Reporter probes

Alternative Probes. Even though C02 possesses a broad range of special virtues, other small molecules could also be used as reporter probes. Once the technique has been refined using C02, photofragmentation reactions that yield N2, CO, HCN, S02, 02, NO, N02 or any of a host of other small molecules should be amenable to similar mechanistic studies using Raman or FTIR spectroscopy. 

FTIR reporter probe, 300 carbon dioxide as a, 303 anharmonicity and, 305 bending frequency and, 306 electrolytic coupling and, 307 intermolecular forces and, 305, 346 intrinsic frequency of, 306 l80C,60 isotopic, 307 peroxide related to, 303 polarizability and, 307 windows and, 301... 

Real-time monitoring of PCR amplification was achieved by sequential CGE after 15, 20, 25, and 30 s (see Figure 9.9) [924]. In addition, PCR of [i-aclin DNA (294 bp) has been integrated with real-time detection at 518 nm using a fluorescent reporter probe [943]. 

A hybridization probe-pair placed over a heterozygous polymorphism is shown in Figure 37-29. The reporter probe is complementary to the normal allele. As the temperature is increased, the mismatched mutant hybrid melts first, giving the first transition, followed by the matched normal hybrid. The melting temperatures of both hybrids are easily seen in the derivative plot generated by numerical Savitzky-Golay... 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

Control Zone Consists of an unmodified DNA oligonucleotide that is complementary 1o liposomal reporter probe... 

Reporter probe-tagged liposomes bind at control zone... 

Fn the presence of target, reporter probe-tagged liposomes hybridize with target which allows complex to bind at capture zone... 

Reporter probe-tagged liposomes mixed with sample containing target molecule... 

Fig. 5. Sandwich-hybridization assay (A) A biotinylated DNA oligonucleotide, which is complementary to one portion of the target sequence, is mixed with streptavidin and applied to form the capture zone 1.5 cm from the base of the membrane. An unmodified DNA oligonucleotide, which is complementary to the reporter probe sequence on the liposomes, is applied to form the control zone 2.5cm from the base of the membrane. (B) In the presence of target, a sandwich hybridization complex forms with dye-encapsulating liposomes resulting in a magenta-colored band at the capture zone. (C) In the absence of target, only the control band is visible as no sandwich complex has formed.

Cholesteryl-TEG modified DNA reporter probe oligonucleotides (Operon Biotechnologies, Inc., Alameda, CA) are diluted to 300 pM with a 1 4 (v/v) mixture of metha-nol/formamide, adding the formamide to the probe first, followed by methanol. The probes are then aliquotted into 50 iL portions prior to storage at -20°C. 

Liposomes tagged with cholesteryl-TEG reporter probe of known optical density or phospholipid concentration. 

Cholesteryl-labeled reporter probe (corresponding to 0.013 mol% of the total lipid input) solution (50pL) is then added to the dissolved lipids and the flask vortexed until visually homogeneous (40). For the preparation of antibody-tagged liposomes, the probe solution is omitted and lipid composition slightly different (see Note 1). 

Add 4 pL hybridization buffer, 1 pL NASBA product, and 2 pL reporter probe conjugated liposomes to the bottom of a 10 x 75 mm culture tube. 

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