Hybridization procedures

Anions of another group were derivatized with formation of gaseous chemiluminescing species. Chemical reaction - gas extraction has been used with chemiluminescence detection in the stream of canier gas in on-line mode. Rate of a number of reactions has been studied as well as kinetic curves of extraction of gaseous products. Highly sensitive and rapid hybrid procedures have been developed for the determination of lO, BrO, CIO, CIO, NO,, N03, CrO, CIO, Br, T, S, 803 with detection limits at the level of pg/L, duration of analysis 3 min. 

All of the hybridization procedures discussed in this section depend on the specific base-pairing properties of complementary nucleic acid strands described above. Perfect matches hybridize readily and withstand high temperatures in the hybridization and washing reac-... 

Although the biocompatibility and biodegradability of these materials were rapidly determined, the bioactivity of Si02-PCL hybrid materials was not studied until recently [99]. In order to provide bioactivity to Si02-PCL hybrid materials, Rhee prepared triethoxysilane end-capped poly(s-caprolactone) which was then cocondensed with tetraethyl orthosilicate and calcium nitrate via the sol-gel method. The Ca-containing PCL/silica hybrid so obtained showed in vitro bioactivity and biodegradability. The hybridization procedure between the a,co-hydroxyl PCL and silica phases was proposed to be as follows ... 

Most protocols that use RNA probes recommend stringent procedures to ensure the absence, as far as possible, of contaminating RNase during the probe preparation and hybridization procedures. RNase is... 

We have developed a simple method of nonisotopically labeling sample nucleic acids, which are then hybridized simultaneously to an array of unlabeled, immobilized probes. This "reversed hybridization" procedure thus provides identification results after a single hybridization reaction. 

Chevrier, D., Rasmussen, S. R., and Gues-Jon, J. L. (1993). PCR product quantification by non radioactive hybridization procedures using an oligonucleotide covalently bount to microwells. Mol. Cell. Probes 7,187-197. 

Compared to genosensors based on GEC, the novelty of this approach is in part attributed to the simplicity of its design, combining the hybridization and the immobilization of DNA in one analytical step. The optimum time for the one-step immobilization/hybridization procedure was found to be 60 min [66]. The proposed DNA biosensor design has proven to be successful in using a simple bulk modification step, hence, overcoming the complicated pre-treatment steps associated with other DNA biosensor designs. Additionally, the use of a one-step immobilization and hybridization procedure reduces the experimental time. Stability studies conducted demonstrate the capability of the same electrode to be used for a 12-week period [66]. 

The hybridization event can be detected both with label-free or enzymatic labelling procedures. The single-point attachment of DNA can be achieved by the immobilization of biotinylated DNA on Av-GEB platform. In this case, a one-step immobilization/hybridization procedure is achieved. The capability of surface regeneration of the biocomposite electrodes allows repeated analyses with the same electrode as... 

Southern Hybridization. Our hybridization procedures are essentially those of Palmer.3 New blots are washed for 1 hr at 65° in 0.1 X SSC, 0.5% (w/v) sodium dodecyl sulfate (SDS) blots that have been used previously are treated with 0.4 N NaOH at 40° for 30 min and rinsed 3 times with 2 X SSC for 10 min each time. If a large number of blots are to be hybridized simultaneously, 10 can be sealed into one bag and 50 ml of hybridization buffer added. Hybridization buffer is 4X SSC, 10 M EDTA, 5X Den-hardt s [50 X Denhardt s is 1.0% (w/v) BSA, 1.0% (w/v) Ficoll 40 (Sigma), and 1% (w/v) polyvinylpyrrolidone (PVP type 400, Sigma)], 0.5% (w/v) SDS, and 25 /tg/ml of calf thymus DNA sonicated and freshly boiled. All except the last component are Millipore (Bedford, MA) filtered before the DNA is added. For heterologous probes we prehybridize and hybridize at 55° for homologous or highly conserved DNA we use 65°. We prehybridize for a minimum of 2 hr, then replace the buffer with fresh solution. The denatured probe DNA (see below) is diluted in 5 ml buffer and added. Probe concentrations should not exceed 50 to 100 ng/ml for buffers that do... 

