Biotinylated probes

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 for nick translation (this chapter). 

Detect biotinylated probe with rabbit anti-biotin. 

Probes tagged with an enzyme signal generating system, such as alkaline phosphatase or horseradish peroxidase, are sometimes used in direct assays. Alternatively, enzymes as reporters may be used with biotinylated probes in a direct affinity assay or in an indirect affinity assay. For the former assay, the... 

Add sufficient biotinylated probe (0.5 pg/mL final concentration) to the hybridization buffer in a microfuge tube and heat to 90°C for 5 min (see Note 6). 

Chan, W. T.-W., Fleming, K. A., and McGee, J. O D. (1985) Detection of subpicogram quantities of specific DNA sequences on blot hybridization with biotinylated probes Nucleic Acids Res. 13,8083-8091... 

Wash in TBS, and develop the signal using NBT/BCIP as described for detection of biotinylated probes. 

Proceed as for APAAP detection of biotinylated probes from step 2 in Section 3.6 3. 

To facilitate our work on plasmids with no known phenotype, we have developed a method for the use and detection of biotinylated probes in colony hybridization. It is suitable both for the detection of rare positive hybridization events over a background of nonreactive colonies and for the detection of nonhybridizing colonies in a population containing sequences homologous to the probe. The latter capability could be useful in such applications as the detection of cured (i.e., plasmid-free) cells in a bacterial population containing plasmids. 

Detection of the sites of hybridization-dependent binding of biotinylated probe to the filters is most readily conducted with commercially available kits. Favorable results have been obtained with the BluGene Nonradioactive Nucleic Acid Detection System from BRL. Follow the manufacturer s instructions when carrying out the following steps. After washing, sequentially expose the filters to streptavidin and biotinylated alkaline phosphatase (or to a conjugate of these two proteins) This causes the immobilization of alkaline phosphatase at sites of positive hybridization... 

McQuaid, S., and Allan, G. M. 1992. Detection protocols for biotinylated probes Optimization using multistep techniques. J. Histochem. Cytochem. 40 569-574. 

Biotinylated DNA can be firmly single-point attached in Av-GEB. In this case, a capture format was used in which the immobilization of the biotinylated probe together with the hybridization was performed in a one-step procedure (Figs. 21.IB and 21.2A) [66]. Briefly, the three-step experimental procedure consists of (i) one-step immobilization/... 

Hybridisation of FITC-labelled target DNA to biotinylated probes on streptavidin-modified SPCEs. Addition of anti-FITC-ALP conjugate Hybridisation of Pt(II) complexed target DNA to biotinylated probes on streptavidin-modified SPCEs... 

Biotinylated probe, then DPV oxidation of accumulated cationic product of substrate hydrolysis by ALP enzyme Amperometric detection of catalytically oxidised DNA... 

Fig. 30.4. Evaluation of 2.9 pmol of IS200 amplicon using a genosensor based on dry-adsorption of the target on GEC transducer 68.2 pmol of biotinylated probe 9.00 /ig of enzyme conjugate. The nonspecific adsorption signal is shown in black (more details in Ref. [2]) (N — 3).

Biotinylated probe (complementary to underlined sequence of the target). The biotin is separated to the first base by 10-T spacer in the 5 -end Biotin-fi -TTT-TTT-TTT-TAT-GGC-TAT-AAA-CAG-AGT-CCA-S. ... 

The genosensors used for the PCR products detection are streptavidin-modified SPCEs with biotinylated probes as sensing phase [1,2]. 

Tris(hydroxymethyl)methylamine (TRIS), sodium chloride, sodium citrate, ethylenediamine tetraacetic acid disodium salt (EDTA), lithium chloride, Tween 20, streptavidin 10 nm colloidal gold labelled, hydrochloric acid (37%), nitric acid, streptavidin-coated paramagnetic beads (MB) with a diameter of 2.8 pm, Dynabeads M-280 Streptavidin (Dynal Biotech, Oslo, Norway) biotinylated probe oligonucleotides which sequences are shown in Table 53.1. 

---