scholarly journals Expanding detection windows for discriminating single nucleotide variants using rationally designed DNA equalizer probes

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guan A. Wang ◽  
Xiaoyu Xie ◽  
Hayam Mansour ◽  
Fangfang Chen ◽  
Gabriela Matamoros ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rural Areas ◽  
Simultaneous Detection ◽  
Dna Nanotechnology ◽  
Quantitative Relationship ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Hybridization Probes

Abstract Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.

scholarly journals Ability of the Digene Hybrid Capture II Test To Identify Chlamydia trachomatis and Neisseria gonorrhoeae in Cervical Specimens

1999 ◽  
Vol 37 (11) ◽  
pp. 3668-3671 ◽  
Author(s):  
Julius Schachter ◽  
Edward W. Hook ◽  
William M. McCormack ◽  
Thomas C. Quinn ◽  
Max Chernesky ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Chlamydia Trachomatis ◽  
Neisseria Gonorrhoeae ◽  
Fluorescent Antibody ◽  
Simultaneous Detection ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Attractive Alternative ◽  
Single Specimen ◽  
Hybrid Capture

The Digene Hybrid Capture II (HCII CT/GC) test is a combination test designed to detect Chlamydia trachomatis andNeisseria gonorrhoeae in a single specimen. It is a nucleic acid hybridization test which uses signal amplification to increase sensitivity. We compared its performance to that of culture on cervical specimens from 1,370 women. Direct fluorescent-antibody assay was used to resolve discrepant results for C. trachomatis. Samples were collected with a proprietary cervical brush or with endocervical swabs. The HCII CT/GC test proved to be sensitive and specific in detecting these organisms. Compared to N. gonorrhoeaeculture, it had a sensitivity of 93% (87/94) and a specificity of 98.5% (1,244/1,263). Compared to C. trachomatis culture, the sensitivity was 97.7% (129/132) and specificity was 98.2% (1,216/1,238). Testing of some specimens with discrepant results by PCR suggested that the test would actually prove to be even more specific if it were compared to a nucleic acid amplification test (NAAT). The sensitivity of C. trachomatis culture was somewhat less, at 88.6% (117/132). The endocervical brush appeared to be better than Dacron swabs for collecting specimens. The HCII CT/GC test offers an attractive format that allows simultaneous detection of C. trachomatis and N. gonorrhoeae with a single specimen. An initial positive result is followed by repeat tests with probes to identify chlamydiae or gonococci. This test is more sensitive than C. trachomatis culture and is at least as sensitive as culture for gonococci. It deserves further evaluation and comparison with NAATs and may well offer an attractive alternative for diagnosis and screening of these infections.

scholarly journals Locked Nucleic Acid Thymine Monomer Probe Identifies Four Single-Nucleotide Variants by Melting Temperature

2017 ◽  
Vol 112 (3) ◽  
pp. 331a-332a
Author(s):  
Judy M. Obliosca ◽  
Sara Y. Cheng ◽  
Yu-An Chen ◽  
Mariana F. Llanos ◽  
Yen-Liang Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Melting Temperature ◽  
Locked Nucleic Acid ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

scholarly journals IsoPCR: An Analytically Sensitive, Nested, Multiplex Nucleic Acid Amplification Method

2013 ◽  
Vol 59 (2) ◽  
pp. 436-439 ◽  
Author(s):  
Martin Jensen Søe ◽  
Mikkel Rohde ◽  
Jens Mikkelsen ◽  
Peter Warthoe
Keyword(s):  
Nucleic Acid ◽  
Candida Glabrata ◽  
Simultaneous Detection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Qpcr Assay ◽  
Nucleic Acid Amplification ◽  
Molecular Diagnostic ◽  
Nucleic Acid Detection ◽  
Amplification Method

BACKGROUND Nucleic acid tests that can simultaneously detect multiple targets with high sensitivity, specificity, and speed are highly desirable. To meet this need, we developed a new approach we call the isoPCR method. METHODS The isoPCR method is a 2-stage nested-like nucleic acid amplification method that combines a single multiplex preamplification PCR with subsequent distinct detection of specific targets by use of isothermal amplification. We compared isoPCR to nested quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and nested LAMP (PCR followed by LAMP), for detection of DNA from Candida glabrata. We evaluated the method's multiplex capability for detecting low copy numbers of pathogens commonly involved in sepsis. RESULTS IsoPCR provided detection of 1 copy of Candida glabrata, an LOD that was 5-fold lower than a nested qPCR assay (5 copies), while the amplification time was simultaneously halved. Similarly, the LOD for isoPCR was lower than that for a LAMP assay (1000 copies) and a nested LAMP assay (5 copies). IsoPCR required recognition of 6 regions for detection, thereby providing a theoretically higher specificity compared to nested qPCR (4 regions). The isoPCR multiplexing capability was demonstrated by simultaneous detection of 4 pathogens with individual LODs of 10 copies or fewer. Furthermore, the specificity of isoPCR was demonstrated by successful pathogen detection from samples with more than 1 pathogen present. CONCLUSIONS IsoPCR provides a molecular diagnostic tool for multiplex nucleic acid detection, with an LOD down to 1 copy, high theoretical specificity, and halving of the amplification time compared to a nested qPCR assay.

scholarly journals Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes

1988 ◽  
Vol 251 (3) ◽  
pp. 935-938 ◽  
Author(s):  
A H al-Hakim ◽  
R Hull
Keyword(s):  
Nucleic Acid ◽  
Blot Hybridization ◽  
Nucleic Acid Hybridization ◽  
Cross Linking ◽  
Dot Blot ◽  
Hybridization Probes ◽  
Target Dna ◽  
Amino Derivatives ◽  
Biotin Molecule ◽  
Long Chain

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.

scholarly journals Molecular Assay for Detection of Ciprofloxacin Resistance in Neisseria gonorrhoeae Isolates from Cultures and Clinical Nucleic Acid Amplification Test Specimens

2015 ◽  
Vol 53 (11) ◽  
pp. 3606-3608 ◽  
Author(s):  
S. W. Peterson ◽  
I. Martin ◽  
W. Demczuk ◽  
A. Bharat ◽  
L. Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Sensitivity ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Nucleotide Polymorphisms ◽  
Molecular Assay ◽  
Nucleotide Polymorphism ◽  
Pcr Assay ◽  
Single Nucleotide ◽  
Ciprofloxacin Resistance

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

scholarly journals Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

2020 ◽  
Author(s):  
Georgina C. Gavins ◽  
Katharina Gröger ◽  
Michael D. Bartoschek ◽  
Philipp Wolf ◽  
Annette G. Beck-Sickinger ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cho Cells ◽  
Fluorescent Dyes ◽  
Coiled Coil ◽  
Dna Nanotechnology ◽  
Nucleic Acid Hybridization ◽  
Landing Platform ◽  
Acid Protein ◽  
Reaction Proceeds ◽  
A Cell

AbstractDNA nanotechnology is an emerging field, which promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress in the area is the ability to create the nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction, which installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled coil peptide tag. Once installed the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness, and achieving reversible labelling by way of toehold mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled coil systems, with EGFR and ETBR, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of EGFR on CHO cells.

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