scholarly journals Enzymatic synthesis of gene-length single-stranded DNA

2017 ◽  
Author(s):  
Rémi Veneziano ◽  
Tyson R. Shepherd ◽  
Sakul Ratanalert ◽  
Leila Bellou ◽  
Chaoqun Tao ◽  
...  
Keyword(s):  
Enzymatic Synthesis ◽  
Nucleotide Composition ◽  
Maximum Length ◽  
Gene Length ◽  
Modified Nucleotides ◽  
Asymmetric Pcr ◽  
Single Stranded Dna ◽  
Well Control ◽  
Homology Directed Repair

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxic ity, yet ssDNA synthesis strategies are limited by the maximum length attainable, as well control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kb using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and imaging applications.

scholarly journals Phenothiazine-linked nucleosides and nucleotides for redox labelling of DNA

2017 ◽  
Vol 15 (33) ◽  
pp. 6984-6996 ◽  
Author(s):  
Anna Simonova ◽  
Luděk Havran ◽  
Radek Pohl ◽  
Miroslav Fojta ◽  
Michal Hocek
Keyword(s):  
Electrochemical Detection ◽  
Enzymatic Synthesis ◽  
Dna Bases ◽  
Modified Nucleotides

New phenothiazine-modified nucleotides were prepared and used for enzymatic synthesis of labelled DNA for electrochemical detection and redox coding of DNA bases.

scholarly journals Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration

2021 ◽  
Author(s):  
Chen-Tran Hsu ◽  
Yu-Hsuan Yuan ◽  
Yao-Cheng Lin ◽  
Steven Lin ◽  
Qiao-Wei Cheng ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Nonhomologous End Joining ◽  
Protoplast Regeneration ◽  
End Joining ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Dna Oligonucleotides ◽  
Vector Construction ◽  
Regenerated Plants ◽  
Low Efficiency

AbstractGenome editing requires insertion of DNA sequences into specific locations. Protocols involving clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins rely on homology-directed repair, require laborious vector construction, and have low efficiency. DNA oligonucleotides can be used as donors for targeted insertion via nonhomologous end joining. Our simple protocol eliminates the need for expensive equipment and vector construction by using polyethylene glycol to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. We achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated plants showed targeted insertions, and 1 contained a precise insertion of a 6-bp EcoRI site. Whole-genome sequencing showed that the DNA inserted only in the targeted positions, and genetic analysis showed that the inserted sequences transmitted to the next generation.

Enzymatic Synthesis of Nucleobase-Modified Single-Stranded DNA Offers Tunable Resistance to Nuclease Degradation

2018 ◽  
Vol 19 (8) ◽  
pp. 3525-3535 ◽  
Author(s):  
Renpeng Gu ◽  
Thomas Oweida ◽  
Yaroslava G. Yingling ◽  
Ashutosh Chilkoti ◽  
Stefan Zauscher
Keyword(s):  
Enzymatic Synthesis ◽  
Single Stranded Dna ◽  
Nuclease Degradation

The staining efficiency of cyanine dyes for single‐stranded DNA is enormously dependent on nucleotide composition

2019 ◽  
Vol 40 (12-13) ◽  
pp. 1708-1714 ◽  
Author(s):  
Xutiange Han ◽  
Erchi Wang ◽  
Yixiao Cui ◽  
Yikai Lin ◽  
Hui Chen ◽  
...  
Keyword(s):  
Cyanine Dyes ◽  
Nucleotide Composition ◽  
Single Stranded Dna

scholarly journals Rapid in vitro production of single-stranded DNA

2019 ◽  
Author(s):  
Dionis Minev ◽  
Richard Guerra ◽  
Jocelyn Y Kishi ◽  
Cory Smith ◽  
Elisha Krieg ◽  
...  
Keyword(s):  
High Purity ◽  
In Vitro Production ◽  
Single Stranded Dna ◽  
Selective Precipitation ◽  
Homology Directed Repair ◽  
Increasing Demand ◽  
User Friendly ◽  
Vitro Production

Abstract There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40–50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 μl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

scholarly journals High-efficiency retron-mediated single-stranded DNA production in plants

2021 ◽  
Author(s):  
Wenjun Jiang ◽  
Gundra Sivakrishna Rao ◽  
Rashid Aman ◽  
Haroon Butt ◽  
Radwa Kamel ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genome Engineering ◽  
High Efficiency ◽  
Plant Biotechnology ◽  
Fold Increase ◽  
Gene Encoding ◽  
Single Stranded Dna ◽  
Homology Directed Repair ◽  
Potential Applications

ABSTRACT Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.

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