Fluorescence based strategies for genetic analysis

Ranasinghe, Rohan T.; Brown, Tom

doi:10.1039/b509522k

Cited by 245 publications

(147 citation statements)

References 113 publications

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“…The modified oligonucleotides were purified by HPLC on a semipreparative RP-C18 column (300 Å, Supelco) using the following conditions: A ) NH 4 OAc buffer (50 mM), pH ) 6.5; B ) acetonitrile; gradient 0-30% B over 50 min, flow rate 2.5 mL/min, UV/vis detection at 260 and 641 nm. The oligonucleotides were lyophilized and quantified by their absorbance in 10 mM sodium phosphate buffer at 260 nm on a Varian Cary 100 spectrometer.…”

Section: Methodsmentioning

confidence: 99%

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

et al. 2009

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The azides 1 and 2 bearing a phenoxazinium and a coumarin fluorophore, respectively, were applied in postsynthetic "click"-type bioconjugation and coupled to oligonucleotides modified with alkyne groups using two alternative approaches: (i) as a nucleotide modification at the 2′-position of uridine and (ii) as a nucleotide substitution using (S)-(-)-3-amino-1,2-propanediol as an acyclic linker between the phosphodiester bridges. The corresponding alkynylated phosporamidites 3 and 6 were used as DNA building blocks for the preparation of alkyne-bearing DNA duplexes. The base pairs adjacent to the site of modification and the base opposite to it were varied in the DNA sequences. The modified duplexes were investigated by UV/vis absorption spectroscopy (including melting temperatures) and fluorescence spectroscopy in order to study the different optical properties of the two chromophores and to evaluate their potential for bioanalytical applications. The sequence-selective fluorescence quenching of phenoxazinium 1 differs only slightly and does not depend on the type of modification, meaning whether it has been attached to the 2′-position of uridine or as DNA base surrogate using the acyclic glycol linker. The 2′-chromophore-modified uridine still recognizes adenine as the counterbase, and the duplexes exhibit a sufficient thermal stability that is comparable to that of unmodified duplexes. Thus, the application of the 2′-modification site of uridine is preferred in comparison to glycol-assisted DNA base surrogates. Accordingly, the coumarin dye azide 2 was attached only to the 2′-position of uridine. The significant Stokes shift of ∼100 nm and the good quantum yields make the coumarin chromophore a powerful fluorescent label for nucleic acids.

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Section: Methodsmentioning

confidence: 99%

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

et al. 2009

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“…Nittaya Gale, 2 Petr Kocalka, 1 charlotte Mardle 1 and Tom Brown 1, was synthesized from propargylamine, iodouracil and 6-aminohexanoic acid. The first two steps were similar to alkyne-T-PNA monomer 1, briefly N-Boc-protected propargylamine 9, 17 was coupled to modified iodouracil 5 via a Sonogashira coupling after which hydroysis of the ethyl ester under basic conditions gave the pure product in 82% yield over two steps.…”

Section: Pna Hybeacons For Analysis Of Human Mutations Related To Stamentioning

confidence: 99%

PNA HyBeacons for analysis of human mutations related to statin-induced myopathy

Gale

Kočalka

Mardle

et al. 2011

Artificial DNA: PNA & XNA

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“…A characteristic fluorescent signal will then be emitted which can be directly linked to the detection of the targeted sequence and/or structure. (20) Most recent reports of DNA-templated reactions applied to oligonucleotide sensing are based on the Staudinger reaction, (21)(22)(23)(24)(25) aldol-type, (26)(27) organomercury-activated (28) or S N Ar (29) reactions.…”

Section: Introductionmentioning

confidence: 99%

Dual Sensing of Hairpin and Quadruplex DNA Structures Using Multicolored Peptide Nucleic Acid Fluorescent Probes

2010

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Synthesis of water-soluble 5-mer Peptide Nucleic Acids (PNAs) functionalized at their 5' and 3'-ends with two original precursors of pentamethine cyanine dye synthesis is reported. The successful use of these PNA probes for sensing DNA hairpin structures in vitro was also demonstrated where specific hairpin formation was associated with the appearance of a characteristic fluorescence signal at 660 nm. A comparative study between three different strategies where PNAs were targeting either the stem or the loop of the hairpin was carried out. Best sensitivity was obtained using PNA sequences complementary to the loop sequence and directing both functional moieties toward the base of loop. Unprecedented proof-of-concept for the simultaneous sensing of hairpin and quadruplex DNAs with a nonoverlapping two-color system (C3 and C5) is also demonstrated.

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Fluorescence based strategies for genetic analysis

Abstract: Synthetic 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.

Cited by 245 publications

References 113 publications

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

Comparison of a Nucleosidic vs Non-Nucleosidic Postsynthetic “Click” Modification of DNA with Base-Labile Fluorescent Probes

PNA HyBeacons for analysis of human mutations related to statin-induced myopathy

Dual Sensing of Hairpin and Quadruplex DNA Structures Using Multicolored Peptide Nucleic Acid Fluorescent Probes

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