Biotinylated probes detection

Detect biotinylated probe with rabbit anti-biotin. 

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

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

Fig. 8.4. Biosensors may become interesting for probe detection. In this example (from Olson et al., 1991), a biotinylated and a fluoresceinated probe are hybridized in solution to the target (I). The primer sequences should be located outside this region for PCR-product detection. Avidin acts as a bridge between the biotinylated probe and the biotinylated surface of the biosensor (II). Fluorescein is detected with a urease-antibody conjugate. The rise in pH, after the addition of urea, is detected with the pH-sensitive biosensor. The total assay time is less than 2 h with a detectability of 30 amol and a coefficient of variation (standard deviation/mean) of less than 10%.

Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.2), but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. 

Biotinylated probes can be detected with avidin, streptavidin or modified avidin (Yehle et al., 1987). Replacing phosphate-buffered saline by SSC during hybridization reduces the background of unmodified avidin (fluoresceinated) making it superior to modified avidin for single-copy detection (McNeil et al., 1991). 

Haptens in the probe are detected using streptavidin, for biotin, or antibody conjugates. The size of a biotinylated probe is critical to keep noise at a low level and the signal/noise ratio is often between... 

A high speed hybridization procedure (total, including fixation and detection, 1 h) has been described by Bourinbaiar et al. (1991). They suspended washed lymphocytes, for the detection of HIV, in a mildly hypotonic medium (1 part medium and 2 parts distilled water) and spread 25 xl ( 10 cells) in 8 mm wells of Teflon-coated multiwell glass slides and fixed the cells in Carnoy s II solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then for 10 min in absolute acetone. Five microliters of biotinylated probes (100 ng/ml) is added to the dried cells and the slides are covered with autoclavable plastic (cut from biohazard bag) and placed in a microwave oven. The position of the slide in the oven and the incubation time are crucial for this hybridization step. In microwave ovens with a carousel, the slide is placed about 6 cm from the center and a 150 ml beaker filled with tap water is placed in the center. The slide is then irradiated for 30 s on Defrost . The probes can be detected after a rinse with 2 x SSC. 

There are currently several methods for analysis of the amplified target DNA. For HIV-1, liquid hybridization with radioactively labeled probes is used (K12). Tests for HLA genes and sickle cell anemia utilize the reverse dot-blot format with a nylon membrane (S3). Each clinical research format has a well-characterized detection method defining the optimum probe concentration, the hybridization times and temperatures, as well as the concentrations of indicator reagents. Table 5 describes the optima and tolerances of a nonradioactive dot-blot assay that uses biotinylated probes and detection by a chemiluminescent substrate and a strepta-vidin-HRP conjugate. 

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