54 research outputs found
Fluorescence based strategies for genetic analysis
No full textSynthetic chemistry has been central to the design of modern methods of genetic analysis. In this article, we discuss the underlying chemistry and biophysical principles that have been used in the development of robust methods for the analysis of DNA in the diagnostic laboratory
Ultrasensitive fluorescence-based methods for nucleic acid detection: towards amplification-free genetic analysis
No full textReal time PCR is the mainstay of current nucleic acid assays, underpinning applications in forensic science, point-of-care diagnostics and detection of bioterrorism agents. Despite its broad utility, the search for new tests continues, inspired by second and third generation DNA sequencing technologies and fuelled by progress in single molecule fluorescence spectroscopy, nanotechnology and microfabrication. These new methods promise the direct detection of nucleic acids without the need for enzymatic amplification. In this feature article, we provide a chemist's perspective on this multidisciplinary area, introducing the concepts of single molecule detection then focussing on the selection of labels and probe chemistry suitable for generating a signal detectable by ultrasensitive fluorescence spectroscopy. Finally, we discuss the further developments that are required to incorporate these detection platforms into integrated ‘sample-in-answer-out’ instruments, capable of detecting many target sequences in a matter of minute
Novel techniques for genetic analysis
No full textEThOS - Electronic Theses Online ServiceGBUnited Kingdo
Naphthalenyl- and anthracenyl-ethynyl dT analogues as base discriminating fluorescent nucleosides and intramolecular energy transfer donors in oligonucleotide probes
No full textFluorescent thymidine analogues functionalised in the 5-position with the moieties naphthalenylethynyl (NeT), anthracenylethynyl (AeT) and anthracenylbuta-1,3-diynyl (AeeT) have been incorporated into oligonucleotides. The modified oligonucleotides undergo significant emission enhancement when hybridised to fully complementary strands and a decrease in fluorescence emission when the modified thymine is paired with guanine. Thus these analogues are potentially useful as base discriminating fluorescent nucleosides (BDFs). When a fluorescein dT monomer is incorporated into the same oligonucleotide strand as the modified base, energy transfer enhances the fluorescein emission, particularly upon duplex formation. These dual-labelled probes may be useful for genetic analysis to detect point mutations and SNPs and could provide multiplexing capability
Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo [2,3-d] pyrimidin-2(7H)-one nucleoside analogues
No full textSubstituted 3H-pyrrolo[2,3-d] pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity compared to T
Kinetics and thermodynamics of biotinylated oligonucleotide probe binding to particle-immobilized avidin and implications for multiplexing applications
No full textIn this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10?12 M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin?biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin?biotin interaction.<br/
Four base recognition by triplex-forming oligonucleotides at physiological pH
No full textWe have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [20-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively.Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, MeP and APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA
Multistep synthesis on SU-8: Combining microfabrication and solid-phase chemistry on a single material
No full textSU-8 is an epoxy-novolac resin and a well-established negative photoresist for microfabrication and microengineering. The photopolymerized resist is an extremely highly crosslinked polymer showing outstanding chemical and physical robustness with residual surface epoxy groups amenable for chemical functionalization. In this paper we describe, for the first time, the preparation and surface modification of SU-8 particles shaped as microbars, the attachment of appropriate linkers, and the successful application of these particles to multistep solid-phase synthesis leading to oligonucleotides and peptides attached in an unambiguous manner to the support surface
Sandy coastlines under threat of erosion
No full textSandy beaches occupy more than one-third of the global coastline1 and have high socioeconomic value related to recreation, tourism and ecosystem services2. Beaches are the interface between land and ocean, providing coastal protection from marine storms and cyclones3. However the presence of sandy beaches cannot be taken for granted, as they are under constant change, driven by meteorological4,5, geological6 and anthropogenic factors1,7. A substantial proportion of the world’s sandy coastline is already eroding1,7, a situation that could be exacerbated by climate change8,9. Here, we show that ambient trends in shoreline dynamics, combined with coastal recession driven by sea level rise, could result in the near extinction of almost half of the world’s sandy beaches by the end of the century. Moderate GHG emission mitigation could prevent 40% of shoreline retreat. Projected shoreline dynamics are dominated by sea level rise for the majority of sandy beaches, but in certain regions the erosive trend is counteracted by accretive ambient shoreline changes; for example, in the Amazon, East and Southeast Asia and the north tropical Pacific. A substantial proportion of the threatened sandy shorelines are in densely populated areas, underlining the need for the design and implementation of effective adaptive measures.Accepted Author ManuscriptCoastal Engineerin
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