Reporter, molecular beacon

Real-time PCR is a quantitative method for measuring amplicons as they are produced by measuring the increase in fluorescence of a dye added to the reaction mixture.12,104,105 Methods using fluorescent reporters, such as SYBR Green,104,106 TaqMan ,107,108 or molecular beacons,9 collect quantitative data at the time when DNA is in the exponential phase of amplification. 

Ionic strength influences are well known with respect to the rate and energetics of nucleic acid hybridization [17]. Charge and ionic radius are both important in terms of stabilizing the structure of the duplex as well as stabilizing the stem portion of the molecular beacon [17]. The stem structure stability was increased when a divalent cation was incorporated into the hybridization buffer solution [17]. It was reported that cations were best at stabilizing the duplex formed upon hybridization in the order Ca2+ > Mg2+ K+ > Na+. The ultimate detection limit of the sensor configuration was calculated to be 1.1 nM [17]. 

APTS (for sugar isomers) [560], RNA molecular beacon probes [337], and YOPRO-1 (for dsDNA) [613]. In one report, a ring of 24 blue LEDs (470 nm) has been used for flood illumination [321]. 

In another report, real-time NASBA was used to amplify RNA in a Si-Pyrex chip containing 50-nL reaction chambers. This is an isothermal (41°C) technique. A fluorescent molecular beacon probe was used to detect the RNA derived from the human papillomavirus (HPV16) [337]. 

Tsourkas et al. (2003) reported dual FRET molecular beacon assays, where the donor probe was labeled with either Eu3+ or Tb3+ complex of DTPA-csl24-ethylenediamine (and no quenchers attached). For the Eu3+ complex, the acceptor probe was Cy5-labeled (and no quenchers attached) and for the Tb complex, the acceptor probe was labeled with Cy3 or ROX as a fluorophore and with dabcyl as a quencher. They demonstrated that these pairs of probes detected DNA targets ( 50-mer) with high S/N. 

Finally, there have been reports that deal with other forms of spectral detection. A platinum (Il)-coproporphyrin reagent has been evaluated for phosphorescent labelling of oligonucleotides. The presence of the label had little effect on conjugation, and labelled primers were effective in PCR reactions. A silicon nanoparticle conjugated to ODNs acted as a luminescent label, and a molecular beacon (see later) has been prepared which contains a photoluminescent dye (Ru(II)(bpy)3) and the luminescent quencher Black Hole Quencher-2 . ... 

Although there are some specific applications of fluorescent analogues (FRET, molecular beacons, single-molecule detection described below) there are many reports that use fluorescence as a means of detection and monitoring... 

Molecular beacons (MBs) are hairpin-shaped oligonucleotides that report the presence of specific nucleic acids. The MBs have been immobihzed by Tan and co-workers [27] onto ultrasmall optical fibre probes through avidin-biotin binding. The MB-DNA biosensor detected its target DNA molecules, in real time, with selectivity for a single base-pair mismatch. This MB-DNA-biosensor was used by Perlette and Tan [28] for real-time monitoring of mRNA-DNA hybridization inside a living cell. 

The fluorescent ddA 5 -triphosphate derivative (163) was shown to be an effective substrate for terminal transferase, and various DNA polymerases. The resultant DNA could then be detected by fluorescence spectroscopy. A molecular beacon DNA probe incorporating a donor and a quencher dye has been reported, " which shows high sensitivity and dynamic range. Such molecular beacons are anticipated to have use in DNA/RNA and pro-tein/DNA/RNA interactions. 

Molecular beacons are oligonucleotide probes designed originally for the detection of nucleic acid sequences (Tyagi and Kramer, 1996). They generally have a hairpin shape, which brings into close contact fluorescence reporters bound... 

So far, there are only few reports about fluorescent oligomers that change their constitution upon addihon of an analyte. In contrast, there are many examples of fluorescent molecules that undergo a conformational change upon addition of certain analytes (e.g., molecular beacons [7]). Although conformationally dynamic receptors are sometimes discussed in the context of DCC [8], these systems will not be described in more detail in the present chapter. 

Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a new approach to detect the presence of Salmonella species using these fluorogenic reporter molecules and demonstrated their ability to discriminate between similar E. coli species in real-time PCR assays. A detection limit of 1 CPU per PCR reaction was obtained. The assays were carried out entirely in sealed PCR tubes, enabling fast and direct detection in semiautomated format. 

A number of modifications to molecular beacons (MBs) have been reported. MBs have been synthesised using PNA (see section 1.2.1) for the detection of DNA mismatches. It is reported that DNA MBs are only able to discriminate matched from mismatched sequences within narrow temperature ranges, but using PNA MBs up to 100-fold selectivity for matched versus mismatched is described. " Sheng et al. have incorporated the unnatural base pair (93) into the stem of a MB. The (93) base pair is specific and neither base will form a base pair with the native DNA bases, therefore... 

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