Solution hybridization

Kapke, G. F et al. (1997). Comparison of the Chiron quantiplex branched DNA (bDNA) assay and the Abbott genostics solution hybridization assay for quantification of hepatitis B viral DNA. J. Viral Hepat. 4,67-75. 

The second stage is the proof of principle In this phase, we take the initial theoretical library idea and begin to apply chemistry experiments to validate experimental designs and potential library schemes at this stage, one also evaluates the method of library production (solid/solution/hybrid phases). In this phase, which is usually the longest phase in any library production process, we will perform the initial experiments, optimize the chemical yields and purities, modify the experiments to generate easily removable by-products, which can be removed by traditional parallel purification methods (i.e. SPE, Resin capture), and determine the most feasible route to the final product. 

The DNA in the sections is denatured by treatment with 70% formamide/2 x SCC for 5 min at 80°C. Ten microliters of the probe solution (hybridization buffer 7 pd, probe 1 pi, and distilled water 2 pi) is placed on the slide and coverslipped. The slide is placed in a microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of 15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear staining. The sections are promptly observed under a fluorescent microscope equipped with epifluorescence filters and a photometric CCD camera. The captured images are digitized and stored in an image analysis program. 

Now the wave equation obviously furnishes one s and three p functions as the solution for the carbon atom in the 5S state however, a linear combination of these four functions will also be a solution (hybridization, hybrid orbital). 

Hybridization solution 45% Formamide, 5X SSC, 5X Denhardt s solution, 20 mMsodium phosphate, pH 6.5, 300 pg/mL freshly denatured, sheared, herring sperm DNA, 200 ng of biotinylated DNA/mL. Before its addition, the biotinylated probe DNA is denatured by incubating for 10 min in a boiling water bath and quickreduce size is unnecessary, since the products generated by nick translation are sufficiently small. Filter and store the hybridization solution as was done for the prehybridization solution. Hybridization solution can be recovered after use and stored at-20°C. The solution can be reused at least 10 times over a time-span of at least 5 mo, without noticeable... 

A DNA optical sensor system was proposed by Cass and co-workers [35] based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for the rapid detection of DNA hybridization. Sandwich solution hybridization uses two sets of DNA probes, one labelled with biotin, the other with an enzyme marker and hybridization is performed in solution where the mobility is greater and the hybridization process is faster, rather than on a surface. The hybrids were bound to the streptavidin-coated magnetic beads through biotin-streptavidin binding reaction. A chemiluminescence fibre-optic biosensor for the detection of hybridization of horseradish peroxidase-labelled complementary DNA to covalent immobilized DNA probes was developed by Zhou and co-workers [36]. 

The Gen-Probe Mycoplasma TC Rapid Detection System (Eurogenetics) employs the principle of nucleic acid hybridization to detect mycoplasma in tissue culture. Using in-solution hybridization of ribosomal RNA it is possible to detect positive samples in 3 h or less. The test kit contains a 3H-labeled DNA probe homologs to mycoplasma or acholeplasma ribosomal RNA. 

Denature the probe by boiling for 5 min and add it immediately to the hybridization solution. Hybridize the nylon filter overnight at 42°C. 

Amplified DNA is identified by solution hybridization of two nonisotopically labeled oligonucleotides to one strand of the amplified DNA. Following hybrid formation DNA is bound to a solid phase and detected by enzyme-labeled specific antibodies. Because only one strand is used for detection, the other one is washed off and constitutes the main source of laboratory contamination and carryover. Take precautions to avoid the spread of these molecules to other rooms (e.g., work in a chemical fume hood, decontaminate used wash buffer with acid or sodium hypochloride, and so on). 

Recently, solution hybridization has been combined with amplification, detection, and quantification and analysis all in the same tube. These homogeneous, closed-tube, realtime assays do not require any additions, washing, or separation steps. 

Methods for homogeneous SNP analysis in a closed-tube system differ greatly in their level of complexity (Figure 37-32). The number of oligonucleotides required varies from 2 to 8. The simpler techniques do not require probes at aU, although some of the more complex techniques require up to three labels or modifications on each probe. All of these methods use fluorescence and solution hybridization. Some of the methods that use melting analysis will detect more than two alleles if present those based on aHele-specific amplification or endpoint analysis are limited to two. 

Homogeneous Solution Hybridization with Fluorescence Resonance Energy Transfer, 625... 

Williams, D.L., T.C. Newman, G.S. Shelness and D.A. Gordon. Measurement of apolipoprotein mRNA by DNA-excess solution hybridization with single-stranded probes. Methods Enzymol. 128 671-689, 1986. 

The fo5 of the acridinium ester in the hybridized probe decreases if mismatches are present. In such experiments, hydrolysis is omitted and RNA/DNA-probe hybrids are adsorbed to hydroxyapatite. Although about 80% may be hybridized, half-lives could be reduced, for some mismatches, to close to that of unhybridized probes (Arnold et al., 1989). Probes with mismatches cannot be used in solution hybridization but can be used in hybridization on solid phase where... 

Wolf et al. (1987) attached DNA to submicrometer hydrazide-functionalized latex beads and observed that hybridization rates of these conjugates were comparable to those in true solution hybridization. These beads (0.19 xm or 0.038 p,m diameter) can be collected and washed in Centricon 10 devices. 

---