scholarly journals Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

2014 ◽  
Vol 12 (39) ◽  
pp. 7758-7773 ◽  
Author(s):  
Saswata Karmakar ◽  
Andreas S. Madsen ◽  
Dale C. Guenther ◽  
Bradley C. Gibbons ◽  
Patrick J. Hrdlicka
Keyword(s):  
Single Nucleotide ◽  
Double Stranded Dna ◽  
Mixed Sequence

Energetically activated double-stranded probes with interstrand arrangements of intercalator-functionalized nucleotides enable recognition of mixed-sequence DNA with single nucleotide fidelity.

scholarly journals Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Zhiwei Hu ◽  
Yannan Wang ◽  
Qian Liu ◽  
Yan Qiu ◽  
Zhiyu Zhong ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Base Editing ◽  
Guide Rna ◽  
Guide Rnas ◽  
Double Stranded Dna ◽  
Target Sites ◽  
Guide Sequence ◽  
Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

Thioflavin T specifically brightening “Guanine Island” in duplex-DNA: a novel fluorescent probe for single-nucleotide mutation

The Analyst ◽  
2019 ◽  
Vol 144 (7) ◽  
pp. 2284-2290 ◽  
Author(s):  
Wei Zhou ◽  
Ze Yu ◽  
Ge Ma ◽  
Tian Jin ◽  
Yunchao Li ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Thioflavin T ◽  
Duplex Dna ◽  
Single Nucleotide ◽  
Single Nucleotide Mutation ◽  
Double Stranded Dna ◽  
Nucleotide Mutation

Here, we found that Thioflavin T (ThT) could specifically bind with a G-GGG unit (named as “Guanine Island”) in double stranded DNA (ds-DNA).

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing

2000 ◽  
Vol 31 (2) ◽  
pp. 107 ◽  
Author(s):  
Tommy Nordstrm ◽  
Mostafa Ronaghi ◽  
Lena Forsberg ◽  
Ulf de Faire ◽  
Ralf Morgenstern ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Direct Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Double Stranded Dna

Selective recognition of G-quadruplexes by a dimeric carbocyanine dye

RSC Advances ◽  
2016 ◽  
Vol 6 (90) ◽  
pp. 87400-87404 ◽  
Author(s):  
P. Chilka ◽  
P. R. Patlolla ◽  
B. Datta
Keyword(s):  
Selective Recognition ◽  
Dna Molecules ◽  
Double Stranded Dna ◽  
Mixed Sequence ◽  
Carbocyanine Dye ◽  
G Quadruplex

A novel dimeric carbocyanine dye is found to recognise G-quadruplex structures selectively compared to mixed sequence or double-stranded DNA molecules.

scholarly journals Sensitive and Selective Detection of DNA Fragments Associated with Ganoderma Boninense by DNA-Nanoparticle Conjugate Hybridisation

2020 ◽  
Author(s):  
Ekta Rani ◽  
Siti Akhtar Mohshim ◽  
Nor Hidayat Yusof ◽  
Muhammad Zamharir Ahmad ◽  
Royston Goodacre ◽  
...  
Keyword(s):  
Colorimetric Assay ◽  
Colour Change ◽  
Dna Fragments ◽  
Label Free ◽  
Ganoderma Boninense ◽  
Large Excess ◽  
Single Nucleotide ◽  
Free System ◽  
Complementary Dna ◽  
Double Stranded Dna

<u><strong> </strong></u><p>A colorimetric assay for the detection of DNA fragments associated with the oil palm pathogen Ganoderma boninense is reported, which is based on the aggregation of DNA-nanoparticle conjugated in the presence of complementary DNA from the pathogen. Here, various designs of DNA-nanoparticle conjugates were evaluated, and it was found that the best design gave a visually observable colour change with as little as 2 pmol of double-stranded DNA analyte even in the presence of a large excess of a mixture of non-complementary DNA. The assay was also able to differentiate analyte sequences with three or more single nucleotide mismatches. Overall, this label-free system is rapid, sensitive, selective, simple in design and easy to carry out. It does not require specialist equipment or specialist training for the interpretation of the results and therefore has the potential to be deployed of agricultural diagnostics in the field.</p><u><strong></strong></u>

scholarly journals High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

2003 ◽  
Vol 49 (6) ◽  
pp. 853-860 ◽  
Author(s):  
Carl T Wittwer ◽  
Gudrun H Reed ◽  
Cameron N Gundry ◽  
Joshua G Vandersteen ◽  
Robert J Pryor
Keyword(s):  
High Resolution ◽  
Mutation Screening ◽  
Melting Curve ◽  
Single Nucleotide ◽  
Mutation Scanning ◽  
Double Stranded Dna ◽  
Closed Tube ◽  
Pcr Products ◽  
Melting Analysis ◽  
Detection Of Mutations

Abstract Background: High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396–406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. Methods: We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), β-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. Results: The six common β-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1–2 min, amplification and analysis were achieved in 10–20 min with rapid cycling conditions. Conclusions: High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

scholarly journals SV40 Large T Antigen Helicase Unwinds Double-Stranded DNA in Single-Nucleotide Steps as Revealed by Nanopore Tweezers

2020 ◽  
Vol 118 (3) ◽  
pp. 72a
Author(s):  
Christopher A. Thomas ◽  
Jonathan M. Craig ◽  
Andrew H. Laszlo ◽  
Ian C. Nova ◽  
Sherry Xie ◽  
...  
Keyword(s):  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Single Nucleotide ◽  
Double Stranded Dna
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