Fluorescent bases, molecular beacon

For studies of hematological malignancies, the sequence-specific fluorescence probe-based systems provide the advantage of an important further test for identification of the sequence of interest by virtue of hybridization of fluo-rescently labeled internal probes to the amplified target sequence. The most commonly used sequence-specific chemistries include the exonuclease (TaqMan), the linear hybridization probe, and the hairpin-based (Molecular Beacon) systems. (See Chapter 37 for further information on nucleic acid techniques.)... 

A simple diagnostic test has been devised for prostate cancer, using a specific molecular beacon mixed with the target DNA on a microscope slide. The DNA is treated to separate the strands and, provided there is the correct correspondence between the bases, in situ combination occurs between the bases on the molecular beacon and those on the strands. Thus, if fluorescence is observed, the DNA sample must contain the base sequence indicative of prostate cancer. 

Molecular beacons have also been attached to glass beads. In buffer solution, the beads (containing FAM and methyl red at the termini) are not fluorescent until exposed to co mplementary DNA. Terminus modifiers based on methox oxalamido (MOX) groups (for example the double MOX modifier attached to either a cyclohexane ring (143) or via a 5 -aminothymidine) have been described. The modifiers allow for post-synthesis functionalisation of ODNs. 

Fig. 5 Two RET"based sensing schemes. A molecular beacon structure with a fluorescent donor (D) and a quenches (Q) enhances emission upon target hybridization. A protease substrate with donor and acceptor conjugated to an amino acid recognition sequence (DEVD) enhances emission upon enzymatic cleavage. (View this art xi color at www.dekker.com.)...

Figure 5.1 Sketch of the DNA molecular beacon. The five bases at the two ends of the beacon are complementary to each other. The size of the loop and its content are varied. The beacon flips between open and closed states with the characteristic rates and k+.Thefluorophore(F)and the quencher (Q) are covalently linked to the two arms of the beacon. In the open state the beacon fluoresces, in the closed state the fluorescence is quenched. Reprinted with permission from Bonnet etal., Kinetics of conformational fluctuations in DNA hairpin-loops. Proceedings of the National Academy of Sciences of the United States of America 95 (1998) 8602-8606. Copyright 1998 NationalAcademy of Sciences, USA.

Several proprietary fluorescence dye-based detection systems that characterize and quantify probe-bound nucleotide sequences have been developed and commercialized in recent years. For example, general and nonspecific DNA dyes can be used that bind with any double-stranded DNA (i.e., the probe-strand complex or otherwise) and are useful in gel electrophoresis. Much more sophisticated systems rely on oligonucleotide probes that incorporate fluorescent dyes that illuminate when a match between complementary strands are made. Included among the latter are TaqMan , molecular beacons, and Scorpion probes. 

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