Detection of PCR Products in Real Time Using Light-up Probes

Svanvik, Nicke; Ståhlberg, Anders; Sehlstedt, Ulrica; Sjöback, Robert; Kubista, Mikael

doi:10.1006/abio.2000.4824

Cited by 77 publications

(41 citation statements)

References 16 publications

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“…[21][22][23][24][25][26][27][28][29][30][31][32][33] Usually, cyanine dyes or other DNA stains [34,35] have been linked to nucleic acids by means of a flexible tether. Previous work by ourselves and others has shown that the tether plays an important role as far as the responsiveness of fluorescence and the stability of probe-target duplexes are concerned.…”

Section: Introductionmentioning

confidence: 99%

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Jarikote

Krebs

Tannert

et al. 2006

Chemistry A European J

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Double‐stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye–nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base‐pair‐specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So‐called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNA⋅DNA duplexes. A detailed analysis of the base‐pair dependence of optical properties is provided and enforced binding of TO is compared with “classical” binding of free TO‐PRO1. UV‐visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting‐curve analyses confirmed site‐specific TO intercalation. Thiazole orange exhibited base‐specific responses that are not observed in noncovalent dye–nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (±60 % variation of the averaged εmax of 57 000 M−1 cm−1) on nearest‐neighbor base pairs. TO signals hybridization, as shown by increases in the steady‐state fluorescence emission. Studies of TO fluorescence lifetimes in FIT–PNA and in DNA⋅DNA and PNA⋅DNA complexes highlighted four different fluorescence‐decay processes that may be closed or opened in response to matched or single‐mismatched hybridization. A very fast decay process (0.04–0.07 ns) and a slow decay process (2.33–3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22–0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.

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Section: Introductionmentioning

confidence: 99%

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Jarikote

Krebs

Tannert

et al. 2006

Chemistry A European J

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“…[29][30][31] YO and TO have also been covalently linked to oligonucleotides and inserted into peptide nucleic acids constructs, which become fluorescent upon hybridisation of the light-up probe to a specific complementary strand. [32][33][34][35][36][37][38] The origin of the very high contrast in emission between the free and the bound form of the dyes has long been thought to be due to an ultrafast decay of the excited state of the free form through a large-amplitude torsional motion around the monomethine bond connecting the benzoxazole, benzothiazole and quinoline moieties, respectively. [39,40] This isomerisation mechanism is blocked upon intercalation of the dyes into DNA and, as a consequence, they light-up by three orders of magnitude.…”

mentioning

confidence: 99%

Structure–Fluorescence Contrast Relationship in Cyanine DNA Intercalators: Toward Rational Dye Design

Fürstenberg

Deligeorgiev

Gadjev

et al. 2007

Chemistry A European J

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The fluorescence enhancement mechanisms of a series of DNA stains of the oxazole yellow (YO) family have been investigated in detail using steady‐state and ultrafast time‐resolved fluorescence spectroscopy. The strong increase in the fluorescence quantum yield of these dyes upon DNA binding is shown to originate from the inhibition of two distinct processes: 1) isomerisation through large‐amplitude motion that non‐radiatively deactivates the excited state within a few picoseconds and 2) formation of weakly emitting H‐dimers. As the H‐dimers are not totally non‐fluorescent, their formation is less efficient than isomerisation as a fluorescent contrast mechanism. The propensity of the dyes to form H‐dimers and thus to reduce their fluorescence contrast upon DNA binding is shown to depend on several of their structural parameters, such as their monomeric (YO) or homodimeric (YOYO) nature, their substitution and their electric charge. Moreover, these parameters also have a substantial influence on the affinity of the dyes for DNA and on the ensuing sensitivity for DNA detection. The results give new insight into the development and optimisation of fluorescent DNA probes with the highest contrast.

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“…In addition, many other fluorescence based chemistries are developed (LUX fluorogenic primers, amplifluor assays, QZyme primers, Light-Up probes, eclipse probes, Pleiades), and new designs are still being proposed (23)(24)(25)(26)(27)(28)(29)(30). These assays are expected to be more adopted in the future.…”

Section: Specific Sequence Detection Chemistrymentioning

confidence: 99%

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

Nestorov¹,

Matić²,

Elaković³

et al. 2013

Journal of Medical Biochemistry

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Summary: Real-time RT PCR has been recognized as an accurate, reliable and sensitive method for quantifying gene transcription. However, several steps preceding PCR represent critical points and source of inaccuracies. These steps include cell processing, RNA extraction, RNA storage, assessment of RNA concentration and cDNA synthesis. To compensate for potential variability introduced by the procedure, normalization of target gene expression has been established. Accurate normalization has become an absolute prerequisite for the correct quantification of gene expression. Several strategies are in use for the normalization of data, including normalization to sample size, to total RNA or to an internal reference. Among these, the use of housekeeping genes as an internal (endogenous) control is the most common approach. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper reference gene for normalization have become increasingly stringent. The aim of this paper is to discuss the concept of normalization in mRNA quantification, as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. By showing that the use of inappropriate endogenous control might lead to incorrect results and misinterpretation of experimental data, we are joining the creators of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) in an attempt to convince scientists that proper validation of potential reference genes is an absolute Kratak sadr`aj: RT-PCR je prepoznat kao precizna, pouzdana i osetljiva metoda za kvantifikaciju transkripcije gena. Me|utim, ovoj metodi prethodi nekoliko koraka koji predstavljaju kriti~ne ta~ke i izvor potencijalnih gre{aka. Ovi koraci uklju~uju obradu }elijskog materijala, ekstrakciju ĩ uvanje RNK, odre|ivanje koncentracije RNK i sintezu cDNK. Da bi se kompenzovala potencijalna varijabilnost nastala tokom procedure, uvedena je normalizacija ekspresije ciljnih gena. Precizna normalizacija je postala apsolutni preduslov za ta~nu kvantifikaciju ekspresije gena. Postoji nekoliko strategija za normalizaciju eksperimentalnih podataka, uklju~uju}i normalizaciju u odnosu na veli~inu uzorka, ukupnu RNK ili internu kontrolu (referencu). Kao interna (endogena) kontrola naj~e{}e se koriste geni sa stabilnom ekspresijom. Imaju}i u vidu veliku osetljivost, reproducibilnost i ve liki dinami~ki opseg PCR metode, zahtevi za odgova raju}im referentnim genima koji }e se koristiti za normalizaciju podataka postali su veoma restriktivni. Cilj ovog rada je da razjasni koncept normalizacije i prokomentari{e nekoliko statisti~kih algoritama koji su razvijeni kako bi pomogli u validaciji potencijalnih referentnih gena. Pokazuju}i da kori{}enje neodgovaraju}ih referentnih gena (endogenih kontrola) mo`e da dovede do neta~nih rezultata i pogre{ne interpretacije eksperimentalnih podataka, mi se priklju~ujemo tvorcima uputstva MIQE (eng. Minimum Information for Publication of Quantitati...

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Detection of PCR Products in Real Time Using Light-up Probes

Cited by 77 publications

References 16 publications

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Exploring Base‐Pair‐Specific Optical Properties of the DNA Stain Thiazole Orange

Structure–Fluorescence Contrast Relationship in Cyanine DNA Intercalators: Toward Rational Dye Design

Gene Expression Studies: How to Obtain Accurate and Reliable Data by Quantitative Real-Time RT PCR / IZUČAVANJE EKSPRESIJE GENA: KAKO DOBITI TAČNE I POUZDANE PODATKE KVANTITATIVNIM RT PCR-OM U REALNOM VREMENU

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