbstractTremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

scholarly journals High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

Viruses ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 684 ◽  
Author(s):  
Toufic Elbeaino ◽  
Armelle Marais ◽  
Chantal Faure ◽  
Elisa Trioano ◽  
Thierry Candresse ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Viral Infections ◽  
Small Scale ◽  
Rt Pcr ◽  
Cyclamen Persicum ◽  
Sequence Variations ◽  
Pcr Products ◽  
Complete Sequences ◽  
Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

scholarly journals High-Throughput Transposon Mutagenesis of Corynebacterium glutamicum and Construction of a Single-Gene Disruptant Mutant Library

2006 ◽  
Vol 72 (5) ◽  
pp. 3750-3755 ◽  
Author(s):  
Nobuaki Suzuki ◽  
Naoko Okai ◽  
Hiroshi Nonaka ◽  
Yota Tsuge ◽  
Masayuki Inui ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
High Throughput ◽  
Single Gene ◽  
Dna Amplification ◽  
Open Reading Frames ◽  
Thermal Asymmetric Interlaced Pcr ◽  
Transposon Insertion ◽  
Cellular Life ◽  
Pcr Products ◽  
Tail Pcr

ABSTRACT A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

scholarly journals A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein–Protein Interactions in an Academic Screening Facility

2017 ◽  
Vol 23 (1) ◽  
pp. 94-101 ◽  
Author(s):  
Andrew F. Voter ◽  
Michael P. Killoran ◽  
Gene E. Ananiev ◽  
Scott A. Wildman ◽  
F. Michael Hoffmann ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Small Molecule Inhibitors ◽  
Klebsiella Pneumonia ◽  
Protein Protein Interactions ◽  
Response Curves ◽  
Single Stranded Dna

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

Sign in / Sign up

Export Citation Format

Share Document