It is difficult to improve on direct DNA sequence comparisons for evolution studies of the repeated DNA sequences. The only drawback is that these studies are relatively labor intensive, limiting the experimental sample to a much smaller one than can be studied using hybridization procedures. As a first step in analyzing any repeated DNA family, however, several independent copies should be sequenced in order to obtain some... 

Two alternative hybridization procedures are described one is a modified sodium phosphate method performed at high temperature,16 and the... 

For replicate copy number determinations of ribosomal RNA genes in maize and broad bean, we obtain a coefficient of variation of 8 -15% using the spectrophotometric method of DNA quantitation with hybridization Procedure A. The coefficient of variation is about 50% using the densito-metric method of quantitation with hybridization Procedure B. 

Hybridization Procedures. Filters are placed in small leakproof vials, preincubated with a medium to block all adsorptive sites not occupied by DNA, then placed within reaction vials containing buffer and the labeled probe. On completion of the incubation, the filters are washed and dried, and the radioactivity is measured. 

Hybridization Procedure. Formamide is deionized by adding 1 g of mixed bed ion-exchange resin (e.g., AG 501-X8, Bio Rad, Hercules, CA) to 10 ml of formamide, which is stirred on a magnetic stirrer for at least 1 hr and then filtered twice through filter paper. Store at —20°. Prehybridize filters in the above buffer for 4-8 hr at 45°, replace the solution with fresh... 

Heterologous Hybridization Procedure. The DNA fragment to be labeled is denatured in a small volume of distilled water (up to 12 fA) at 100° for 10 min, quick-cooled on ice, and spun down to collect the condensate. Set up a labeling reaction with 2 fA of 10 X primer buffer, 3 /A of dNTP mix, 2 fA (20 ftCi) of [a-32P]dCTP (3000 G/mmol, aqueous solution), and 12 fA of denatured DNA, on ice. Start the reaction by adding 1 /A of 2 U /fA Klenow enzyme (DNA polymerase I, large fragment) and incubate at 37° for 1 hr. Stop the reaction by adding 2 fA of 0.2 M EDTA (pH 8.0). It is optional to separate the labeled probe from unincorporated nucleotides, for example, on a 1-ml Sephadex G-50 column. 

The second scheme, which is more generally used, involves a hybrid procedure patterned after the methodology of Section 2.11. Here one distinguishes between pure condensed phases, indexed by the symbol s, and components forming homogeneous mixtures, indexed by the symbol j. For the pure condensed phases one adopts Eq. (3.6.2) in the specification of the chemical potential for species in solution it is conventional to introduce Eq. (3.5.21). The equilibrium condition for the reaction = 0 is now specified by... 

Bake the membranes in a vacuum oven (20-30 PSI) at 80°C for 30 min. Store membranes at room temperature in a plastic bag or container until they are used in the hybridization procedure (membranes can be stored for several weeks without loss of signal). Hybridize for HBV DNA as described in Subheading 3.3.4. 

FIGURE 3 Intersection approach procedure for 2D microfluidic microarray analysis. (A) Probe line printing with the radial channel plate. (B) Hybridization procedure with die spiral channel plate. Hybridization occurring at the intersections of the spiral channels and radial probe lines, shown as colored patches in die right-most disc. Used with permission from Wang et al. (30). 

Kafatos, F.C., C.W. Jones and A. Efstratiadis. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 24 1541 — 1542, 1979. 

Superparamagnetic beads, with covalently linked streptavidin, are also commercially available (Dynal, Inc., Oslo, Norway, or Great Neck, NY Advanced Magnetics, Inc., Cambridge, MA Promega). Hornes and Korsnes (1990) found that hybridization is complete within a few minutes and that it is not necessary to have a large excess of capture probe (even at 1 1, the efficiency is close to 100%). A typical hybridization procedure, excluding detection step, takes... 

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